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In course of the recent results from Wendelstein 7-X, stellarators are on the brink for assessing their maturity as a fusion reactor. To this end, stellarator specific transport regimes need detailed exploration both with appropriate systematic experimental investigations and models. A way to enhance the efficiency of this process is seen in an systematic evaluation of existing experimental data. We propose appropriate tools developed in information theory for examining large datasets. Information entropy calculations, that have proven to assist the systematic assessment of datasets in many other scientific fields, are used for novelty detection.
Potentially, as a first use-case of this holistic process, this thesis attempts to link and to develop approaches to examine the stellarator specific core-electron-root-confinement (CERC) regime. The specific interest for CERC emerges from the behavior of the radial electric field. While ion-root conditions exhibit negative radial electric fields, CERC’s positive field in the very core of fusion grade plasmas adds an outward thermodynamic force to high-Z impurities and could add to potential actuators to control impurity influx as to be examined for full-metal wall operation in large stellarators. Recently, this feature received revived intent for reactor scale stellarators.
Also, in this work, parameter regions close to the transition from ion-root to CERC are
examined. At lower rotational transform (a characteristic feature of the magnetic field confining fusion grade plasmas), transitions were detected when the plasma current evolved. As in smaller stellarators, it is concluded that low-order rationals and magnetic islands are related to the transitions. This is widely supported by extensive MHD simulations which finally provide indications for the role of zonal flow oscillations. As one of the outcomes, gyrokinetic instabilities are seen interacting for the first time with the neoclassical mechanisms in experiments.
In order to cope with the vast number of highly sampled spatio-temporal plasma data, new
techniques for novelty detection are required. Fundamental prerequisites for the detailed
physics investigations were the feasibility study of entropy-based data analysis techniques, and their adaptation to detect previously unrevealed transition mechanisms. These tools were applied to multivariate bulk plasma emissivity data, which allowed the exploration of large parameter spaces and provided insights in the spatio-temporal dynamics of CERC transitions.
In this manner, this research highlights the feasibility of information flow measure analysis in fusion studies. Applications of different entropy-based complexity measures are explored and this work sheds light on the capabilities, added value and limitations of these techniques. This investigation presents the integration of information flow measures to gain deeper understanding of plasma transport phenomena, by providing an approach to fast systematic data mining suited for real-time analysis. This work paves the way for further development and implementation of information-theoretic methods for plasma data analysis.
In summary, this research highlights the gained insight on CERC transitions, while showcasing the feasibility, added values and limitations of information flow measure analysis for fusion studies, to induce theory based analysis revealing new insights in fundamental, stellarator-specific transport mechanisms.
Global change is one of the major challenges our society faces in recent times and is becoming increasingly noticeable in all aspects of our lives. In the last ten years, reports about droughts in Europe increased, contrary to expected natural climate variations and are attributed as indicators of climate change. Droughts resulted in a severe decrease in water levels of lakes, rivers and reservoirs, posing socio-economic and environmental challenges. Climate scenarios by the Intergovernmental Panel on Climate Change (IPCC) project increasing temperatures, more frequent, longer and/or more intense heat waves and warm spells, and an increase in aridity with short-term droughts in the upcoming decades for Western and Central Europe. Some areas – such as Northeast Germany – are already affected by negative water balances and the lowering of lake and groundwater levels. Additionally to possible challenges in water availability, excess nutrients and heavy metals from industrial emissions, agricultural fertilisers and land use changes lead to declining water quality. In the past century, extensive eutrophication and environmental pollution have become major water quality issues in many freshwater bodies.
Nonetheless, water and its availability in a sufficient quantity and quality are prerequisites for life and must be prioritised in future development. The European Union aims for a good status in all surface and groundwater bodies by 2027 regarding their ecological, chemical and quantitative status. However, a profound understanding of eutrophication, pollution sources, and water bodies' reference conditions – referring to pre-anthropogenic conditions – should be available for each system to apply integrated restoration strategies. Moreover, an in-depth understanding of long-term climate variability and its dynamics is indispensable to approach these climate change challenges and reliably predict water availability.
During the past decades, numerous paleoenvironmental studies have been carried out on Northern German sediment archives, using mainly lacustrine sediments to reconstruct hydroclimatic variability, often inferring lake-level variations as key indicators. However, most studies were carried out in areas affected by more maritime or continental climate. Studies from the transition zone are rare. Only few existing studies offer high-resolution records and/or robust chronologies, which limits the understanding of past environmental changes significantly. Besides, the Northern German lowlands have been anthropogenically affected since at least the Neolithic (~5.6 ka cal BP) and, in particular, forest composition and density have recently been shown to have at least partially an impact on lake-level variations. However, a reliable distinction between climatic impacts and anthropogenic interferences is widely missing, which is a problem because many studies were conducted on rather small lacustrine systems in which expected anthropogenic signals are higher, and single events may overprint the climatic signals. These biases lead to an incoherent picture of the past hydroclimatic variability in Northern Germany during the Holocene. To overcome this situation, it is inevitable to identify a suitable sedimentary archive from the transition zone – preferably a large lacustrine system in which natural (supra-)regional paleoenvironmental signals are expected to be not overprinted by single events. Moreover, it is necessary to establish robust chronologies and apply high-resolution methods to infer past environmental changes in a high temporal resolution. Taken together, this could contribute to an enhanced understanding of past environmental and climatic changes in Northern Germany.
This thesis consolidates the evidence for Schweriner See to act as a suitable sedimentary archive in Northern Germany for (supra-)regional climate reconstructions. Schweriner See is a large lowland lake in Northern Germany located within the transition zone from maritime to continental climate. In the first step, (paleo)lacustrine landforms, i.e. beach ridges, subaerial nearshore bar, and a silting-up sequence, are investigated along the north-eastern shoreline using a combined approach of sedimentology (e.g. grain size variations) and the relatively novel method of luminescence profiling offering relative age determinations to understand depositional processes and their chronological framework. Absolute age information is mainly inferred by OSL dating. Secondly, an important prerequisite to interpreting information obtained from lacustrine sediment archives is a thorough understanding of processes controlling sedimentation. Schweriner See is characterized by a complex morphometry, which influences in-lake processes, i.e. i) in-lake productivity, ii) carbonate precipitation and iii) wind- and wave-induced processes, resulting in a distinct spatial heterogeneity. This thesis shows that it is crucial first to understand sedimentary depositional processes and controlling mechanisms to i) select suitable coring location(s) and ii) reconstruct paleoenvironmental and hydroclimatic variations reliably.
Based on bathymetric considerations and inferred in-lake processes, two main coring locations were identified to infer i) the anthropogenic impacts and ii) hydroclimatic variations. Short sediment records from the shallow water areas (< 15 m water depth) cover the most recent environmental history of Schweriner See. A well-dated sedimentary record (210Pb/137Cs and 14C dating) links distinct sedimentary and geochemical changes with historical events. Schweriner See was extensively affected by lake-wide eutrophication and contamination, closely related to sewage and population dynamics within the catchment. The water quality only improved after the German Reunification in 1990 CE when sewage was precluded from Schweriner See. Contamination trends at Schweriner See showed similar trends to different archives along the southern Baltic Sea, implying a common regional driving mechanism, e.g. environmental legalisation.
A well-dated sediment record from the profundal zone (52 m water depth) allowed the reconstruction of large-scale atmospheric conditions during the past 3 ka cal BP by inferring winter temperature variability, the moisture source region and/or evaporative lake water enrichment, which resemble variations in the North Atlantic Oscillation (NAO). The NAO greatly influences the Central European climate, affecting, for example, surface air temperature, precipitation or storm tracks. During 3-2.8 ka and 2.1-0.8 ka cal BP, predominantly positive NAO conditions are reconstructed, which are characterized by warmer winter temperatures, moisture conditions bringing isotopically enriched precipitation from the southern/central North Atlantic to Northern Central Europe and/or warmer temperatures that may result in a higher evaporative isotopic lake water enrichment as a result of northwards displaced westerlies. Conversely, during 2.8-2.1 ka and 0.8-0.1 ka cal BP, results correspond to predominantly negative NAO phases influenced by southwards displaced westerlies. Frequent atmospheric blocking allows for the intrusion of northerly or easterly winds, resulting in colder winter temperatures, isotopically depleted precipitation from the Northern Atlantic and Arctic region and/or a lower evaporative lake water enrichment. In addition to these long-term changes in atmospheric conditions, short-term hydroclimatic variations have been reconstructed, mainly reflecting lake-level variations in conjunction with precipitation variability, with the proxy signal being additionally amplified by wind speed and wave motion. Comparisons with other archives support these results.
So far, the paleoenvironmental reconstruction is limited to the Late Holocene, but initial dating results imply possible interferences until the Late Pleistocene. Therefore, future studies should focus on extending the profundal record from Schweriner See further back in time, providing a high-resolution record covering both the Holocene and possibly the Late Pleistocene.
Pregnancy involves adaptations of the cellular composition in utero to establish a functioning fetal-maternal interface. Different subsets of leukocytes populate the endometrium and contribute to tolerance of the fetal allograft while protecting it from potentially threatening infections or rejection. ¬¬Innate lymphoid cells are recently discovered immune cells that, besides the gut, lung and skin, possess immunoregulatory functions in the female reproductive tract, especially during gestation. Although present at the fetal-maternal interface, the dynamics of ILC migration during pregnancy remains poorly investigated. The involvement of homing receptors in ILC migration to the uterus was the main subject of the present work.
First, the expression of homing receptors on ILCs from miscellaneous organs was assessed across the course of murine pregnancy in vivo by means of flow cytometry. Then, their migratory capacity towards pregnancy-relevant chemokines was investigated in vitro. The impact of pregnancy related hormones on the migration and homing of ILCs was then analysed in vitro via migration assays.
The results confirm altered proportions of ILCs in utero and the altered expression of homing receptors in ILCs in pregnancy. Different murine lymphoid organs showed augmented expression of chemokine receptors and decreased levels of homing integrin α4β7 in the first trimester, suggesting enhanced migration patterns of ILCs during early pregnancy. Subsequently, migration assays were used to demonstrate the role of different chemokine ligands in enhancing ILC migration.
Eventually, the alterations in homing receptor expression were correlated with female pregnancy hormones. Progesterone treatment caused similar effects on homing receptor expression in ILCs as observed during early gestation. These results represent the first study evaluating the effect of sex steroid hormones on ILC chemokine receptor distribution.
Taken together, our results indicate the involvement of pregnancy-relevant chemokines, including CCL4, CCL20 and CCL28, in the recruitment of ILCs to the uterus during pregnancy. The data highlight an endocrinological-immune crosstalk in the regulation of ILC homing to the female reproductive tract. Gestation alters chemokine receptor expression in order to regulate the access of immune cell subsets to the fetal-maternal interface. An adequate regulation is highly needed, as a lack or abundance of different subgroups could result in pregnancy complications, including fetal loss, pre-eclampsia or pre-term birth. Thus, the role of ILC chemotaxis to the pregnant uterus and its regulation are of interest in the understanding, prevention and treatment of the clinically relevant obstetric diseases.
The combination of the Layer-by-Layer (LbL) method, a nano-material such as carbon nanotubes (CNTs), and charged polyelectrolytes (PEs) is a reliable approach to produce highly functionalized surface coatings. These coatings are stable, controllable, ultra-thin, and most importantly, biocompatible. The ability to tune their properties by varying the preparation conditions and the terminating layer opens up a wide range of applications in the fields of biology and medicine. Here, the goal was to create electrically conductive coatings on which cells grow and proliferate. To achieve this goal, a coating with a stable conductive film structure, a suitable film surface topography, and suitable surface potential (and 𝜁-potential) must be prepared.
At the beginning of this thesis, the focus was on the fabrication of electrically conductive multilayer films, whose electrical properties should be stable and adjustable in a controlled manner (Article 1). The combination of chemically modified CNTs as polyanions, a strong linear polycation like poly(diallyldimethylammonium chloride) (PDADMA), and the LbL-method allowed us to prepare such films. Their characterization was carried out in air at ambient conditions. Since PDADMA is non-conductive, the charge transfer within the film and thus the electrical conductivity itself depends mainly on the CNTs and their arrangement. It was found that four CNT/PDADMA bilayers (BL) were always necessary to create a lateral network structure with multiple CNT crossing points to enable and support electron transport within the film. Moreover, additional CNT/PDADMA BL resulted in decreasing sheet resistance, while the conductivity remained constant at ≈ 4 kS/m regardless of the number of bilayers. Increasing the PDADMA molecular weight (Mw) from 44.4 kDa to 322 kDa did not affect film properties such as thickness or electrical conductivity.
However, increasing the CNT concentration from 0.15 mg/ml to 0.25 mg/ml in the deposition suspension resulted in thicker and less conductive films. This is attributed to a faster adsorption process of the CNTs leading to more adsorption sites for the polycation. We found an increased PDADMA monomer/CNT ratio compared to films prepared with the lower CNT concentration in the deposition suspension. The electrical conductivity decreased by a factor of four down to 1.1 kS/m, which can be attributed to fewer contact points between the CNTs. Overall, we were able to prepare stable and electrically conductive multilayer films. Additionally, by varying the preparation conditions tuning of the electrical conductivity is possible.
To fulfill requirements regarding i.e., medical implants, film properties not only have to be stable and controllable in a dry state (described in Article 1) but also in a biological aqueous environment. Therefore, in Article 2 we immersed our coated samples in three different solutions usually employed in biological research and compared their properties with their dry state, respectively. Also, hydration/swelling effects that normally occur for polyelectrolyte multilayer films (PEMs) in solutions were investigated.
For the film preparation, PDADMA (Mw = 322 kDa) and a deposition suspension of modified CNTs with two different concentrations (0.15 mg/ml and 0.25 mg/ml), which aged for two years, were used. Independent of the CNT suspension concentration, it turned out that the film thickness of the samples, prepared from the aged suspension, decreased significantly compared to the film thickness previously measured in Article 1. As a cross-check a new and fresh CNT suspension was made, which allowed us to reproduce the film thickness described in Article 1.
These results indicated that something happened with the CNT suspension over a two-year period. An analysis via X-ray photoelectron spectroscopy (XPS) showed a decrease in the percentage of functional groups in the CNTs from the aged suspension. The loss of functional groups resulted in less negatively charged CNTs and thus in fewer adsorption sites for the polycation PDADMA. Consequently, the PDADMA monomer/CNT ratio decreased, which lowered the thickness per bilayer by a factor of three, compared to films prepared with a freshly prepared CNT suspension. The lower linear charge density of the aged CNTs also enhanced their hydrophobicity, which is, in combination with the electrostatic forces, another important factor for multilayer cohesion. In contrast to PEMs made from polycations and polyanions, no swelling of the films occurred when immersed in solutions. This can be attributed to the fact that the increased hydrophobicity of the CNTs and the hydrophobic nature of the PDADMA backbone prevent the incorporation of water into the multilayer film. In solution, the films slightly shrink (by ≈ 2 nm), which makes them even more compact. Yet they remain stable. The result is an increased electrical conductivity from 9.6 kS/m, in the dry state, up to 15.3 kS/m immersed in solutions. To summarize, we showed that by tuning the interpolyelectrolyte forces the swelling and the ensuing decrease of the electrical conductivity of the films can be prevented.
Regarding the application in biology and medicine, we must consider that long-term exposure of cells to nano-materials like CNTs could lead to damage and inflammation of adjacent tissue. Therefore, it is necessary to prevent direct contact between the electrically conductive multilayer, i.e., CNT/PDADMA film, and the cells. The solution to this problem is a biocompatible top film that covers the CNT/PDADMA multilayer completely and still provides a lateral surface structure that supports cell adhesion and proliferation. Additional layers consisting solely of PEs could provide such a top film.
In Article 3 we investigated the self-patterning of PEM films as function of deposition steps. After preparation in water, the films were dried, characterized in air, and in vacuum. The films were built with high and low molecular weight PEs. PDADMA was used as polycation and poly(styrene sulfonate) sodium salt (PSS) as polyanion. The observation via Atomic Force Microscopy (AFM) showed that films prepared with high molecular weight PEs are laterally homogeneous and form no patterns, due to the chain immobility. The flat surfaces are ineligible as a substrate for cell adhesion.
In contrast, films built with a short PSS, especially at Mw, PSS = 10.7 kDa, began to self-pattern after seven deposited PDADMA/PSS bilayers. With each additionally deposited bilayer, the surface got more and more structured, from grooves over stripes to circular domains. Increasing film thickness led to an increased lateral mean distance between the surface structures. Scanning Electron Microscopy (SEM) images showed that exposure to a vacuum resulted in a decrease in the film thickness attributed to water removal, while the mean distance between the domains increased. Thus, by using this self-pattering process we are able to prepare PEMs with a highly structured surface. By adding PDADMA/PSS bilayers, not only the CNT/PDADMA film can be covered completely, but also a suitable surface morphology for cells can be created. Controlling the number of deposited bilayers allows the preparation of suitable coatings for cells.
To further improve the interaction of the cell and coated substrate not only the lateral structure but also the interacting electrostatic forces between cells and substrate are important for the nature of cell adhesion, function, and proliferation. In Article 4 we investigated PEMs, consisting of strong PEs with a low (PDADMA) and high (PSS) linear charge density. We performed asymmetric force measurements with the help of the colloidal probe technique (CP). Here, the forces between a PEM-covered surface and a colloidal probe (silica sphere) glued to a cantilever were investigated. The colloidal probe was either bare or covered with polycation poly(ethylenimine) (PEI). The surfaces were immersed in NaCl solutions with different ionic strengths (INaCl), starting with deionized water, then enriched up to 1 mol/L NaCl. The interaction force between a CP and the surface was measured. Thus, insight into the surface potential/charge was obtained.
During film preparation, two growth regimes (parabolic and linear) exist. These regimes and the terminating layer determine the surface force of the PEM. PEMs with a terminating PSS layer are predominantly flat and negatively charged when the ion concentration is low and the film is in the parabolic growth regime (between 1 and ≈ 15 BL). This indicates charge reversal on PSS adsorption. At the transition point between the parabolic and linear growth regimes, the ratio between polyanion and polycation monomers starts to switch and some cationic monomers are neutralized not by anionic monomers but by monovalent ions. Therefore, the surface charge density in diluted NaCl solutions changed from slightly positive near the transition to positive in the linear growth regime. At the lowest ionic strengths (INaCL) the range of the surface potential goes from – 40.5 mV (9 BL, parabolic) up to + 50 mV (19 BL, linear).
In contrast, polycation (PDADMA) terminated films are overall positive in diluted NaCl solutions. At the beginning of the parabolic growth regime, the layers are more compact and flat. However, with each additional layer deposited, the film becomes less compact and the chains begin to loosen. The now more loosely bound chains start to protrude into the solution and form pseudo-brushes. This could already be observed for 10.5 BL.
It intensifies in the linear growth regime (begin at ≈ 15 BL) and results in steric surface forces. Changing the surrounding INaCl affects this behavior and the pseudo-brushes scale as polyelectrolyte brushes.
By controlling the number of bilayers (thus the growth regime), the surrounding ionic strength, and the conformation of PEs at the PEM surface, it is possible to prepare a suitable range of surface properties i.e., for cell adhesion and proliferation. To prove that these multilayers can provide a suitable surface and have a positive effect on cell behavior, we coated in Article 5 titanium-covered samples with PEMs. Investigated was the cell interaction with the surface at different zeta(ζ) - potentials, a parameter for dynamic surface potential. Here the cell activity is measured by the mobilization of calcium (Ca2+) within the cell as a function of the ζ - potential of the substrate and the externally applied electrical potential. The cell activity indicates if the ζ - potential, provided by the sample surface, is suitable or not for the cells. The favorable interaction with the substrate is also reflected in the cell morphology and proliferation. The results showed that highly negative ζ - potentials between - 90 and - 3 mV led to a decreasing/reduced Ca2+ mobilization which correlates with reduced cell activity. Nearly neutral to moderate positive surfaces (ζ - potential + 1 to + 10 mV) i.e., PSS-terminated PEMs are able to promote cell adhesion and growth as demonstrated by an increased Ca2+ mobilization. The access to the intracellular Ca2+ stores, provided by the external stimulus, is now more effective and suggests a higher cell activity. Increasing the ζ - potentials up to ≈ + 50 mV (highly positive), i.e., PDADMA - terminated PEMs with pseudo-brushes, resulted in restricted cell viability and impaired Ca2+ mobilization, which led to a disturbed cell morphology and proliferation. In conclusion, only surfaces, terminated with i.e., PEI, with moderate positive charges (ζ - potential + 1 to + 10 mV) are able to improve the Ca2+ mobilization and thus the cell activity and proliferation. PEMs with a PSS termination provide negative 𝜁−potentials, onto which cells adhere, and proliferate. Therefore, they are a good alternative for surface functionalization for implant surfaces. In summary, the objective set at the beginning of the thesis is addressed within articles written as part of this thesis. It is possible to fabricate PEMs with modified CNTs to produce coatings that are electrically conductive with tunable sheet resistance, whether dry in air or immersed in an aqueous solution (Articles 1 and 2). Also, for pure PEMs, it is shown that with the right molecular weight of PEs and a certain number of bilayers, a suitable surface structure for cell adhesion can be produced (Article 3). Additional surface properties such as a suitable surface charge density can be provided by PEMs which can improve the cell activity as monitored with Ca2+ mobilization (Articles 4 and 5). The next step is to combine the knowledge gained from Articles 1 – 5 and link it to the application of external electrical fields to cells.
During infections, innate immune cells are crucial for initiating a pro-inflammatory immune response and clearing the invading pathogen. Delay in pathogen clearance or initiation of an immune response due to impaired functionality of immune cells can result in devastating consequences. The cellular compartment of the innate immune system comprises an array of specialized cell types: Macrophages are tissue-resident professional phagocytes that clear cellular debris, pathogens, and foreign objects. Dendritic cells (DCs) are immune sentinels specialized in antigen uptake and subsequent T cell priming. They are primary sources of cytokines in response to infection. Neutrophils are efficient effector cells that respond rapidly to infection and clear bacteria by different mechanisms. If effector mechanisms of these cells are affected by either bacterial or other factors, infections might not be resolved and can spread throughout the host. Cobalt-chromium-molybdenum biomaterial is widely used in arthroplasty. Implant-derived wear particles and ions lead to macrophage-driven adverse local tissue reactions: Such reactions have been linked to an increased risk of periprosthetic joint infection after revision arthroplasty. While metal-induced cytotoxicity is well characterized in human macrophages, direct effects on their functionality remain elusive. In Paper I, we show that local peri-implant tissue is exposed to Co and Cr in situ. Influx of macrophages is also evident. Exposure of isolated human monocytes/macrophages to Cr3+ in vitro had only minor effects. However, exposure of monocytes/macrophages to pathologic concentrations of Co2+ significantly impaired both phenotype and functionality. High concentrations of Co2+ induced loss of surface markers, including CD14 and CD16. Both Co2+ and Cr3+ impaired macrophage responses to Staphylococcus aureus infection. Co2+ -exposed macrophages, in particular, showed decreased phagocytic activity. These findings demonstrate the immunosuppressive effects of locally elevated metal ions on the innate immune response. Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild to severe necrotizing soft tissue infections (NSTIs). In the host environment hypervirulent GAS variants carrying mutations within the genes encoding for control of virulence (Cov)R/S two component system are enriched. This adaptation is associated with loss of SpeB secretion. In Paper II, we show that in vitro infections with hyper-virulent GAS variants harboring dysfunctional CovR/S suppress secretion of IL-8 and IL-18 by human monocytic cells. This phenotype was mediated by a caspase-8 dependent mechanism. Knockout of streptococcal SLO in a GAS strain carrying functional CovR/S even increased secretion of IL1β and IL-18 by moDCs. Of 67 fully sequenced GAS NSTI isolates, 28 contained covS or covR mutations that rendered the TCS dysfunctional. However, no differences in systemic IL-8 and IL-18 were detected in these patients. GAS isolates recovered from patients often display a mixed phenotype, consisting of SpeB positive (SpeB+ ) and SpeB negative (SpeB- ) clones. Irreversible loss of SpeB expression is often caused by loss of function mutations in regulatory components (CovR/S, RopB). Loss of SpeB is often associated with hyper-virulence. In Paper III, we show that the host environment induces transiently abrogated secretion of SpeB by GAS. Tissue inflammation, neutrophil influx, and degranulation correlated with increased frequencies of SpeB- GAS clones. Isolates recovered from tissue expressed but did not secrete SpeB, which was reversible. Neutrophilderived ROS were identified as the main factor responsible for abrogated SpeB secretion. Hyper-virulent SpeB- clones also exhibit better survival within and induce excessive degranulation of neutrophils.
In their idealized forms, enzymes can facilitate complex reactions with extreme specificity and selectivity. Additionally, in this imaginative form, they only require mild reaction conditions, resulting in low energy consumption, and they are biodegradable, efficient, reusable, and sustainable. Unfortunately, this idealized form often deviates significantly from reality, where enzymes are more likely to be associated with marginal stability and low reaction rates, leaving them less than desirable for many industrial applications. As such, if we could master the process of engineering the configuration of a protein towards a given task, the implications could be staggering.
This thesis aims to contribute to the process of protein engineering, mainly how computational tools can be used to make the protein engineering process more efficient and accessible.
Article I explores the current state of the art in machine learning-guided directed evolution and serves as a foundation for Article II, which is a concrete application of these techniques to an engineering campaign. Despite successfully improving overall activity and selectivity, we also observe limitations and constraints within the methodology. Article III then delves into these drawbacks and attempts to lay the foundation for a more generalizable and, more importantly, efficient engineering workflow, balancing the strengths and weaknesses of computational techniques with advances in gene synthesis. We then validated this novel pipeline in Article IV, where we show the potential of this methodology. Article V describes a more standard protein engineering campaign on squalene-hopene cyclases for potentially interesting products in the flavor and fragrance industry. Lastly, Article VI outlines a PyMol plugin for molecular docking.
Materials and Methods:
Literature search was conducted using electronic MEDLINE/ Cochrane databases, relevant references, citations and hand search was conducted. Academic Colleges were contacted to identify relevant studies and full texts. Inclusion Criteria were Randomized clinical trials, human cohort and case series reporting increasing vertical dimension and restoring worn dentition in adults suffering from tooth wear using fixed, minimal invasive and adhesive techniques.
The search period spanned from 2000 up to January 2023. Of the 550 articles identified, 111 went throw full text screening for eligibility and 12 studies were included in our study.
Failure, follow-up period for all the studies were assessed. Attrition , Bruxism, increase of vertical dimension rates and mean time of failure were calculated using random effect models.
Results:
Tooth wear was reported equally in anterior and posterior region, and restorations were done in direct and indirect manners, mostly with pre-evaluation of the needed increase of vertical dimension. The mean of increased vertical dimension was 2.3 mm. The mean observation time of the restorations was 41.3 months with a minimum observation period of 12 months and a maximum of 84 months. Failure rate of all the included studies was 9.9% (95% CI: 91.00 % to 95.80%) high heterogeneity was detected in Failure rate with I2 of (93.85). Time to fail was calculated to be 37.5 months for 2458 cases (95% CI:32.32 to 91.13%). A fixed effect model was performed to calculate the event of increasing vertical dimension , the event rate was 81.72% (95% CI: 0.00 to 88.10%).
Conclusion:
The performance of direct and indirect restorations is satisfactory, and the failure rate is low, which leads us to conclude that these non-invasive restorations are a reliable and cost-effective middle-term treatment method to restore vertical dimension in moderate to severe worn dentition.
Conflict of interest: None
Our study examined whether potentially critical indications from depression questionnaires, interviews, and single items on suicidal ideation among partici-pants in a large prospective population-based study are related to short-term sui-cides within one year. For this purpose, we studied the association between (a) the severity of depressive symptoms according to the M-CIDI and the PHQ-9, BDI-II, and CID-S depression screening and (b) elevated scores on single sui-cidal ideation items and mortality according to claims databases.
In the baseline cohort, the frequency of depressive symptoms measured by CID-S was 12.90% (SHIP-START-0). The frequency for “Moderate” to “Severe de-pression” measured by the PHQ-9 (≥ 10 points) and BDI-II (≥ 20 points) ques-tionnaires ranged from 5.40% (SHIP-LEGENDE) to 8.80% (SHIP-TREND Morbid-ity follow-up). The 1-month prevalence of unipolar depression, measured by the M-CIDI in SHIP LEGENDE, was 2.31%.
Between 5.90% (SHIP-TREND Morbidity follow-up) and 6.60% (SHIP-LEGENDE) of respondents showed a certain degree of suicidal ideation in the two weeks preceding the assessment, according to BDI-II and PHQ-9.
Our results show the high frequency of depressive symptoms in the study region, with women being affected more frequently than men, especially in the higher categories. Furthermore, women were more frequently affected by suicidal idea-tion, although this difference was not evident in the highest categories.
There was one potential suicide in the year after a SHIP examination.
From our results, we cannot conclude that severe self-reported symptoms from depression questionnaires should be reported back to participants of an obser-vational population-based study to prevent suicide deaths within one year.
Clear cell renal cell carcinoma is the most frequent malignant kidney tumor in adults. It is often associated with biallelic VHL mutations. We characterized our cell lines according to their HIF expression pattern. Cell lines RCC4, UOK-220 and CaKi-2 were assigned to subtype H1H2, cell lines 786-O and A-498 were assigned to subtype H2 and cell lines CaKi-1 and Rc-124 were assigned to subtype VHLwt.
Previous work of our group had shown, that p53 can be activated in ccRCC following irradiation but is not able to induce apoptosis. An important link to p53 activity with strong antiapoptotic qualities is the NFB pathway, which can be activated e. g. by irradiation.
We irradiated the three subtypes previously established as well as two control cell lines, SAOS-2 (p53 negative cell line) and HEK 293 (contains functioning p53) with 2 Gy and 10 Gy and analyzed several proteins of the pathway by using western blots. Several target genes with antiapoptotic qualities were analyzed by using rt-PCR.
We found, that out of the cell lines analyzed, both control cell lines (SAOS-2, HEK 293) showed the strongest response (activation of the NFB pathway) following irradiation. Among the three ccRCC subtypes the VHLwt cell lines showed the strongest response. H2 cell lines showed almost no response at all.
A connection between the missing ability of p53 to induce apoptosis and an induction of transcription factors by the NFB pathway could not be determined. We also could not determine biological differences between the subtypes.
We analyzed, whether any NFB proteins were present in the nucleus following irradiation and found, that only p50 homodimers were present in the nucleus. This might point towards p50 homodimers, which have been described to have different characteristics than heterodimers. More research is needed to analyze this important finding.
With high prevalence and mortality, myocardial infarction constitutes a social and economic burden in Germany and worldwide. Current guidelines for MI treatment require prompt reperfusion to salvage heart tissue and minimize short- and long-term complications. However, there are currently no treatments available to attenuate reperfusion injury. Ischemic as well as pharmacological post-conditioning have been identified as important clinical strategies to improve outcome. Membrane stabilizers, like Poloxamer 188 (P188), have been shown to improve myocardial ischemia reperfusion (IR) injury and mitochondrial function but have not yet been proven to directly offer mitochondrial protection. Mitochondrial function is crucial for cardiomyocyte function, and mitochondrial dysfunction plays an important role in myocardial injury.
In this study, hearts from 79 Sprague Dawley rats were isolated and perfused ex-vivo with oxygenated Krebs Buffer for 20 min before 30 min of no-flow ischemia. Hearts were reperfused for 10 min with Krebs buffer or 1 mM P188. Cardiac mitochondria were isolated with 1 mM P188 vs 1 mM polyethylene glycol (PEG) vs vehicle by differential centrifugation. Mitochondrial function was assessed as adenosine triphosphate (ATP) synthesis, oxygen consumption and calcium retention for complex I and II substrates of the respiratory chain.
An improvement of myocardial function with 10 min P188 post-conditioning could not be shown. Direct mitochondrial protection of P188 or PEG could not be observed in this model either. Further research is needed to ascertain whether P188 has a direct protective effect on mitochondria and, if so, on what pathways of IR injury it acts.
The maintenance of protein homeostasis in muscle by degradation systems, e.g. the autophagy lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS), is of great importance. It prevents the accumulation of nonfunctioning and not properly folded proteins, which can lead to protein aggregate myopathies (PAMs) and several other protein storage diseases. Degradation by the UPS depends on the transfer of ubiquitin to a target protein. This happens in a cascade of E1-E2-E3 proteins. This process is also involved in protein location and regulation of protein activity. E3 ligases are often tissue specific. Muscle RING-finger proteins (MuRFs) are a family of really interesting new gene (RING)-Finger E3 ubiquitin ligases, that are almost exclusively expressed in the striated muscle. They play a role in muscle wasting, but are also important for the maintenance of the structure of striated muscle. MuRF proteins are also involved in the regulation of the striated muscle energy metabolism. Previous work has demonstrated that MuRF1/MuRF3 DKO mice show a protein surplus myopathy characterized by an accumulation of myosin heavy chain proteins in striated muscles and a reduction in function of both heart and skeletal muscle. The aim of this study was to test the hypothesis that the myopathic phenotype of MuRF1/MuRF3 DKO mice is mediated by a disturbed energy homeostasis in the heart and skeletal muscle, with focus on mitochondrial function. Because sex-specific differences have not been investigated in these mice so far, a further aim was to investigate any differences between male and female mice.
To test these hypotheses, we measured the weight of the heart and the hindlimb muscles tibialis anterior and soleus to detect a possible hypertrophy in the DKO mice. Hematoxylin and eosin staining of histological cross sections of the tibialis anterior were performed to investigate protein accumulations. Muscle function was quantitated via grip strength and specific force measurements. Possible changes in protein amounts were detected via mass spectrometry analyses and western blot analyses. Changes in gene expression were investigated by qRT-PCR. Coimmunoprecipitation was used to determine direct interactions between proteins. Protein stability and ubiquitination were investigated by cycloheximide (CHX) and ubiquitination assays, respectively.
DKO mice showed an increase in heart and skeletal muscle weights. Grip strength assays revealed limb weakness of DKO mice. H&E staining of histological cross sections of the tibialis anterior muscle (TA) showed protein aggregates within myofibers. Mass spectrometry analyses of proteins isolated from TA and heart muscle revealed an increase of muscle stress markers and structural proteins in DKO mice, while proteins involved in the energy metabolism were reduced. Especially interesting here were the proteins of the mitochondrial electron transport chain (ETC), which play a major role in the energy production of the mitochondria by catalyzing the phosphorylation of ADP to ATP, the universal energy carrier in all living organisms. These changes were more pronounced in TA compared to heart. Western blot and qRT-PCR results of ETC subunits supported our proteome data. They also revealed a sex-specific difference, in which the reduction ETC subunits was more pronounced in females than males. In female
TA NDUFB8, SDHB, UQCRC2, MTCO1 and ATP5 were significantly reduced compared to controls, while only UQCRC2 and ATP5 were decreased in male TA compared to controls. A significant reduction in gene expression of Ndufb8, Sdhb, Mtco1 and Atp5 was detected in TA of female mice compared to controls, while only Ndufb8, Sdhb and Atp5 were decreased in male TA compared to controls. We observed the same pattern in Heart of male (protein: NDUFB8; mRNA: Mtco1) and female (protein: UQCRC2, MTCO1, ATP5; mRNA: Sdhb, Mtco1) DKO mice compared to their controls. The reduction in ETC subunits was paralleled by a reduction in complex I and complex III activity in the TA of DKO mice, but not in heart. However, this was only significant in the TA of female but not male mice. Mechanistical analyses using coimmunoprecipitation, cycloheximide chase and ubiquitination assays showed that MuRF1 physically interacted with the transcriptional repressor histone deacetylase 5 (HDAC5), mediated its ubiquitination as well as its UPS-dependent degradation. The absence of MuRF1 and MuRF3 in DKO mice let to an increase in the amounts of HDAC5 in TA. Because HDAC5 binds to PGC-1α, the master regulator of mitochondrial biogenesis (encoded by Ppargc1a), we investigated its gene expression in DKO muscle and found it to be reduced.
These data connect MuRF1 and MuRF3 directly to the striated muscle energy metabolism, by regulating mitochondrial function. The results provide insights into the development of PAMs and possibly other protein storage diseases, where a decrease of mitochondrial function has already been described.
Responses of bovine and human neutrophils to members of the Mycobacterium tuberculosis complex
(2023)
PMN are one of the most important cells of the innate immune system and are responsible for fast clearance of invading pathogens in most circumstances. The role of human PMN during mycobacterial infection have been widely studied. Nevertheless, there are contradicting results regarding their role in protection or pathology during TB. Similar studies focusing on bovine PMN and their role in M. bovis infection remain understudied. Also, not much is known about attenuation of M. tb in cattle and responses of PMN to this MTBC member.
The major aims of this study were to i) gain insights into bovine PMN biology and the cellular processes triggered by challenge with virulent mycobacteria and to ii) find out whether interspecies differences result in different outcomes upon in vitro challenge. In the first part of the work, a new isolation method for bovine PMN from whole blood was developed. Human and bovine PMN have different buoyant properties and hence need to be isolated using different procedures. The magnetic isolation method developed within this thesis is robust and results in very good yields of highly pure, viable bovine PMN populations. This is extremely advantageous and indispensable for downstream functional assays that are required to be performed on a single day.
The second goal of this study was to compare and contrast the functional differences between bovine and human PMN upon BCG infection. The findings reveal for the first time that human PMN phagocytose more BCG in comparison to bovine counterparts. Non-opsonized bacteria were internalized via the lectin-like C-domain, require cholesterol and an active cytoskeleton in human PMN, whereas opsonized bacteria entered cells via the CR3 and, in particular, CD11b. It remains unresolved why bovine PMN reacted differently, notably phagocytosis remained unaltered, to various treatments, including blocking monoclonal antibodies to CD11b and chemical inhibitors altering the cell membrane. Nonetheless, the increased uptake of BCG by human PMN correlates to more potent response of these cells in functional assays in comparison to bovine PMN. No PMN intrinsic differences were found in the basal cholesterol content. Comparative assays with the virulent strains would be essential in order to generalize these observations.
The third aim was to investigate the responses of bovine PMN to BCG, M. tb and M. bovis. While there was no difference in uptake between BCG and M. tb, serum opsonized BCG was taken up at a higher amount. This finding suggests differential binding of bacterial epitopes to host cell receptors which modulates mycobacteria uptake. However, between the virulent strains M. tb and M. bovis, the human-adapted bacillus was phagocytosed at a higher rate which hints towards the possibility of rapid recognition and clearance of M. tb in bovine host thereby possibly preventing pathology. The release of selective cytokines by PMN post infection with the virulent strains offers baseline information relevant for processes that probably occur in vivo. This work for the first time provides insights into responses of bovine PMN to mycobacteria in a two-tier approach: by cross-species analysis of PMN responses to selected mycobacterium and by head-to-head analysis of bovine PMN to animal-adapted and human-adapted mycobacteria.
As a prospect for future research in bovine PMN biology in the context of mycobacterial infection, it would be highly advantageous to compare the subcellular localization of M. tb and M. bovis in bovine PMN using confocal and/or electron microscopy. This analysis would confer proof on attachment or internalization of mycobacteria by PMN and identify the features of the mycobacteria-containing compartments. Also, in-depth investigations of additional entry pathways for the pathogen in bovine cells would be informative for unlocking downstream cell signaling events. In addition, PMN viability studies will be meaningful particularly in bovine PMN challenged with M. bovis and M. tb, given the impact of death patterns on tissue pathology. Current results and follow up studies will contribute to the understanding of the roles of PMN in controlling elimination or growth of M. bovis and M. tb in cattle.
In this work, 2-dimensional measurements in the THz frequency range with self-made spintronic THz emitters were presented. The STE were used to optimize the spatial resolution and determine the magnetization in geometric shapes. At the beginning, various combinations of FM and NM layers were produced and measured to achieve an optimal composition of the STE. The layer thickness of the ferromagnetic CoFeB layer and the nonmagnetic PT layer was also varied. The investigations have shown that a layer combination of 2 nm thick CoFeB and 2 nm thick Pt, applied to a fused silica glass substrate and covered with a 300 nm thick SiO2 layer, emits the highest THz amplitude. Based on these, a structured sample, consisting of an STE and an additional layer system of 5 nm Cr and 100 nm Au, was produced. Further, three wedge-shaped structures were removed from the gold layer by an etching process so that the THz radiation generated by the STE can pass through these areas. This enables the optimization of the resolution of the system. For this purpose, the sample was moved perpendicular to the laser beam by two stepping motors with a step size of 5 μm and imaged 2-dimensionally. By reducing the step size to 0.2 μm, the beam diameter could be measured at the edge of the structure using the knife-edge method. Based on this measurement, the resolution of the system could be determined as 5.1 ± 0.5 μm at 0.5 THz, 4.9 ± 0.4 μm at 1 THz, and 5.0 ± 0.5 μm at 1.5 THz. These results are confirmed by simulations considering the propagation of THz wave packets through the SiO2. The expansion of the FWHM of the waves, passing through the 300 nm thick layer, is about 1%. Only a SiO2 layer with a thickness in the μm range occurs an expansion of around 10%. This shows that it is possible to perform 2-dimensional THz spectroscopy with a resolution in the dimension of the exciting laser beam by using near-field optics. Afterward, the achieved spatial resolution was used to investigate the influence of external magnetic fields on the STE and the emitted THz radiation. By implementing a pair of coils above the sample, an external magnetic field could be applied parallel to the pattern. The used sample was designed in such a way that only certain geometric areas on the fused silica glass substrate were coated with an STE so that THz radiation is emitted only in those areas. The 2-dimensional images show the geometric structures for f = 1.0 THz and f = 1.5 THz clearly. By applying a permanent, positive magnetic field (+M), a positive course of the THz amplitude can be seen. A rotation of the magnetic field by 180° (-M) leads to a reversal of the orientation of the emitted THz radiation, whereby the magnetic field does not influence the corresponding frequency spectrum. By using minor loops, the sample was demagnetized by the constant reduction of the magnetic field strength with alternating magnetic field direction. The 2-dimensional representation of the pattern with a step size of 10 μm shows that the sample was demagnetized since both, positively and negatively magnetized structures, could be imaged. In addition, in the 2nd row from the top, a completely demagnetized circle and a rectangle with a division into two domains can be seen. These structures have both positive and negative magnetized areas, which are separated by a domain wall. To investigate this in more detail a 2-dimensional measurement of the divided regions was made with a step size of 2.5 μm. These images confirm the division of the structures into positive and negative domains, separated by a domain wall, which was verified by Kerr-microscope measurements. Both data show a similar course of the domains and the domain wall. However, to be able to examine the domain wall more precisely using 2-dimensional THz spectroscopy, the resolution of the system must be improved to a range of a few nm, because the expected domain wall width is between 𝑙𝑊 = 12.56 nm and 𝑙𝑊 = 125.6 nm. The improved resolution would make it possible to image foreign objects, such as microplastics in biological cells or tissue. For this purpose, different plastics, such as polypropylene, polyethylene, and polystyrene, were investigated in the THz frequency range up to 4 THz. While no specific absorption could be determined for PP, characteristic absorption peaks were found for PE and PS. The energy of the photons with a frequency of about 2.2 THz excites lattice vibrations in the PE. Therefore, this frequency is specifically absorbed, and the intensity in the transmission spectrum is lower than for other frequencies. PS absorbs especially THz radiation with a frequency of 3.2 THz. In addition, all of the investigated plastics are mostly transparent for THz radiation, which makes imaging of these materials feasible. Based on these basic properties, it will be possible to image and identify these types of plastic.
Until today, more than 17% of the population in Mecklenburg Western-Pomerania suffer from chronic kidney disease (CKD) which was revealed by the SHIP study (Study of Health in Pomerania). 20% of CKD cases can be traced back to glomerulopathies. One common characteristic of glomerulopathies is the morphologic change of the glomerular filtration barrier which consists of endothelial cells, the glomerular basement membrane and podocytes. Under healthy conditions, the foot processes of the podocytes interdigitate with the foot processes of the neighboring podocytes with a filtration slit in between. Apart from the slit membrane protein nephrin, typical adherens junction proteins like occludin or JAM-A are also expressed at this cell-cell junction. This junction is therefore considered to be a specialized type of adherens junction, necessary to maintain the size-selectivity of the filtration barrier. During podocyte injury, podocyte foot processes lose their characteristic morphology and the typical meandering filtration slit becomes linearized, a process which is described as foot process effacement.
Since morphological change is directly linked to change or loss of function, ultrastructural analysis of the foot processes is necessary for diagnostics and research. By using 3D-structured illumination microscopy (3D-SIM), we quantified these morphological changes as well as studied a possible biomarker, the tight junction protein claudin 5 (CLDN5). Our study showed a spatially restricted up-regulation of CLDN5 in effaced filtration slit areas in biopsies of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in mice after NTS injection and in the uninephrectomy DOCA-salt mouse model. CLDN5/nephrin ratios of biopsies from patients with glomerulopathies and of tissue received from NTS-treated mice were significantly higher compared to controls. We found that in patients the CLDN5/nephrin ratios were negatively correlated with the filtration slit density. Since CLDN5 up-regulation was observed in several areas of high filtration slit density, we hypothesized that CDLN5 upregulation preceded visible foot process effacement. Taken together, we suggest that CLDN5 could be a helpful biomarker to identify an early change of the foot process morphology in addition to filtration slit density measurement. Additionally, correlation analysis of foot process effacement with patient data showed a significant negative correlation of the filtration slit density with proteinuria in MCD patients.
Tafazzin is an acyltransferase with key functions in remodeling of the mitochondrial phospholipid cardiolipin (CL) by exchanging single fatty acids species in CL. Tafazzin-mediated CL remodeling determines the actual CL compositions and has been implicated in mitochondrial morphology and function. Thus, any deficiency of tafazzin leads to altered fatty acid composition of CL which is directly associated with impaired mitochondrial respiration and ATP production. Mutations in the tafazzin encoding gene TAZ, are the cause of the severe X-linked genetic disease, BARTH syndrome (BTHS).
Previous work provided first hints on a linkage of CL composition and subsequent limitations in the cellular ATP levels which may contribute to the restriction of growth. However, in C6 cells ATP levels remained unaltered due to compensatory activation of glycolysis. Moreover, it has been demonstrated that the substantial changes in CL composition are similarly resulting from knocking down either cardiolipin synthase (CRLS) or TAZ. This has also been shown in C6 glioma cells. Most notably only the knock down of TAZ, but not that of CRLS, compromised proliferation of C6 glioma cells. Therefore, a CL- independent role of TAZ in regulating cell proliferation is postulated.
In this study, any linkage of the lack of tafazzin to cellular proliferation should be investigated in more detail to allow first insight into underlying mechanisms.
The results of the current study demonstrate that the tafazzin knockout in C6 glioma cells show changes in global gene expression by applying transcriptome analysis using the- microarray Clarion S rat Affymetrix array. Out of 22,076 total number of genes detected, 1,099 genes were differentially expressed in C6 knockout cells which were either ≥2 and ≥4 fold up or down regulated genes. Furthermore, expression of selected target genes was validated using RT-qPCR. We have hypothesised that the changes in TAZ dependent gene expression is via PPAR transcription factor. According to eukaryotic promoter database (EPD) for selected target genes, exhibited at least one putative binding site for PPARG and PPARA transcription factors. However, pioglitazone and LG100268, synthetic ligands of PPARG and RXR, could not show any effect on changes in gene expression in C6 TAZ cells. Another class of cellular lipids, oxylipins were found to occur in significantly higher amounts in C6 TAZ cells compared to C6 cells which makes them candidates for mediating cellular effects and regulating gene expression via PPARs. A computational tool CiiiDER was used to for the prediction of transcription factor binding site. The transcription factors enriched in TAZ- regulated genes were found to be HOXA5 and PAX2, binding sites of which could be detected in 100 % of TAZ- regulated genes (>2-fold). By applying IPA to the differentially expressed genes we could identify lipid metabolism, and cholesterol superpathway in particular as the most affected pathway in C6 TAZ cells. This pathway consists of 20 genes, of which all (20/20) appeared to be differentially regulated in C6 TAZ cells. Of all the 20 genes, 4 of the differentially expressed genes were selected for further validation by RT-qPCR. By IPA it was possible to identify the upstream regulators that might be responsible for the differential expression of genes in C6 deficient cells. Some of the genes ACACA, HMGCR, FASN, ACSL1, 3 and, 5 identified was decreased by predicted activation and inhibition of the regulators. Further we have analysed the levels of cellular cholesterol content in C6 and C6 TAZ (w/o Δ5 and FL) cells. In C6 cells cholesterol is present more in its free form. C6 TAZ cells have increased amount of cholesterol compared to C6 cells. However, Δ5 and FL expressed C6 TAZ cells showed less amount of cholesterol.
Previous work established that knockout of tafazzin in C6 cells showed decreased cell proliferation in the absence of any changes in ATP content. To understand this phenomenon cellular senescence associated β-galactosidase in C6 and C6 TAZ cells was performed. C6 TAZ cells showed increased percentage of β-gal positive cells compared to C6 cells. Moreover, senescent associated secretory phenotype (SASP) represented by e.g. CXCL1, IL6, and IL1α was determined using RT-qPCR. Gene expression of these SASP factors was significantly upregulated in C6 TAZ cells.
Several human tafazzin isoforms exists due to alternate splicing. However, whether these isoforms differ in function and in CL remodelling activity or specificity, in particular, is unknown. The purpose of this work was to determine if specific isoforms, such as human isoform lacking exon 5 (Δ5), rat full length tafazzin (FL) and enzymatically dead full length tafazzin (H69L), can restore the wild type phenotype in terms of CL composition, cellular proliferation, and gene expression profile. Therefore, in the second part, it was demonstrated that expression of Δ5 to some extent and rat full length tafazzin can completely restore CL composition, in C6 TAZ cells which is naturally linked to the restoration of mitochondrial respiration. As expected, a comparable restoration of CL composition could not be seen after re-expressing an enzymatically dead full-length rat TAZ, (H69L; TAZ Mut). Furthermore, re-expression of the TAZ Mut largely failed to reverse the alterations in gene expression, in contrast re-expression of the TAZ FL and the Δ5 isoforms reversed gene expression to a larger extent. Moreover, only rat full length TAZ was able to reverse proliferation rate. Surprisingly, the expression of Δ5 in C6 TAZ cells did not promote proliferation of the wild type. Different effects of Δ5 and FL on CL composition and cell proliferation points to the specific and in part non-enzymatic functions of tafazzin isoforms, but this certainly requires further analysis.
A long-standing controversy in emotion research concerns the question whether stimuli must be conceptually interpreted, or semantically categorized, to evoke emotional reactions. According to the semantic primacy hypothesis, the answer to this question is positive; whereas according to the affective primacy hypothesis, it is negative: Emotions can also be, and perhaps often are, elicited by preconceptual stimulus representations, such as particular shapes or color patterns.
In the present dissertation project, the semantic primacy hypothesis was tested in eight experiments using different latency judgment paradigms in which the perceptual latencies of object recognition and affect onset were measured and compared. The chronometric measurement methods comprised temporal judgments (temporal order judgments and simultaneity judgments: Publication A, Experiments 1–4; the rotating spot / rotating clock hand method: Publication B, Experiments 1–2) and speeded reaction time measurements (Publication C, Experiments 1–2). To elicit affective responses, pictures of pleasant (e.g., cats, children) and unpleasant objects (e.g., spiders, moldy food) from everyday life were presented.
According to the semantic primacy hypothesis, object recognition is a necessary partial cause of affect. This implies the following three predictions that were tested in the studies: (1) Because causes must precede their effects, the time of the onset of object recognition must precede the time of the onset of affect. (2) The longer it takes a person to recognize an object, the longer it should also take them, other factors constant, to experience affect; therefore, the latencies of the two mental events should be positively correlated across individuals. (3) An experimental manipulation that delays the onset of object recognition (in this case a moderate blurring of the pictures) should also delay the onset of affect, and the effect of the manipulation on affect latency should be mediated by the delay in object recognition.
In agreement with Prediction 1, regardless of the chronometric method used, the latency of object recognition consistently proved to be shorter than the latency of affect onset. According to the meta-analytically integrated latency differences estimated in the temporal judgment experiments, affect followed object recognition with a delay of 117 ms. This result was obtained for both pleasant and unpleasant stimuli and was independent of task order. Supporting Prediction 2, the latencies for object recognition and affect onset were positively correlated across participants (meta-analytic r = .50). Supporting Prediction 3, delaying object recognition by blurring the affective pictures was found to also delay the onset of affect and the effect of blurring on the latency of affect was found to be partly mediated by delayed object recognition.
Two additional predictions tested and confirmed in Experiment C2 were: (4) False-coloring the affective pictures delays the onset of affect but not object recognition, and this effect is mediated by reduced affect intensity. (5) Judgments of the valence of the stimuli (i.e., whether the imaged object is pleasant or unpleasant) take more time than reports of object recognition, but less time than affect onset reports, for which valence judgments have often been used as a substitute in previous studies.
Taken together, the results of the eight experiments provided consistent support for semantic primacy in the generation of pleasant and unpleasant feelings evoked by affective pictures: Object recognition can be considered a necessary partial cause of affect in the reported experiments. The results are compared to previous findings, possible reasons for deviant response patterns found in a small minority of the participants are considered, and several implications of the findings for emotion research are derived. Possible adaptations of the chronometric approach to investigate other questions of emotion research are suggested. Finally, limitations of the dissertation project are pointed out and possible ways to address these in future research are proposed.
The present study deals with the spread and population genetics of the invasive Asian bush
mosquito Ae. japonicus in Europe and Germany. Since the first detection of Ae. japonicus
in Europe in 2000, the species spread rapidly through Europe, either actively by flying or
passively by human activities. In 2017, four confirmed populations of Ae. japonicus existed
in Europe. The largest population covered western Germany, parts of France, Switzerland,
Liechtenstein, Austria and Italy. The most northern population around Hanover, Germany,
did not spread since 2013. A very small population existed in Belgium and the second largest
population covered parts of Austria, Italy, Slovenia, Croatia and Hungary. By 2019, Ae.
japonicus had established in 15 European countries.
Most of the monitoring programmes in Europe dealing with the distribution and spread of
Ae. japoncus investigate cemeteries for juvenile stages. However, activities are not
harmonised, e.g. regarding numbers of investigated collection sites and declaration of
negative sites, making data comparison between different studies difficult. Therefore,
suggestions for a standardised Ae. japonicus monitoring method have been developed and
provided.
In the present study, 445 individuals of Ae. japonicus originating from five different
European countries were investigated for population genetic analyses by sequencing parts
of the nad4 gene and genotyping seven polymorphic microsatellite loci. In total, 16 different
nad4 haplotypes were identified with haplotype H1 being the most common and widespread
one through all populations.
Within Germany, Ae. japonicus has been spreading immensely over the last decade. Even
though the present results (2017) demonstrate incipient genetic admixture of populations as
compared to previous studies (2012-2015), no complete genetic mixture has taken place yet.
The populations of Ae. japonicus still fall into two genetic clusters, but the genetic diversity
on individual level had increased considerably (from three nad4 haplotypes in 2012 to 12
according to the present thesis). Both additional introductions and mutation are possible
reasons, but determining the origin of the German populations is not possible anymore.
In the years following the invasion of Germany, Ae. japonicus spread to southeastern
Europe. In 2013, it established in Croatia, in 2017 in Bosnia and Herzegovina and in 2018
in Serbia. In the current study, immature stages of Ae. japonicus were found at 19 sites in
Croatia, two sites in Bosnia and Herzegovina and one site in Serbia. The population genetic
analyses indicate at least two independent introductions in that area. Aedes japonicus collected west of Orahovica (Croatia) seemed to be genetically similar to samples previously
investigated from Southeast Germany/Austria and Austria/Slovenia. By contrast, samples
from east of Orahovica, together with those from Serbia and Bosnia and Herzegovina, were
characterised by another genetic make-up, but their origin could not be determined.
In 2021, individuals of Ae. japonicus were detected at two collection sites in the Czech
Republic for the first time: Prachatice close to the Czech-German border and Mikulov on
the Czech-Austrian border. Population genetics and comparison of genetic data showed a
close relationship of the Prachatice samples to a German population, while for Ae. japonicus
from Mikulov close relatives could not be identified.
In the future, the global spread and establishment of invasive mosquitoes through
international trade and travel will increase. Potential vectors, like the Asian bush mosquito
Ae. japonicus, can become a problem in Europe and Germany, especially in the course of
global warming which supports pathogen transmission. Monitoring the known populations
and identifying introduction and migration routes are therefore essential for vector
managing.
The need for the diversification of utilised species has emerged in the present aquaculture
production environment. Shifts in consumer interest, climate change-induced temperature
increases, and major fish disease outbreaks have put a strain on this industry. In this context,
the pikeperch (Sander lucioperca) has become a new target species for aquaculture in Central
Europe. This new aquaculture focus species exhibits high numbers of offspring, fast growth,
and high consumer acceptance. It can also effectively deal with higher temperatures and turbid
water. However, the rate of successful rearing is still low, as various developmental
transformations and environmental effects commonly lead to high mortality rates during the
early ontogenetic stages. The aim of this doctoral project was thus to obtain insight into
embryonic to larval developmental changes during pikeperch ontogeny. Specifically, the times
of change that influence survival were of focus. Based on the available literature, particular
attention was paid to general growth patterns and the connected developmental changes, the
determination of myogenesis gene marker expression changes, and the support of animal
welfare efforts for pikeperch rearing procedures. To achieve the aims of the study, a methodical
setup consisting of morphometric and developmental observations was combined with
transcriptome gene marker analysis for the different ontogenetic stages.
Three developmental phases were differentiated during the embryo-larval transition. Each of
these possessed distinct growth patterns with different growth rates. The intermediate
threshold phase showed internal organ development that focused on digestive, neuronal, and
heart tissues. Three activity phases of myogenesis were determined: during early embryonic
development, before hatching, and after hatching during the larval stages. Therefore, muscle
development seemed to be regulated to balance energy expenditures. Additionally, two
coinciding skeletogenic phases were found. Furthermore, a cell line from whole embryos was
developed to support the replacement of animals in future experimental setups. A software
system for video analyses was developed to support rearing procedures in aquaculture
facilities. This prototype can be used to automate the counting of specimens and thus allows
for faster responses to increasing mortalities. Based on the results of this thesis project, further
insights into the early development of pikeperches were obtained. This will facilitate the design
and adaptation of raising and husbandry protocols, which can help to further establish
pikeperch as an aquaculture species and support its application in modern recirculatory
systems.
The dissertation looks at bioeconomy innovation at different levels through the lens of economic geography. By progressing from the meta to the micro-scale, it tries to find answers to how the interrelated concepts of bioeconomy and innovation are embedded in these respective contexts while consecutively concretising bioeconomy and de-fuzzing it. To do that, it adopts a mixed-methods approach that starts general and ends specific, going from the meta-scale of literature over the macro-scale of three distinct areas in which bioeconomy is discussed to the meso-level of central actors of a European funding network before, lastly, considering case studies at the micro-scale. Throughout, the thesis aims to spatialise the bioeconomy by shedding light on the term and its drivers across multiple geographic layers. It thereby not only offers new insights into dimensions of innovation in the bioeconomy but also contributes to the discipline of economic geography by applying some of its essential theoretical ideas to an emerging political framework.
Interplay of reactive oxygen species with the mechanical properties of cells and mitochondria
(2023)
Cell mechanical properties are a popular label-free method for understanding basic cellular processes. In this thesis, I used Real-time deformability cytometry (RT-DC), a high-throughput microfluidic technology, to investigate the mechanical properties of cells and mitochondria under various conditions such as increased reactive oxygen species (ROS) levels and the application of different ligand coated gold nano-particles (Au-Nps) effect on cells. Initially, we showed the possibility to measure organelles, cells, and tissue-like structures (spheroids) in a single system by constructing a virtual fluidic channel. We investigated a potential application using cytochalasin D (cyto D) treatment, which revealed increased deformation and decreased stiffness in both the normal and virtual channels. Using mechanics as a marker, I investigated the effect of excessive ROS on the mechanical properties of human myeloid precursor cells (HL60). My findings suggest that the mechanical response of HL60 cells to increased ROS levels is mediated by re-localization of microtubules toward the cell center and F-actin to the cell periphery. Interestingly, I also observed intracellular acidification, which is a largely unexplored mechanism that may have contributed to our findings. I then extended our ROS and mechanics assay to investigate cell-AuNP interactions, demonstrating that cell properties vary depending on the cell culture media and ligand coating. The results showed that dextran coated gold nano-particels (Au-Nps) had low cytotoxicity, lower ROS release, and no change in cell mechanics, indicating a potential application for dextran Au NPs. Finally, I expanded our assays to include high-throughput microfluidic characterization of isolated mitochondria. Using both exogenously and endogenously induced ROS, we found an increase in mitochondrial deformation and a decrease in their size, which could have implications on mitochondrial function, i.e., fission and fusion. We believe that advanced applications of RT-DC technology will improve the comparability of results across different sample sizes while also promoting it as a disease detection technique.
Seas and oceans are essential for the global ecosystem. Entire societies, economies and countless livelihoods rely on their good environmental status. Yet, pressures on marine environments are increasing. An extensive assessment and monitoring of marine habitats is a vital precondition for understanding these systems and their sustainable conservation. Remote sensing methods can temporally accelerate the mapping, improve the spatial resolution and support the interpretation of large areas. Hydroacoustic becomes the method of choice for areas deeper than the coastal zone as optical signals are limited by strong attenuation in the water column. Apart from depth measurements for the creation of bathymetric charts, the recording of backscatter strength is useful for the characterization of the seafloor surface. The direct influence of the inhabiting benthic community on the backscattered signal is rarely considered, although it can be utilized for the detection of benthic life. Information about habitat-specific backscatter responses or a hydroacoustic remote sensing catalog for benthic habitats is missing so far.
The multibeam echosounder (MBES) has the advantage of recording both, bathymetry and backscatter strength simultaneously with related incidence angle. Further, recent technological developments allow to change between frequencies. Angular range curves supported the quantification of backscatter strength of different frequencies. Acoustic data sets were complemented by ground truthing in form of sedimentological and biological samples as well as video profiles. Study areas were located offshore the island of Sylt in the North Sea as well as in vicinity to Oder Bank and close to the coast offshore Hohe Düne/Rostock, both in the Baltic Sea. Investigated habitats included sand areas inhabited by tubeworms, loose mussel clusters on top of sand areas, seagrass meadows, coarse sand and gravel areas, and a reef covered by mussels.
Multifrequency backscatter maps, combining frequencies between 200 kHz and 700 kHz, illustrate small-scale features at the seafloor not visible in monofrequent maps. Key habitats showed a specific backscatter response, which can partly be related to macrobenthic flora and fauna. Data sets recorded with a (partly calibrated) MBES in three different month (May, August, October) revealed that backscatter strength can further detect spatial as well as temporal habitat dynamics. Alterations in the sediment composition at the seafloor surface of the ecologically valuable coarse sand and gravel areas were caused by seasonal changes in local hydrodynamics.
A newly developed 3D seismic lander has the ability to support hydroacoustic remote sensing as an additional, non-destructive ground truthing method utilizing a high frequency of 130 kHz to image the shallow subsurface. Buried objects, e.g., stones, shells, fruit gummy worms, as well as sediment disturbances could be detected and visualized in a laboratory experiment. The 3D seismic lander is likely to improve the investigation of volume scatter contribution to backscatter strength and is potentially applicable for the imaging of bioturbation.
Until today, more than 100 years after its first description in Italy, the highly pathogenic avian influenza virus (HPAIV) has not lost its fearsome character for wild birds, poultry and humans. On the contrary, the number of outbreaks with high casualty rates in wild birds and poultry has multiplied in recent years and cases of zoonotic infections are also increasingly reported from HPAI endemic areas. The epidemiology of these infections is complex and also involves surface water and possibly sediments of shallow standing waters, which could play a role as a vector medium and/or virus reservoir. The goal of this project was to expand current knowledge of the influence of water on the spread of AIV. As part of this project, we were able to ...
1. ...improve AIV detection methods using real time RT-PCR in terms of sensitivity and breadth of viruses detected. In addition, we succeeded in economizing the procedure so that fewer resources are required and results are obtained faster (publication I: [173]).
2. ...develop an ultrafiltration-based enrichment method for AIV from surface water and evaluate it with field samples from HPAI outbreak areas in wild bird habitats (Wadden Sea coast of Schleswig-Holstein) and previously unaffected regions (Antarctic Weddell Sea) (publication II: [174]). Furthermore, protocols for testing different environmental sample matrices for AIV screening were tested and compared to results of passive monitoring by dabbing diseased or dead wild birds. AIV was detected in more than half (61%) of 44 water samples. We received additional sediment samples from 36 of the 44 water samples. In 18 of 36 of the sediments tested, as well as in 4.16% of 1705 fecal samples tested AIV was detected. However, the studies of the environmental samples mostly yielded only generic AIV detections, with viral loads in the range of the detection limit. This massively hampered further investigations for sub- and pathotyping. In contrast, 79.41% of 68 samples from passive monitoring showed high to very high HPAIV viral loads which also allowed sub- and pathotyping.
3. ...demonstrate in animal experiments that even very low titers (0.1 TCID50 ml-1) of HPAI viral infectivity in water can induce productive infection in susceptible but clinically largely resistant mallard ducks (publication III: [175]). Furthermore, we were able to develop evidence that there is a difference in virus spread that depends on the type of (contaminated) water source. This means that infections on poultry farms with inverted or nipple drinkers may follow a different course than infections in the wild, which are mediated via larger surface waters.
Overall, the results of this project highlight the important role of surface and drinking water, as well as aquatic sediments, in the spread of AIV. The methods developed here for AIV detection extend the possibilities for surveillance of AIV infections; however, passive remains superior to active surveillance of HPAIV infections in several aspects. Examination of various environmental samples did not yield a significant advantage in terms of an early warning system that would indicate the presence or spread of HPAIV in wild bird habitats prior to the occurrence of lethal infections in wild birds.
The deep geological underground represents an important georesource for the short-
term storage of renewable energy and the long-term reduction of greenhouse gas emissions. To ensure the economic viability and safety of any subsurface storage project, detailed characterisation of the quality and integrity of the reservoir and its cap rock is required. This characterisation includes the accurate determination of the petrophysical properties, such as porosity and permeability, as well as the potential mineral reactions, such as the dissolution of reactive phases, which may occur during the lifespan of such a project. Clay minerals are common components of many reservoir systems and, depending on their type and structure, can have a significant impact on storage and transport properties. These processes are, however, currently not well understood. In order to address these issues, the main focus of this thesis is on mineralogical analyses using X-ray diffraction (XRD) and microstructural studies using focused ion beam scanning electron microscopy (FIB-SEM) together with micro X-ray computed tomography (µXCT) to gain a better understanding of the influence of clay minerals on reservoir and cap rock properties.
A central part of this thesis focuses on the analysis of clay minerals and pore structures of the Bebertal Sandstone of the Parchim Formation (Early Permian, Upper Rotliegend), which is considered a natural analogue for the tight reservoir sandstones of the North German Basin. Two illite polytypes with a variety of characteristic structures have been identified in the Bebertal sandstone. Disordered 1Md illite forms the majority of the observed structures, which include omnipresent grain coatings, altered permeable feldspar grains and pore-filling meshwork structures. Trans-vacant 1M illite represents the second and youngest generation of authigenic illite and occurs as fibrous to lath-shaped particles that grew into open pore spaces and led to a significant reduction in porosity and permeability during late diagenesis. Based on these results, a model for the formation of illite polytypes in the aeolian layers of the Bebertal sandstone was developed that describes the temporal and spatial evolution of porosity and permeability during diagenesis. Information from this model was then used to improve the prediction of permeability of the Bebertal sandstone based on µXCT pore space models and direct numerical simulations. To achieve this, a micro-scale pore space model was created that allowed the simulation of permeability reduction by clay minerals by including nanoporous illite domains based on a novel morphological algorithm. By performing Navier-Stokes-Brinkman simulations, more accurate predictions of permeabilities with respect to experimentally determined values were obtained compared to conventional Navier-Stokes simulations.
The detailed characterisation of the Bebertal sandstone has shown that natural reservoir rocks are usually complex heterogeneous systems with small-scale variations in texture,
composition, porosity and permeability. Flow-through experiments on the Bebertal sandstone revealed that the coupled geochemical and hydrodynamic processes that occur during the dissolution of calcite could not be predicted by reactive transport models. Therefore, as part of this thesis, a novel approach for developing synthetic sandstones at low temperatures based on geopolymer binder was developed. It is shown that simpler and more homogeneous porous materials can be produced with porosity and permeability values in the range of natural sandstones. These can be used to better understand the dynamic and coupled processes relevant to the storage of renewable energy in reservoir rocks through improved experimental constraints.
The final part of this thesis reports on a detailed clay mineral and pore space study of
three shale formations and one mudstone that were identified as potential seals for the Mt. Simon sandstone reservoir in the Illinois Basin. During the Illinois Basin - Decatur Project, this reservoir was used for the sequestration of one megaton of supercritical carbon dioxide. In order to better assess the quality of the sealing units and to better understand the role of the intergranular clay mineral matrix as potential pathway for fluid migration, a multi-scale evaluation was conducted that included thin section analysis, quantitative evaluation of minerals by scanning electron microscopy (QEMSCAN), mercury intrusion capillary pressure (MICP) measurements, quantitative XRD and high-resolution FIB-SEM. The results allow for the classification of the studied formations into primary and secondary seals and emphasise the importance of three-dimensional clay-mineral-related pore structure characterisations in cap rock studies. XRD proved the most reliable method for the identification and quantification of clay minerals in the studied cap rocks and mudstones. In contrast, FIB-SEM and QEMSCAN provided the spacial constraints for reconstructing fluid flow pathways within the clay mineral matrix.
Overall, this thesis highlights the importance of the precise identification of clay minerals in geological reservoirs and their cap rocks. It also illustrates the need for three-dimensional characterisation and modelling of the associated small pore structures for an improved understanding of the rocks diagenetic history as well as the prediction of the transport and storage properties of these crustal reservoir systems.
Background: Intact socio-cognitive abilities, such as theory of mind (ToM), facial emotion recognition (FER), social decision making (SDM) and visual perspective taking (VPT), are essential for human well-being and quality of life. Impairment in social cognition can have major implications for health in affected individuals and society as a whole. Evidence for changes in social cognition in healthy and pathological aging processes, such as subjective cognitive decline (SCD) and mild cognitive impairment (MCI), is currently either sparse or inconclusive. It is important to determine how social cognition changes in healthy and pathological aging and provide grounds for targeted and early assessment and intervention. The aims of this thesis were to investigate social cognition across four domains, in particular, ToM, FER, SDM and VPT, in healthy young and older individuals, as well as in individuals with cognitive deficits, such as SCD and MCI. In the case of a decline, further goals were to investigate the degree of impairment and the domains affected.
Methods: A systematic literature search was conducted in four major academic databases, MEDLINE, Web of Science Core Collection, CENTRAL, and PsycInfo, for studies investigating social cognition in healthy young and old individuals as well as individuals affected by SCD and MCI which met the inclusion criteria. The primary outcome was ToM and secondary outcomes were FER, SDM and VPT. After a systematic review was performed, studies eligible for meta-analysis were divided according to comparison groups and outcomes. Random-effects meta-analyses were conducted using standardized mean differences (SMD). Risk of Bias was assessed using the “Tool to assess risk of bias in cohort studies” modified for the present study design.
Results: After a thorough systematic literature search, 86 studies containing 88 comparisons were included in the systematic review, of which 47 were eligible for quantitative analysis. The meta-analysis revealed a progressive decline in ToM and FER abilities from young adulthood to MCI. Varying effect sizes demonstrated different trajectories of change for specific domains. Due to a lack of research, data investigating SDM and VPT, as well as SCD were insufficient for quantitative analysis.
Conclusion: ToM and FER decline gradually from healthy to pathological aging. Therefore, assessment of social cognition is important and should be incorporated in routine neurocognitive testing, so that targeted interventions can be introduced when needed. With this information in mind, future research should focus on the development of new assessment tools, as well as preventive and treatment strategies. This review also identified research gaps in certain populations (e.g. SCD, middle age, MCI-subtypes) as well as domains (VPT and SDM) that need to be addressed in the future.
The skull is an extremely informative part of the vertebrate body. Skulls are
involved to hunt, feed and drink, to nurse, fight, dig, and to many other activities.
Also, main sensory organs are situated on the head in order to enable a given
animal to see, smell, taste, feel, listen, equilibrate and think; hence, the head is the
main connection to the external world. It follows that a skull, with and without soft
tissue, can tell a lot about its owner. Each skull consists of many individual bones
constituting regions (e.g., snout and braincase) that represent different aspects of an
anatomical mosaic, which in turn allows deeper (palaeo)biological insights.
In the past three centuries, palaeontologists dug out countless fossils from all
over the world and from many preserved periods and groups, including dinosaurs.
Hence, public and private collections house numerous fossil skull specimens. To
further enlighten our understanding of palaeoecological, physiological and
phylogenetic affinities of dinosaurian representatives belonging to different groups,
and in order to reveal new aspects on their (neuro)anatomy, behaviour, ontogeny
and evolution, a thoroughly examination with modern techniques is the aim of this
thesis.
In order to get a phylogenetically broad understanding, fossil remains from at
least four extinct species, including Irritator challengeri (a theropod: mostly bipedal
carnivores) from the Early Cretaceous of northeastern Brazil, Europasaurus holgeri
(a sauropod: long-necked, quadrupedal herbivores) from the Late Jurassic of Lower
Saxony, Emausaurus ernsti together with an unnamed taxon from the Early Jurassic
of Mecklenburg-Western Pomerania, and Struthiosaurus austriacus, Late
Cretaceous of eastern Austria (the latter three are thyreophorans: armoured, mostly
quadrupedal herbivores), were in closer focus. To document and digitally reconstruct
cranial bones and cavities therein, the material was examined with micro computed
tomography (microCT). On this basis, the full morphology of the preserved anatomy
was revealed, described and contextualized, for example, in conjunction with
comparative anatomy and biomechanical considerations. During this process, further
methods were used to investigate and depict individual fossils: macro- and microphotography,
photogrammetry and phylogenetic analyses, each encompassing
multiple sub-tasks and being supported by 3D prints.
As part of the result, it was possible to formulate reasoned assumptions about
the lifestyle of the taxa in focus. For instance, the neuroanatomy and the osteological
characteristics of the spinosaurid Irritator challengeri implicate that this taxon was an
agile hunter with a habitually inclined snout that was specialized in catching relatively
small prey with a robust dentition and a comparably weak - but fast - bite, with a
remarkable jaw mechanism which enabled the animal to kinetically widen the
pharynx during lower jaw depression. The (neuro)anatomy of I. challengeri, S.
austriacus, E. ernsti and E. holgeri presented here, enrich our knowledge about a
plethora of (lifestyle-related) aspects of these animals, their closer relatives and the
prehistoric world they lived in.
External effects of agglomeration and human capital is a more than a century old research topic. Their theory and empirics have evolved over time to interact with more economic factors and to dig more deeply into heterogeneous effects, whose empirical evidence remains scarce, especially for developing countries. Furthermore, local human capital and agglomeration are often introduced in separate models rather than a single model in explaining productivity. These limitations motivate the implementation of this thesis. To achieve this, firm-level panel data from Vietnam is employed. This developing country provides an interesting case because its level of agglomeration and human capital is still low but its socio-economic conditions are highly dynamic with the high growth rates of urbanization and university-educated labor force (production inputs) as well as income (production output), in comparison with developed countries. The heart of this thesis lies on its chapter 2 and chapter 3, which are summarized as follows.
Chapter 2 aims at finding which agglomeration forces play the dominant role in affecting firms’ productivity and how agglomeration induces unequal influences across various firm characteristics. To achieve this, a six-year panel data set is employed, and the estimation is based on a production function that the left-hand side is firm’s total factor productivity while the righthand side is local technology which contains the agglomeration terms. In the first step of regression, consistent values of productivity are obtained following a strategy that combines the control function approach with the instrumental variables technique to tackle endogeneity caused by a possibility that firms choose their production inputs based on their productivity. In the second step, log of productivity is regressed on agglomeration proxies and controls, using multiple fixedeffects terms to control for unobserved factors and local shocks. Estimated results show that urbanization rather than specialization has a positive impact on productivity. Besides, the agglomeration effects are stronger for foreign-owned, small-sized, or young firms.
Chapter 3 shows attempts to find evidence of human capital externalities along with urbanization economies, given that the two external terms are rarely placed together in a single specification in literature. The estimation is implemented based on a production function whose context is an unique spatial equilibrium resulting from migration behaviors of entrepreneurs and workers. In this function, the externalities play the role of a region-specific productivity shifter. The model is regressed primarily with the instrumental variables technique to tackle possible identification problems. Between the two external terms, the resulting estimates confirm only the existence of urbanization economies. However, human capital externalities are found to be strong and significant in high-tech industries, implying that the effects of local human capital are very heterogeneous across different technological levels.
Objectives:
This study investigates the effectiveness of the Cold AP on the alteration of the enamel surface
without using acid etchant by using Conventional photo-activated resin bond to bond the
orthodontic brackets.
Materials and Methods:
One hundred and twenty-five Enamel specimens are prepared from disinfected bovine
mandibular incisors are divided into five groups. Group I: brackets are attached on the enamel
surface with the standard adhesive technique (etch + primer +bond). Group II: the brackets are
attached with the Standard Orthodontic adhesive technique without etching. Group III: the
enamel surface is conditioned with pure Argon Cold atmospheric plasma before the application
of the primer without using an acid etchant. Group IV: the enamel surface is conditioned with
the admixture of Argon Cold atmospheric plasma with 0.5 % Oxygen before the application of
the primer without using acid etchant Group V: after the application of Argon Cold atmospheric
plasma with 0.5 % Oxygen the surface is rewetted by deionized water before the application
of the primer and adhesive. After that, the samples are exposed to thermal cycling. The shear
bond strength of the samples is tested by the universal testing machine which measured the
maximum force at which the brackets are deboned from the tooth surface at a speed of
1mm/minute is measured.
Results:
Significant intergroup differences were found. Group V showed the highest shear bond
strength followed by Group I, VI, III, II respectively. There isn’t a statistical difference in the
values of The Shear bond strength values between Group III and IV.
Conclusions:
this study implies that Cold Atmospheric Plasma is a safe method to change the chemical
surface characteristics of the enamel surface.in addition to the significant importance of plasma
treatment followed by water rewetting, which could enhance adhesion between the orthodontic
attachments and the enamel layer
Podocytes are highly specialized kidney cells that are attached to the outer aspect of the glomerular capillaries and are damaged in more than 75% of patients with an impaired renal function. This specific cell type is characterized by a complex 3D morphology which is essential for proper filtration of the blood. Any changes of this unique morphology are directly associated with a deterioration of the size-selectivity of the filtration barrier. Since podocytes are postmitotic, there is no regenerative potential and the loss of these cells is permanent. Therefore, identification of small molecules that are able to protect podocytes is highly important. The aim of this work was to establish an in vivo high-content drug screening in zebrafish larvae. At first, we looked for a reliable podocyte injury model which is fast, reproducible and easy to induce. Since adriamycin is commonly used in rodents to damage podocytes, we administered it to the larvae and analyzed the phenotype by in vivo microscopy, (immuno-) histology and RT-(q)PCR. However, adriamycin did not result in a podocyte-specific injury in zebrafish larvae. Subsequently, we decided to use a genetic ablation model which specifically damages podocytes in zebrafish larvae. Treatment of transgenic zebrafish larvae with 80 µM metronidazole for 48 hours generated an injury resembling focal and segmental glomerulosclerosis which is characterized by podocyte foot process effacement, cell depletion and proteinuria. Following this, we established an in vivo high-content screening system by the use of a specific screening zebrafish strain. This screening strain expresses a circulating 78 kDa eGFP-labeled Vitamin D-binding fusion protein, which passes the filtration barrier only after glomerular injury. Therefore, we had an excellent readout to follow podocyte injury in vivo. We generated a custom image analysis software that measures the fluorescence intensity of podocytes and the vasculature automatically on a large scale. Furthermore, we screened a specific drug library consisting of 138 compounds for protective effects on larval podocytes using this in vivo high-content system. The analysis identified several initial hits and the subsequent validation experiments identified belinostat as a reliable and significant protective agent for podocytes. These results led to a patent request and belinostat is a promising candidate for a clinical use and will be tested in mammalian podocyte injury models.
Methane (CH4) is a potent greenhouse gas with rising atmospheric concentrations.
Microorganisms are essential players in the global methane cycle. In fact, the largest part of methane emissions derives from microbial production by methanogenic Archaea (methanogens). Microorganisms do not only produce methane: methanotrophs can also oxidize the methane produced by methanogens. In addition, soil methanotrophs are the only biological methane sink, oxidizing up to 30-40 Tg of this potent greenhouse gas per year worldwide.
However, intensified management of grasslands and forests may reduce the methane sink capacity of soils.
In general, the interaction of methanogens and methanotrophs determines whether a soil is a source or a sink for methane. It is, therefore, crucial to understand the microbial part of the methane cycle and which factors influence the abundance and activity of methane-cycling microbes. However, capturing the soil microbiome's abundances, activity, and identity is
challenging. There are numerous target molecules and myriad methods, each with certain
limitations. Linking microbial markers to methane fluxes is therefore challenging. This thesis aimed to understand how methane-cycling microbes in the soil are related to soil methane fluxes and how soil characteristics and human activity influence them.
The first publication investigated the biotic and abiotic drivers of the atmospheric methane sink of soils. It assessed the influence of grassland land-use intensity (150 sites) and forest management type (149 sites) on potential atmospheric methane oxidation rates (PMORs) and the abundance and diversity of CH4-oxidizing bacteria (MOB) with qPCR in topsoils of three temperate regions in Germany. PMORs measured in microcosms under defined conditions were approximately twice as high in forest than in grassland soils. High land-use intensity of grasslands negatively affected PMORs (−40%) in almost all regions. Among the different aspects of land-use intensity, fertilization had the most adverse effect reducing PMORs by 20%.
In contrast, forest management did not affect PMORs in forest soils. Upland soil cluster (USC)α was the dominant group of MOBs in the forests. In contrast, USCγ was absent in more than half of the forest soils but present in almost all grassland soils. USCα abundance had a direct positive effect on PMOR in forests, while in grasslands, USCα and USCγ abundance affected PMOR positively with a more pronounced contribution of USCγ than USCα.
In the second publication, we used quantitative metatranscriptomics to link methane-cycling microbiomes to net surface methane fluxes throughout a year in two grassland soils. Methane fluxes were highly dynamic: both soils were net methane sources in autumn and winter and net methane sinks in spring and summer. Correspondingly, methanogen mRNA abundances per
gram soil correlated well with methane fluxes. Methanotroph to methanogen mRNA ratios were higher in spring and summer when the soils acted as net methane sinks. Furthermore, methane uptake was associated with an increased proportion of USCα and γ pmoA and pmoA2 transcripts. High methanotroph to methanogen ratios would indicate methane sink properties.
Our study links the seasonal transcriptional dynamics of methane-cycling soil microbiomes for the first time to gas fluxes in situ. It suggests mRNA transcript abundances as promising indicators of dynamic ecosystem-level processes.
We conclude that reduction in grassland land-use intensity and afforestation can potentially increase the methane sink function of soils and that different parameters determine the microbial methane sink in forest and grassland soils. Furthermore, this thesis suggests mRNA transcript abundances as promising indicators of dynamic ecosystem-level processes. Methanogen transcript abundance may be used as a proxy for changes in net surface methane emissions from grassland soils.
This work first sets out to find if economic, ecological, or social incentives drive consumers towards or against dietary decisions (Contribution A). It then develops a framework of TCA for food to describe economically conveyed incentives that are tied to ecological and social indicators within the food market (Contribution B). The framework is subsequently enhanced and broadened to include a deeper understanding and broader field of indicators for more holistic TCA calculations (Contributions C and D). Lastly, based on these calculations, TCA of food is implemented in a factual use case as the framework and calculations are deployed for commodities of a German supermarket chain; then consumer, as well as expert feedback is used for the discussion on socially responsible campaigning and policy change (Contribution E).
Emerging infectious diseases are among the greatest threats to human, animal and plant health as well as to global biodiversity. They often arise following the human-mediated transport of a pathogen beyond its natural geographic range, where host species are typically not well adapted due to a lack of co-evolutionary host-pathogen dynamics. One such pathogen is the fungus Pseudogymnoascus destructans (Pd), which causes White-Nose disease in hibernating bats. While Pd was first observed in North America where it has led to mass-mortalities in some bat species, the pathogen originates from Eurasia where infection is not associated with mortality. Most of the Pd research has focused on the invasive North American range, which likely underestimated the genetic structure of the pathogen and the role it might play in the disease dynamics.
In my work, I therefore evaluated the genetic structure of Pd in its native range with the aim of uncovering cryptic diversity and further use population genetic data to address some key ecological aspects of the disease dynamics. With an extensive reference collection of more than 5,000 isolates from 27 countries I first demonstrated strong differentiation between two monophyletic clades across several genetic measures (multi-locus genotypes, full genome long-read sequencing and Illumina NovaSeq on isolate pools). These findings are consistent with the presence of two cryptic species which are both causative agents of bat White-Nose disease (‘Pd-1’, which corresponds to P. destructans sensu stricto, and ‘Pd-2’). Both species exist in the same geographic range and co-occur in the same hibernacula (i.e., in sympatry), though with specialised host preferences. I further described the fine-scale population structure in Eurasia which revealed that most genotypes are unique to single hibernacula (more than 95% of genotypes). The associated differences in microsatellite allele frequencies among hibernacula allowed the use of assignment methods to assign the North American isolates (exclusively Pd-1) to regions in Eurasia. Hence, a region in Ukraine (Podilia) is the most likely origin of the North American introduction.
To gain further insights into the spatial and temporal dynamics of White-Nose disease on a localised scale, several hibernacula were sampled with high intensity (artificial hibernaculum in Germany and natural karst caves in Bulgaria). Low rates of Pd gene flow were observed even among closely situated hibernacula. This indicates that Pd does not remain viable on bats over summer or it would be frequently exchanged among bats (and hence hibernacula) resulting in a homogenous distribution of genotypes. Instead, bats need to become re-infected each hibernation season to explain the yearly re-occurrence of White-Nose disease. Given the distribution and richness of Pd genotypes on hibrnacula walls and infected bats of the same hibernacula, bats become infected from the hibernacula walls when they return after summer. This means that environmental reservoirs exist within hibernacula (i.e., the walls) on which Pd spores persist during bat absence and which drive the yearly re-occurrence of White-Nose disease. In an experimental setup, I confirmed the long-term viability of Pd spores on abiotic substrate for at least two years and furthermore discovered temporal variations in Pd spores’ ability to germinate. In fact, these variations followed a seasonal pattern consistent with the timing of bats absence (reduced germination) and presence (increased germination) and could indicate adaptations of Pd to the bats’ life-cycle. The infection of bats from environmental reservoirs hence seems to be a central aspect of White-Nose disease dynamics and Pd biology.
Pds ability to remain viable for extended periods outside the host increases its risk of being anthropogenically transported and might have played a role in the emergence of White-Nose disease in North America. The existence of a second species (Pd-2) poses a great additional danger to North American bats considering that its introduction there could lead to deaths and associated population declines in so-far unaffected species given what is known about differing host species preferences in Eurasian bats. Even within the native range of Pd, the movement of Pd between differentiated fungal populations could facilitate genetic exchanges (e.g., through sexual reproduction) between genetically distant genotypes. Such genetic exchanges could lead to phenotypic jumps in pathogenicity or host-species preferences and should hence be prevented.
The native range of a pathogen holds great potential to better understand the genetic and ecological basis of a (wildlife) disease. My work informs about the dangers associated with the accidental transport of Pd (and other pathogens) and highlights the need for ‘prezootic’ biosecurity-oriented strategies to prevent disease outbreaks globally. Once a pathogen has arrived in a new geographic range, and particularly if it has environmentally durable spores (as demonstrated for Pd), it will be difficult/impossible to eradicate. Furthermore, a pathogen’s ability to remain viable outside the host and infect them from environmental reservoirs has been associated with an increased risk of species extinctions and needs to be considered when designing management strategies to mitigate disease impact.
Increasing environmental changes primarily due to anthropogenic impacts, are affecting organisms all over the planet. In general, scientists distinguish between three different ways in which organisms can respond to environmental changes in their habitat: extinction, dispersal and adaptation. An example of organisms which are highly adaptable and can easily cope with new and changing environments are invasive species which are able to colonize new habitats with only few individuals. To successfully survive in their new environment, invasive species adapt fast to novel abiotic and biotic parameters, such as different temperature regimes. Phenotypic plasticity which enables organisms to quickly modify their phenotype to new environmental conditions, explains the success in adaptation of invasive species.
While underlying mechanisms of phenotypic plasticity are not fully understood, one possible “motor” of phenotypic plasticity is epigenetics. Especially DNA methylation could explain the fast changes of the organism’s phenotype due to plasticity when experiencing changing environments, as invasive species do. DNA methylation could even contribute to the adaptation of invasive species via phenotypic plasticity, especially with clonally reproducing species. Methods such as common garden experiments with clonally reproducing species are a useful tool to differentiate between phenotypic plasticity and genetic adaptation because the confusing effects of genetic variation are lowered in clonally reproducing species.
Our overall goal was to evaluate the genetic adaptive potential of New Zealand mud snail (Potamopyrgus antipodarum) populations from Europe since they went through an extreme bottleneck after colonizing Europe only 180-360 generations ago. Seemingly, two different clonal lineages colonized Europe because two 16 s rRNA and cytochrome b haplotypes were found across different European countries, haplotypes t and z. The NZMS is a highly successful invasive species that is nowadays nearly globally distributed. The shells of the NZMS show a habitat-dependent high variability and are a fitness-relevant trait. The high variability in shell morphology is due to both genetic variation and phenotypic plasticity. To disentangle genetic from environmental effects on the shell morphology NZMS, we conducted a common garden experiment. We kept asexually reproducing females from eleven European populations in climate cabinets with three different temperatures to produce offspring. We compared shell size and shape across three generations using the geometric morphometrics approach. Furthermore, we estimated reaction norms, maternal effects, broad-sense heritability, the coefficient of genetic variation (CVA) and evolvability (IA) in shell size and shape across different temperature conditions. Additionally, we investigated the reproductive rate of the parental generation.
Results showed that the shell morphology of the parental generation differed across populations. In contrast, the shell morphology of offspring generations became more similar. The reaction norms of the F1 generation were rather variable across the three temperatures. However, we were able to observe a haplotype-dependent pattern across the reaction norms suggesting a restricted genetic differentiation among NZMS in Europe. We detected high heritability values in size indicating a high genetic influence. Heritability values for shape were lower than in size. Generally, heritability varied slightly depending on temperature. Size seemed to have a higher evolvability than shape. However, the values of all our calculations were very low which indicates that the European NZMS populations are genetically diminished. The reproductive rate of the parental generation was rather haplotype than temperature dependent. In summary, we were able to display that the NZMS is capable to plastically adapt its shell morphology to different temperatures showing significant differences between the two haplotypes. Nevertheless, the low evolvability values indicate that little genetic variation has formed since the arrival of the NZMS in Europe and therefore, European NZMS seem to have a reduced ability to react to selection.
These results implied that phenotypic plasticity is important for the adaptation to different environmental conditions in the NZMS and maybe other molluscan species. Since classical experimental approaches can only describe the resulting phenotypes, we also intended to shed more light on the mechanistic side of environmentally induced phenotypic modifications using DNA methylation analysis. Although molluscs represent one of the most diverse taxa within the metazoan and are found in many different habitats, our knowledge of the DNA methylation in molluscs is scarce. Therefore, we aimed at deepening and summarizing our understanding about DNA methylation in molluscs. Publicly available molluscan genomic and transcriptomic data of all eight mollusc classes was downloaded to search for DNA methyltransferases (DNMTs 1-3) responsible for DNA methylation. Additionally, we estimated the normalized CpG dinucleotide content (CpG o/e) indicating the presence/absence and the frequency of DNA methylation in the genome. The CpG o/e ratio refers to the level of DNA methylation in the genome. Based on the sensitivity of methylated cytosines to mutate into thymine residues, species having a high germline methylation in genomic regions over evolutionary time, also have a lower CpG content, which is called CpG depletion. In contrary, species with a limited germline methylation in genomic regions over evolutionary time, show a higher CpG content and lack CpG depletion. The presence or absence of CpG depletion can be calculated with the CpG o/e ratio. Ultimately, the goal of our analyses was to gain insight into the evolution of methylation in molluscs.
We detected DNMTs in all eight mollusc classes and in most of the species. It is therefore plausible that the last common ancestor of molluscs has already had the enzymatic machinery which is needed for DNA methylation. However, various species did not possess the complete DNMT toolkit indicating evolutionary modification in DNA methylation. In general, we found a wide distribution of the bimodal CpG o/e pattern in six mollusc classes, resulting from CpG depletion. The genes in these groups seem to be divided into genes with a high degree of methylation and genes with a lower degree of methylation. This implies that DNA methylation seems to be rather common in molluscs. Species of Solenogastres and Monoplacophora were not or only sparsely methylated. It seems that those mollusc groups have undergone a reduction in DNA methylation. We hope that our investigations will demonstrate the lacking knowledge in epigenetics of molluscs and encourage scientist to execute and continue genetic studies on molluscs.
As the animal-to-human interface becomes increasingly narrow, transmission events of zoonotic pathogens between animals and humans become more and more probable. While SARS-CoV-2 already accomplished a spillover infection to humans and is responsible for the current pandemic, the bat H9N2 IAV with so far unknown zoonotic potential was only recently discovered. In order to identify I) the role and potential of a newly discovered, potentially pre-pandemic virus, such as the bat H9N2, or II) possible future prevailing virus mutant variants of an already known pandemic virus, such as SARS-CoV-2, it is important to characterize these emerging viruses in vivo as soon and as good as possible.
The first objective in this dissertation (Publications I and II) therefore deals with the characterization of bat H9N2 and the estimation of its zoonotic or even pandemic potential.
In Publication I, a general susceptibility of directly inoculated Egyptian fruit bats to bat H9N2 was confirmed by successful seroconversion, although exhibiting only moderate viral shedding. All three contact animals remained seronegative, though one contact bat showed slight lesions in the histopathological analysis.
Publication II further addressed the question of the zoonotic potential of this virus. Inoculation of day-old turkey hatchlings demonstrated moderate susceptibility to bat H9N2 infection with a measurable seroconversion, while day-old chicken hatchlings were not susceptible to bat H9N2. Ferrets proved to be highly susceptible to bat H9N2 with high viral shedding, a transmission efficiency rate of 100% to direct contact animals at 2 days post contact, but with only minimal clinical signs. Importantly, the virus demonstrated the ability to evade the MxA-restriction factor and to replicate efficiently in human lung tissue explants. Furthermore, seasonal IAV- and standard IAV-vaccines showed no cross reactivity against the bat-N2 protein in humans. Therefore, further research on such viruses is urgently needed in order to prevent a renewed pandemic situation in the future as caused by SARS-CoV-2.
The second objective in this dissertation dealt with the identification and characterization of emerging SARS-CoV-2 Variants of Concern (VOCs).
Therefore, in Publication III, competitive infection experiments were performed using the Syrian golden hamster, the ferret, and transgenic mouse models (K18-hACE2 and hACE2-KI). These studies revealed replicative and transmissive predominance of Alpha VOC over Beta VOC, but not over SARS-CoV-2 WT in the hamster model, although Beta VOC substantially replicated in the lungs of donor animals. In contrast, the Alpha VOC had an unambiguous replication and transmission advantage over WT SARS-CoV-2 in the ferret and both mouse models. A recombinant SARS-CoV-2 WT-SAlpha virus helped to assign the fitness advantage of this variant particularly to the spike protein-associated mutations.
In Publication IV, in vitro results inferred an early replicative fitness advantage of Omicron BA.1 over Delta VOC, although the opposite was observed in competitively inoculated hamsters, ferrets and naive hACE2-KI mice. In addition, Publication IV demonstrated a disadvantage in transmission for the VOC Omicron BA.1 over the Delta VOC and a lack of susceptibility of ferrets after a single infection with the VOC Omicron BA.1. An mRNA vaccination of K18-hACE2 mice caused a drastic reduction of infectious virus particles in organ material following an infection with a recombinant SARS-CoV-2 WT-SDelta, but not when challenged with the SARS-CoV-2 SOmicron BA.1 clone.
This dissertation includes numerous, comprehensive experimental studies that are generally important for the characterization of emerging, potentially pre-pandemic viruses and may provide crucial information about the future dominance of certain virus variants in an ongoing pandemic. Here, the need for the use of a variety of animal models becomes apparent. By characterizing and classifying potentially zoonotic strains, these methods will help to better prepare for potentially upcoming pandemics and, in the case of a zoonotic or even pandemic event, to better detect and understand the circulating strains and their evolution.
The respiratory epithelium acts as both, a barrier of the respiratory tract to Nipah virus (NiV) entry and at the same time as a significant determinant of virus shedding. Both, for humans and pigs, replication in the respiratory tract epithelia is considered a major factor in transmission to other hosts. To understand why the virus constitutes a high-risk pathogen for livestock and humans, knowledge about
viral replication and host responses in relevant cells and tissues is crucial. Most in vitro studies, however, have been performed in conventional cell lines or non-differentiated lung cells. Only a few examples exist where Henipavirus infections have been investigated in fully-differentiated lung
epithelial cell models.
Thus, one aim of this thesis was to investigate infection, replication, spread and host protein dynamics of NiV in primary bronchial epithelial cells (BEC) cultivated at the air-liquid-interphase (ALI). By
immunofluorescence imaging, the NiV infection dynamics in BEC-ALI cultures were monitored over a 12 day time course, in order to provide detailed information about the infection process in the
respiratory epithelium of pigs and ferrets. Compared to undifferentiated primary BEC, the specific infectivity of NiV in BEC-ALI cultures was low. Infections remained focal and complete infection of the
cultures was not observed, even at 12 dpi. Analysis of viral titers and viral mRNA indicated a limited
virion release from the infected ALI-cultures while most of the newly synthesized NiV-RNA remained
cell associated. Immunofluorescence analysis of cross sections from infected ALI-cultures revealed
large infected areas that exhibited a strong cytopathic effect (CPE). Disruption of the epithelium
resulted in apical release of virus antigen-positive cell detritus while ciliated areas and basal cells were
less affected. From these data it was concluded, that NiV transmission could be supported by
exhalation of cell debris associated NiV and thus may contribute to rapid spread of infection in swine
populations.
A second aim was to explore the dynamics of host responses to NiV infection in differentiated BEC-ALI
culture and to assess whether this differs to conventional cell line data available from literature. Even
though strong CPE appeared in later phases of NiV infection, at least the porcine PBEC-ALI cultures
remained robust enough to allow protein sampling over 12 days infection course. Subsequent MS-based proteomics enabled unprecedent insight in complex cell culture response upon NiV infection.
Previous reports indicated a lack of efficient interferon type I induction in non-differentiated pig or
human BEC which were considered a prerequisite for efficient replication in the respiratory epithelium
and virusspread. In contrast to non-differentiated pig BEC (PBEC), in PBEC-ALI cultures multiple factors
involved in interferon responses were upregulated upon NiV infection. Thereby it was demonstrated
that NiV infection induced a robust innate immune response upon infection with elevated components of antigen processing and presentation resulting in the conversion from the constitutive proteasome to the immunoproteasome. In contrast to previous reports about NiV-infected non-differentiated
PBEC or endothelial cells, incomplete immunoproteasome formation and limitations in interferon
response could be excluded. Thus, a model is proposed in which NiV infection and spread in differentiated PBECs is slowed by potent innate immune responses to the virus infection. Overall, the
findings highlight the important role of the respiratory epithelium not only as a physical barrier to virus
infections but also indicate itsrole as a primary site of adaptive immune induction through NiV induced
antigen processing and MHC I presentation.
Finally, to allow functional studies of Henipaviruses at the BSL-2 biosafety level a recombinant CedPV
was generated and rescued. An imaging based screening and quantitative analysis pipeline was established to investigate the role of cellular factors and to screen for potential virus and host gene
directed inhibitory factors. Accordingly, different host and viral genes were targeted with a siRNA-pool
either targeting virus or selected cellular mRNAs followed by the infection with the CedPV and the
quantification of infected cells. With proof of concept of the siRNA screening pipeline, the recombinant
CedPV clone was used as a backbone to insert variousfluorescence reporter genesin order to optimize
the analysis workflow by allowing direct virus quantification in live, unstained samples. Consequently,
this thesis provides a valuable proof for future approaches related to the function of virus proteins,
influence of host-factors and virusreplication and Henipavirus-inhibitorscreens at low biosafety levels.
The role of cell-penetrating peptides in the induction of T cell responses by virus-like particles
(2023)
Many viral structural proteins can self-assemble into virus-like particles (VLPs). VLPs can serve as an effective vaccine or be used as a vaccine platform. One of these structural proteins is the hepatitis B virus core antigen (HBcAg), which appears to be suitable as an antigen carrier due to its high immunogenicity. HBcAg has a major immunodominant region (MIR) that is presented on the surface of the VLPs after self-assembly. Foreign antigens can be inserted into this region. Since HBcAg VLPs, unlike the Hepatitis B virus (HBV), do not have an envelope, they are not able to penetrate cell membranes efficiently. As an extracellular antigen, HBcAg VLPs primarily induce a strong humoral immune response.
In the present study, we investigated the extent to which HBcAg can be modified to also elicit an enhanced cellular, particularly a cytotoxic, immune response. A cytotoxic CD8+ T cell response is predominantly induced by intracellular antigens. Therefore, our goal was to increase the cell penetration capacity of VLPs. We aimed to achieve this by fusing cell-penetrating peptides (CPPs) to HBcAg. CPPs can spontaneously penetrate cell membranes to enter the cytoplasm of cells. To guarantee that the CCPs were localized to the surface of the VLPs, we fused CPPs to the N-terminus of HBcAg. The CCPs were followed by a tag to allow the purification of VLPs. The T cell epitopes, against which the induced CTL should be directed, were derived from the Large T antigen and inserted into the MIR of HBcAg. Finally, we fused fluorescent proteins to the C-terminus of HBcAg to track the entry of VLPs into cells.
Modifications of HBcAg may lead to reduced stability or altered structure of VLPs. To analyze the stability of VLPs, we used nanoscale differential scanning fluorimetry (nanoDSF) analysis. This revealed that the N-terminal fusion of CPPs or the tag to HBcAg does not reduce VLP stability. However, some peptides incorporated into the MIR had a significant effect on the structure and stability of the VLPs. While the incorporation of a Flag-tag or a peptide from ovalbumin had no negative effect on VLP stability, the incorporation of peptides representing T cell epitopes of Large T antigen interfered with VLP formation. Denaturation and reassembly of the aggregates significantly improved the homogeneity of the VLPs, and the C-terminal addition of arginine-rich domains enhanced stability.
Using live cell imaging and flow cytometry, we demonstrated that HBcAg VLPs functionalized with CPP exhibited up to 40% more efficient penetration into professional antigen-presenting cells (JAWS II) than HBcAg VLPs without CPP. This resulted in the increased presentation of integrated T cell epitopes by dendritic cells. In vivo, we detected significantly increased induction of SV40 Large T antigen-specific CTL in mice immunized with CPP-conjugated VLPs compared to unconjugated VLPs.
In this study, we demonstrated that a stronger cellular immune response can be induced by CPP-functionalized HBcAg VLPs than with the unmodified HBcAg VLPs in vitro as well as in vivo. This discovery may have positive implications for future vaccine development where an enhanced cellular component of the immune response is desirable.
Underground hard coal mining operations irreversibly disrupt the pre-existing mechanical equilibrium of the geological media. The employment of high-recovery methods modifies the stress field of the sedimentary sequence, generating movement and faulting of the rock layers above and below mined seams. These new fracture zones do affect the original conditions of the hydrogeological system by modifying flow pathways and increasing the permeability of the rock sequence. Moreover, the surface area of rock exposed to air and water is increased, conditioning the water-rock interaction. Despite this rather clear conceptualization, flow and reactive transport processes in fractured overburdens are rarely modeled simultaneously. Discrete setups that consider fractures and porous matrix require extensive characterization of both media, which is impractical for regional case studies. As a result, most post-mining models explicitly ignore fracture structures by employing the equivalent porous approach or even both media with lumped parameter models. However, omitting either medium represents a delicate simplification, considering that mining-related fractures control the rate and direction of water flow within moderately permeable but relatively highly porous rock sequences.
In this dissertation, the specific contribution of fractured and matrix continua to the transient discharge and water quality of a post-mining coal zone is quantified and evaluated. For this purpose, dual and multiple interacting continua models are employed to simulate fluid flow and reactive mass transport in fractured and variable water-saturated rock sequences. The effectiveness of the models is evaluated by simulating the origin, generation and transport of acid mine drainage (i.e., water with elevated concentrations of hydrogen, iron, sulfate and chloride) within the shallow overburden of the Ibbenbüren Westfield. Compared to other coal districts in Germany, this area is strongly delimited by the local geology and topography, resulting in a well-defined hydrogeological system to test the models. Petrographic and chemical analyses performed on core samples from the area show the strong influence of mining-derived fractures on the water-rock interaction within the Carboniferous sequence. The presence of oxidized pyrite along with amorphous iron hydroxide phases in weathering fronts on both sides of the fractures demonstrates the exchange of solutes and gases between the fractured and the porous matrix media.
Based on the previous evidence, the TOUGHREACT software is employed to characterize flow and reactive transport processes in the Westfield. However, each of the two processes is simulated at separate stages to have more control in the adjustment of sensitive parameters for which little information is available. For the flow component, a dual continuum model, with Richard’s equations is used to characterize the unsaturated water flow in both fractured and matrix media. Under this approach, the model adequately reproduces the bimodal flow behavior of the discharges measured in the mine drainage for the years 2008 and 2017. Simulation results show how the fractured continuum generates intense discharge events during the winter months while the rock matrix controls smooth discharge limbs in summer, when water is slowly released back to the fractures. With the flow component calibrated, the second part of the study incorporates the geochemical processes into the model based on actual data from the rock samples. Their simulation requires extending the two-continuum setup to a multiple continua model with five nested block strings: one for the fractures and four for the rock matrix. This further subdivision prevents under-representations of kinetic reactions with short equilibrium length scales and numerical instabilities due to lack of chemical and flow gradients. As a result, the new multiple continua model provides good agreement with respect to long- and short-term concentrations and discharge trends measured in the mine drainage. The flow of oxygen and meteoric water through the fractured continuum leads to a high and steady release of hydrogen, iron and sulfate ions derived from pyrite oxidation in the matrix continua closest to the fractures. Moreover, high chloride concentrations result from the mixing and gradual release of relatively immobile solutes in the matrix as they interact with percolating water in the fracture. Both findings are equally congruent with the reactive pyrite oxidation and iron hydroxide precipitation fronts identified in the fractured core samples.
In the end, the multiple continua models, the simulation procedure and the results of the benchmark and sensitivity analysis scenarios developed for the Westfield pave the way for the application of the approach in other mining zones. The first candidate emerges in the Ibbenbüren Eastfield, where a coupled elemental-isotopic approach included in this thesis has confirmed that water-conducting fracture zones are primary elements for solute generation and transport in the first 300 meters of the overburden. In the latter case, calibration and verification of the models can be complemented with measurements of δ34S in sulfates and δ18O, δ2H, and Tritium in water.
To what extent do norms of regional Intergovernmental Organizations (IGOs) have an impact on member states’ borders and their permeability? International agreements and regional integration measures quite often highlight how harmonization of mechanisms and procedures related to cross-border interaction within specific communities takes place. As these agreements and measures contain mutual expectations about appropriate behavior, a form of convergence in bordering practices – and therefore effects – is implied. This leads to the assumption that cross-border interaction is gradually increasing and eventually leading to a ‘borderless’ realm that allows for the free movement of goods, services, persons, and capital. However, the nature of borders or a deeper understanding of the bordering process itself is often not central to the studies of international relations. They represent mere fixtures of international interaction and appear in the public discussion only if sudden restrictions are implemented or if large-scale changes in the international environment affect their functioning.
Specific literature on borders is relatively new and located mainly within an interdisciplinary setting that largely lacks coherency in its ontological concepts or deals exclusively with individual cases. The literature on the normative capacity of (regional) IGOs on the other hand is well matured and one of the cornerstones of international studies. However, here a blatant neglect of borders and bordering is apparent. This situation is aggravated by the circumstance that the field of International Relations by definition is dealing with cross-border interaction.
Apart from the general ontological issue of what borders and bordering processes are from a political science perspective, three main gaps in conjunction with the above question could be identified within the literature. The first relates to the general efficacy of the normative influence of regional IGOs on enhancing cross-border interaction through the issuing of specific normative provisions. On this, the relevant literature is divided. Arguments range from an all-permeating relevance of norms as intersubjective understandings that create specific expectations of appropriate behavior to the primacy of rational choices that are targeted to create benefits in most economic and security- related matters. The second gap relates to the relevance of domestic precognition in cross-border interaction. It is not clear to what extent dominant normative conceptions at the national level create obstacles to the efficacy of IGO provisions in shaping cross- border interaction. Finally, the impact of critical junctures – as kind of catalysts – that shift member state preferences in following communal obligations are being analyzed. Here, the assumption is that these junctures may either enhance or negate IGO provisions dependent on the dominant domestic preferences. Essentially, the approach taken here is a layered one, where each identified gap provides the basis for the following ones. The analysis itself is divided into a quantitative and a qualitative part. For the former, a new dataset has been created that specifically lists all normative provisions targeting the free movement of goods, services, persons, and capital of the two IGOs selected for this research project. This is an important departure from the usual dichotomous perception of IGO influence used throughout the literature. In conjunction with specific data related to each of the four freedoms, multiple variations of a gravimetric Poisson Pseudo-Maximum Likelihood model are specified. The qualitative approach draws upon descriptive and matching approaches to analyze the impact of dominant domestic norms and critical junctures on the bordering process. For this purpose, a corpus of source material is created that includes government declarations and media articles, as well as the specific IGO provisions on bordering to each of the four freedoms that are central to this research.
The quantitative findings indicate a strong and consistently positive impact of regulative IGO norms while evaluative IGO norms do not provide similar results. The qualitative findings point in a similar direction. As long as IGO norms have a more regulative direction and are obligatory, the effect is less likely to be diminished by contesting dominant domestic norms or critical junctures vis-`a-vis more evaluative IGO norms.
The results and additional findings of this research have implications for further research. Central to this is the capacity of regional IGOs to exert normative influence on the bordering practices of their member states. An additional finding relates to the interrelatedness of normative provisions. The more these provisions are cross-referencing each other – or are interlocked – the more robust they seem to get. This is a trait that is relevant for policy-makers and IGO bureaucracies if they want to enhance the compliance of their agents. On a methodological level, the here introduced data set on normative IGO provisions provides other researchers with a substantially more fine- grained approach to investigating the impact of specific IGO measures on a particular dimension of cross-border interaction.
Amid the current global biodiversity crisis, being able to accurately monitor the changing state of biodiversity is essential for successful conservation actions and policy. Despite the pressing need for reliable and cost-effective monitoring methods, collecting such data remains extremely difficult for elusive species, such as temperate zone bats. Although bats are important indicators of environmental changes, monitoring bat populations is challenging because they are nocturnal, volant, small, and highly sensitive to human activities and disturbance. Thus far, population trends of temperate zone bats have been mainly based on visual surveys, including winter hibernation counts at underground sites. However, as bats may not always be roosting in visible locations within the hibernacula, it is currently unknown how these estimates relate to actual population sizes.
Infrared light barriers combined with camera traps are a novel method to monitor bats at underground sites. When installed at the entrance of hibernacula, infrared light barriers have the potential to estimate site-level population sizes more accurately than visual surveys, by counting all bats flying in and out of the site. Moreover, camera traps, consisting of a digital camera and white flash, can be used for species-level identification. However, for this new method to be applicable as a large-scale bat monitoring technique, it is important to characterize it with regard to three main criteria: is the method minimally invasive, is it accurate, and is it scalable in terms of spatial and temporal resolution? Therefore, the purpose of this thesis was to investigate the invasiveness and accuracy of this novel bat monitoring method, and to develop standardized and automated data analysis pipelines, both for the light barrier and camera trap data, to support the deployment of this method at scale.
In Publication I, we used light barrier data, infrared video recordings and acoustic data from an experimental field study to investigate whether the white flash of the camera trap has any measurable short- or long-term effect on bat activity and behavior. The flash of the camera trap was turned on and off every week at each site, which allowed us to compare the activity and behavior of bats between flash-on and flash-off nights. We found that despite the high sensitivity of bats to disturbance, they did not change their nightly activity patterns, flight direction, echolocation behavior, or long-term site use in response to the white flash of the camera trap. Based on these results, we concluded that camera traps using a white flash are a minimally invasive method for monitoring bat populations at hibernacula, providing high quality images that allows species-level identification.
In Publication II, we used infrared video surveillance to quantify the accuracy of infrared light barriers, and we described a standardized methodology to estimate population sizes and trends of hibernating bat assemblages using light barrier data. We showed that light barrier accuracy varies based on the model and location of the installation relative to the entrance, with the best combination achieving nearly perfect accuracy over the spring emergence phase. When compared to light barrier-based estimates, we found that visual counts markedly underestimated population sizes, recovering less than 10% of the bats at the most complex hibernacula. Moreover, light barrier-based population trends showed regional patterns of growth and decline that were not detectable using the visual count data. Overall, we established that the light barrier data can be used to estimate the population size and trends of hibernating bat assemblages with unprecedented accuracy and in a standardized way.
In Publication III, we described a deep learning-based tool, BatNet, that can accurately and efficiently identify bat species from camera trap images. The baseline model was trained to identify 13 European bat species or species complexes using camera trap images collected at 32 hibernation sites (i.e., trained sites). We showed that the baseline model performance was very high across all 13 bat species on trained sites, as well as on untrained sites when the camera angle and distance from the entrance were comparable to the training images. At untrained sites with more atypical camera placements, we demonstrated the ability to retrain the baseline model and achieve an accuracy comparable to the trained sites. Additionally, we showed that the model can learn to identify a new species, while maintaining high classification accuracy for all original species. Finally, we established that BatNet can be used to accurately describe ecological metrics from camera trap images (i.e., species diversity, relative abundance, and species-specific phenology) that are relevant for bat conservation.
We conclude that infrared light barriers and camera traps offer a minimally invasive and accurate method to monitor site-level bat population trends and species-specific phenological estimates at underground sites. Such remote data collection approaches are particularly relevant for monitoring large, complex hibernation sites, where traditional visual surveys are not feasible or account only for a small fraction of the actual population. Combining this automated monitoring method with a deep learning-based species identification tool, BatNet, allows us quickly and accurately analyze millions of camera trap images resulting from large-scale, long-term camera trap studies. As a result, we can gain unprecedented insights into the behavior and population dynamics of these enigmatic species, drastically improving our ability to support data-driven bat conservation.
Foraging behavior, neuroanatomy and neuroplasticity in cursorial and stationary hunting spiders
(2023)
The central nervous system (CNS) is the integration center for the coordination and regulation of
all body activities of animals and the source of behavioral patterns, behavioral plasticity and
personality. Understanding the anatomy and the potential for plastic changes of the CNS not only
widens the knowledge on the biology of the respective species, but also enables a more
fundamental understanding of behavioral and ecological patterns. The CNS of species with
different sensory ecologies for example, will show specific differences in the wiring of their CNS,
related to their lifestyle. Spiders are a group of mesopredators that include stationary hunting
species that build webs for prey capture, and cursorial hunting species that do not build capture
webs. These distinct lifestyles are associated with major differences in their sensory equipment,
such as size of the different eyes.
In this thesis, I aimed to answer if a cursorial mesopredator would change its behavior due to
different levels of perceived predation risk, and if this behavior would be influenced by individual
differences (chapter 1); how the visual pathways in the brain of the cursorial hunting jumping
spider Marpissa muscosa differs from that of the nocturnal cursorial hunting wandering spider
Cupiennius salei (chapter 2); to what degree the visual systems of stationary and cursorial hunting
spiders differ and whether CNS areas that process vibratory information show similar differences
(chapter 3); and finally if the CNS in stationary and cursorial hunting spiders shows different
patterns of neuroplasticity in response to sensory input and deprivation during development
(chapter 4).
In chapter 1, I found that jumping spiders adjust their foraging behavior to the perceived level of
risk. By favoring a dark over a light substrate, they displayed a background-matching strategy.
Short pulses of acute risk, produced by simulated bird overflights, had only small effects on the
behavior. Instead, a large degree of variation in behavior was due to among-individual differences
in foraging intensity. These covaried with consistent among-individual differences in activity,
forming a behavioral syndrome. Our findings highlight the importance of consistent amongindividual
differences in the behavior of animals that forage under risk. Future studies should
address the mechanisms underlying these stable differences, as well as potential fitness
consequences that may influence food-web dynamics.
In chapter 2, I found that the visual pathways in the brain of the jumping spider M. muscosa differ
from that in the wandering spider C. salei. While the pathway of the principal eyes, which are
responsible for object discrimination, is the same in both species, considerable differences occur
in the pathway of the secondary eyes, which detect movement. Notably, M. muscosa possesses
an additional second-order visual neuropil, which is integrating information from two different
secondary eyes, and may enable faster movement decisions. I also showed that the tiny posterior
median eye is connected to a first-order visual neuropil which in turn connects to the arcuate body
(a higher-order neuropil), and is thus not vestigial as suggested before. Subsequent studies should
focus on exploring the function of the posterior median eyes in different jumping spider species,
Foraging behavior, neuroanatomy, and neuroplasticity in cursorial and stationary hunting spiders
as they show considerable inter-specific size differences that may be correlated with a differing
connectivity in the brain.
In chapter 3, I described all neuropils and major tracts in the CNS of two stationary (Argiope
bruennichi and Parasteatoda tepidariorum) and two cursorial hunting spiders (Pardosa amentata
and M. muscosa). I found major differences in the visual systems of the secondary eyes between
cursorial and stationary hunting spiders, but also within the groups. A. bruennichi has specialized
retinula cells in two of the secondary eyes, which connect to different higher-order neuropils. P.
tepidariorum has only a single visual neuropil connected to all secondary eyes, and lacks
recognizable mushroom bodies. The neuroanatomy of CNS areas that process mechanosensory
information on the other hand, is remarkably similar between cursorial and stationary hunting
species. This suggests that the same major circuits are used for the processing of mechanosensory
information in both cursorial and stationary hunting spiders. Future studies on functional aspects
of sensory processing in spiders can build on the findings of our study.
In chapter 4, I found that developmental neuroplasticity in response to sensory input differs
between a cursorial (M. muscosa) and a stationary hunting spider (P. tepidariorum). While
deprivation of sensory input leads to a volume increase in several visual and mechanosensory
neuropils M. muscosa, neither sensory deprivation nor sensory enrichment had an effect on the
volume of neuropils in P. tepidariorum. However, exposure to mechanical cues during
development had an effect on the allometric scaling slope of the leg neuropils in both M. muscosa
and P. tepidariorum. Future studies should focus on the genetic and cellular basis of
developmental neuroplasticity in response to sensory input in order to explain the observed
patterns.
Autoclaved aerated concrete (AAC) is a building material that combines heat insulation
properties with sufficient mechanical strength for masonry construction. Compared to
ordinary concrete, the matrix is highly porous (>50%) and hardened by a hydrothermal curing
process at 150°C - 200°C. During this process, quartz sand and portlandite react to form first
calcium silicate hydrates (C-(A)-S-H) with Ca/Si ratios <1.3 and then tobermorite. Especially
tobermorite, which has a much larger crystallite size than C-(A)-S-H, provides improved
mechanical strength. This reaction sequence is influenced by many parameters and
additives of which calcium sulfate is probably the most important. Despite several attempts to
investigate these hydrothermal reactions, the actual reaction mechanism involved when
adding sulfate ions is not fully understood. It has been suggested that the addition of ca.
1.5 wt% significantly improves the mechanical properties due to the enhanced formation and
arrangement of tobermorite in the porous matrix. Since the sulfate content in AAC waste is
exceeding regulatory threshold for low-quality reuse in some countries, the aim of this study
was to investigate in detail the reaction mechanisms involving sulfate addition. Such
knowledge may open up the possibility to improve AAC production and to avoid the need for
sulfate addition. To achieve this goal, this research work focused on investigating the
hydrothermal curing process to determine the sequence of hydrothermal reactions and the
spatial distribution of the phases formed. For this purpose, a new setup for in situ X-ray
diffraction was specifically designed to study hydrothermal reactions and to conduct time
intensive experiments on a normal laboratory diffractometer. In order to quantitatively
evaluate the in situ measurements by Rietveld analysis using TOPAS, it was also necessary
to develop atomistic structure models for C-(A)-S-H phases. This was made possible by
adopting a supercell approach that was previously used to describe turbostratically stacked
clay minerals. The structure models, derived from tobermorite, are placed in an otherwise
empty supercell to simulate the C-(A)-S-H nanostructure. Adopting these methodological
advances, it was possible to obtain absolute phase quantities from in situ data and to track
the reaction kinetics of the hydrothermal curing process. These results were then combined
with ex situ X-ray diffraction and scanning electron microscopy. Confirming previous studies,
the major effect of sulfate ions was the formation and decomposition of hydroxylellestadite. It
was further revealed that C-(A)-S-H formation was delayed during hydroxylellestadite
formation, which is supposed to support the silicate ion diffusion and hence the tobermorite
formation at a stage critical for improved hardening of AAC. This can be linked to the
formation of lower amounts of capillary pores in the range of 1-5 µm, as observed by
scanning electron microscopy, and therefore a lower concentration of inherent defects that
resulted in the improved mechanical properties. This research work highlights how important the spatial distribution of crystallites is for the properties of a building material and how this
distribution can be influenced by small alterations in reaction chemistry.
The effect of interdental cleaning on progression of caries, periodontitis and tooth loss is a highly discussed topic in dental research since these conditions are among the most common infectious diseases of mankind. Caries is a multifactorial disease defined by a demineralization process of the dental hard tissue, caused by bacteria, which, if untreated ultimately results in tooth decay and tooth loss. A study published in 2015 confirmed that untreated caries in permanent teeth is still the most prevalent condition worldwide. Gingivitis, an acute inflammation of the gingival tissue, caused by substances deduced from the microbial plaque can develop into the clinical picture of an acute periodontitis. Severe periodontitis is still the sixth-most prevalent condition globally with a prevalence of 11.2% between 1990-2010. Progression of periodontitis leads to bone loss which as well ultimately results in tooth loss, if left untreated. In our study we want to examine the use of IDA in relation to caries and periodontal diseases, thus tooth retention to gain more detailed and long-term results about the effect of IDA and therefore prevent, counteract and understand these oral diseases better.
Using data from SHIP-TREND, a population-based observational cohort study conducted in Western Pomerania (Germany), we examined effects of daily usage of interdental cleaning aids on follow-up (SHIP-TREND-1) values of oral outcomes comprising caries (DFS, interdental DFS, non-interdental-DFS), gingivitis (plaque, BOP), chronic periodontitis (mean PD, mean interdental PD, mean non-interdental PD, mean CAL, mean interdental CAL, mean non-interdental CAL, CDC/APP case definition) and tooth loss (number of missing teeth) using comprehensively adjusted linear and ordinal logistic regression models. In total, data from over 2,000 participants with a follow-up time of approximately seven years were utilized. Based on interviews, participants were asked about their habit and the regularity of using interdental aids as a cleaning aid at home. Furthermore, the type of IDA was then analyzed and differentiated into groups of IDA non-users, wooden stick users, floss users and interdental brush users.
Regular interdental aids usage was associated with reduced levels of periodontitis severity (mean PD and mean CAL) and gingivitis variables (plaque and BOP). The beneficial effect was more pronounced in participants using dental floss or interdental brushes regularly. After seven years of follow-up, odds of having higher mean PD levels were halved (Odds Ratio 0.49; 95% confidence interval (CI) 0.35;0.66) comparing dental floss users with non-users. Respective ORs were 0.61 (95%CI 0.45;0.83) for mean CAL, 0.52 (95%CI 0.36;0.77) for BOP and 0.36 (95%CI 0.24;0.54) for plaque. Similarly, ORs for interdental brush users were 0.75 (95%CI 0.55;1.02) for mean PD, 0.64 (95%CI 0.41;0.97) for BOP and 0.55 (95%CI 0.39;0.77) for plaque, compared to non-users. For wooden sticks non-significant associations were found, which does not allow any statement to be made regarding possible effects on oral health. Caries variables (DF-S) and the number of missing teeth were non-significantly associated with interdental aids usage.
In conclusion, results suggest that interdental cleaning aids usage may contribute to healthier gums and reduced inflammation, if combined with daily toothbrushing and regular dental checkups. Specifically, dental flossing and interdental brushing might notably reduce gingival inflammation and therefore prevent chronic periodontitis. These findings contribute to a more distinct picture of how IDA might help to prevent oral diseases and must be properly integrated into our daily oral hygiene program.
Graphene is a strictly two-dimensional honeycomb lattice of carbon atoms whose low-energy charge-carrier dynamics obey the massless pseudospin-1/2 Dirac-Weyl equation (or chiral Weyl equation) where the chiral centers (or valleys) are the corners K and K‘ of the Brillouin zone. The linear spectrum near the Dirac nodal points lends graphene its exotic and ultra-relativistic properties.
However, condensed matter systems can possess fermionic excitations with linear dispersions that have no analog in high-energy physics since the crystal space group - instead of the Poincare group - constrains the energy dispersions. Perhaps the first example in this regard is the T_3 lattice (Dice Gitter), a honeycomb-like lattice with an extra atom placed at the center of each hexagon and coupled to only one of the sublattices. The spectrum features a strictly flat band that crosses the two conical intersections of the Dirac cones at K and K' inherited from graphene. The enlarged pseudospin-1 Dirac-Weyl equation describes the low-energy dynamics. By rescaling the transfer amplitude of the additional atoms in the T_3 lattice with a parameter 0<α<1, the resulting α-T_3 lattice continously interpolates between graphene and the T_3 lattice.
In this work, we explore the behavior of generalized Dirac-Weyl quasiparticles in external magnetic and valley-dependent pseudoelectromagnetic fields induced by out-of-plane strain. First, we studied Dirac-Weyl quasiparticles in external fields confined to circular quantum dots by generalizing the infinite-mass boundary condition to the α-T_3 lattices. We verified the analytically derived valley-anisotropic eigenstates of the quantum dot by numerically solving the tight-binding lattice-model in closed (isolated) and open (contacted) systems.
Second, we considered strain fields in the α-T_3 lattices to modify the low-energy transport properties by an effective pseudo-gauge field with opposite signs at the K and K‘ valley. In particular, we showed that the inhomogeneous pseudomagnetic field generated by Gaussian out-of-plane strain at the center of a four-terminal Hall bar setup acts as a valley filter. Most interestingly, the valley polarization is most dominant when incoming electrons are excited to pseudo-Landau level subbands. These bands are linked to different iso-field orbits encircling the lobes of the pseudomagnetic field. Addittionaly, any intermediate α breaks the inversion symmetry of the α-T_3 lattice and thus splits the pseudo-Landau levels into sublattice-polarized bands.
Third, we equipped the out-of-plane strain with a time-periodic drive to induce a valley-dependent pseudoelectric field perpendicular to the pseudomagnetic field. We assessed the steady-state transport properties and found – besides the static regime for small energies – two α-dependent valley-filtering regimes due to the periodic drive. Firstly, we found an additional valley-polarization plateau at the Floquet-zone boundary between the central and first Floquet copy that also displayed a “flower”-like pattern in the local density of states. Secondly, we detected a series of transmission gaps at the center of every Floquet sideband 2mΩ related to the Floquet coupling of the flat band with the central Floquet copy. Under certain strain parameters, a novel valley-filtering regime appears near the transmission gaps where the incoming K electrons are focused through the bump by the pseudoelectric field, instead of encircling the lobes of the pseudomagnetic field. A stability analysis demonstrated that the polarization regimes are tunable by the driving frequency.
Lastly, we demonstrated that the flat band in the Haldane-dice lattice modified by a uniaxial strain along the zigzag orientation remains singular at all band crossings where the model undergoes a topological phase transition between C=+-2 and C=0. To show this, we computed the compact localized eigenstates and the quantum distance of the Bloch wave function around the band-touching points. We derived the resulting non-contractible loop states and an extended state whose components are tunabe by the system parameters.
Mass spectrometry-based Proteome analysis of porcine cells infected with African swine fever virus
(2023)
ASFV, a highly contagious, pathogenic and lethal pathogen of swine, poses a major threat to domestic and wild suids worldwide as neither vaccines nor treatments are available. Compared to other well-characterized similarly complex viruses like herpesviruses or adenoviruses, the understanding of ASFV biology is poor.
To improve the understanding of ASFV biology, following the establishment of a robust protocol for the isolation of primary monocyte-derived porcine macrophages (moMΦ) and their infection with ASFV for mass spectrometry (MS)-based proteome analysis was performed.
Under both conditions, naïve and infected, the isolated cells showed cell type-specific characteristics like phagocytosis and antigen presentation and protein expression patterns, including the expression of swine leucocyte antigens and CD markers. Furthermore, moMΦ could be reproducibly infected with ASFV isolates of different genotypes and pathogenicity.
The ASFV protein expression patterns in moMΦ correlate well with those observed in established cell lines at transcript and protein level. The expression of 27 ASFV proteins was confirmed at the protein level. Among them, 9 members of multi-gene families (MGF) and 12 novel open reading frames (nORFs) were recently predicted based on transcription start site mapping.
The direct comparison of closely related ASFV genotype II isolates revealed no virulence-associated protein expression patterns beyond those expected based on the genome sequences of the isolates.
Using different MS quantification strategies, it was shown that ASFV affects both static protein expression levels and protein synthesis. These changes in protein expression impact proteins and pathways known to be targeted by ASFV, including CD-markers, ER-stress and cell death pathways, and cellular antiviral responses. Beyond these observations that further validated the moMΦ infection model, novel effects of the ASFV infection on the cellular proteome were noticed.
These effects include the decreased expression levels of cathepsins, especially cathepsins D (CTSD), H (CTSH) and L (CTSL) as well as the transient activation of MAPK14/p38 prior to its strong downregulation. In addition to MAPK14/p38 further members of the MAPK14/p38 signaling pathway, like MAPKAPK2, were affected by ASFV infection.
As these modulations of the cellular proteome would in general result in decreased pro-inflammatory responses, it did stand out that the synthesis of interferon-response related genes including MX1 and ISG15 evaded the ASFV-induced global reduction of protein synthesis. In contrast, the synthesis of genes involved in RNA processing and splicing was significantly impaired. In total, the regulations of individual host proteins assessed in the context of the whole cellular proteome integrate well with each other and other cellular responses to ASFV infection and may help to improve the understanding of host-virus interactions.
Overall, this thesis provides novel insights into the expression of ASFV-encoded ORFs of different isolates and the host response to ASFV infection. It points out that the current knowledge of the ASFV coding capacity, temporal protein expression patterns, protein functionality, post-translational modifications and host interactions is still sketchy as many aspects of ASFV replication have yet to be understood. The established moMΦ-model to study ASFV infections in vitro provides a powerful tool for future applications to increase the understanding of ASFV biology.
Statistical Methods and Applications for Biomarker Discovery Using Large Scale Omics Data Set
(2023)
This thesis focuses on identifying genetic factors associated with human kidney disease progression, with three articles presented. Article I describes the identification of loci associated with UACR through trans-ethnic, European-ancestry-specific, and diabetes-specific meta-analyses. An approximate conditional analysis was performed to identify additional independent UACR-associated variants within identified loci. The genome-wide significance level of 𝛼=5×10−8 is used for both primary GWAS association and conditional analyses. However, unlike primary association tests, conditional tests are limited to specific genomic regions surrounding primary GWAS index signals rather than being applied on a genome-wide scale.
In article II, we hypothesized that the application of 𝛼=5×10−8 is overly strict and results in a loss of power. To address this issue, we developed a quasi-adaptive method within a weighted hypothesis testing framework. This method exploits the type I error (𝛼=0.05) by providing less conservative SNP specific 𝛼-thresholds to select secondary signals in conditional analysis. Through simulation studies and power analyses, we demonstrate that the quasi-adaptive method outperforms the established criterion 𝛼=5×10−8 as well as the equal weighting scheme (the Sidak-correction). Furthermore, our method performs well when applied to real datasets and can potentially reveal previously undetected secondary signals in existing data.
In article III, we extended our quasi-adaptive method to identify plausible multiple independent signals at each locus (a secondary signal, a tertiary signal, a signal of 4th, and beyond) and applied it to the publically available GWAS meta-analysis to detect additional multiple independent eGFR-associated signals. The improved quasi-adaptive method successfully identified additional novel replicated independent SNPs that would have gone undetected by applying too conservative genome-wide significance level of 𝛼=5× 10−8. Colocalization analysis based on the novel independent signals identified potentially functional genes across the kidney and other tissues.
Overall, these articles contribute to the understanding of genetic factors associated with human kidney disease progression and provide novel methods for identifying secondary and multiple independent signals in conditional GWAS analyses.
In this thesis, I was able to provide answers to transport processes in lipid monolayers, which are ultimately, all of biological relevance. In particular, I was interested in lipid oxidation and dynamic compression/expansion processes of surfactant monolayers at the air-water interface:
Lipid oxidation was shown to be a consequence of the formation of a high concentration of reactive oxygen species (ROS) during cell respiration, which finally can lead to severe cell damage. It is not yet understood clearly, which part of the lipid molecules is especially prone to a ROS attack. I was particularly interested in the role of the double bonds of the acyl chains of the lipid molecules during oxidation. Further, I wanted to know the time scales of lipid interaction with the ROS.
Compared to lipid vesicles, lipid monolayers have the advantage that many parameters of the system can be adjusted easily. In our system, I made use of this by setting the lateral pressure to low values during H2O2 treatment, which facilitated the ROS to reach the double bonds in the acyl chains.
A prime example of biological systems out of thermal equilibrium was given in the alveolus surface, which is covered with a surfactant monolayer. During breathing, these monolayers undergo such a highly dynamic compression and expansion. Arising flows from breathing could disrupt a film and consequently, it would lose its protective role. One of my goals was to understand flows and their influence on domain shape. Dependent on the strength of the flows, I expected different growth regimes, with differing prevailing transport processes. Once understanding the underlying mechanisms in domain shaping would allow me to draw conclusions on biological systems.
In order to address these questions, I established two systems, both based on the compression of lipid monolayers. I used isotherms to study the phase behavior of the lipids:9 During compression, the lipids can undergo phase transitions from the gaseous phase to the liquid expanded phase (LE-phase) and further from the LE-phase to the liquid condensed phase (LC-phase). A coexistence regime is observed in between the LE-phase and the LC-phase, characterized by a flat increase of lateral pressure with decreasing molecular area. Some lipids exhibited LC-phase domains. These were further investigated with Brewster angle microscopy (BAM). The used BAM was equipped with an integrated Scheimpflug optics, enabling an overall focused image plane. Furthermore, time-resolved observation of the growth of the domains was possible by recording videos (20 frames per seconds).
The first system enabled the investigation of lipid peroxidation, when the lipids were exposed to ROS. I chose DMPC, POPC, DOPC and PLPC, since these are phospholipids differing in the number and position of double bonds in acyl chains, but not in the head group. I used a H2O2 enriched phosphate buffered saline (PBS) solution, which served as a precursor for more reactive ROS, like hydroxyls (.OH). PBS was chosen, since it resembles the cell environment best. During defined waiting times of H2O2 treatment, the ROS diffused vertically from the subphase towards the monolayer. The lipid molecules were in the LE-phase, which facilitated the ROS molecules to reach also the double bonds of the acyl chains. The oxidized monolayers were then compressed at constant compression speed. Since the corresponding isotherms could be measured with high precision, the relative area increase δA/A between oxidized and non-oxidized monolayer along the isotherm proved to be a good measure for lipid peroxidation. The area increase δA in the molecular area of the oxidized molecules was explained by the eventually added, more hydrophilic −OOH group at the position of a carbon atom adjacent to a double bond in the unsaturated acyl chain. The −OOH group is drawn to the hydrophilic head group of the lipid. This leads to a kink in the acyl chain, which increases the molecular area A by δA. A model, which explained this peroxidation process in lipid vesicles, could be adopted to monolayers.
I compared the oxidation of phospholipids, differing in the number and position of the double bonds of their acyl chains. I found that δA/A increased with the growing number of double bonds in one acyl chain. However, a comparison of DOPC with POPC also showed the importance of the position of the acyl chain. I determined a slow reaction kinetic. It could be estimated by a √t dependence of the number density N_surface, which denominates the ROS sticking on the monolayer. The transport of ROS towards the monolayer was found to be diffusive, because it was the slowest process in the reaction. This interpretation was reinforced by a comparison of the temperature dependence of the relative area increase δA/A with the Stokes-Einstein diffusion coefficient of water molecules. The initial ROS concentration c_0 in the trough could be traced back (c_0~ 50 nM), which is indeed a realistic value found in human cells.
Concluding, our results can be understood as a feasibility study. The complexity of the monolayer can be arbitrarily increased, for example by the addition of proteins, allowing the investigation of other oxidative processes occurring in the cell membrane.
The second system allowed the investigation of growth of LC domains during fast compression processes of monolayers. I chose erucic acid monolayers, due to its low line tension and a continuous nucleation phase, enabling the formation of fractal domains. The monolayers were investigated with isotherms and BAM videos. Since v_C (compression speed of the monolayer) was continuous over the whole compression time, I had a system with well-defined hydrodynamic conditions. This allowed me a complete analysis of the system, starting with descriptive features of the observed domains to a classification of the observed growth regimes by means of hydrodynamic theory, through to the distinction and quantification of different kind of flows and supersaturations, involving Ivantsov theory:
Dependent on the compression speed v_C, I observed seaweed or dendritic domains. The LE/LC phase transition pressure pi_t was slightly increased compared to pi_inf of the equilibrium isotherm. A high compression speed v_C induced a supersaturation Δc. I introduced the excess lateral pressure Δpi=pi-pi_inf as an appropriate quantity to describe the supersaturation Δc. I showed a linear behavior of Δc on Δpi. Δc is a macroscopic quantity since it is averaged over the whole monolayer area. I characterized the domains of the seaweed and dendritic regime with respect to tip radii, branch lengths, side branch separations and fractal dimensions. I calculated the growth speed of the main branches. A roughly doubling of the growth speed of dendritic domains, compared to seaweed domains was observed. This was an evidence of adjunctive (Marangoni) flow in the subphase.
For each monolayer, I observed drifts during domain growth, which I explained by an anisotropy in the LE-phase, caused by the continuous nucleation of the domains. These kind of surface flows were superimposed to bulk flows in the subphase. Since I had a well established system, I could analyze the influence of these surface flows on domain shape, in terms of magnitude, direction and duration of the surface flows. I therefore used FFT spectra and directionality histograms. At low flows, the FFT showed six-fold symmetry. Higher drifts exhibited incisions in the FFT, eventually leading to dumbbell shaped FFTs at very high drifts. The domains grew preferentially in the direction parallel to the incision.
I used directionality histograms to analyze the angular distribution of the growing domains. They showed that the drift direction always correlated with a minimum in the histogram. In order to analyze drift duration, I split the domain in downstream and upstream side. I could show that for small drift durations, downstream growth was preferred. However, for longer drift durations, the flows got more isotropic and consequently growth was more balanced then.
I could observe only a weak correlation between drift velocity v_D and compression speed v_C. However, dendrites were formed when the compression speed v_C was high, while seaweed domains were formed when v_C was small. Domain distortion occurred in the same way, independent if seaweed or dendritic domains were considered. I further showed that hydrodynamic flows in the subphase and surface flows are superimposed and scale differently. Consequently, they have different impact on domain shape: hydrodynamic flows act on μm scale and influence the domain morphology (distance between side branches, and tip radius) and the growth speed of the main branches. Surface flows act on the mm to cm scale, cause an anisotropic flow in the LE phase surrounding the domain, and thus affect the overall domain shape.
The anisotropy in the LE-phase led to a locally different degree of supersaturation. To take this into account, I introduced a local normalized supersaturation Δ, based on the Ivantsov solution. Therefore, I calculated Péclet numbers p of measured quantities of the system. I obtained values of 0.88 ≤Δ≤0.90 for the seaweed regime (p<5) and 0.93 ≤Δ≤0.96 for the dendritic regime (p>6). Since the Ivantsov solution can only be applied for purely diffusive processes, I applied a modified Ivantsov solution Δ_mod, which calculates Δ at a distance 𝛿 ahead of the dendrite tip. I was able to determine the progression of the diffusive layer 𝛿, however a quantitative determination failed.
Applying hydrodynamic theory allowed me to classify the two growth regimes with respect to the Boussinesq number Bq. Since for both growth regimes, I achieved values of Bq<1, bulk viscous losses dominated over surface viscous losses. Further, a cross-over length 𝜉 was calculated, from which one can distinguish, whether advective transport dominates over diffusion.
I further connected the two defined supersaturations Δ and Δc via the excess lateral pressure Δpi. From this, I saw differences in the seaweed and dendritic growth regimes: The local normalized supersaturation Δ of seaweed growth seemed to be quite stable for a further increase of the lateral excess pressure Δpi, whereas it reacted quite sensitive in the dendritic regime. This was found to be an indication of a non-equilibrium regime, caused by the strong coupling of the monolayer to the subphase. It reinforces therefore the theory of Marangoni-flow.
The findings of this thesis emphasize the importance of understanding highly dynamic compression/expansion processes arising in surfactant monolayers. Using the example of the compression of the alveolus surface, it can be seen that a more realistic model of the pulmonary alveolus is not only enabled by increasing the complexity of the surfactant monolayer (e.g. by adding specific proteins or lipid mixtures to the monolayer). Equally important is the understanding in transport processes and the consequences for the monolayer structure. By the analysis of domain shapes, I presented a method, which is suitable for such a study.
Study of the effect of the podocyte-specific palladin knockout in mice with a 129 genetic background
(2023)
Worldwide, chronic kidney disease is one of the leading public health problems. Podocytes, highly specialized postmitotic cells in the filtration unit of the kidney glomerulus, are essential for the size selectivity of the filtration barrier. Loss of the complex 3D morphology of their interdigitating foot processes, effacement and detachment of the cells from the capillaries lead to proteinuria and often loss of kidney function.
Since the morphology of podocyte foot processes is highly dependent on an intact actin cytoskeleton and actin-binding proteins, we investigated the role of the actin-binding protein palladin in podocytes from mice with a 129 genetic background, that is more susceptible to kidney injury. PodoPalld129-/- mice were examined at 6 and 12 months of age using immunofluorescence staining, electron and 3D super-resolution microscopy as well as qRT-PCR.
Our analysis of PodoPalld129-/- mice at 6 and 12 months of age showed that podocyte- specific knockout of palladin results in dilation of the capillary tuft accompanied by loss of mesangial cells, indicating the influence of palladin on glomerular tuft formation. Besides, we observed morphological abnormalities such as an enlarged sub-podocyte space, cyst formations and an increased number of cell-cell contacts between podocytes and parietal epithelial cells in PodoPalld129-/- mice compared to controls. Moreover, palladin knockout resulted in downregulation of the slit diaphragm protein nephrin as well as an age-dependent significant increase in podocyte foot process effacement. Although there was a significant change in foot process morphology, we did not detect albuminuria in PodoPalld129-/- mice of both age groups. However, we found an increase of trefoil factor 1 (Tff1) in the urine of the mice, indicating an altered, more permeable filtration barrier.
Considering that palladin has several binding sites for important actin-binding and regulatory proteins, we studied the expression of Lasp-1, Pdlim2, VASP and Klotho in dependence on palladin. We found a remarkable reduction in, for example, phosphorylated Lasp-1 as well as Klotho, which could influence the morphology of podocyte foot processes.
Compared with PodoPalldBL/6-/- mice, PodoPalld129-/- mice showed stronger glomerular tuft dilation and developed podocytes with increased morphological abnormalities, underlining the importance of the genetic background.
In conclusion, these results demonstrate the essential role of palladin for podocyte morphology in mice with a 129 genetic background.
Hepatitis E virus (HEV) is emerging worldwide as a zoonotic pathogen that has remained largely undetected for decades, if not centuries. Its enormous success can be attributed to the wide range of host species, which can transmit the virus to humans, depending on the viral genotype. As a result, HEV is likely to remain a challenge even when the remaining hepatitis viruses (HAV, HBV, HCV), which are transmitted exclusively between humans, are under control. Although millions of HEV infections occur each year, little is known about this puzzling pathogen. One major issue in HEV research is the lack of reliable model systems. Established animal models are inefficient, expensive, or simply not representative of human HEV. On the other hand, cell culture systems are limited by the slow growth of the virus and inefficient replication and infection. The aim of this work is to with deepen the understanding of zoonotic HEV in animal hosts in Germany. For this purpose, a molecular and phylogenetic characterization of HEV sequences from rabbits and swine was conducted. A novel subtype of the zoonotic genotype HEV-3 was identified in a rabbit sample, further emphasizing the role of rabbits as HEV host species and possible reservoir of zoonotic HEV infections in Germany. On the other hand, a molecular biological screening of pigs and wild boars in Mecklenburg-Western Pomerania indicates a wide range of HEV-3 subtypes circulating in swine in north-east Germany. Furthermore, an optimized replicon system was established in order to enable characterization of various HEV sequences by reverse genetics. As a proof of concept, two rabbit HEV derived replicons were compared with two established, cell culture adapted HEV strains. The influence of different regions of the nonstructural protein on HEV replication was determined and quantified. In particular, a system was established, to reproducibly compare different strains and genotypes. This refined replicon system will enable the characterization of further HEV sequences and thus expand the knowledge on the determinants of the viral life cycle.
In vitro assays play a crucial role in the biopharmaceutical assessment of drugs. During the past two decades, biorelevant media became an indispensable tool to forecast the in vivo solubility and dissolution of pharmaceutical drug candidates, and to assess absorption risks like low solubility or drug precipitation. Nevertheless, in vitro set-ups are still a simplification of the conditions in the human GI tract. This thesis aimed to shed light on some of the remaining open questions, aiming at providing a better understanding of the effects of biorelevant media on solubility, dissolution, and precipitation processes, and providing guidance for a more streamlined usage in the future. The results of this work can be outlined in brief as follows: First, a new design of experiment-based method development was introduced which increased the robustness and accuracy of derivative UV spectrophotometric methods for drug quantification in biorelevant precipitation assays. Second, based on this new approach, the impact of SIF powder aging on the supersaturation and precipitation behavior of the model drug ketoconazole was investigated. Recommendations on the use of biorelevant media for precipitation assays were developed to further improve the reproducibility of transfer experiments and to enhance data reliability. Third, it was investigated under which circumstances the physiological bicarbonate buffer should be applied to Fasted State Simulated Intestinal Fluid medium for in vitro solubility, dissolution, and precipitation testing to resemble the in vivo conditions.
An already existing shortage of nurses was exacerbated by the COVID-19 pandemic. Inactive (former) nurses were regarded as a so-called silent reserve and were called upon by various agencies to volunteer for nursing. The question arose as to what factors might encourage or hinder such volunteering and facilitate deployment.
First, inactive nurses were asked via an online survey whether they had registered for deployment or not and what the reasons were for this decision. Further information on professional background was collected, including the reason for having left the profession in the first place. Based on the results of the online survey, focus group discussions were conducted with registered and unregistered inactive nurses, with nurses who had returned to the profession permanently, and with care home managers.
Only one third of the participants in the online survey said they had registered for a temporary assignment during the pandemic. The main reasons for registering were that inactive nurses ‘wanted to do their bit’ to manage the crisis, felt it was their duty and/or felt a sense of belonging to the nursing profession. The main reasons given for not having registered was that respondents ‘could not see a reason at the moment’, had health concerns, and ‘other relevant job commitments’. The majority of respondents still had jobs related to health, care or nursing.
The topics covered in the focus group discussions included the following: perception of the pandemic as a crisis, identity as a nurse and sense of professional commitment, role of current occupation in the decision to register, winning over inactive nurses with a very negative attitude towards returning to care during a crisis situation, support measures and offers regarding a deployment in nursing.
Both in the online survey and in the focus group discussions, a sense of belonging to the nursing profession was evident among many participants. However, this identity does not necessarily lead to a willingness to return to nursing during a crisis situation. Weighing up the risk of deployment against the positive or negative experiences gained during the active period can influence willingness. However, the possibility of taking a break from current work and returning to nursing at short notice is not always given. Many inactive nurses continue to work in the health sector and fulfil equally important tasks during a crisis situation which render them unavailable for deployment.
Different kinds of support for those willing to return to nursing during a crisis situation and communication on conditions of deployments need to be implemented and continuously improved to offer the inactive nurses the greatest possible security and to enable a largely unbureaucratic deployment.
Convolutional Neural Network-based image classification models are the current state-of-the-art for solving image classification problems. However, obtaining and using such a model to solve a specific image classification problem presents several challenges in practice. To train the model, we need to find good hyperparameter values for training, such as initial model weights or learning rate. However, finding these values is usually a non-trivial process. Another problem is that the training data used for model training is often class-imbalanced in practice. This usually has a negative impact on model training. However, not only is it challenging to obtain a Convolutional Neural Network-based model, but also to use the model after model training. After training, the model might be applied to images that were drawn from a data distribution that is different from the data distribution the training data was drawn from. These images are typically referred to as out-of-distribution samples. Unfortunately, Convolutional Neural Network-based image classification models typically fail to predict the correct class for out-of-distribution samples without warning, which is problematic when such a model is used for safety-critical applications. In my work, I examined whether information from the layers of a Convolutional Neural Network-based image classification model (pixels and activations) can be used to address all of these issues. As a result, I suggest a method for initializing the model weights based on image patches, a method for balancing a class-imbalanced dataset based on layer activations, and a method for detecting out-of-distribution samples, which is also based on layer activations. To test the proposed methods, I conducted extensive experiments using different datasets. My experiments showed that layer information (pixels and activations) can indeed be used to address all of the aforementioned challenges when training and using Convolutional Neural Network-based image classification models.
Ecological Impacts and Phenotypic Plasticity of a Global Invasive Cactus, Opuntia ficus-indica
(2023)
Biological invasions by non-native species pose one of the major threats to biodiversity, the way ecosystems function, and the well-being of humans. These invasions can occur through various means, including accidental or intentional introductions by humans, natural dispersal, and climate change. Non-native species can harm the native species and ecosystems, by homogenizing plant communities, competing for resources, changing how the ecosystem operates, and eventually causing native species to go extinct. Even though not all non-native species become invasive, changes in climate and ecosystems can cause the successful establishment and spread of non-native species. Despite the advancements in our understanding of biological invasions in recent years, research has been biased towards temperate regions, whereas arid and semi-arid regions that are highly impacted by climate change are underrepresented. Thus, particularly focusing on the impacts of biological invasions in subtropical arid and semi-arid regions, the goal of this PhD project was to explore the effects of an invasive cactus on the local native communities and investigate the mechanisms of its successful invasion. Certain species are found to take advantage of the ever-drying climates in the arid/ semi-arid regions of the world. Opuntia ficus-indica, native to Mexico, is an exceptionally successful drought-tolerant invasive cactus that successfully grows in these regions. O. ficus-indica, a most widespread invasive cactus, is considered an ecosystem engineer as it modifies the habitats of indigenous plant species and dependent animals. This project aimed to identify the ecological impacts of O. ficus-indica in the highlands of Eritrea, the competitive potential of O. ficus-indica and the plastic changes that enabled its spread and invasion (Chapters I-III). For this purpose, field observations and common garden experiments were carried out throughout the project.
We investigated the effects of Opuntia ficus-indica on the spatial diversity of native plant communities (Chapter I), its competitive ability against native species (Chapter II) and the phenotypic plasticity of O. ficus-indica (Chapter III). To investigate the main ecological effects of O. ficus-indica on the native community, field data was collected from the highlands of Eritrea and comparisons were made between O. ficus-indica invaded and noninvaded areas (Chapter I). The study aimed to understand the effects of O. ficus-indica by examining species composition, richness, and diversity across vegetation layers and revealed that O. ficus-indica homogenises the species composition of the native ecosystem. This provides evidence that the presence of O. ficus-indica reduces landscape-level heterogeneity or spatial diversity. However, O. ficus-indica did not influence the species richness and diversity of the local communities. The mechanisms of the successful homogenisation of the local communities by O. ficus-indica were attributed to the potential competitive abilities of O. ficus-indica against the native species, and the plastic and adaptive traits it developed in the non-native ranges. The first assumption was tested by setting up a common garden competition experiment between two native Eritrean species, Ricinus communis and Solanum marginatum (Chapter II). The experiment used two water availability treatments, wet and dry, and categorized plants into intraspecific (native or invasive only) and interspecific (native and invasive) competition. The study evaluated the impacts by comparing the growth of O. ficus-indica alone to the growth alongside native species which revealed the weak competitive potential of O. ficus-indica. However, O. ficus-indica was observed to outgrow the native species in several folds which can be attributed to its successful invasion. The second assumption of the successful spread of O. ficus-indica was attributed to the phenotypic plastic traits adapted by O. ficus-indica in the non-native ranges (Chapter III). The phenotypic plasticity of O. ficus-indica was assessed by exposing it to water stress across dry and wet environments. The species were cultivated from a diverse set of 12 populations, encompassing its native range in Mexico with three cultivars and nonnative ranges in Africa (Algeria, Eritrea, Ethiopia), the island of Madeira off the coast of Africa, and in Europe, Italy with two cultivars and in Portugal from three sites. In Mexico and Italy, we collected various cultivars to ensure a wide representation of genotypes. We found that the species originating from the native range of O. ficus-indica exhibited lower plasticity to conditions of elevated water availability. Furthermore, a trial gradient experiment on O. ficus-indica was conducted to determine the appropriate watering levels for the species and the experiment revealed not only the species' capacity to endure a lack of water for nine months but also its ability to withstand prolonged waterlogged conditions.
This thesis illustrates the fact that invasive species are a major threat to biodiversity and ecosystem functioning worldwide, especially in rarely studied regions with dry climates and limited resources. How can invasive plants spread and cause negative impacts on native ecosystems (Chapter I), despite their weak competitive abilities (Chapter II)? This thesis explored these questions by examining the case of O. ficus-indica, an invasive species in arid/ semi-arid climates (Chapter I). It showed that O. ficus-indica has a high growth potential that allows it to overcome resource limitations, that its growth is not affected by competition from native species (Chapter II), and that it exhibits adaptive plasticity that enhances its invasion success in different environments (Chapter III). This thesis revealed the complex mechanisms and consequences of biological invasions in dry climates and contributes to the understanding of invasive species. It also suggests that more research is needed in understudied regions to assess the impacts of O. ficus-indica or invasive species in general on native biodiversity and ecosystem services and to identify the factors that influence the competitive and adaptive potentials.
Marine algae are essential for fixation of carbon dioxide, which they transform into complex polysaccharides. These carbohydrates are degraded e.g., by marine Bacteroidetes and the understanding of their decomposition mechanism can expand our knowledge how marine biomasses can be accessed. This understanding then gains insights into the marine carbon
cycle. This thesis summarizes the current knowledge of marine enzymatic polysaccharide degradation in review Article I and extents a previously discovered ulvan degradation pathway in Article II with the description of a novel dehydratase involved in the ulvan degradation pathway. This enlarged ulvan-degradation pathway can be used to generate fermentable sugars from the algal derived polysaccharide ulvan. A potential biorefinery process is proposed in Article III, where B. licheniformis was engineered to degrade ulvan, thus establishing the initial steps for a microbial cell factory development. In addition to ulvan, also plenty of other complex carbohydrate sources are present in the ocean. The enzymatic elucidation principles previously developed were thus adapted towards a new marine carbohydrate. In Article IV a xylan utilization pathway was elucidated, using enzymes present in Flavimarina Hel_I_48 as model bacterium. The Flavimarina genome contains two separated genome clusters which potentially targets xylose containing polymers reflecting the diversity and adaptions towards different marine xylan-like substrates. Besides, marine Bacteroidetes are adapted towards decomposition of methylated polysaccharide, e.g., porphyran, via demethylation catalyzed by cytochrome P450 monooxygenases. This reaction results in the formation of toxic formaldehyde and thus the marine Bacteroidetes require formaldehyde detoxification principles. The analysis of potential formaldehyde detoxification mechanisms revealed a marine RuMP pathway (Article V) and a novel auxiliary activity of an alcohol dehydrogenase of which the encoding gene is adjacent to the demethylase cluster (Article VI).
This dissertation focuses on the characterization of novel enzymes and metabolic pathways that fulfill crucial functions during marine carbohydrate degradation by Bacteroidetes and thus contributes to an advanced understanding of the global carbon cycle. Depolymerization and utilization of marine polysaccharides by Bacteroidetes requires a tremendous repertoire of enzymes with a wide range of functions. For instance, during the breakdown of the marine red algal polysaccharide porphyran, an oxidative demethylation of the methoxy sugar 6-O-methyl-D-galactose (G6Me) by cytochrome P450 monooxygenases occurs. This reaction produces huge amounts of cytotoxic formaldehyde, marine bacteria capable of degrading porphyran must therefore possess suitable formaldehyde detoxification pathways. Consequently, Article I focus on the identification of possible formaldehyde detoxification pathways in marine
Flavobacteriia, which led to the discovery of the ribulose monophosphate pathway as specific pathway for the detoxification of formaldehyde in certain Bacteroidetes like Zobellia galactanivorans. Furthermore, it was demonstrated in Article II that alcohol dehydrogenases play an essential role in the microbial utilization of G6Me and therefore possess a function in porphyran degradation. Discovering novel enzymes, entire enzymatic cascades or biotechnologically important microorganisms that can metabolize these marine carbohydrates also contributes to the utilization of marine polysaccharides as feedstock for potential biotechnological applications. A prospective biorefinery process was proposed in Article III by the identification of Bacillus licheniformis as promising utilizer of marine carbohydrate-derived monosaccharides and the creation of a microbial cell factory capable of growing on ulvan, a marine carbohydrate obtainable from algal bloom-dominating green algae, enabling an industrial use of the renewable and abundant algal biomass in future.
Can established parties influence the electoral success of new parties? To answer this research question, the author examined the relationships of 168 new parties in 18 highly developed democracies with their established competitors based on their respective election programmes and election results. His analysis of the textual similarity of these election manifestos shows that established parties can influence their competitors' election results by selectively changing the emphasis of their policies. However, competition among the parties must also be taken into account. This study thus contributes to a better understanding of the dynamics of party competition and the opportunities offered by computer-assisted textual analysis in the social sciences.
Emerging zoonotic viruses are a constant threat to human and animal health. Therefore, knowledge about the host factors influencing viral pathogenicity is highly welcome as a basis for developing treatment or vaccine strategies. In order to identify host factors that potentially determine the
pathogenicity of three highly pathogenic (’high consequence’) zoonotic viruses, the interactomes of
selected viral proteins were analysed in parallel with the interactomes of the homologous proteins from closely related viruses which lack high pathogenicity. For this purpose, affinity purification mass spectrometry (AP-MS) was performed with the virus proteins as baits and lists of candidate proteins were generated that may determine the pathotype and warrant follow-up studies to characterise their function concerning the viral life cycles. In detail, the interactomes of virus pairs from the arenaviruses, filoviruses and henipaviruses were studied. The following protein homologues were selected: for filoviruses, the transcription factor VP30, the co-transcription factor VP35 and matrix protein VP40 of the non-pathogenic Reston virus
(RESTV, species Reston ebolavirus), the pathogenic Ebola virus (EBOV, species Zaire ebolavirus),
and, in addition, the Lloviu virus (LLOV, species Lloviu cuevavirus); in case of the arenaviruses
the nucleoprotein (NP), matrix protein (Z) and glycoprotein (GP) of the pathogenic Junín virus (JUNV, species Argentine mammarenavirus) and the non-pathogenic Tacaribe virus (TCRV, species Tacaribe mammarenavirus); and for the henipaviruses, the fusion protein F of the apathogenic Cedar virus (CedV, species Cedar henipavirus) and the pathogenic Nipah virus (NiV, species Nipah henipavirus). The experimental approach was to express the tagged bait proteins in human cells by transfection with appropriate constructs, purify the interactomes by affinity enrichment and analyse their protein content by MS. Quantitation was performed by labelling with stable isotopes or by label-free quantification (LFQ). High-confidence interactions for the LFQ approach were identified using the Mass Spectrometry interaction STatistics (MiST) scoring tool. Qualitative and quantitative data were used to identify a limited number of candidates for follow-up research. Additionally,
the interactomes were analysed with bioinformatical tools like term enrichment analysis and network analysis to identify cellular pathways which are possibly impacted by the expression of viral proteins. A novel specific interactor of EBOV VP30 was identified, ubiquitin carboxyl-terminal hydrolase7
(USP7, also known as HAUSP), and the interaction was partially characterised. The interaction was confirmed by reverse-pull-down experiments, and the Kd value (determined by Microscale Thermophoresis, MST) was found to be lower than for the interaction of USP7 with the RESTV VP30.
This work adds insight into virus protein interactomes, especially for the often neglected low pathogenic virus species. Furthermore, the pathogenicity of the viruses was refl ected to some degree
in the interactomes of their proteins. The generated interactome data for the different virus species
create a basis in the search for interactions that determine pathogenicity.
Recent experimental campaigns in the Wendelstein 7-X stellarator, a
plasma-confining device designed to investigate the Magnetic Confinement Fusion
(MCF) approach to generating electrical power, have shown that the injection of
fuelling pellets had an unexpected and considerable impact on the performance of
the plasma. Rather than simply refuelling the device and `diluting' the plasma
energy, pellet injection is followed by a significant increase in the ratio of
the ion temperature to the electron temperature. It has been suggested that this
is not merely due to the improved confinement following the reduction of
turbulent transport after the pellet material has homogenised with the bulk
plasma, but also due to a direct transfer of energy from electrons to ions. The
proposed mechanism for this energy transfer is the ambipolar expansion of the
pellet plasmoid, the localised plasma structure produced by the
ionisation of ablated pellet material, along magnetic field lines.
Early work on pellet plasmoid expansion predicted that half the heating power
deposited in plasmoid electrons by collisions with hot ambient electrons is
transferred to plasmoid ions in the form of flow velocity as the plasmoid
expands. The complicated nature of the system of the pellet plasmoid embedded in
the ambient plasma, particularly the behaviour of electrons, which experience
many collisional and collisionless phenomena on multiple disparate timescales,
means that early models of the expansion were not wholly self-consistent, but
rather made use of strong approximations that apply in some regions of the
plasmoid but not in others. For example, only electrons and ions associated with
the plasmoid were rigorously treated, meaning that the framework was one of
`expansion into vacuum'. Combined with the assumption of Maxwellian electrons,
this led to an electric potential that was unbounded at infinity. Naturally, the
validity of the conclusions of such a model are called into question because the
approximations lose their validity far from the plasmoid and as time advances,
yet predictions about the final state of the plasma are desired. A deeper
investigation is required: careful consideration of the phenomena in question
and the timescales (and lengthscales) on which they act must be made in order to
rigorously construct a model that is valid throughout the entire expansion.
The first two papers presented in this thesis iterate on the model established
in the paper that first predicted the electron-to-ion energy transfer; their aim
was to find out how the character of the expansion changes with a more
sophisticated and accurate description of various phenomena, while remaining
within the existing framework of expansion into vacuum. Ultimately, we find that
the qualitative character is unchanged, and that approximately half the heating
power deposited in plasmoid electrons is transferred to ions.
Two other papers in this thesis address the limitations of the original model.
This is achieved by properly considering the electron kinetic problem in a
plasmoid. One paper considers the electron kinetic problem when electrons are
highly isotropised. In this case the kinetic equation can be integrated to
remove all but two independent variables, which is the maximum possible
reduction considering it is a time-dependent problem. The full nonlinear
integro-differential Landau self-collision operator is integrated exactly and
few approximations are made, leading to a rather general kinetic equation.
However, for fuelling pellets some anisotropy in the electron distribution is
expected. Another paper considers the electron kinetic problem (and the entire
plasmoid expansion) allowing for electron anisotropy. Careful consideration of
the ordering of timescales of electron phenomena in a pellet plasmoid leads to a
steady-state kinetic problem that we call collisional quasi-equilibrium (QE). QE
appears in many ways similar to the collisional steady-state characterising a
true thermal equilibrium. It was found that the time-dependent kinetic problem
of the earlier paper, with isotropic electrons, produces the QE distribution
function, corroborating the existence of the QE state. We then take moments of
the electron kinetic equation that is valid on the expansion timescale, assuming
that the electron distribution is that given as the solution to the QE kinetic
problem. This is completely analogous to what is done to obtain the Braginskii
equations or any Chapman-Enskog theory. The result is a set of equations for the
long-term evolution of the macroscopic quantities that describe the distribution
function existing in a quasi-steady-state at each point in time. It is from this
point that one may feasibly describe the plasmoid expansion with an accurate
picture of the electron kinetics and finally obtain the electron-to-ion energy
transfer so desired in a rigorous model of the expansion.
From a broader point of view, the two frameworks provided by these rigorous
investigations of the electron kinetic problem serve as a basis for the future
study of plasmoids. Such a `first-principles' approach to plasmoid dynamics is
novel and interesting in its own right, but it will be demonstrated that such an
approach is essential for pellet plasmoids owing to the fact that they are
poorly described by the `standard tools' of plasma physics.
Using the QE framework it was found that, once more, about half the heating
power experienced by plasmoid electrons is transferred to plasmoid ions. The
incredible robustness of the prediction of such an energy transfer is, in the
author's opinion, the result of the self-similar nature of the expansion found
as a solution to the original model. As a rule, the profiles of self-similar
solutions tend to be attractors for the `real', more complicated, system, and
the qualitative predictions involving no parameters, of which the
electron-to-ion energy transfer is one, tend to be very sturdy.
Aside from fuelling pellets, composed of hydrogen or deuterium, one paper in
this thesis investigates the physics of high-Z pellets that are designed to
terminate the plasma safely in the event of a `disruption', where much of the
magnetic field energy is channelled into a runaway electron beam with
potentially disastrous consequences if the beam encounters a plasma-facing
component. The paper draws on the work carried out in the paper concerning the
kinetic problem of isotropised electrons in a plasmoid.
This thesis is `cumulative'; the vast majority of the work carried out is
described within a set of Papers, labelled A-E, placed at the back of the text.
There is a preceding `wrapper text' (given in numbered Sections) tasked with
introducing the reader to the topic, guiding the reader through the papers, and
expounding some of their main results. Some amount of material not present in
the papers is also provided in the wrapper text. Naturally, the wrapper text
mainly focusses on the results of the papers which are under my first
authorship. In the course of publishing papers over an extended period of time
the nomenclature is bound to vary. Although it is mostly consistent between the
papers, a few difference do arise, and the section `Common symbols and
subscripts' is provided in the frontmatter to alleviate confusion. Particular
care should be taken with the symbols x and z; both can refer to the
coordinate parallel to the magnetic field line, but in papers where z is used
for this purpose x tends to have another definition. In the wrapper text the
choice of symbols is generally chosen to reflect those in the corresponding
paper.
This dissertation explores and tries to unravel the fundamental basis of G-quadruplex end-folding as well as G-quadruplex interactions with small molecules by thermodynamic and structural approaches. Selective targeting of G-quadruplexes with ligands remains elusive, either because the ligand has
considerable binding affinity for other DNA structures or because it fails to discriminate between different G-quadruplex topologies. Unique structural motifs on the G-quadruplex may enhance or inhibit ligand binding to the G-quadruplex. For such aspects, it is necessary to understand the effect of G-quadruplex motifs or elements on the end-folding in order to better tune certain G-quadruplex topologies as model systems. Importantly for targeting G-quadruplex with ligands, motifs called Quadruplex-duplex (QD) junctions and interfaces are shown to be a binding hotspot
for various G-quadruplex ligands containing an intercalator motif. Binding affinity and selectivity of the ligands are discussed with the support of the NMR structures.
In den Weltmeeren findet rund die Hälfte der jährlichen globalen Kohlenstofffixierung statt, davon ein großer Anteil in küstennahen Regionen. Hier kommt es zu wiederkehrenden saisonalen Algenblüten, die durch eine zeitlich begrenzte explosionsartige Vermehrung von Mikroalgen (hauptsächlich Diatomeen und Coccolithophoren) charakterisiert sind. Vor allem Frühjahrsblüten (März-Mai) haben aufgrund ihrer zeitlichen und räumlichen Vorhersagbarkeit einen hohen Stellenwert als Modellsysteme, anhand deren sich der Kohlenstoffkreislauf der Meere untersuchen lässt.
Mikroalgen produzieren eine große Vielfalt an Makromolekülen, die für die mit ihnen vergesellschafteten Bakterien als Nahrungsgrundlage dienen. Besonders im Fokus stehen hier die für den Kohlenstoffkreislauf relevanten Polysaccharide. Im Gegensatz zu anderen natürlichen Makromolekülen wie DNA oder Proteinen können Polysaccharide aus vielen verschiedenen Monomeren mit unterschiedlichsten Bindungen bestehen. Zusätzlich finden sich an diesen Zuckermonomeren viele Modifikationen wie Acetylierungen, Methylierungen oder Sulfatierungen, die die Komplexität weiter erhöhen. Diese Variabilität bedingt eine hohe strukturelle und funktionale Diversität. So können Polysaccharide Speicherstoffe, Zellwandbestandteile oder Teile der extrazellulären Matrix darstellen.
Komplementär hierzu besitzen Polysaccharid-verwertende Bakterien entsprechend komplexe, enzymatische Abbaumechanismen. Besonders hervorzuheben sind hier die Bakterien des Phylums Bacteroidota, die sich in verschiedensten Nischen auf den Abbau von Polysacchariden spezialisiert haben. Sie finden sich in Bodenproben, als Teil der menschlichen Darmflora, oder eben auch als bedeutende Begleiter von Algenblüten.
Bacteroidota (und in marinen Systemen hauptsächlich die zu ihnen gehörenden Flavobakterien) besitzen zum Abbau diverser Polysaccharide sogenannte Polysaccharide utilization loci (PULs), genomische Inseln, die alle notwendigen Proteine zur Aufnahme und Abbau eines bestimmten Polysaccharids codieren. Hierzu gehören hochspezifische Enzyme (Carbohydrate-active enzymes, CAZymes), transkriptionelle Regulatoren sowie Transportersysteme, die initial gespaltene Oligosaccharide über die Membran in das Bakterium transportieren, wo sie von weiteren Enzymen vollständig abgebaut werden. Diese Co-Lokalisation der benötigten Gene und deren gemeinsame Regulation stellt einen enormen Selektionsvorteil der Bacteroidota dar und ist der Grund, warum sie, ähnlich wie Algen, einer jährlich wiederkehrenden Sukzession folgen, die sich gut untersuchen lässt.Die Forschungsartikel, die Teil dieser Doktorarbeit sind, untersuchen das Zusammenspiel von Polysaccharid-produzierenden Algen mit den Bakterien, die sie abbauen, aber auch darauf basierende Beziehungen der Bakterien untereinander. Die erste Publikation beschäftigt sich mit dem weit verbreiteten Speicherpolysaccharid α-Glucan, für das der Großteil der blütenbegleitenden Bakterien einen spezifischen aktiven PUL besitzt. Eine Untersuchung der in der Blüte vorhandenen Algenarten bestätigte, dass die Blüte von β-Glucan-produzierenden Algen dominiert wird. Da Bakterien aber selbst α-Glucane als Speicherpolysaccharide verwenden, konnte gezeigt werden, dass nicht die Algen selbst, sondern die Bakterien Hauptproduzent dieser Polysaccharide während einer Phytoplanktonblüte sind. Bakterielle Proteine, die dem Abbau von Algen-β-Glucan und dem daraus folgenden Aufbau von bakteriellem α-Glucan dienen, waren in Umweltproben und in Laborkulturen unter ähnlichen Bedingungen abundant. Die Untersuchung von extrahiertem bakteriellem Polysaccharid bewies, dass dieses nicht nur α-Glucan enthält, sondern dass dieses Polysaccharid auch in der Lage war, α-Glucan PULs mariner Bakterien zu induzieren. Hier zeigte sich ein innerhalb des marinen Kohlenstoffkreislaufs bisher wenig berücksichtigter Kreislauf, indem Bakterien Polysaccharide anderer Bakterien nutzen, die z.B. durch Viren lysiert wurden.
Die anderen zwei Artikel dieser Arbeit befassen sich mit dem Abbau von Zellwandpolysacchariden durch blütenassoziierte Modellbakterien. In einer der Studien wird detailliert der Abbau eines β-Mannans (ein Polysaccharid das hauptsächlich aus dem Monosaccharid Mannose besteht) durch ein Bakterium des Genus Muricauda beschrieben. Die PUL-Struktur dieses Bakteriums kam in mehreren anderen Phytoplanktonblüten-assoziierten Bakterien vor. Diese Beobachtung wies darauf hin, dass es sich hier um ein Mannan mit zusätzlichen Galactose- und Glucose-Substitutionen handelte. Proteom-Untersuchungen bestätigten, dass das Bakterium derartige Substrate unter Induktion des β-Mannan-PULs nutzen können. β-Mannan konnte durch Antikörpermarkierung in Blütenproben sowie spezifischen Mikroalgenarten (Chaetoceros, Coscinodiscus) nachgewiesen werden. Die in dieser Publikation charakterisieren β-Mannan-PUL-codierten Enzyme waren in der Lage, dieses Signal zu löschen, was bewies, dass Muricauda sp. Mannan-basierte Zellwandpolysaccharide bestimmter Arten von Mikroalgen abbauen kann.
Die dritte Studie geht näher auf den Abbau von Xylanen (bestehend aus Xylose) durch ein blütenassoziiertes Bakterium des Genus Flavimarina ein. In diesem Bakterium wurden anhand der enthaltenen Xylanasen zwei putative Xylan-PULs annotiert. Wachstumsexperimente und Proteom-Untersuchungen zeigten, dass einer dieser PULs hauptsächlich bei Wachstum auf Glucoronoxylan induziert wird, während der andere PUL aufArabinoxylane stärker reagierte. Untersuchung der PUL-CAZymes bestätigte diese Ergebnisse durch Charakterisierung mehrerer Xylanasen sowie Glucoronidasen und Arabinofuranosidasen. Zusätzlich codierten beide PULs für Esterasen, die eine Modifikation der natürlichen Substrate durch Acetylierungen oder Methylierungen nahelegen. Da all diese Merkmale von terrestrischen Xylanen geteilt werden und in Blütenproben aus Küstennahen Regionen Xylane nachgewiesen wurden, ist es möglich, dass Bakterien aus solchen Regionen sowohl Xylane terrestrischen Ursprungs (z.B. durch Flusseinspeisung) sowie marinen Ursprungs abbauen können.
In this thesis, new catalysts as well as unprecedented approaches for the
valorization of sustainable carbon sources were investigated. The first part deals with the design of catalysts for photocatalytic CO2 reduction (Articles I&II). The promiscuous activity of phenolic acid decarboxylase from Bacillus subtilis (BsPAD) was found to catalyze CO2 reduction (Article I). This cofactor-free enzyme could facilitate the replacement of (noble) metal catalysts regularly employed in CO2 reduction. Based on these findings, additional enzyme catalysts were identified for photocatalytic CO2 reduction. The second part (Articles III-VII) focuses on the valorization of resources obtained from biomass, such as olive mill waste water or lignin, by the promising acyltransferases/hydrolase PestE from Pyrobaculum calidifontis VA1 (Articles IV-VII). The potential of PestE for the valorization of sustainable sources has been demonstrated by enzyme engineering and use in (chemo)enzymatic cascade reactions leading to value-added products.
Background
This study aimed to analyze the impact of low-value medications (Lvm), that is, medications unlikely to benefit patients but to cause harm, on patient-centered outcomes over 24 months.
Methods
This analysis was based on longitudinal data of patients with dementia. The impact of Lvm on health-related quality of life (HRQoL), hospitalizations, and health care costs were assessed using multiple regression models.
Results
Over 24 months, Lvm was highly prevalent and significantly increased the risk of hospitalization, increased health care costs, and reduced patients' HRQoL.
Conclusion
More than every second patient received Lvm, negatively impacting patient-reported HRQoL, hospitalizations, and costs. Innovative approaches are needed to encourage prescribers to avoid and replace Lvm in dementia care.
Posttranslational modifications are involved in the regulation of virtually all cellular processes, including immune response, nevertheless, they are also targets manipulated by invading pathogens. The first investigated example is protein citrullination which is an important posttranslational modification that acts on a multitude of processes like supervision of cell pluripotency and rheumatoid arthritis. Citrullination of targeted arginine residues is performed by the Peptidylarginine deiminase. Within the first published manuscript, being part of this thesis, it was possible to show the use of this posttranslational modification by the human pathogen Porphyromonas gingivalis to facilitate innate immune evasion at three distinct level. P. gingivalis was demonstrated to citrullinate proteins by Porphyromonas peptidylarginine deiminase resulting in diminished phagocytosis and subsequent killing by neutrophils. Furthermore, it was shown that citrullination of histone H3 enables P. gingivalis to survive in neutrophil extracellular traps and incapacitate the lysozyme-derived peptide LP9.
The second investigated posttranslational modification is ubiquitination and its role in respiratory tract infections. Ubiquitination is the covalent attachment of a small protein that consisting of only 76 amino acids to the ε-amino group of lysine residues to posttranslational modify proteins. Acute infections of the lower respiratory tract such as viral and bacterial co-infections are among the most prevalent reasons of fatal casualties worldwide. Therefore, the interactions between host and pathogens resulting in the impairment of the hosts immune response and immune evasion of the pathogens, need to be elucidated. To get new insights in the infection driven changes in protein polyubiquitination and alterations in the abundance of ubiquitin E3 ligases involved in ubiquitination, cellular proteomes were monitored in detail by high resolution mass spectrometry. Therefore, the epithelial cell lines 16HBE14o- (Manuscript II) and A549 (Manuscript III) were co-infected with influenza A virus H1N1 and Streptococcus pyogenes or Staphylococcus aureus or with influenza A virus H1N1 and Streptococcus pneumoniae, respectively. Here, it could be shown in 16HBE14o- cells that co-infection of epithelial cells is not characterized by decreased cell survival and that observable effects on the proteome and ubiquitinome are mostly additive rather than synergistic. S. pyogenes infection affected the mitochondrial function, cell-cell adhesion, endocytosis and actin organization. Viral infection affected mRNA processing and Rho signaling. Viral and bacterial co-infection was detected to affect processes that were already affected by both of the corresponding single infections. No further pathways were strongly affected by the co-infection. A similar result has been observed in A549 cells co-infected IAV and S. pneumoniae. Overrepresented gene ontology terms depict the sum of those observed in the viral and bacterial single infection. Moreover, no significant change in cell survival upon co-infection compared to single bacterial infection was noticed for A549 cells either. This led to the suggestion that co-infection of investigated epithelial cells under examined conditions possesses additive rather than synergistic effect and thus, may not worsen the outcome of the infection within the studied conditions. Infections in other systems, may provide varying results and thus should be examined in future studies.
Impact of proteostasis and the ubiquitin proteasome system on myeloid cell function in the CNS
(2023)
Cellular protein homeostasis (proteostasis) maintains a functional proteome and thus proper cell function. Proteostasis is facilitated by the ubiquitin-proteasome system (UPS), an intracellular protein turnover machinery ensuring clearance of damaged, misfolded, old and/or unneeded regulatory proteins. This is particularly important in the central nervous system (CNS), where it is linked to neurodegeneration. Disruptions of the proteostasis systems cause the accumulation of misfolded proteins which are commonly seen in progressive neurodegenerative diseases also linked to neuroinflammation. Proper UPS function can protect cells from the accumulation of defective proteins, neurodegeneration and neuroinflammation. Furthermore, it has been found that loss of function mutations in the genes encoding UPS components are linked to systemic inflammation including neuroinflammation and/or neurodevelopmental disorders. Proteasome defects in patients suffering from these disorders cause decreased proteasome activity, accumulation of proteins, activation of proteotoxic stress responses and systemic inflammation. However, the molecular link between proteotoxic stress and the initiation of inflammatory signalling remained unclear. In Article 2, we summarized the importance of the UPS in immune cell proteostasis and function including activation of innate and adaptive immune responses. Although UPS function is notably important in innate immune signalling, the current understanding of the role of UPS in myeloid cell function in the CNS is limited. We also indicated the involvement of impaired UPS function in sterile systemic inflammation including neuroinflammation as well as tumour diseases and pathogen manipulation of immune cells.
To investigate the molecular link behind proteasome impairment and systemic inflammation in the brain, we focused on microglia cells as the only immune residents of the CNS. In Article 1, we used a pharmacological inhibitor called bortezomib which targets β5 and β5i/LMP7 subunit activities in standard proteasome (SP) and immunoproteasome (IP), respectively. We showed for the first time on the molecular level that inhibition of proteasome activity by bortezomib triggers the accumulation of ubiquitylated proteins, proteotoxic stress responses and innate immune signalling activation depending on the induced proteotoxic stress response called unfolded protein response (UPR) in murine microglia. In particular, activation of the inositol-requiring protein 1α arm of UPR upon bortezomib treatment leads to systemic inflammation as indicated by type I interferon (IFN) response.
IP enhance the proteolytic capacity of UPS by rapid clearance of proteins upon immune signalling activation. Microglia, like other immune cells, exhibit constitutive expression of IP as well as SP to maintain their cellular proteostasis. In Manuscript 3, we studied the particular impact of IP impairment on microglial cellular function. We showed accumulation of ubiquitin-modified proteins and activation of proteotoxic stress responses in IP-impaired mouse and human microglia models. Moreover, we identified possible IP substrates in microglia using β5i/LMP7 knockout mice as an IP deficiency model and, examined how IP deficiency affects microglia function. IP deficient microglia affected the ubiquitylation levels of proteins involved in multiple pathways such as immune responses, energy metabolism, cytoskeleton organisation, cell cycle and ribosome function. Based on the molecular analysis, we confirmed sterile activation of innate immune signalling mechanisms in IP impaired microglia. This is driven by the proteotoxic stress sensor protein kinase R (PKR). In addition, we were able to show that IP impairment altered levels of the microglial activation markers, which are also involved in motility, adhesion and phagocytosis of microglia.
In this thesis, we highlight that UPS function is necessary to maintain microglial proteostasis and, that impairment of proteasome activities triggers sterile inflammation in microglia via activation of proteotoxic stress responses. The described activation of innate immune signalling mechanisms in microglia upon proteasome impairment may be considered as new therapeutic targets for patients suffering from rare protesomapathies or other disorders linked to dysregulated immune signalling.
Background
Previous work has focused on speckle-tracking echocardiography (STE)-derived global longitudinal and circumferential peak strain as potential superior prognostic metric markers compared with left ventricular ejection fraction (LVEF). However, the value of regional distribution and the respective orientation of left ventricular wall motion (quantified as strain and derived from STE) for survival prediction have not been investigated yet. Moreover, most of the recent studies on risk stratification in primary and secondary prevention do not use neural networks for outcome prediction.
Purpose
To evaluate the performance of neural networks for predicting all cause-mortality with different model inputs in a moderate-sized general population cohort.
Methods
All participants of the second cohort of the population-based Study of Health in Pomerania (SHIP-TREND-0) without prior cardiovascular disease (CVD; acute myocardial infarction, cardiac surgery/intervention, heart failure and stroke) and with transthoracic echocardiography exams were followed for all-cause mortality from baseline examination (2008-2012) until 2019.
A novel deep neural network architecture ‘nnet-Surv-rcsplines’, that extends the Royston-Parmar- cubic splines survival model to neural networks was proposed and applied to predict all-cause mortality from STE-derived global and/or regional myocardial longitudinal, circumferential, transverse, and radial strain in addition to the components of the ESC SCORE model. The models were evaluated by 8.5-year area-under-the-receiver-operating-characteristic (AUROC) and (scaled) Brier score [(S)BS]and compared to the SCORE model adjusted for mortality rates in Germany in 2010.
Results
In total, 3858 participants (53 % female, median age 51 years) were followed for a median time of 8.4 (95 % CI 8.3 – 8.5) years. Application of ‘nnet-Surv-rcsplines’ to the components of the ESC SCORE model alone resulted in the best discriminatory performance (AUROC 0.9 [0.86-0.91]) and lowest prediction error (SBS 21[18-23] %). The latter was significantly lower (p <0.001) than the original SCORE model (SBS 11 [9.5 - 13] %), while discrimination did not differ significantly. There was no difference in (S)BS (p= 0.66) when global circumferential and longitudinal strain were added to the model. Solely including STE-data resulted in an informative (AUROC 0.71 [0.69, 0.74]; SBS 3.6 [2.8-4.6] %) but worse (p<0.001) model performance than when considering the sociodemographic and instrumental biomarkers, too.
Conclusion
Regional myocardial strain distribution contains prognostic information for predicting all-cause mortality in a primary prevention sample of subjects without CVD. Still, the incremental prognostic value of STE parameters was not demonstrated. Application of neural networks on available traditional risk factors in primary prevention may improve outcome prediction compared to standard statistical approaches and lead to better treatment decisions.
Age is the single biggest risk factor for most major human diseases. As such, understanding the intricate molecular changes that drive biological aging holds great promise in attempting to slow
the onset of systemic diseases and thereby increase the effective health-span in modern societies.
This thesis explores several computational approaches to capture and analyze the molecular biological alterations triggered by intrinsic and extrinsic aging using skin as a model tissue to deliver genes and pathways as potential targets for intervention strategies.
Publication 1 demonstrates the utility of multi-omics data integration strategies for aging research, leading to the identification of four latent aging phases in skin tissue through an integrated cluster analysis of gene expression and DNA methylation data. The four phases improved the detection of molecular aging signals and were shown to be associated with sunbathing habits of the test subjects. Deeper analysis revealed extensive non-linear alterations in various biological pathways particularly at the transition into the fourth aging phase, coinciding with menopause, with potentially wide-reaching functional implications. Publication 2 describes the development of a novel type of age clock, that provides a new level of interpretability by embedding biological pathway information in the architecture of an artificial neural network. The clock not only generates meaningful biological age estimates from gene expression data, but further allows simultaneous monitoring of the aging states of various biological processes through the activations of intermediate neurons. Analyses of the inner workings of the clock revealed a wide-spread impact of aging on the global pathway landscape. Simulation experiments using the transcriptomic clock recapitulated known functional aging gene associations and allowed deciphering of the pathways by which accelerated aging conditions such as chronic sun exposure and Hutchinson-Gilford progeria syndrome exert their effects. Publication 3 further explores the molecular alterations caused by the pro-aging effector UV irradiation in the skin. The multi-omics data analysis of repetitively irradiated skin revealed signs of the immediate acquisition of aging- and cancer-related epigenetic signatures and concurrent wide-spread transcriptional changes across various biological processes. Investigations into the varying resilience to irradiation between subjects revealed prognostic biomarker signatures capable of predicting individual UV tolerances, with accuracies far surpassing the traditional Fitzpatrick classification scheme. Further analysis of the transcripts and pathways associated with UV tolerance identified a form of melanin-independent DNA damage protection in individuals with higher innate UV resilience.
Together, the approaches and findings described in this thesis explore several new angles to advance our understanding of aging processes and external drivers of aging such as UV irradiation in the human skin and deliver new insight on target genes and pathways involved.
Gram-negative bacteria secrete lipopolysaccharides (LPS), leading to a host immune
response of proinflammatory cytokine secretion. Those proinflammatory cytokines are
TNF-α and IFN-γ, which induce the production of indoleamine 2,3-dioxygenase (IDO). IDO production is increased during severe sepsis, and septic shock. High IDO
levels are associated with increased mortality. This enzyme catalyzes the degradation of tryptophan (TRP) to kynurenine (KYN) along the kynurenine pathway (KP).
KYN is further degraded to kynurenic acid (KYNA). Increased IDO levels accompany
with increased levels of KYNA, which is associated with immunoparalysis.
Due to its central role, the KP is a potential target of therapeutic intervention.
The degradation of TRP to KYN by IDO was intervened by 1-Methyltryptophan (1-
MT), which is assumed to inhibit IDO. By administering 1-MT, the survival of
1-MT-treated mice suffering from sepsis increased compared to mice not treated with
1-MT. The levels of downstream metabolites such as KYN and KYNA were
expected to be decreased. Surprisingly, in healthy mice and pigs, an increase in KYNA
after 1-MT administration was reported. Those unexpected metabolite alterations after 1-MT administration, and the mode of action, were not the focus of recent
research. Hence, there is no explanation for KYNA increase, while KYN did not change.
This thesis aims to postulate a possible degradation pathway of 1-MT along the KP
with the help of ordinary differential equation (ODE) systems.
Moreover, the developed ODE models were used to determine the ability of 1-MT to
inhibit IDO in vivo. Therefore, a multiplicity of ODE models were developed, including
a model of the KP, an extension by lipopolysaccharide (LPS) administration, and 1-MT
administration.
Moreover, seven ODE models were developed, all considering possible degradation pathways of 1-MT. The most likely degradation pathway was combined with the ODE model
of LPS administration, including the inhibitory effects of 1-MT.
Those models consist of several dependent equations describing the dynamics of the KP.
For each component of the KP, one equation describes the alterations over time. Equations for TRP, KYN, KYNA, and quinolinic acid (QUIN) were developed.
Moreover, the alterations of serotonin (SER) were also included. All together belong
to the TRP metabolism. They include the degradation of TRP to SER and to KYN,
which is further degraded to KYNA and QUIN. Every degradation is catalyzed by an enzyme. Therefore, Michaelis-Menten (MM) equations were used employing the substrate
constant Km and the maximal degradation velocity Vmax. To reduce the complexity of
parameter calculation, Km values of the different enzymes were fixed to literature values.
The remaining parameters of the equations were determined so that the trajectories of
the calculated metabolite levels correspond to data. The parameters of different models were determined. To propose a degradation pathway of 1-MT leading to increased
KYNA levels, seven models were developed and compared. The most likely model was
extended to test whether the inhibitory effects of 1-MT on IDO can be determined.
Three different approaches determined the ODE model parameters of the different hypothesis of 1-MT degradation. In the first approach, ODE model parameters were fixed
to values fitted to an independent data set. In the second approach, parameters were
fitted to a subset of the data set, which was used for simulations of the different hypotheses. The third approach calculated ODE model parameters 100 times without
fixed parameters. The parameter set ending up in trajectories of the TRP metabolites,
which have the smallest distance to the data, was assumed to be the most likely. The
ODE model parameters were fitted to data measured in pigs. Two different
experimental models delivered data used in this thesis. The first experimental model
activates IDO by LPS administration in pigs. The second one combines the IDO
activation by LPS with the administration of 1-MT in pigs.
The most likely hypothesis, according to approach 1 was the degradation of 1-MT to
KYNA and TRP. For the second data set the most likely one was the direct degradation of 1-MT to KYNA. With approach 2 the most likely degradation pathways were
the combination of all degradation pathways and the degradation of 1-MT to TRP and
TRP to KYNA. With approach 3 the most likely way of KYNA increase was given by
the direct degradation of 1-MT to KYNA. In summary, the three approaches revealed
hypothesis 2, the direct degradation of 1-MT to KYNA most frequently. A cell-free
assay validated this result. This experiment combined 1-MT or TRP with or without
the enzyme kynurenine aminotransferase (KAT). KAT was already shown to degrade
TRP directly to KYNA. The levels of TRP, KYN and KYNA were measured. The
highest KYNA levels were yielded with an assay adding KAT to 1-MT, corresponding
to hypothesis 2. The models describing the inhibitory effects of 1-MT revealed that
the model without inhibitory effects of 1-MT on IDO was more likely for all three approaches.
The correctness of hypothesis 2 has to be confirmed by further in vitro experiments. It
also has to be investigated which reactions promote the degradation of 1-MT to KYNA.
The missing inhibitory properties of 1-MT on IDO, determined by the in silico ODE
models, align with previous research. It was shown that the saturation of 1-MT was too
low, e.g. in pigs, to inhibit IDO efficiently.
In this study, the first possible degradation pathway of 1-MT along the KP is proposed.
The reliability of the results depends on the quality of the experimental data, and the
season, when data were measured. Moreover, the results vary between the different
approaches of parameter fitting. Different approaches of parameter fitting have to be
included in the analysis to get more evidence for the correctness of the results.
Although the outcome of patients with acute myeloid leukemia (AML) has
improved in the past decades, the overall survival is below 50% [1, 2] and there
is still an unmet need for the development of new therapeutic strategies. Here,
we aimed to identify functional vulnerabilities in AML and investigated the
therapeutic potential of target structures involved in proteostasis, cell polarity and
RNA-binding molecular pathways.
We determined that genetic deletion of the cell fate determinant and polarity
regulator Scribble delays AML development, however, its deletion also seems to
affect the proliferative capacity of normal hematopoietic cells, lowering its value
as a therapeutic target. In contrast, inactivation of YBX1 (a pleiotropic protein with
DNA/RNA binding capacity that excerpts post-transcriptional control on its
targets) and PSMB8/LMP7 (a catalytic subunit of the immunoproteasome multiprotein
complex that belongs to the ubiquitin-proteasome system (UPS)) inhibit
leukemic cells without influencing normal hematopoietic stem and progenitor cell
function, establishing these targets as potential novel therapeutic strategies
against AML.
Genetic deletion of YBX1 caused reduced proliferation and colony forming
capacity in leukemic cells independent of the oncogenic driver mutation and
delayed AML development in vivo. The role of Ybx1 in leukemia maintenance
was investigated using a conditional knockout model, confirming the functional
requirement of Ybx1 in AML maintenance. Mechanistically, YBX1 recruited
oncogenic transcripts to polysomes, increasing their translation. Displacement of
these transcripts from polysomes after YBX1 deletion decreased their protein
expression.
Genetic and pharmacologic inhibition of PSMB8/LMP7 decreased proliferation
and colony forming capacity selectively in KMT2A (MLL)-rearranged leukemic
cells. In vivo treatment with a PSMB8/LMP7 inhibitor delayed disease
development in KMT2A-rearranged leukemic mice or patient derived xenografts
(PDX). We identified the transcriptional corepressor BASP1 as a functional
effector of the immunoproteasome. BASP1 was enriched after PSMB8/LMP7
inhibition and it was found binding to KMT2A-target genes. Moreover,
pharmacologic inhibition of PSMB8/LMP7 led to decreased expression of bonafide
KMT2A-fusion target genes and enrichment for genes deregulated by
inhibitors of the KMT2A complex partners DOT1L and MEN1. This prompted us
to investigate a potential synergism between MEN1 inhibition and
immunoproteasome inhibition. Combination treatment in AML cells revealed
decreased proliferation in vitro and increased survival in vivo as compared to the
single treatments, demonstrating the therapeutic potential of combining
immunoproteasome and MEN1 inhibitors.
Background & Aim: Person-Centered-Care (PCC) requires knowledge about patient preferences. Among People living with Dementia (PlwD) only limited evidence about patient preferences, more specifically quantitative preferences, is available. Additionally, data on congruence of patient preferences with physicians’ judgements are missing. Information on patient preferences and their congruence with physicians’ judgements is expected to support Shared Decision-Making and respectively support the implementation of PCC in dementia. The aim of this dissertation was to analyze patient preferences and physicians’ judgements for PCC, including an assessment of their congruence, based on data from the mixed-methods PreDemCare-study. (Funding: Doctoral Scholarship from the Hans & Ilse Breuer-Stiftung.)
Methods: Development and conduct of a cross-sectional Analytic Hierarchy Process (AHP) survey with n=50 PlwD and n=25 physicians. Individual AHP-weights were calculated with the principal right eigenvector method and aggregated per group by Aggregation of Individual Priorities (AIP) mode. Individual consistency ratios (CRs) were calculated and aggregated per group. Group differences were analyzed descriptively by AIP-derived means and standard deviations of AHP-weights, resulting ranks, and boxplots. Additionally, differences between groups were investigated with independent paired t-tests or Mann Whitney-U tests. The sensitivity of AHP-results at the level of criteria was tested by an exclusion of inconsistent respondents in both groups, with an accepted threshold of the individual CR at ≤ 0.3 for PlwD and ≤ 0.2 for physicians.
Results: Contrary to expectation, PlwD’s and physician’s ranking of AHP-elements did not differ meaningfully. Memory Exercises was the only AHP-criterion, for which a significant difference in AHP-weights could be identified (p-value = 0.01). After inconsistent participants had been excluded, no rank reversals occurred. At the level of criteria, the mean CR for PlwD was 0.261 and 0.181 for physicians, id est (i.e.) below the
defined threshold.
Conclusion: In the selected study setting of the PreDemCare-study, patient preferences and physicians’ judgements for elements of PCC in dementia aligned well, contrary to expectations. Subject to restrictions by small sample sizes, the findings may form a basis to guide the implementation of preference-based, person-centered dementia care.
Liver dysfunctions are commonly associated with diabetes and mortality in the general
population. However, previous studies lack to define these disorders with hepatic markers from
MRI, which have been shown to be more accurate and sensitive than hepatic ultrasound and
laboratory markers. Further, previous studies defining different categories of prediabetes by oral
glucose tolerance states revealed controversial findings. Hence, this dissertation contributed to
understand the associations of liver dysfunctions with glucose intolerance states and all-cause
mortality in the general population.
In the first part of the dissertation, the associations of MRI-related hepatic steatosis and hepatic
iron overload with prediabetes were investigated. Prediabetes was categorized into IFG, IGT,
(alone or in combination) or previously unknown type 2 diabetes mellitus using OGGT data, as
suggested by the ADA. For analyses, we included 1632 subjects with MRI who participated in
an OGTT and reported no type 2 diabetes mellitus. We found that hepatic steatosis was
positively associated with continuous markers of glucose metabolism. Similarly, subjects with
hepatic steatosis as defined by MRI had a higher relative risk ratio to be in the prediabetes
groups (i-IFG, i-IGT and IFG + IGT) or having undiagnosed diabetes than individuals without
this condition. The observed associations were more obvious for MRI-derived hepatic steatosis
compared to ultrasound. In comparison to hepatic steatosis, we found that MRI-assessed hepatic
iron overload was positively associated only with both 2-hour plasma glucose and the combined
IFG + IGT category. There were no significant associations between hepatic iron overload and
other glucose tolerance states or biomarkers of glucose metabolism, regardless of possible
confounding factors.
In the second part, the associations of liver volume and other markers of hepatic steatosis with
all-cause mortality in the general population were investigated. We included 2769 middle-aged
German subjects with a median follow-up of 8.9 years (23,898 person-years). Serum liver
enzymes and FIB-4 score were used as quantitative markers, while MRI measurements of liver
fat content and total liver volume included as qualitative markers of hepatic steatosis. Compared
to other markers of hepatic steatosis, larger liver volumes were significantly associated with a
nearly three-fold increase in the long-term risk of all-cause mortality. Furthermore, this
association was consistent across all subgroups considered (men vs. women; presence or absence
of metabolic syndrome or type 2 diabetes at baseline). A positive association between FIB-4
score and all-cause mortality was found both in the entire cohort and in women. Likewise,
positive associations of higher serum AST and GGT levels with all-cause mortality were found
in the entire cohort and in men.
To conclude, this dissertation acknowledges the fact that prevention and early intervention of
liver dysfunction has major impact to reduce the burden of public health problems. Thus, our
findings suggest that hepatic markers contributes to an increased risk of prediabetes and all-cause
mortality, which might be helpful to identify high risk groups who need closer attention with
respect to prevention of liver disorders and diabetes.
Objective
Alexithymia is associated with various mental and physical disorders. Some rare evidence also suggested high alexithymia to affect the HPA axis based on small and selective samples. It was aimed to investigate the impact of alexithymia on basal cortisol levels in a large population-based cohort.
Methods
In a sample of N = 3444 individuals from the Study of Health in Pomerania (SHIP-TREND-0), the effect of alexithymia on basal serum cortisol levels was investigated in a cross-sectional design.
Multiple linear regressions utilizing cortisol levels as the response variable and alexithymia as the predictor of interest were calculated, while adjusting for conven-tional confounding covariates including depression. Multiple stratified, moderation and mediation analyses were performed to validate the results.
Results
Alexithymia was not significantly associated with basal cortisol levels (b = 0.23, 95 percent confidence interval (CI) of [-0.24, 0.69]; sr2 = 0.00, CI: [-0.00, 0.00]).
Sex- and age-stratified regression analyses as well as dichotomized models of non-alexithymic and alexithymic individuals substantiated the non-significance.
Additional mediation analyses with (1) depression and (2) physical health (R2 > 1 in both cases) and moderation analysis regarding the interaction of physical health and alexithymia (b = -1.45, 95 percent confidence interval (CI) of [-6.13, 3.32]; sr2 = 0.00, CI: [-0.00, 0.00]) corroborated the results.
Conclusion
This study does not support previous findings as it shows no association between alexithymia and basal cortisol; however, a consideration of the circadian rhythm, stress exposure or specific sample compositions heeding the methodological design should be the subject of further research.
Antimicrobial resistance (AMR) is of paramount importance in the context of One Health, an integrated and unifying approach that aims to achieve a sustainable balance in the well-being of people, domestic and wild animals, plants, and their shared environments. Whenever bacteria become resistant to the therapeutic effects of antibiotics, they can cause infections that are difficult to treat effectively, increasing the risk of severe disease progression and death. Although AMR can develop naturally over time and is per se “ancient”, the excessive use of antibiotics in human and veterinary medicine over the past century has significantly accelerated its emergence and spread. Opportunistic Gram-negative enterobacteria, particularly Escherichia coli (E. coli ) and Klebsiella pneumoniae (K. pneumoniae) strains, increasingly exhibit resistance to multiple classes of clinically used antibiotics, thus presenting multidrug-resistant (MDR) phenotypes. To make matters worse, some of these strains combine multidrug resistance with high-level virulence, posing a threat to both immunocompromised and healthy individuals. Consequently, MDR E. coli and K. pneumoniae have been designated as high-risk pathogens by the World Health Organization, underscoring the urgent need for new antibiotic development.
This thesis is motivated by the fact that only a limited number of international high-risk clonal E. coli and K. pneumoniae lineages stand out across all One Health dimensions and dominate the broad pool of MDR enterobacteria. While we only know little about the underlying drivers and contributing factors impacting their occurrence, emergence, and adaptation across different ecologies, this thesis employs a diverse range of bioinformatics and phenotypic approaches to identify the key factors important for the success of these lineages, also in rather under-explored settings. It includes three main components: (i) the analysis of genomic survey data of MDR E. coli isolates from ecologies in sub-Saharan Africa, (ii) the application of functional genomics and phenotyping techniques to characterize bacterial virulence and assess its clinical relevance in a food-borne E. coli strain, and (iii) the investigation of evolutionary pathways that promote the development of resistance to a novel drug combination and exploring compensatory mechanisms in a K. pneumoniae strain. To achieve these objectives, this research integrates genomics and transcriptomics with molecular biology and functional studies encompassing a comprehensive set of in vitro and in vivo virulence and resilience assays to explore MDR bacteria in-depth.
We provide compelling evidence for the broad occurrence of successful high-risk clonal lineages in the One Health context and their circulation among clinics, wildlife, and food in international locations. In the first study, we isolated extended-spectrum β-lactamase (ESBL)-producing E. coli strains from houseflies collected from various wards at the University Teaching Hospital of Butare (Rwanda). In a follow-up study, we then examined in-depth the genomes of additional ESBL-producing E. coli from the same clinic and obtained from hospitalized patients, their caregivers, associated community members, and pets. The analyses revealed that the sample sets from this sub-Saharan African context consisted predominantly of globally recognized E. coli lineages, including sequence types (ST)131, ST167, ST410, and ST617. They play a pivotal role in the further dissemination and stabilization of AMR across diverse habitats within the One Health context. Moreover, our genomic results emphasize that these One Health-related high-risk clonal lineages exhibit the ability to successfully combine multidrug resistance with high-level bacterial virulence.
To gain a more detailed understanding of the sophisticated interplay of virulence and AMR, we developed and refined a set of in vitro and in vivo methods for virulence phenotyping. These methodologies enabled us to characterize pathogens based on crucial clinical aspects such as biofilm formation, siderophore secretion, resistance to complement-mediated killing, and their capacity to cause mortality in Galleria mellonella larvae. By using a food-borne E. coli strain from an internationally recognized high-risk clonal lineage, we verified the remarkable combination of a MDR phenotype with clinically significant virulence properties, including synthesis of curli fibers and cellulose as part of biofilm formation, extensive secretion of siderophores, resilience against complement-containing human serum and pronounced mortality in the infection model.
Nevertheless, the success of One Health-related high-risk clonal lineages does not rely solely on an “ideal” synergistic interplay between bacterial virulence and AMR. It also depends on their ability to rapidly mitigate the fitness costs associated with AMR acquisition, as these costs manifest in the form of reduced competitiveness and virulence in the absence of antibiotics. However, this is at odds with the observation of the global distribution of One Health-related high-risk clonal lineages across various One Health dimensions, even in environments with expectedly low selection pressures. To comprehensively address this, we conducted experimental evolution studies selecting for ceftazidime-avibactam-resistant mutants, which illuminated the rapid adaptations to changing environments. The adaptations and compensatory mechanisms were seemingly driven by major bacterial regulators, including the envelope stress response regulator RpoE on genomic and transcriptomic levels.
In conclusion, the results of this thesis shed light on the fundamental principles that govern the character and interplay between AMR and bacterial virulence and advance our understanding of the contributors and drivers of successful MDR international high-risk clonal lineages in the One Health context. This is also important for effective and alternative intervention strategies to prospectively further address the global threat of AMR.
Plus‐strand RNA [(+)RNA] viruses are the largest group of viruses, medically highly relevant human pathogens, and are a socio‐economic burden. The current global pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) shows how a virus has been rapidly spreading around the globe and that– without an antiviral treatment– virus trans mission is solely dependent on human behavior. However, other (+)RNA viruses such as rhino‐, noro‐, dengue‐ (DENV), Zika, and hepatitis C virus (HCV) are constantly spreading and expanding geographically. As in the case of hepatitis C, since its first identification in the 1970s, it took more than 30 years to understand the HCV structure, genome organiza t ion, life cycle, and virus‐host interplay leading to the cure of a chronic and life‐threatening disease. However, no vaccination or antiviral treatment exists for most (+)RNA viruses. Con sequently, a precise and comprehensive analysis of the viruses, their life cycles, and parasitic interactions with their hosts remains an important field of research. In the presented thesis, we use mathematical modeling to study the life cycles of (+)RNA viruses. We analyze replication strategies of closely related (+)RNA viruses, namely HCV, DENV, and coxsackievirus B3 (CVB3), to compare their life cycles in the presence and ab sence of the host’s immune response and antiviral drug treatment and consider different viral spreading mechanisms. Host dependency factors shape the viral life cycle, contribut ing to permissiveness and replication efficiency. Our mathematical models predicted that host dependency factors, such as ribosomes, and thus the virus’ ability to hijack the host cell’s translation machinery play an essential role in the viral genome replication efficiency. Furthermore, our mathematical model suggested that the availability of ribosomes in the vi ral life cycle is a crucial factor in disease outcome: the development of an acute or chronic disease. Even though the host developed strategies to attack the virus, e.g., by degrading the viral genome, blocking the viral protein production, and preventing viral spread, viruses found strategies to countermeasure those so‐called host restriction factors derived from the immune system. Our mathematical models predicted that DENV might be highly effective in blocking the cell’s attempts to recognize the invader. Moreover, we found ongoing HCV RNAreplication even with highly effective antiviral drugs that block processes in the viral life cycle. Furthermore, we found alternative pathways of infection spread, e.g., by HCV RNA carrying exosomes, which may be a possible explanation for reported plasma HCV RNA at the end of treatment, found in a subset of patients. Hence, the mathematical models presented in this thesis provide valuable tools to study the viral replication mechanism in detail. Even though being a simplification of reality, our model predictions confirm and explain known and suggest novel biological mechanisms. In the pre sented thesis, I will summarize and discuss key findings and contextualize model predictions in the broader scientific literature to improve our understanding of the viral dynamics and the virus‐host interplay.
Introduction
The concept of thermal ablation has proven a minimally invasive alternative or accompaniment to conventional tumour therapy. Patients with hepatic primary tumours or metastases are able to profit from it. Several modalities of thermal ablation exist, including radiofrequency ablation, microwave ablation and laser ablation. They differ in regards to their indications and their physical backgrounds, yet they all share the same aim: the hyperthermic ablation of tumorous target tissue.
At this point in time the maximum ablation diameter attained in a singular session using a singular applicator is about 30 mm. The maximum attainable volume is about 23 cm3. However, the mean and median of hepatic lesions exceed that number with about 50 mm. Most hepatic tumours therefore cannot be easily ablated in toto.
One of the main limitations of thermal ablation is the periprocedural transformation of vital tissue into a boundary layer. This boundary layer prevents efficient energy transmission into peripheral tissue and thus limits the potential of thermal ablation. The boundary layer is usually located centrally around the ablation applicator. In laser ablation the formation of this boundary layer is called carbonisation.
A technically simple, yet potentially effective approach to delay or prevent the formation of this boundary layer is the usage of a spacer. This perfused spacer cools the central zone surrounding the applicator. Therefore central temperatures remain beyond the point of carbonisation.
Methods
The development of two spacer prototypes took place in cooperation with the AG “Experimentelle Radiologie” of the University Clinic Charité. The first fully closed prototype featured an internal circulation of cooling fluid without tissue perfusion. The second open prototype perfused into tissue through an opened tip.
The conduct of this study included ex vivo experiments on bovine livers (n = 15) by means of laser ablation. Ablation diameter and ablation volume were recorded through MR-guided volumetry and manual displacement volumetry. The mean values of diameter and volume that were recorded when the stand-alone applicator system was used were then compared to the mean values recorded when using the closed spacer-supported applicator system and the open spacer-supported applicator system. The difference in values between the three applicator types were then examined for statistical significance using SPSS.
To exclude covariates a preliminary experiment was conducted which aimed to maximise power input of the laser and time interval while minimising the chance of carbonisation. For that, one of the variables was increased in intervals and the ablation diameter of all three applicator types was measured until carbonisation occurred.
Results
In the preliminary experiment it was found that following the increase of the pre-set power input of the laser a proportional increase of ablation diameter followed. However when increasing power input above 25 Watt almost instantaneous carbonisation of the central tissue occurred. This was the same for all three applicator types.
When increasing the time interval > 10 minutes the stand-alone applicator system showed central carbonisation, which was not the case when using the closed spacer-supported applicator system or the open spacer-supported applicator system. The two spacer prototypes only experienced carbonisation when a time interval of > 25 minutes was set. Thus the comparison of all three applicator types was conducted at 25 Watt and 10 minutes, whereas the comparison between the closed spacer-supported applicator system and the open spacer-supported applicator system was conducted at 25 Watt and 25 minutes.
During the first experiment the stand-alone applicator system achieved mean values of 37.50 mm ablation diameter and 23.61 cm3 ablation volume. This was a statistically significant (p < 0.001) increase to the values either spacer was able to attain: the closed spacer-supported applicator system recorded a mean value of 28.67 mm ablation diameter and 18.12 cm3 ablation volume, whereas the open spacer-supported applicator system recorded a mean value of 31.00 mm ablation diameter and 18.49 cm3 ablation volume. However, setting a longer time interval was not possible when the stand-alone applicator system was used for ablation. Due to this, a second experiment comparing mean ablation diameter and volume between the two spacer prototypes followed.
During the second experiment with a time interval of 25 minutes the closed spacer-supported applicator system attained a mean value of 52.07 mm ablation diameter and 75.25 cm3 ablation volume. These values showed a statistically significant (p < 0.001) difference in comparison to the open spacer-supported applicator system with mean values of 47.60 mm ablation diameter und 72.20 cm3 ablation volume.
Discussion
Within the framework of this study it was proven that the presence of a spacer between laser applicator and hepatic tissue was able to achieve a significant increase in ablation diameter and ablation volume. By using a closed spacer an increase in volume by a 3.19 factor of change was possible. The open spacer obtained an increase in volume by a 3.06 factor of change. The concept of using a spacer in thermal ablation as a proof of concept study is therefore valid and suitable for further pre-clinical studies.
Ion traps such as Paul traps and MR-ToF (multi-reflection time-of-flight) devices are indispensable tools at radioactive ion beam facilities for the preparation of high-quality radioactive ion beams for subsequent experiments or for precise measurements of the properties of radioactive ions, such as nuclear binding energies or nuclear charge radii.
Within the work of this thesis, Doppler- and sympathetic cooling is implemented in a linear Paul-trap cooler-buncher enabling a reduction of the longitudinal emittance of radioactive ion beams resulting in a significant improvement of the ion beam quality. Moreover, a next-generation MR-ToF device is conceptualized in order to achieve isobaric pure beams with a higher ion intensity than state-of-the-art MR-ToF devices can provide. Once fully constructed and commissioned, it will operate at an unprecedented ion beam energy of 30 keV. Both of these advances are expected to become important for a wide range of experimental programs pursued at low-energy branches of RIB facilities ranging from fundamental symmetry studies, nuclear structure, rare isotope studies with antimatter, searches of physics beyond the standard model to material science and the production of medical isotopes.
The next-generation MR-ToF mass separator is based on MIRACLS’ 30-keV MR-ToF device for highly sensitive and high-resolution collinear laser spectroscopy. By storing the ions in the Multi Ion Reflection Apparatus for Collinear Laser Spectroscopy (MIRACLS), the same ion bunch is probed by a spectroscopic laser for thousands of times compared to a single passage in traditional collinear laser spectroscopy (CLS). Dedicated simulation studies show that the accuracy and resolution will be close to traditional single-passage CLS while the sensitivity is significantly enhanced. Hence, measurements of nuclear properties via fluorescence-based CLS of very rare radionuclides as well as highly sensitive and high-precision measurements of electron affinities via laser-photodetachment-threshold spectroscopy of negatively-charged (radioactive) ions will become possible.
First measurement campaigns employing MIRACLS’ 1.5-keV MR-ToF device confirm the outstanding boost in signal sensitivity and provide confidence in the application of the MIRACLS technique for the measurement of scarcely produced radioactive ions that have been so far beyond the reach of conventional techniques. Furthermore, the electron affinity of 35Cl was measured, which is in perfect agreement with the literature value. These measurements will serve as important benchmarks for modern atomic and nuclear theory, especially in its description of nuclear charge radii.
In summary, the implementation of Doppler and sympathetic cooling at RIB facilities, the conceptualization of a 30-keV MR-ToF apparatus for highly selective and high-flux mass separation as well as for highly sensitive and high-resolution fluorescence-based laser spectroscopy and the expansion of the MIRACLS technique for the study of negatively-charged ions will enable unprecedented new measurement opportunities at RIB facilities.
This thesis presents the production of polyanionic clusters within two ion storage devices:
Considering a Penning trap, the accessible range of polyanionic aluminium clusters has been expanded up to the 10th charge state. In particular, abundance curves for clusters with 5 to 9 excess electrons have been measured for the first time and analysed with respect to their lifetime-dependent appearance sizes. These sizes reveal a nearly quadratic dependency on the charge state for experimentally accessible lifetimes.
Additionally, the production of polyanionic clusters has been enabled in a radiofrequency ion trap. Therefore, the transition from a harmonic to a digital 2- and 3-state guiding signal has been investigated with respect to the ion storage. The passing of electrons through the trap during field-free periods of the guiding signal led to the first production of polyanionic clusters within a radiofrequency ion trap.
Research into nuclear physics has enjoyed a long and rich history since the earliest experiments began investigating atomic constituents. The discovery of the atomic nucleus in the early 20th century started a complex field of research that has undergone many transformations with the advancements of modern technology. Today, atomic nuclei are not only studied to advance our understanding of the strong force but also to gain more information on the synthesis of elements in the universe, to exploit nuclear decay to investigate the weak interaction, and to search for physics beyond the standard model.
In this work, we will study the strong force in atomic nuclei, i.e. the way nucleons (protons and neutrons) arrange themselves in a many-body system governed by the repulsive Coulomb interaction and the attractive strong interaction. In particular, we will focus on nuclear structure near nuclei with a "magic number" of Z protons and N neutrons, so-called doubly-magic nuclei, exhibiting a particularly stable configuration with respect to neighboring nuclei.
Within the nuclear shell model, similar to the atomic shells, the magic numbers indicate shell closures accompanied by energy gaps. Nuclei at double-shell closures and their direct vicinity provide an important playground to benchmark nuclear theories and models that aim to predict the intricate interplay of the nucleons that lead to enhanced nuclear binding energies, significant changes in charge radii and transition strengths, etc.
Of particular interest are nuclear isomers, long-lived excited states, in which the nucleon configuration with respect to its ground state is altered, resulting in a modification of their properties despite having the same number of protons and neutrons.
The main part of this work consists of three publications, which report on nuclear structure investigations through mass measurements and laser spectroscopy near the doubly magic nuclei nickel-78, tin-100, and lead-208.
The nuclides investigated in this work include neutron-deficient indium isotopes, neutron-rich zinc isotopes, and neutron-rich mercury isotopes.
This work investigates turbulence in the core plasma of the optimised stellarator
Wendelstein 7-X. It focuses on experimental characterisation and
evaluation of the electrostatic micro-instabilities, which drive turbulent fluctuations,
and the saturation of turbulence by zonal flows. Expectations for
Wendelstein 7-X are formulated by reviewing theoretical work and with
the help of gyrokinetic simulations. The experimental analysis centres on
line-integrated density fluctuation measurements with the phase contrast
imagining diagnostic in electron cyclotron heated hydrogen discharges. An
absolute amplitude calibration was implemented, and a method for reliable
determination of dominant phase velocities in wavenumber-frequency
spectra of density fluctuations has been developed. Line-averaged density
fluctuation levels are observed to vary between magnetic configurations.
The wavenumber spectra exhibit a dual cascade structure, indicating fully
developed turbulence. The dominant instability driving turbulent density
fluctuations on transport relevant scales is identified as ion-temperaturegradient-
driven modes, which are mainly localised in the edge region of the
confined plasma. Despite the line-integrated nature of the measurement, the
localisation of density fluctuations is shown by comparing their dominant
phase velocity with the radial profile of the E × B rotation velocity due to
the ambipolar neoclassical electric field. Nonlinear gyrokinetic simulations
and a simplified plasma rotation model within a synthetic diagnostic confirm
the localisation. Oscillations of the dominant phase velocity indicate
the existence of zonal flows as a saturation mechanism of ion-temperaturegradient-
driven turbulence. A direct effect on turbulent density fluctuation
amplitudes and radial transport is observed.
The layer-by-layer method is a robust way of surface functionalization using a wide range of materials, e.g. synthetic and natural polyelectrolytes (PEs), proteins and nanoparticles. Thus, this method yields films with applications in diverse areas including biology and medicine. Sequential adsorption of different oppositely charged macromolecules can be used to prepare tailored films with controlled molecular organization. In biomedical research, electrically conductive coatings are of interest. In manuscript 1, we investigated films sequentially assembled from the polycation poly (diallyldimethyl-ammonium) (PDADMA) and modified carbon nanotubes (CNTs), with CNTs serving as the electrically conductive material. We assume that charge transport occurs through CNT contacts. We showed that with more than four CNT/PDADMA bilayers, the electrical conductivity is constant and independent of the number of CNT/PDADMA bilayers. A conductivity up to 4∙10^3 S/m was found. It is possible to control the conductivity with the CNT concentration of the CNT deposition suspension. A higher CNT concentration resulted in thicker CNT/PDADMA bilayers, but in a lower conductivity per bilayer. We suspect that an increased CNT concentration leads to a rapid CNT adsorption without the possibility to rearrange themselves. If PDADMA then adsorbs on the disordered CNTs in the next deposition step, the average thickness of the polymer layer is thicker than on the more ordered CNT layer from the dilute solution. This leads to an increased PE monomer/CNT ratio and lower conductivity. More polycations between the CNT layers leads to less CNT contacts. Thus, the controlled composition of films can be used to fulfill specific requirements.
For many applications of polyelectrolyte multilayers (PEMs), cheap PEs with a broad distribution of molecular weights are used. It was unknown whether the distribution of molecular weights of the PE in the adsorption solution is maintained during the adsorption process and hence in the film. To investigate this, the PSS adsorption solution in article 2 consisted of a binary mixture of short and long poly (styrene sulfonate) (PSS). A good model system to study layered films in terms of composition are PDADMA/PSS multilayers. Neutron reflectivity and in-situ ellipsometry measurements were carried out to determine the PSS composition in the film and the growth regimes. At a mole fraction of long PSS of 5 % or more in solution, the exponential growth (which is characteristic of short PSS) is totally suppressed, and only long PSS is deposited in the resulting multilayer. Variation of adsorption time of PSS showed that short PSS first adsorbs to the surface but is displaced by long PSS. Between 0 and 5 % of long PSS in the adsorption solution exponential growth occurs. The fraction of short PSS in the film continuously decreases with the increase of long PSS in the adsorption solution. In the assembly of films prepared from binary PSS mixtures, the short PSS leaves the film through adsorption/desorption steps both during PSS adsorption and during PDADMA adsorption (as PDADMA/PSS complexes). Both techniques show that the composition of the film does not correspond to that of the deposition solution. The composition and thus the properties of the resulting multilayer are influenced by the choice of adsorption time. Moreover, we conclude that a multilayer grown from a polydisperse polyelectrolyte contains fewer mobile low molecular weight polymers than the deposition solution.
In manuscript 1 and article 2, the composition of multilayers was studied. In manuscript 1 adsorption kinetics were important for the arrangement of CNTs on the surface. In article 2, the adsorption kinetics, i.e. the diffusion of the polyelectrolytes to the surface, was also investigated. In article 3, we investigated the influence of the composition of the film as well as the preparation condition on the mobility of PEs in the film. The molecular weight of the polycation PDADMA and the NaCl concentration of the deposition solution were varied. The vertical PSS diffusion constant D_PSS within the PDADMA/PSS multilayers was measured using neutron reflectivity. The salt concentration of the preparation solution defines the polymer conformation during deposition. The molecular weight of the polycation determines the degree of intertwining. Together, both parameters determine the polyanion-polycation coupling and thus the PSS mobility within the network. Log−log display of D_PSS vs the molecular weight of PDADMA and fits to two power laws (D_PSS ∝ X_n(PDADMA)^(-m) ∝ M_w(PDADMA)^(-m)) reveals for films built from 10 or 200 mM NaCl a kink. Below and above the kink, the dependence of D_PSS on M_w(PDADMA) can be described by different power laws. For Χ_n(PDADMA) < X_n,kink(PDADMA) ≈ 288, the exponents are consistent with the predictions of the sticky reptation model. X_n(PDADMA) ≈ 288 is the entanglement limit. For Χ_n(PDADMA) > X_n,kink(PDADMA) ≈ 288, the decrease of D_PSS with M_w(PDADMA) is larger than below the entanglement limit, which is indicative of sticky reptation and entanglement. The PSS diffusion constant of films built from 100 mM NaCl drops three orders of magnitude when increasing the molecular weight of PDADMA from 45 kDa to 72 kDa. To figure out if an immobile PSS fraction exists in the film built from 72 kDa PDADMA (beyond the entanglement limit), the film was annealed at different conditions in article 4: both temperature and salt concentration were varied. For data analysis, the simplest model with two PSS fractions with different diffusion constants was used. These diffusion constants increase as the temperature of the surrounding solution is increased. As assumed in article 3, an immobile PSS fraction exists when annealing at room temperature. At higher annealing temperatures, at least two diffusion processes must be distinguished: the diffusion of the highly mobile PSS fraction through the entire film and a slow PSS fraction, mowing in a limited way. The choice of preparation conditions determines whether a polyelectrolyte multilayer can intermix completely. It is not clear if complete intermixing will ever occur for films built with PDADMA beyond the entanglement limit. It is possible that the diffusion is more complex. Long-term measurements will clarify this question. Calculating scattering length density profiles with subdiffusive behavior would be interesting and is a challenge for the future. Furthermore, immobile fractions are only visible with long annealing times. We hypothesize that an immobile or nearly immobile fraction is present whenever the dependence of D_PSS on the molecular weight of PDADMA cannot be described by the sticky reptation. To verify this hypothesis, further studies are necessary.
All results presented and discussed in the manuscript and articles show that by varying the preparation conditions, tailored films can be built. The composition of the film is also determined by the adsorption kinetics. In addition, the mobility of the PEs within the multilayers can be controlled by varying the conformation, mingling and entanglement of the chains within the film. The influence of the salt concentration in the preparation solution on the growth regimes during film formation is part of our future research. It is planned to investigate films built of different PDADMA molecular weights under varied annealing conditions to better understand the mobile and immobile fractions.
In vitro and in vivo analyses of mono- and mixed-species biofilms formed by microbial pathogens
(2022)
Microbial biofilms can be defined as multicellular clusters of microorganisms embedded in a self-produced extracellular matrix (ECM), which is primarily composed of polymeric biomolecules. Biofilms represent one of the most severe burdens in both industry and healthcare worldwide, causing billions of dollars of treatment costs annually because biofilms are inherently difficult to prevent, treat, and eradicate. In health care settings, patients suffering from cystic fibrosis, or patients with medical implants are highly susceptible to biofilm infections. Once a biofilm is formed, it is almost impossible to quantitatively eradicate it by mechanical, enzymatical, chemical, or antimicrobial treatment. Often the only remaining option to fully eradicate the biofilm is removing of the infected implant or body part. The primary reasons for the inherent resistance of biofilms against all forms of antimicrobial treatment are (I) a reduced metabolic activity of biofilm-embedded cells climaxing in the presence of metabolic inactive persister cells, as well as (II) the protective nature of the biofilm matrix acting as a (diffusion) barrier against antimicrobials and the host immune system. Consequently, there is an urgent need to better understand microbial biofilms from a structural and (patho-) physiological point of view in order to be able to develop new treatment strategies.
Therefore, the aims of this study were to investigate fundamental physiological properties of different clinically relevant single and multi-species biofilms, both in vitro and in vivo. Furthermore, the effectiveness of a novel treatment strategy using cold atmospheric pressure plasma was evaluated in vitro to treat biofilms of the pathogenic fungus C. albicans.
In article I, the intracellular and ECM protein inventory of Staphylococcus aureus during in vitro biofilm growth in a flow reactor was analyzed by liquid-chromatography coupled to tandem mass-spectrometry (LC-MS/MS) analysis combined with metabolic footprint analysis. This analysis showed that anaerobiosis within biofilms releases organic acids lowering the ECM pH. This, in turn, leads to protonation of alkaline proteins – mostly ribosomal proteins originating from cell lysis as well as actively secreted virulence factors – resulting in a positive net charge of these proteins. As a consequence, these proteins accumulate within the ECM and form an electrostatic network with negatively charged cell surfaces, eDNA, and metabolites contributing to the overall biofilm stability.
In article II, the in vivo metaproteome of the multi-species biofilm community in cystic fibrosis sputum was investigated. To this end, an innovative protocol was developed allowing the enrichment of microbial cells, the extraction of proteins from a small amount of cystic fibrosis sputum, and subsequent metaproteome analysis. This protocol also allows 16S sequencing, metabolic footprint analysis, and microscopy of the same sample to complement the metaproteome data. Applying this protocol, we were able to significantly enhance microbial protein coverage providing first insights into important physiological pathways during CF lung infection. A key finding was that the arginine deaminase pathway as well as microbial proteases play a so far underappreciated role in CF pathophysiology.
In articles III and IV, a novel treatment strategy for biofilms formed by the important fungal pathogen Candida albicans was evaluated in vitro. Biofilms were treated with two different sources of nonthermal plasma (with the Nonthermal Plasma Jet “kINPen09” as well as with the Microwave-induced plasma torch “MiniMIP”) and the effect on growth, survival, and viability was assessed by counting colony-forming units (CFU), by cell proliferation assays, as well as by live/dead staining combined with fluorescence microscopy, confocal laser scanning microscopy, (CLSM) and atomic force microscopy (AFM). These tests revealed that biofilms were effectively inactivated mostly on the bottom side of biofilms, indicating a great potential of these two plasma sources to fight biofilms.
Under the influence of human activities, increased climate variability induces changes in
multiple marine environments. Especially vulnerable are the coastal ecosystems where organisms
must cope with constant extreme changes of environmental drivers, such as temperature, salinity, pH,
and oxygen content. In coastal areas, brachyuran crabs are important animals that have a high impact
on ecosystem functioning and serve as a link in food webs and pelagic-benthic coupling. Larval stages
of crabs are crucial for population persistence and dispersal. They are generally more vulnerable to
changes of environmental drivers and failure to adapt to new conditions may result in population
collapse. To analyse the effects of multiple environmental drivers on larval performance and to
elucidate interspecific and intraspecific difference, this project examined larval performance in the
European shore crab Carcinus maenas. In this study, larvae of C. maenas from three native
populations (Cádiz: Cádiz Bay, Helgoland: North Sea, Kerteminde: Baltic Sea) were reared in a
factorial design consisting of different temperature (15-24 °C) and salinity treatments (20, 25, 32.5
PSU). Results demonstrated how descriptors of larval performance (growth, physiological, and
developmental rates, and survival) were affected by combined environmental drivers. Larval
responses to temperature and salinity showed contrasting patterns and differed among native
populations originating from distant or contrasting habitats, as well as within the populations. The
highest overall performance was recorded in the Cádiz population, while the Kerteminde population
had the lowest performance in most of tested traits. The interactive effects of multiple drivers differed
among the populations. In the Cádiz and Helgoland populations, higher temperatures mitigated the
effect of lower salinity while the Kerteminde population showed a maladaptive response when
exposed to lower salinity. Differences in performance showed better locally adapted populations (e.g.
Cádiz) that could acclimate faster, have better adaptive mechanisms or stronger dispersive abilities.
Because of their wider tolerance to increased temperature and decreased salinity, interactive effects
in particular populations may favour some populations in a changing climate, especially in coastal
habitats. Variation in larval performance showed complex interactions in larval performance and
highlighted the necessity to quantify inter-population responses to climate-driven environmental
change where responses of species should not be generalised. This study emphasizes the need for
inclusion of multiple traits, drivers, and populations in experimental studies to properly characterize
performance of marine coastal animals.
As the effects of anthropogenic climate change become more pronounced, it is critical to understand if and how species can persist in novel environments. Range-expanding species provide a natural experiment to study this topic: by studying the factors contributing to successful colonization of new habitats, we can gain insight into what influences organisms’ adaptive potential. The wasp spider, Argiope bruennichi, has expanded its range from warm, oceanic and Mediterranean climate zones (populations in this region are referred to as “ancestral” or “core”) into a new thermal niche, the continental climate of the Baltic States and Scandinavia (referred to as “expanding” or “edge”) within the last century. Past work demonstrated that the expanding populations are European in origin, but are more diverse than the ancestral populations, due to genetic admixture. This discovery led to the following questions, which are investigated in this dissertation: (i) Was the successful colonization of colder, more continental northern climates due to phenotypic plasticity or genetic adaptation? (ii) If A. bruennichi’s establishment of northern latitudes can be attributed to genetic adaptation, did selection act on standing genetic variation, on genetic variation introduced via admixture/introgression, on specific genomic regions, or on novel mutations? (iii) Is there a role of the microbiome in the A. bruennichi range expansion?
In Chapter 1, we assembled a chromosome-level genome for the species: the first such high-quality genome for a spider, which we made use of as a resource to provide the genomic context of single nucleotide polymorphisms in our primary study on genetic adaptation and phenotypic plasticity (Chapter 3). The genome assembly also opened the door to many new projects, such as the study presented in Chapter 2. In Chapter 2, the chromosome-level resolution of our assembly allowed us to identify the sex chromosomes in A. bruennichi. Due to the X1X20 sex chromosome system, where males have one copy of two X chromosomes, and females have two copies, the X chromosomes have a lower effective population size, and lower recombination rate, than autosomes. These characteristics give rise to the theoretical prediction of increased evolutionary rates in sex chromosomes. Knowing the identity of the sex chromosomes in our A. bruennichi genome assembly will allow us to test if there is stronger differentiation between populations on the X chromosomes.
Chapter 3 represents the central study of this dissertation. We performed a reciprocal transplant common garden experiment to assess plasticity and adaptation in cold tolerance traits, using spiderlings from the core of the range in France, and the edge of the range in Estonia. We combined this with data on clinal variation in adult phenotypes (body size, pigmentation, and fecundity) and genotypes in a transect across the European range. This study revealed a strong signature of genetic adaptation for increased cold tolerance in edge populations, and clear genetic differentiation of ancestral and expanding populations over a very short geographic distance, despite gene flow. We provide genome-wide evidence for adaptive introgression, and conclude that the A. bruennichi range
expansion was enabled by adaptive introgression, but has reached a poleward range limit.
Interactions with microbes shape all aspects of eukaryotic life. Endosymbiotic bacteria have been shown to alter the thermal tolerance of arthropod hosts, and influence dispersal behavior in spiders. With this background, in Chapter 4, we asked whether the microbiome might play a role in the rapid range expansion of A. bruennichi. We characterized the microbiome in various dissected tissues of spiders from two populations. Although we found no obvious differences between populations or tissues, this study yielded the discovery of a novel, dominant, vertically transmitted symbiont with astoundingly low similarity to all other sequenced bacteria. Since that discovery, we have found evidence of the unknown symbiont in A. bruennichi populations across the Palearctic (unpublished data), making it relatively unlikely to play a role in the range expansion.
By studying the establishment and subsequent differentiation of core versus edge populations of A. bruennichi following range expansion, we were able to gain insight into the evolutionary and ecological processes that allowed this species to successfully cope with novel environments. The rapidity with which local adaptation arose in A. bruennichi suggests that evolutionary adaptation to novel environments is possible over short time periods. However, this may only be possible in species with sufficient standing genetic variation, or with genetic variation introduced via admixture, as in A. bruennichi, which has important implications for our understanding of species responses in the face of ongoing global climate change.
Kinetic modeling and infrared spectroscopy of charge carriers across the plasma-wall interface
(2022)
In this thesis, charge transport at the plasma-wall interface is investigated theoretically, on a semiclassical, microscopic level. Based on the Boltzmann and Poisson equations a set of equations is derived and numerically solved to model charge carriers both within a semiconducting wall and a gaseous plasma in front of it. While the plasma is considered collision-free, within the solid, phonon collisions, as well as recombination processes between conduction band electrons and valence band holes are considered. This results, for the first time, in a self-consistent modeling of both the gaseous electron-ion plasma and the electron-hole plasma in the solid on the same footing. Utilizing specific approximations for different physical scenarios, numerical solutions are presented both for the floating and the electronically contacted (biased) interface. In the latter case, the current voltage characteristic is calculated and shown to heavily depend on the charge kinetics within the wall.
Furthermore, we present optical methods to measure the wall charge noninvasively. These utilize the influence of the deposited surplus charges on the optical reflection coefficient of the surface. By calculating the optical response of these charges, we show that the magnitude of the surface charge can be inferred from the change in the reflectivity of the surface caused by the presence of the plasma. While nonlocal effects are considered, it is shown analytically and numerically that these can be neglected at the scales of the considered physical systems.
Potential of nitrous oxide sedation in pedodontics to reduce the need of dental general anesthesia
(2022)
Aim: To investigate the possible reduction of the need for dental general anesthesia (DGA) through nitrous oxide sedation (NOS), when is used in combination with behavior management techniques among patients younger than 12 years of age referred to a specialized pedodontics practice due to the high dental treatment need and poor cooperation level.
Materials and methods: Retrospective analysis was performed of the digital medical records of all children treated under NOS in a specialized pedodontics clinic between 2012 and 2017. The potential reduction of the need for dental general anesthesia was assessed depending on the success rate of NOS on patient-level with relations to multiple related factors such as age, reason of referral and treatment need.
Results: out of 510 patients, 228 meet the inclusion criteria. Nitrous oxide was used in 406 dental treatment sessions on 228 pre-cooperative and/or anxious patients aged 3-12 years (mean 6.4±1.7; 43.4% female). 91.9% of the NOS sessions were successful in achieving the intended dental treatment. Complete oral rehabilitation was possible for 84% of the patients using NOS without the need of DGA. Regarding the age, DGA reduction among pre-school children was lower than in schoolchildren (77.8% and 87.9%, respectively).
Conclusion: A high fraction of anxious or semi-cooperative children with high dental treatment needs can be treated without the use of DGA when a comprehensive concept of caries management is combined with the use of NOS and behavior management techniques. NOS should, therefore, always be considered as an option for dental treatment of semi-cooperative children with high dental treatment needs before making a decision towards DGA.
Despite the extensive ongoing research, there still exist plenty of diseases whose mechanisms have not yet been fully understood, one such example being proteasome-related disorders. Over the last few years, an increasing number of studies have been initiated
to elucidate their driving pathophysiological mechanisms. Determining the systematic effects of genomic alterations occurring in genes encoding 19S proteasome subunits is a key to comprehend the molecular basis of syndromic intellectual disability (ID) pathogenesis and
the subsequent design of new targeted therapies. Therefore, the main objective of my research was to contribute to the identification of potential drivers of syndromic ID, and thereby pave the way for the development of new targeted therapy approaches. In this regard, my aim was to characterize tissue, proteomic and metabolomic changes in cells from patients with PSMC5 mutations and uncover a potential dysregulation of various biochemical and/or inflammatory pathways.
To this end, I undertook a comparative examination of control and patient T cells expanded from peripheral blood mononuclear cells (PBMCs). First, I assessed the proteasome composition in these samples (both in its denaturized and native form), by means of
SDS-PAGE, native PAGE and western-blotting. Moreover, I determined proteasome chymotrypsin-like activity by measure of Suc-LLVY-AMC peptidase activity assay. In addition, I analysed the activation status of the ER stress and mTOR pathway by RT-PCR and SDS-PAGE /western-blotting prior to a subsequent analysis of T-cell markers.
The data show that the investigated p.(Pro320Arg) and p.(Arg201Trp) de novo heterozygous missense mutations in the PSMC5 gene do not cause haploinsufficiency as the steady-state expression level of the PSMC5/Rpt6 full-length protein does not vary between control and patient cells. Further analysis of control and patient T cells under non-reducing conditions revealed that PSMC5/Rpt6 mutants were less efficiently incorporated into 26S proteasome complexes than their wild-type counterparts. The failure to assemble PSMC5/Rpt6 into fully mature proteasomes was associated with a reduced proteasome chymotrypsin-like activity in patient T cells, as determined by in-plate assays. These data unambiguously demonstrate that both of the p.(Pro320Arg) and p.(Arg201Trp) PSMC5 mutations identified in patients suffering from syndromic ID are loss-of-function mutations. Interestingly, my data further show that proteasome dysfunction in these patients was accompanied by abnormalities in mTOR signalling and T-cell differentiation, as determined by western-blotting and flow cytometry, respectively.
Altogether, our data identified for the first time PSMC5 as a disease-causing gene for
a syndromic form of ID. How proteasome dysfunction caused by PSMC5 variants contributes to disease pathogenesis, remains to be fully determined.
The order of bats (Chiroptera) account for ~20% of all mammalian species and attracted immense global attention due to their identification as important viral reservoir. Bats can harbour a plethora of high-impact zoonotic viruses, such as filoviruses, lyssaviruses, and coronaviruses without displaying clinical signs of disease themselves. Given this striking diversity of the bat virome, their ability of self-powered flight, and global distribution, understanding chiropteran immunity is essential to facilitate assessment of future spillover events and risks.
However, scarcity of bat-specific or cross-reactive tools and standardized model systems impede progress until today. Furthermore, the richness of species led to generation of isolated datasets, hampering data interpretation and identification of general immune mechanisms, applicable for various chiropteran suborders/families. The key to unlocking bat immunity are coordinated research approaches that comprehensively define immunity in several species. In this work, an in-depth study of innate and adaptive immune mechanisms in the fructivorous Egyptian Rousette bat (Rousettus aegyptiacus, ERB) is presented.
Detailed stability analyses identified EEF1A1 as superior reference gene to ACTB, and GAPDH, which rendered unstable upon temperature increase or presence of type-I-IFN. Since the body core temperatures of pteropid bats reach from 35°C to 41°C and it has been postulated that bats display constitutive expression of IFNs, a suitable reference gene has to be stable under these physiologically relevant conditions. To study cellular innate immunity in detail, cell lines from the nasal epithelium, the olfactory compartment and the cerebrum were generated. To include immune responses of epithelia cells, essential for immunity at sites of primary viral infection, primary epithelia cells from the nasal epithelium, trachea, lung and small intestine were generated. Cellular identities were determined by comprehensive analyses of transcripts and proteins expressed by each cell line. The capacity of each cell line to produce type-I- and III-IFNs was assessed at 37°C and 40°C upon stimulation with viral mimetics. This revealed cell type-dependent differences is the capability to express IFNs upon stimulation. Furthermore, the constitutive expression of type-I- and III-IFNs was significantly elevated in higher temperatures and quantified at mRNA copy levels. To characterize ERB innate immunity upon infection with high-impact zoonotic viruses, cells from the nasal epithelium, the olfactory system, and the brain were infected with several lyssaviruses. This revealed striking differences in susceptibility: cells from the nasal epithelium rendered least whereas cells from the olfactory epithelium rendered most susceptible to viral infection and replication. Additionally, due to a lack of IFN expression in infected cells, it could be shown that LBV possibly possesses advanced strategies to ensure successful replication in ERB cells. Since the current SARS-CoV-2 pandemic put bats even further in the focus of zoonotic research, primary epithelial cells and animals were infected with this virus to monitor ERB-specific immune transcripts in cells and tissues. These studies revealed a notably early IFNG expression in the respiratory tract of infected individuals.
To understand immunomaturation in bats, the immune cell landscape in periphery and various tissue in adult and juvenile ERB was analyzed by flow cytometry and scRNA-seq, revealing intriguing, age-dependent variations in the abundance of granulocytes and lymphocytes. Flow cytometry revealed a significantly higher number of granulocytes in adults, as well as higher numbers of B cells in juveniles. scRNA-seq allowed detailed identification of different leukocyte subsets, uncovering the presence of highly-abundant NKT-like cells and a unique PLAC8 expressing B cell population. A functional characterization of phagocytic cells and lymphocytes derived from adult and juvenile ERB revealed no significant differences in cellular functionality.
In conclusion, the presented work demonstrated suitability of all established ERB cell lines to study bat immunity in vitro, which led to striking findings regarding IFN expression at steady state, or upon stimulation or viral infection. In addition, established qRT-PCR protocols allowed definition of constitutive and temperature-dependent elevation of IFN expression magnitudes, as well as insights into expression of immune-related transcripts in SARS-CoV-2 infected ERB. Finally, based on optimized scRNA-seq technologies and flow cytometry, frequencies and absolute cell counts could be determined in ERB of different ages, revealing e.g. age-dependent variations in leukocyte profile compositions.
Quality of Life (QoL) is a core patient-reported outcome in healthcare research. However, a conceptual, operational, and psychometric elaboration of QoL in the context of telemedical care (TM) was needed, as standardised instruments to assess QoL do not comprehensively represent essential aspects of intended outcomes of TM. Therefore, the overall aim of this thesis was to conceptualise QoL in the context of TM and to develop an instrument that can adequately assess QoL in TM.
Gynaecologic immunologic research aims to answer an important question: How does the immune system manage to protect both mother and unborn child while not harming the semi-allogeneic and thus partially unaccustomed fetus? Several distinct adaptions in both the innate and adaptive immune system take place during pregnancy. Alterations in these processes can cause dramatic consequences like pregnancy loss. Here, molecules with immunomodulatory functions can provide possible treatment options. One molecule with the described features emerged as a candidate: The transmembrane molecule mCD83 as well as its soluble form, sCD83. As mCD83 overexpressing cells and cells from pregnant mice showed similar behaviour regarding interleukin-10 secretion and B-cell (BC) development, a contribution of mCD83 in immunologic pregnancy adaptions is possible. Additionally, the soluble form could be a future therapeutic agent in pregnancy disorders, regarding its already shown benefits in therapy of various autoimmune diseases in animal models.
The aim of this work is to evaluate the expression, release and regulation of CD83 in its membrane bound and soluble forms during normal and disturbed pregnancies in mouse models.
The semi-allogeneic pairing of two inbred stems, C57Bl6/J×BALB/c, results in healthy pregnancy and was used to investigate the expression in different stages of pregnancy. Pairing CBA/J females with DBA/2J males results in resorption of fetal units and represents a poor pregnancy outcome mating (PPOM). This model in comparison with CBA/J×BALB/c pairings (presenting a good pregnancy outcome mating (GPOM)) is a model for immunologic pregnancy disturbance. It was used to detect alterations in mCD83 expression and sCD83 release during disturbed pregnancy.
During normal murine pregnancy, mCD83 expression increased with a peak on day 14 of pregnancy on B- and T-cells, while the amount of mCD83 positive cells was elevated at the end of pregnancy. PPOM mice showed higher mCD83 expression and mCD83 positive cell count on various lymphocyte subtypes in comparison to GPOM, while sCD83 levels were lower in PPOM pregnancies. Splenocytes released sCD83 in cell culture, whereby the main part under unstimulated conditions was produced by BC. Progesterone treatment of splenocytes led to a dose dependent mCD83 upregulation on T-cells and reduced mCD83 expression as well as sCD83 release from BC. Culture of splenocytes with tissue inhibitor of metalloproteinases 1 (TIMP1) resulted in elevated sCD83 release and mCD83 expression on BC. Progesterone reduced TIMP1 expression on BC in vitro.
mCD83 expression and sCD83 release showed various alterations during normal murine pregnancy as well as when comparing PPOM with GPOM. Noticeable are in particular a higher mCD83 expression on splenic BC on day 14 of pregnancy. In BC from PPOM, mCD83 expression is higher than on BC from GPOM, while PPOM mice show a lower sCD83 serum level, hinting a problem in the shedding mechanism during PPOM.
Progesterone regulates mCD83 expression on BC via TIMP1 and a yet unknown proteinase, resulting in degradation of mCD83 with lower mCD83 expression and sCD83 release. Here, the resulting expression level may vary depending on the BC surroundings and cell compartmentation.
The results thereby suggest a CD83 involvement in pregnancy and encourage further research on mCD83 expression at the feto-maternal interface as well as sCD83 in human blood and tissue. Especially the sCD83 alterations are of clinical interest, indicating the molecule as potential therapeutical option for pregnancy disturbances.
Literature shows that people with a migration background (PwM) with dementia are an especially vulnerable group. Data on the number of PwM with dementia in Germany is scarce meaning the healthcare system faces a challenge of an unknown magnitude. They are mostly not part of the healthcare landscape and lack knowledge about dementia and healthcare services. Healthcare professionals and services do not seem to be culturally sensitive enough and not adequately equipped to take care of PwM with dementia. Therefore, this work focuses on a) estimating the number of PwM with dementia broken down by country of origin and federal state; b) exploring the caregiving experience, barriers of healthcare utilisation and measures to increase utilisation; and c) determining the scope of culturally sensitive information and healthcare services as well as projects on dementia and migration in Germany. A combination of quantitative and qualitative research methods as well as a scoping review are applied to examine the research focus.
Calculations show that an estimated 96,500 PwM have dementia, presumably mostly originating from Poland, Italy, Turkey, Romania, and the Russian Federation. The majority of affected PwM live in North Rhine-Westphalia, Baden-Württemberg, and Bavaria. Family members experience similar challenges and consequences as non-migrants in the care of a person with dementia. PwM lack sufficient knowledge of dementia and information regarding the available healthcare services. These are only two of the reasons why the healthcare system is not utilised. To increase utilisation, services should be culturally sensitive and information easily accessible. In addition, easier navigation and the expansion of existing healthcare structures is needed. The scoping review identified 48 culturally sensitive healthcare and information services and projects for PwM with dementia. The majority are located in North Rhine-Westphalia, Baden-Württemberg, Bavaria, and Hesse, which mirrors the distribution of PwM with dementia in Germany. For the most part, these services offer counselling in different languages. These results confirm that PwM (with dementia) and healthcare professionals need in-depth education on this topic. There should be a focus on the design of information and healthcare services that are tailored in a culturally sensitive way. This dissertation further indicates that culturally sensitive healthcare services, personalised for individual situations on site, should be expanded and also facilitated by not only healthcare professionals and service providers but also by law- and decision-makers. Furthermore, there is a need for cooperation between researchers, healthcare professionals, service providers, healthcare systems, law-makers, and other stakeholders in the field on a national and an international level.
Introduction:
The amniotic fluid – as the medium surrounding the fetus, it is holding a crucial role in the maintenance and development of a successful pregnancy. While providing mechanical protection to the fetus, it also offers considerable immunological defense. In fact, it is known that the amniotic fluid plays a significant role in the innate immune system, as many of its corresponding substances show substantial antimicrobial function. Also, components of the adaptive immune system, including B cells, have been described within the amniotic fluid. An increase of immune cells in the amniotic fluid in cases of intra-amniotic infection indicates their involvement in inflammation-related pathologies of pregnancy. However, especially B cells in the amniotic fluid have not yet been thoroughly investigated.
The aim of this work is a deeper examination of the B-lymphocytes within the amniotic fluid. Based on the analysis of surface molecules this includes their phenotype, origin and func-tion. In the long term this could substantiate our understanding of intraamniotic inflammation and or infection, which are casually linked with preterm birth, fetal inflammatory response syndrome and fetal morbidity.
This, in turn, could pave the way for potential diagnostic methods and treatments.
Methods:
For all experiments 8-12-weeks-old pregnant mice were sacrificed at day 14 of pregnancy. The amniotic fluid was collected and specific cell subsets were isolated using MACS cell separation. Cells were then co-cultured with a bone marrow stromal cell line and stimulated in vitro.
The analysis of the population distribution and cytokine production was performed by flow cytometry. To analyze IgM-levels in the supernatant of the co culture, ELISA was used. Statistical analysis was performed using GraphPad Prism software.
Results:
The amniotic fluid contains different developmental stages of B cells, which most likely are of fetal origin. This is supported by the expression of paternal surface markers. An extensive proliferation and switch towards a more mature phenotype upon co-culture shows that the immature subsets of amniotic fluid B cells are able to expand and mature in vitro. Amniotic
fluid B cells spontaneously produce IgM and show functional adaption upon in vitro stimula-tion as evidenced by the increase of cell activation markers.
Conclusion:
For the first time a deep investigation of B-cells within the amniotic fluid was performed, covering phenotype and cell functionality. This work shows that there is a B cell compartment within the amniotic fluid, which, to a certain extent, is able to mature and gain functionality when exposed to external stimuli. This supports the hypothesis of the amniotic fluid as crucial immunological line of defense against inflammatory and infectious challenges during pregnancy.
Clostridioides difficile is the leading cause of antibiotic-associated diarrhea referring to infections of the gastrointestinal tract in the course of (broad-spectrum)antibiotic therapy. While antibiotic therapy, preferentially with fidaxomicin or vancomycin, often stops the acute infection, recurrence events due to remaining spores and biofilm-associated cells are observed in up to 20% of cases. Therefore, new antibiotics, which spare the intestinal microbiota and eventually clear infections with C. difficile are urgently required. In this light, the presented work aimed at the evaluation and characterization of three natural product classes, namely chlorotonils, myxopyronins and chelocardins, with respect to their antimicrobial activity spectrum under anaerobic conditions and their potential for the therapy of C. difficile infections. Briefly, compounds of all three classes were screened for their activity against a panel of anaerobic bacteria. Subsequently, the systemic effects of selected derivatives of each compound class were analyzed in C. difficile using a proteomics approach. Finally, appropriate downstream experiments were performed to follow up on hypotheses drawn from the proteomics datasets. Thereby, all three compound classes demonstrated significant activity against C. difficile. However, chelocardins similarly inhibited the growth of other anaerobes excluding chelocardins as antibiotic candidates for C. difficile infection therapy. In contrast, chlorotonils demonstrated significantly higher in vitro activity against C. difficile and close relatives compared to a small panel of other anaerobes. In addition, it could be shown that chlorotonils affect intracellular metal homeostasis as demonstrated in a multi-omics approach. The data led to speculate that chlorotonils eventually affect cobalt and selenate availability in particular. Moreover, a metaproteomics approach verified that oral chlorotonil treatment only marginally affected the intestinal microbiota of piglets on taxonomic and functional level. Furthermore, the proteome stress response of C. difficile 630 to myxopyronin B, which similarly showed elevated activity against C. difficile compared to a few other anaerobes, indicated that the antibiotic inhibited early toxin synthesis comparatively to fidaxomicin. Finally, evidence is provided that C. difficile 630 responds to dissipation of its membrane potential by production and accumulation of aromatic metabolites.
Background: A large body of research indicates that the cognitions individuals have
about their own age and aging, so called self-perceptions of aging (SPA), predict health and
wellbeing in later life. However, much less is known about associations of SPA with
developmental correlates such as personality. Some initial studies have found cross-sectional
and longitudinal associations of the Big Five traits (openness to experience,
conscientiousness, extraversion, agreeableness, and neuroticism) with SPA. Building on these
findings, this thesis aimed at advancing knowledge on associations of personality with SPA.
To this end, cross-sectional associations of the meta-traits of agency, i.e., a focus on the self,
and communion, i.e., a focus on others, with SPA were examined in study 1, and longitudinal
associations of agentic and communal personal values with SPA were examined in study 2.
Study 3 aimed at expanding findings of previous studies on associations of SPA with selfreported
physical function to an objective indicator of physical function, namely, gait pattern.
In all studies, SPA were treated as a multidimensional construct comprising gains and losses.
Methods: Study 1 was based on data of 154 adults aged 75 and older that were
recruited in hospital. Data was collected one month after recruitment. In regression analyses,
associations of agentic and communal traits with SPA beyond health were examined. Study 2
was based on data of 6,089 adults aged 40 and older enrolled in the German Ageing Survey
(DEAS). Multiple regression analyses were used to test whether personal value priority
predicted change in SPA over three years beyond age stereotypes. For study 3, latent profile
analysis was employed to detect gait patterns based on data of 150 adults aged 70 and older
collected via an automated walkway at participants’ regular speed and individual maximum
speed. In a next step, associations of SPA with gait patterns beyond personality traits were
investigated in binary logistic regressions.
Results: Agentic and communal personality traits were associated with gain-, but not
loss-related SPA when controlling for health (study 1). In study 2, the value priority of
openness to change (self-direction, stimulation) predicted more gain-related SPA three years
later, while the value priority of conservation (tradition, security) was negatively associated
with gain-related SPA. The value priority of self-enhancement (achievement, power) was
associated with more loss-related SPA three years later. Finally, the value priority of selftranscendence
(universalism, benevolence), i.e. a concern for the well-being of others, was
associated with more gain- and less loss-related SPA at follow-up. In study 3, latent profile
analyses distinguished two groups with different gait patterns in both gait speed conditions.
One group exhibited a slower and less well-coordinated gait pattern, which reflected
functional limitations. The other group exhibited a faster and well-coordinated gait pattern,
which reflected better physical function. More loss-, but not gain-related SPA were associated
with higher likelihood to exhibit a functionally limited gait pattern at regular speed.
Conversely, gain- but not loss-related SPA were associated with higher likelihood to exhibit a
fit gait pattern at individual maximum speed.
Conclusion: Results of this thesis have three main implications for research on SPA.
First, agency and communion may constitute useful dimensions for further investigating SPA
domains, as both were associated with SPA in study 1. Second, findings of study 2 point to
the role of motivation for SPA that needs to be further explored. Third, findings of study 3
indicate that SPA are not only associated with self-reported, but also objectively measured
physical function, which stresses the importance of SPA for health in later life. As a practical
implication, the findings presented here suggest that interventions on SPA should consider
participants’ personality, both on the level of traits and values.
Geometric T-Duality
(2022)
From a physicists point of view T-duality is a relation connecting string
theories on different spacetimes. Mathematically speaking, T-duality should be a symmetric relation on
the space of toroidal string backgrounds. Such a background consists of: a smooth manifold M; a torus bundle E over M - the total space modelling spacetime; a Riemannian metric g on E - modelling the field of gravity; a U(1)-bundle gerbe G with connection over E - modelling the Kalb-
Ramond field.
But as of now no complete model for T-duality exists. The three most notable
approaches for T-duality are given by the differential approaches by Buscher in the form of the Buscher rules and by Bouwknegt, Evslin and Mathai in the form of T-duality with H-flux on the one hand, and by the topological approach given by Bunke, Rumpf and Schick which is known as topological T-duality. In this thesis we combine these different approaches to form the first model for T-duality over complete geometric toroidal string backgrounds and we will introduce an example for this geometric T-duality inspired by the Hopf bundle.
Cell mechanical properties reveal substantial information on cell state and function. Utilizing mechanics as a label-free biomarker allows for investigation of fundamental cellular processes as well as biomedical applications, e.g., disease diagnosis. High-throughput methods for accessing the elastic properties of cells in suspension from hydrodynamic deformation in a microfluidic constriction are available with real-time analysis rates of up to 1000 cells per second. However, accessing elastic as well as viscous properties of cells and multicellular systems in suspension as well as adhered to surfaces at high throughput has not been possible so far. In this thesis, I approached this question and developed as well as applied microfluidic and holographic technologies to analyze the viscoelastic properties of single cells and multicellular aggregates, respectively.
First, I demonstrated that real-time deformability cytometry (RT-DC) can be applied in transfusion medicine, where the highest quality standards have to be maintained while blood product release is time-critical. We showed for platelet and red blood cell concentrates as well as for hematopoietic stem cells that their mechanical properties can be used for label-free quality assessment. The results have been published in Lab on a Chip (Aurich et al. 2020).
For RT-DC and many other methods based on hydrodynamic deformation, the constriction size has to be adapted to the objects of interest to allow for a shear-induced deformation. We introduced virtual fluidic channels, which are established by two co-flowing aqueous polymer solutions. Virtual fluidic channels can be precisely adjusted in their cross section, allowing for mechanical phenotyping of single cells as well as cell clusters or tissue spheroids in one microfluidic system. Importantly, measurements can also be performed in standard microfluidic geometries beyond soft lithography, e.g., in the cuvette of a flow cytometer. For cell spheroids as a model system for multicellular aggregates, we show a 10-fold lower Young's modulus of the tissue compared to single-cell mechanics, suggesting cell-cell and cell-matrix interactions being potential contributors to the mechanics of multicellular aggregates. Our work on virtual fluidic channels has been published in Nature Communications (Panhwar et al. 2020).
Within this thesis, I expanded the high-throughput elastic phenotyping performed by RT-DC towards viscoelastic cell properties by developing dynamic real-time deformability cytometry (dRT-DC). Dynamic tracking of cells while passing the microfluidic constriction allows to access steady-state (elasticity) and time-dependent (viscosity) material properties for a complete viscoelastic characterization of cells in suspension at high throughput. I introduced a shape mode decomposition based on a Fourier transformation, which allows to disentangle the superimposed stress responses to an extensional stress at the channel inlet and a constant shear stress in the channel. These hydrodynamic stress distributions are present in almost every microfluidic channel geometry. From the separated stress responses, viscoelastic material properties can be determined independent of cell shape.
We demonstrated experimentally the sensitivity of dRT-DC to cytoskeletal alterations and confirmed the validity of the method by reference measurements on calibrated hydrogel beads. In our work, we also presented a viscoelastic fingerprint of the major subpopulations of peripheral blood: erythrocytes, granulocytes, and peripheral blood mononuclear cells (PBMCs) (e.g., lymphocytes and monocytes), all characterized by the same method. The technique and the results have been published in Nature Communications (Fregin et al. 2019).
In cell mechanical methods based on hydrodynamic deformation, cell shape is usually monitored while a stress is applied. For extraction of material properties as well as for studying shape dynamics, it is essential to describe cell shape yielding highest strain differences for a given microfluidic system and experimental setting. Using dRT-DC, I compared nine different shape descriptors to analyze cell deformation in an extensional as well as shear flow. A relaxation time analysis was performed on different levels of data aggregation from single cells to an ensemble scale. I demonstrated that the steady-state deformation can be predicted from stress response curves without them reaching the steady-state. This is important for cell mechanical measurements in microfluidic systems as the characteristic times are unknown in general and as the channel length is fixed. In addition, by introducing a cut-off criterion for how much of the response trace needs to be captured within the channel, the analysis time per cell can be reduced while material properties can still be extracted. Performing simulations, I compared the accuracy of relaxation times extracted from ensemble and single-cell studies under experimental conditions. Introducing a scoring system to evaluate which combinations of shape descriptors and analysis strategies provide biggest effect size, we concluded that single-cell analyses in an extensional flow are most sensitive to cytoskeletal modifications independent of shape parametrization. The manuscript was submitted to the Biophysical Journal.
Finally, I translated the fast non-contact cell mechanical probing from suspension to adherent cells. No such technology has been available and with the majority of cells being adherent, a robust label-free method for mechanophenotyping at high-throughput is required. Within this thesis, I have introduced and realized a new concept: holographic vibration spectroscopy (HVS), where adherent cells are mechanically excited on a vibrating surface while their height oscillations are measured optically. Analysis is done in an interferometric heterodyne setup by using frequency multiplexing and time-averaged holography in off-axis configuration. Based on interference images captured by a high-speed complementary metal-oxide-semiconductor (CMOS) camera, I established a mathematical model to reconstruct the vibration amplitude of adherent cells as well as their retardation phase compared to the exciting vibration. From the amplitude and phase response, viscoelastic parameters can be derived, which have to be investigated in subsequent studies.
In summary, I introduced in my work two high-throughput methods for the viscoelastic characterization of suspended as well as adherent cells while highlighting applications in tissue mechanics and transfusion medicine that are relevant not only in basic but also in translational research.
Ethics of Genetically Determined Chronic Diseases – Hereditary Chronic Pancreatitis as an Example
(2022)
Background: To deliver appropriate support to patients and their families, it is central to know
the needs of those affected by a disease. As a chronic disease, Hereditary Chronic Pancreatitis (HCP) usually accompanies those
affected for their lifetime and can lead to substantial psychological and social consequences
for the individuals affected and their families. Since the subjective experience of individuals
living with HCP has not yet been investigated, the current PhD thesis examines the ethical and
social issues which arise in the context of HCP.
Methods: To get a comprehensive overview of the ethical and social issues involved, different
methods were combined. A qualitative study with patients and their relatives was conducted
to acquire an understanding of living with HCP. Based on the issues identified, ethical and
conceptual analyses and a systematic review were conducted to supplement the empirical
findings.
Results: Twenty-four individual interviews and one focus group were conducted. The
participants described HCP as a continuous but unstable part of their lives. The ‘shifting
perspectives model’ by Paterson covers this experience adequately, but due to the shifting
character of HCP, the participants reported pathologization as a problematical issue in their
lives. Additionally, the study demonstrates that genetic testing has a wide influence in familial
contexts and is accompanied by normative issues, e.g. related to reproductive decisions. The
study revealed the broad range of ethical and social issues that those affected by HCP face. In
this context, patient advocacy organizations (PAOs) are seen as an important source of support by patients and their families.
Discussion: Given the various tasks ascribed to PAOs, it is unclear what roles PAOs have and
what responsibilities they bear. Although the conceptual analysis and the systematic review
provided an orientation about PAOs’ responsibility, no general answer can be given. Instead,
each PAO and its specific situation must be evaluated individually. Responsibility as a
relational concept can help to structure these situations and to understand the role of PAOs
in the healthcare sector and in current bioethical debates better.
Conclusion: The thesis provides empirical and conceptual findings on the ethical and social
issues in the context of a rare, genetically determined, chronic disease. It is important to
recognize these three dimensions and their interplay to deliver optimal care to those affected.
The results build a comprehensive starting point for healthcare professionals managing
genetically determined, chronic diseases, but more research is needed to bring the results of
this thesis into practice.
Avian influenza viruses (AIVs) have their natural reservoir in wild aquatic birds but occasionally
spread to terrestrial poultry. While AIVs of subtypes H5 and H7 are well known to evolve highly
pathogenic avian influenza viruses (HPAIVs) during circulation in domestic birds, non-H5/H7
subtypes exhibit only a low to moderate pathogenicity. Furthermore, spillover events to a broad
range of mammalian hosts, including humans, with self-limiting to severe illness or even fatal
outcomes, were reported for non-H5/H7 AIVs and pose a pandemic risk. The evolution of high
virulent phenotypes in poultry and the adaptation of AIVs to mammalian hosts are predominantly
linked to genetic determinants in the hemagglutinin (HA). The acquisition of a polybasic cleavage
site (pCS) is a prerequisite for the evolution of HPAIVs in poultry, while changes in the receptor
binding preference and virus stability are essential for adaptation of AIVs to mammals.
In August 2012, an H4N2 virus with the pCS motif 322PEKRRTR/G329 but preserved trypsin
dependend replication and low pathogenicity in chickens was isolated on a quail farm in California.
In the first two publications, we followed different approaches to investigate virulence factors and
the potential risk for the transition of H4N2 to high virulence in chickens. The loss of N-terminal
glycosylations in the vicinity of the pCS resulted in decreased binding to avian-like receptors and
dramatically decreased virus stability. On the other hand, one deglycosylation increased virus
replication and tissue tropism in chicken embryos but did not alter virulence or excretion in
chickens. Furthermore, additional basic amino acids in the natural pCS motif improved the trypsin-independent
cleavage of HA and caused slightly increased tissue tropism in chickens. However,
the engineered motifs alone did not affect virulence in chickens. Intriguingly, they even had a
detrimental effect on virus fitness, which was restored after reassortment with segments of HPAIV
H5N1. Together, the results show the importance of HA glycosylations on the stability of H4N2 and
reveal the important role of non-HA segments in the transition of this virus to high virulence in
poultry.
The transmission of another non-H5/H7 AIV of subtype H10N7 from birds to seals resulted in mass
deaths in harbor seals in 2014 in northern Europe. The third publication describes nine mutations
in the HA1 subunit of seal isolates compared to avian H10Nx viruses. We found that some of these
mutations conferred a dual specificity for avian and mammalian receptors and altered
thermostability. Nevertheless, the H10N7seal remained more adapted to avian host cells, despite
of the alteration in the receptor binding specificity.
Altogether, this thesis demonstrates that naturally evolved AIVs beside H5 and H7 subtypes
support a highly pathogenic phenotype in the appropriate viral background and alter virulence and
host receptor specificity by few amino acid substitutions in the HA. These findings improve our
knowledge of the potential of non-H5/H7 AIVs to shift to high virulence in birds and the adaptation
in mammals.
Chronic alcohol abuse is one of the most common addictions and one of the most substantial public health problems as it affects millions of people physically as well as mentally around the world. Globally more than 3 million deaths are assignable to alcohol intake each year. Chronic alcoholism is a multi-component disease and its development is associated with both environmental as well as genetic factors. However, the key mechanisms underlying an addiction, especially on a cellular and physiological basis, are still unknown. Bio-medically an influence of chronic alcohol consumption on synaptic plasticity in the brain of humans as well as rodents has been proven.
On the dendritic shaft of nervous brain cells, small membrane protrusions called dendritic spines can be found. These spines possess the capacity to change their morphology and quantity and are thought to play an important role in learning and memory forming, and seem to be impaired in multiple neurological disorders. These dynamics are called synaptic plasticity. Most of these studies however, were carried out on the cortex. These previous observations raise the question whether such alterations in synaptic plasticity can also be observed in regions of the brain that contribute to the limbic system and therefore to the processing of emotional responses, learning and decision making. The amygdala is of special interest when trying to understand the neurobiology and pathophysiology that lead to the emergence and up keeping of an alcohol addiction. In this thesis a closer look has been taken at possible alterations in synaptic plasticity within different amygdaloid nuclei by the help of a rat model. These rats were put into the so called postdependent state, one of the most common animal models to investigate excessive ethanol intake in rodents. The postdependent state is a model in which the key driving force to obtain alcohol as part of a preserved addiction cycle is based on negative affect. Studies showed differences in the behavioural outcome of those animals that were exposed to chronic intermittent alcohol consumption compared to a control group, so it was of special interest to see whether those behavioural changes also show on a cellular basis.
In the study, a morphological comparison of the spine length as well as the spine density of alcohol dependent rats with a comparable control group has been made. The medial, the central, the lateral and the basolateral amygdaloid nucleus were of special interest in this research project.
The results showed no significant difference of the spine densities in any of the four amygdaloid regions. When comparing the spine morphology within the ethanol and the control group, differences showed in the lateral amygdaloid nucleus. In this region the spines of the ethanol group were significantly smaller. This leads to the conclusion that chronic alcohol intake can have an influence on the spine morphology and hence alter anatomical brain structures.
The aim of this study has twofold. First, to investigate characteristics of paediatric patients seeking emergency dental care and the according treatment in a specialised dental service. The second aim, to propose a data-based guidance for management of dental emergency in children including pain management without traumatizing the child and with a clear differentiation of dental emergency patients.
Records of the demographic and clinical characteristics of 332 children (mean age 8.2 ±4.2 years) who attended the Department of Preventive and Paediatric Dentistry at the University of Greifswald in 2018 without an appointment seeking emergency dental care were collected. Statistical analysis was carried out using descriptive statistics and Chi-square tests. Based on the data a proposal for management of emergency/pain patients was performed.
Pain patients were high caries risk children (mean: 4.59 ±3.65 dmft, 1.65 ±2.73 DMFT). The main clinical diagnoses of pain patients in all different age groups were dental caries and caries-related complications such as reversible and irreversible pulpitis or periapical periodontitis (63.6%), while dental trauma contributed (8.4%). In general, the most frequent treatment was extraction (41.0%), most of these extractions needed to be rendered under either nitrous oxide sedation or general anaesthesia (57.3%).
This study has shown a clear association between the dental emergency management plan (delay of the dental treatment) and other factors such as, the required treatment (invasive or not invasive) P < 0.05, the need of sedation P < 0.05 and child’s previous dental experience (first visit to the dental clinic) P < 0.001. Most of the extraction procedures were not rendered immediately in the emergency/pain visit, rather they were delayed to be performed in the next treatment session (70%), with the majority of these procedures planned to be rendered under sedation. On the other hand, most of the restorations (65.5%) were performed in the emergency/pain visit, with need of sedation for only (8.3%) of these procedures. In addition, the majority of treatments of children who were visiting the dental clinic for the first time were postponed (71.6%; n=63). The correlation analysis shows a higher tendency to postpone the treatment when the required treatment is invasive, need to be rendered under sedation or when
it was the child’s first visit to the dental clinic. These findings indicate the strong impact of child’s cooperation on the emergency management plan.
Based on these data the guidance for management of peadiatric dental emergency patients was developed including pain management without causing negative affect of the child’s future cooperation
Participating in an election is by far the most prevalent form of political participation in modern democracies. Turnout rates, however, not only vary considerably between countries but also over time: By trend, in many Western democracies turnout levels have declined over the last decades. Electoral systems depict a prominent factor that has always been discussed with respect to its impact on turnout. In this respect, a high number of empirical studies found aggregate turnout predominantly to be higher in countries using proportional representation compared to countries using a less proportional electoral system. Based on these findings, one should expect turnout to increase when the electoral system changes towards higher proportionality. However, empirical evidence of the actual lasting impact of changes in electoral institutions on voter turnout is all but conclusive. In this dissertation, I aim at answering the following question: What are the consequences of electoral system change for voter turnout? I argue that it is necessary to examine the relationship between electoral systems and turnout more detailed as most studies did to date by taking the level of electoral constituencies and the temporal dimension of electoral system change into account.
To assess the impact of electoral systems and further proposed causal factors associated with electoral systems, party system size for instance, on turnout empirically, I make use of a comparative research design, analyzing longitudinal data with time-series cross-sectional regression models. These data, being the basis for my empirical analyses, represent a unique data set covering 9.639 electoral districts from 146 national legislative elections in eleven European countries. The dissertation generally finds an increasing district magnitude to boost turnout, while a decreasing magnitude has negative consequences for electoral participation. The positive effect of district magnitude on turnout seems to depend on the size of the population in the respective district, however. In addition, the analyses show that a higher number of parties in a district, respectively an increase in the number of parties in a district, has a negative impact on turnout.
Myxomycetes or Myxogastria (supergroup Amoebozoa) are one of several Protistean groups dispersing via airborne spores. The model organism for the group, so far exclusively studied in a laboratory environment, is Physarum polycephalum. Here, molecular evolution, distribution and the ecology of spores dispersal was investigated for the non-model species Physarum albescens. This nivicolous myxomycete fruits with snow melt in most mountain ranges of the northern hemisphere and disperses via spherical, dark-colored and melanin-rich spores. Fruit body development and subsequent spore dispersal occurs within a short time window of a few days. At this time, the fruiting plasmodium is fully exposed to the harsh environment if the protecting snow melts away. The spores, with a diameter of 10–13 µm of the typical size for myxomycetes, can potentially reach all suitable habitats worldwide, which led to the assumption that not only Ph. albescens but most myxomycete species should be ubiquitously distributed over the world.
In the first part of this study (article 1), the question was, if spore dispersal can realize a gene flow sufficient to meet the above-mentioned assumption. A total of 324 accessions of Ph. albescens, collected all over the northern hemisphere, was sequenced for 1-3 genetic markers (SSU, EF1A, COI), and 98 specimens were further analyzed using the genotyping by sequencing technique. As a result, at least 18 reproductively isolated units, which can be seen as cryptic biological species, emerged as phylogroups in a three-gene phylogeny, but as well in a SNP-based phylogeny and were confirmed by a recombination analysis between the three markers. However, this evolutive radiation is not simply caused by geographic fragmentation due to low dispersal capability: within a certain region, multiple phylogroups coexisted next to each other, although some appeared to be regional endemics. Most likely, mutations in mating-type genes, as shown to exist for the cultivable Ph. polycephalum, are the main drivers of speciation. This challenges the hypothesis of ubiquitous distribution of Ph. albescens and corroborates the results of the few available studies for other myxomycete species. In addition, groups of clonal specimens, mostly but not always restricted to a certain slope or valley indicated that sexual and asexual reproduction coexists in the natural populations of Ph. albescens.
In the second part (articles 2), the fundamental niche for Ph. albescens was described using all available records for the species. The resulting set of 537 unique occurrence points was subjected to a correlative spatial approach using the software MaxEnt. In dependence on the predictor variables three species distribution models emerged which differed only in details. The first consisted of only 19 bioclimatic variables and an elevation map from the WorldClim dataset. The second was corrected for pseudo-absences resulting from missing survey activities, and the third was expanded with an additional categorical environment variable on snow cover. High mean AUC (area under the curve) values above 0.97 could be reached with all three models. Variables for snow cover, precipitation of the coldest quarter (of the year), and elevation correlated highly to predict the distribution of Ph. albescens. Only in mid-northern latitudes, elevation alone was a good predictor, but it would cause false-positive predictions in arid mountain ranges and failed to explain occurrence in lowland sites at higher latitudes. Mountains in humid climates showed the highest incidences, confirming recent studies that long-lasting snow covers combined with mild summers are crucial for the ecological guild of nivicolous myxomycetes, with Ph. albescens as a typical species.
Spore size is crucial for dispersal ability and should thus be a character under strong selection. In addition, spores carrying two nuclei with opposite mating types should have a colonization advantage. This was the hypothesis for the last part of this study (articles 3 and 4), which investigated this trait in a quantitative manner. This required a method to analyze thousands of spores automatically (article 3) and with high precision for size and the number of nuclei enclosed. Human errors should be excluded, to reveal even subtle differences in the resulting spore size distributions. Two challenges had to be met for this approach. First, a preparation technique was developed to reduce false segmentations due to overlaying spores by aligning spores on one common plane with a high-frequency vibration device. Second, the segmentation process was automated to allow separating spores that are densely packed in the respective images. A machine learning algorithm was set up and trained to reliable identify and measure dark-colored spores. The technique produced consistent results with high accuracy, and the large number of spores allowed to compile spore size distributions, to check for the constancy of this character, which is impossible with manual measurements limited to low numbers.
The resulting spore size distributions, obtained from over 80 specimens (article 4), were mostly narrow, which is in accordance with our hypothesis. Spore size was as well fairly constant within fructifications from one colony. However, mean spore size within different accessions of Ph. albescens showed large variation (ca. 10%, a range often indicated to key out different morphospecies of myxomycetes), and this was explained only by a minor part with differences between biospecies. Not much smaller (8%) was the variation within a group of clonal specimens collected within 25 m distance. This points to a strong influence of environmental factors even at a micro spatial scale, perhaps caused by microclimatic differences and high phenotypic plasticity for spore size. The influence of large-scale covariates like altitude or latitude was negligible. However, spore size correlated with the variance in this trait, indicating that oversized spores may be caused by detrimental environmental conditions. Two aberrations in spore development were found: First, a few specimens showed a multimodal distribution for spore size with two or even three discernible spore populations. The estimated volumes of those populations correspond to a multiple of the first and most abundant conspicuous spore size population. Second, not all spores were uninucleate as to be expected for meiotic products. This was revealed by fluorescence signals from staining the same spores with DAPI, with a second machine learning algorithm trained to identify the nuclei in a spore. A few specimens showed a significant proportion of binucleated spores in the size range of normal-sized ones, and these specimens were not the ones with multimodal spore size distributions. This indicates that the negative impacts (inbreeding) of multinucleate spores should outweigh a possible colonization advantage and is in accordance with the high genetic diversity found in the worldwide population of Ph. albescens, indicating predominantly sexual reproduction in wild populations of myxomycetes.
The thesis develops a scholarly-artistic modular method of analysis for literary studies, film studies and comic studies. Artistic modules of analysis are combined with established research methods used by the humanities on order to deepen the level of understanding of the analysed comic/literary/cineastic work. Martin Rowson's comic adaptation of Laurence Sterne's novel "The Life and Opinions of Tristram Shandy, Gentleman" is analysed with the scholartistic method and treated as an addative literary adaptation.
A significant fraction of the decaying algal biomass in marine ecosystems is expected to be mineralized by particle-associated (PA) heterotrophic bacterial communities, which are thus greatly contributing to large-scale carbon fluxes. Whilst numerous studies have investigated the succession of free-living (FL) marine bacteria, the community structure and functionality of PA bacterial communities remained largely unexplored and knowledge on specific contributions of these microorganisms to carbon cycling is still surprisingly limited. This has mostly been due to technical problems, i.e., caused by the enormous complexity of marine particles and the high abundance of eukaryotic microorganisms within these particles. This thesis presents (a) an optimized metaproteomics protocol for an in-depth characterization of marine PA bacteria, (b) an application example with FL and PA communities sampled during a spring phytoplankton bloom in 2009 in the North Sea, which confirmed the reliability of the optimized metaproteomic workflow, (c) the metaproteomic analysis of particulate communities sampled during a spring phytoplankton bloom in 2018, resulting in an as yet unprecedented number of identified protein groups of the bacterial response bloom and (d) a proteomic analysis of a PA bacterial isolate grown on the two naturally abundant marine polysaccharides laminarin and alginate. The observed succession of bacterial clades during metaproteomic analyses of the investigated blooms highlights individual niche occupations, also visible on genus level. Additionally, functional data shows evidence for the degradation of different marine polysaccharides e.g., laminarin, alginate and xylan supporting the important role of PA bacteria during the turnover of oceanic organic matter. Furthermore, most of the identified functions fit well with the current understanding of the ecology of an algal- or surface-associated microbial community, additionally highlighting the importance of phytoplankton-bacterial interactions in the oceans. More detailed insights into the metabolism of PA bacteria were gained by the proteomic characterization of a selected PA bacterial isolate grown on laminarin and alginate. Functional analyses of the identified proteins suggested that PA bacteria employ more diverse degradation systems partially different from the strategies used by FL bacteria.