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- Institut für Botanik und Landschaftsökologie & Botanischer Garten (28)
- Institut für Biochemie (27)
- Kliniken und Polikliniken für Innere Medizin (18)
- Abteilung für Mikrobiologie und Molekularbiologie (17)
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- Zoologisches Institut und Museum (15)
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- Wiley (58)
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Plasma medical oncology: Immunological interpretation in head and neck squamous cell carcinoma
(2020)
Over the past several years, various important articles focusing on cancer therapy
approaches in head and neck squamous cell carcinomas (HNSCCs) using cold
atmospheric plasma (CAP) have been published (SEMMLER et al. 2020 [53],
METELMANN et al. 2018 [44], KEIDAR et al. 2011 [33]). This doctoral thesis presents
selected results from a prospective observational clinical study in CAP therapy of
palliative HNSCC patients, carried out at the Department of Oral and Maxillofacial
Surgery/Plastic Surgery of the Greifswald University Medicine. For oral and
maxillofacial surgeons, ulcerated surfaces of locally advanced head and neck squamous
cell carcinomas (UICC IV) offer a challenging treatment assignment with microbial
contamination and tumour progression. The clinical attempt appears to eradicate
microbial contamination and to initiate tumour regression. This doctoral thesis will
describe the processes of human tumour biology and tumour immunology in HNSCCs
and the extent of present knowledge concerning plasma medical oncology as an
anticancer modality. In the introduction of the doctoral thesis clinical results of plasma
therapy in locally advanced HNSCCs (UICC IV) are set out. This mainly includes the
investigation of a therapeutic concept, the treatment phases, the tumour size development
and the morphological changes of the infected tumour surface following cold atmospheric
plasma therapy. In the main part, a detailed immunological interpretation is proposed on
the basis of present preclinical and clinical immunological knowledge. Finally,
unexplored questions in plasma medical oncology are highlighted. This is highly
significant for future plasma research and clinical anticancer therapy.
This paper presents continuous, high resolution fossil pollen and microcharcoal records from Bo Langvlei, a lake in the Wilderness Embayment on South Africa’s southern Cape coast. Spanning the past ~1300 years and encompassing the Medieval Climate Anomaly (MCA; c. AD 950–1250) and the Little Ice Age (LIA; c. AD 1300–1850), these records provide a rare southern African perspective on past temperature, moisture and vegetation change during these much debated periods of the recent geological past. Considered together with other records from the Wilderness Embayment, we conclude that conditions in the region during the MCA chronozone were – in the context of the last 1300 years – likely relatively dry (reduced levels of Afrotemperate forest pollen) and perhaps slightly cooler (increased percentages of Stoebe-type pollen) than present. The most significant phase of forest expansion, and more humid conditions, occurred during the transition between the MCA and the most prominent cooling phase of the LIA. The LIA is clearly identified at this locality as a period of cool, dry conditions between c. AD 1600 and 1850. The mechanisms driving the changes observed in the Bo Langvlei pollen record appear to be generally linked to changes in temperature, and changes in the influence of tropical circulation systems. During warmer periods, moisture availability was higher at Bo Langvlei, and rainfall was perhaps less seasonal. During colder periods, precipitation resulting from tropical disturbances was more restricted, resulting in drier conditions. While increased precipitation has been reported during the LIA from Verlorenvlei in the Western Cape as a result of an equatorward displacement of the westerly storm-track at this time, the opposing response at Bo Langvlei suggests that any increased influence of westerlies was insufficient to compensate for the concurrent reduction in tropical/local rainfall in the region.
In recent years, negative impact of pharmaceutical products on natural environment became an issue of high public interest. Pharmaceutical residues are detected in various ecosystems worldwide. Due to increasing production and consumption of medicines this problem is intensified. Therefore, an efficient way to restrain release into the world’s water system is required.
This work presents an enzymatic approach for the degradation of pharmaceuticals in wastewater treatment plants, using laccase and cytochrome P450 — two enzymes of high biotechnological and industrial potential. Laccase genes from fungi Trametes versicolor and Pycnoporus cinnabarinus were isolated and overexpressed in the non-conventional yeast Arxula adeninivorans. This organism served also as cytochrome P450 gene donor.
Recombinant laccase Tvlcc5 was purified by immobilized-metal ion affinity chromatography and biochemically characterized using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate for enzyme activity assays. The optimal temperature and pH were found to be 50 °C and 4.5–5.5, respectively. The half-life of Tvlcc5 at 60 °C was around 20 min. It was demonstrated that the presence of copper ions is essential for the synthesis of active protein. Moreover, negative impact of chloride anions on laccase activity was shown.
Cultivation conditions for the Tvlcc5 producing strain A. adeninivorans G1212/YRC102-TEF1-TVLCC5-6H were optimized. It was found that maintaining the pH at a constant level between pH 6.0 and 7.0 is essential for the production of active enzyme. Optimal cell growth and laccase accumulation were reached at 20 °C and in medium supplemented with 0.5 mM CuSO4. Performed fed-batch cultivation resulted in a laccase activity of 4986.3 U L-1.
Factors influencing the synthesis of Tvlcc5 leading to increased production of this protein were investigated. It was found that using three non-native signal peptides (cutinase 2 from A. adeninivorans (ACut2), α-mating factor from S. cerevisiae (MFα), and acid phosphatase from P. pastoris (PHO1) signal peptides) enhances the secretion of active enzyme by 20–80%. Besides that, additional overexpression of copper transporters positively affects laccase production.
Finally, it was proven that recombinant Tvlcc5 is a promising agent for the degradation of certain pharmaceuticals. After 24 h of incubation, the concentration of diclofenac and sulfamethoxazole decreased to 46.8% and 51.1%, respectively. Furthermore, it was shown that the addition of the redox mediator ABTS significantly shortens the degradation time of these substances.
Mathematical phylogenetics provides the theoretical framework for the reconstruction and analysis of phylogenetic trees and networks. The underlying theory is based on various mathematical disciplines, ranging from graph theory to probability theory.
In this thesis, we take a mostly combinatorial and graph-theoretical position and study different problems concerning phylogenetic trees and networks.
We start by considering phylogenetic diversity indices that rank species for conservation. Two such indices for rooted trees are the Fair Proportion index and the Equal Splits index, and we analyze how different they can be from each other and under which circumstances they coincide. Moreover, we define and investigate analogues of these indices for unrooted trees.
Subsequently, we study the Shapley value of unrooted trees, another popular phylogenetic diversity index. We show that it may fail as a prioritization criterion in biodiversity conservation and is outcompeted by an existing greedy approach. Afterwards, we leave the biodiversity setting and consider the Shapley value as a tree reconstruction tool. Here, we show that non-isomorphic trees may have permutation-equivalent Shapley transformation matrices and identical Shapley values, implying that the Shapley value cannot reliably be employed in tree reconstruction.
In addition to phylogenetic diversity indices, another class of indices frequently discussed in mathematical phylogenetics, is the class of balance indices. In this thesis, we study one of the oldest and most popular of them, namely the Colless index for rooted binary trees. We focus on its extremal values and analyze both its maximum and minimum values as well as the trees that achieve them.
Having analyzed various questions regarding phylogenetic trees, we finally turn to phylogenetic networks. We focus on a certain class of phylogenetic networks, namely tree-based networks, and consider this class both in a rooted and in an unrooted setting.
First, we prove the existence of a rooted non-binary universal tree-based network with n leaves for all positive integers n, that is, we show that there exists a rooted non-binary tree-based network with $n$ leaves that has every non-binary phylogenetic tree on the same leaf set as a base tree.
Finally, we study unrooted tree-based networks and introduce a class of networks that are necessarily tree-based, namely edge-based networks. We show that edge-based networks are closely related to a family of graphs in classical graph theory, so-called generalized series-parallel graphs, and explore this relationship in full detail.
In summary, we add new insights into existing concepts in mathematical phylogenetics, answer open questions in the literature, and introduce new concepts and approaches. In doing so, we make a small but relevant contribution to current research in mathematical phylogenetics.
An experimental investigation of particle parallel flows has been carried out at Wendelstein 7-X (W7-X), one of the most advanced stellarators in the world. The studies are restricted to the outermost plasma region, the scrape-off layer (SOL), which is shaped to tackle the exhaust problem in vision of future fusion reactors based on plasma magnetic confinement. The aim of the measurements is to set the basis for a physics analysis of the SOL dynamics by obtaining direct information on convective heat transport, together with the assessment of the predominant flow directions of the main plasma ions and of fusion-products or wall-released impurities. In this way, a better comprehension of the interplay between the transport parallel and perpendicular to the SOL field lines can be achieved, contributing to the understanding of the effectiveness of the island divertor configuration.
The chosen instrument for the experimental studies is the Coherence Imaging Spectroscopy (CIS) diagnostic, a camera-based interferometer capable of measuring 2D Doppler particle flows associated with a selected visible line from the plasma. The diagnostic is distinguished by its high time resolution and spatial coverage, allowing the visualisation and measurements of flow velocities for a full module of W7-X simultaneously. A CIS diagnostic has been fully designed for W7-X with an improved level of accuracy achieved thanks to the implementation of a new calibration source, a continuous-wave-emission tunable laser. The laser allowed a full characterization of the diagnostic and a frequent precise calibration, making the CIS system reliable for parallel flow investigations during the operational campaign OP1.2. The validity and importance of the CIS measurements have been further confirmed with dedicated simulation of the SOL plasma parameters by the EMC3-EIRENE code, and by comparisons with other edge diagnostics. The CIS results show the effects related to dynamical changes in the SOL due to impurity gas puffs or the development of a plasma current. Moreover, CIS can be used as a powerful tool to test the limits of the current theoretical models, for example in the case of forward and reversed field experiments.
The non-renewable energy sources coal, oil and natural gas that contribute the major share of the world's energy, will be running out in the next 40-80 years. With the growing energy demands especially in developing countries, which is likely to surpass that of the developed countries in next 50 years, an alternate energy source is the need to the hour. The nuclear fusion energy is foreseen as one of the potential candidates to solve the current global energy crisis. One of the major challenges faced by the fusion community is the problem of power exhaust. With the larger fusion devices to be built in the future, the heat load on the plasma facing components are expected to grow higher. The present work explores two numerical studies performed on the Wendelstein 7-X, the world's largest stellarator type fusion device, to cope with this problem.
The first project on `'Numerical Studies on the impact of Connection Length in Wendelstein 7-X'' identifies magnetic configuration with long connection lengths, which could bring down the peak heat fluxes onto the divertor to manageable levels, by greater role of cross-field transport which may assist to get a wider heat deposition profile. The second project on `'Development of Heating Scenario to Reduce the Impact of Bootstrap Currents in Wendelstein 7-X'' advocates a novel self-consistent approach to reach high plasma density at full heating power without overloading the divertor during the transient phase of the evolution of the toroidal plasma current, by controlling two parameters; density and power. The aim of both the projects is to contribute to tackling the challenge of the tremendous power exhaust from fusion plasma which, if solved, will be a large step closer to a fusion power plant.
Research on the science and the fiction of supercritical fluid chromatography (SFC) has been ongoing for more than five decades. Today, packed column SFC promises speedy solutions to chiral and semi-preparative separation problems, but academia has been reluctant to incorporate SFC into its curriculum, as doubts linger concerning its practicability. This work sought to explore the merits of SFC in hyphenation with electrospray ionization--single quadrupole mass spectrometry (ESI-MS) and supercritical fluid extraction (SFE) in various aspects of medicinal chemistry and bioanalysis within an academic setting.
SFC was investigated for its usefulness in assessing the purity and the stability of synthesis products, and the quantification of chiral and achiral metabolites - domains conventionally occupied by high performance liquid chromatography (HPLC).
Confronted with analytes prone to hydrolysis (cyclic polysulfides) and UV-induced configurational changes (aza-stilbenes), fast elution by water-free SFC-MS proved complementary to traditional chromatographic techniques.
The quantification of antidepressant ketamine metabolites presented an opportunity to assess supercritical fluid techniques within a bioanalytical context. While SFC hyphenated to single quadrupole MS did not reach the sensitivity levels of HPLC coupled to triple quadrupole MS/MS, exploitation of supercritical CO2 reduced analysis times more than six-fold (60 minutes by HPLC vs 10 minutes by SFC). When coopted for both extraction and analysis, SFE-SFC-MS simplified sample preparation and promoted the transition from off- to on-line bioanalysis. Similar results were obtained when SFC was applied to acidic and basic metabolites of the controversial anodyne flupirtine. Again, SFC featured shorter run times but also expanded the target metabolite spectrum covered within one run.
Finally, a tiered approach to validation demonstrated the reliability achievable by SFC. Critical applications such as quantification of the newly approved antidepressant ketamine or the recently withdrawn analgesic flupirtine were comprehensively validated according to guidelines on bioanalytical method validation by the European Medicines Agency. Notably, this included the first fully validated chromatographic methods for the putative antidepressant (2R,6R)-6-hydroxynorketamine, and the first report of EMA-conforming quantification by on-line SFE-SFC-MS from urine.
Separation scientists find themselves confronted with diverse problems and tools. Although parsing only a microscopic subsection of the available chemical and analytical space, the results obtained here suggest SFC to be a fast and versatile addition to conventional chromatographic methods employed at the intersection of medicinal chemistry and bioanalysis.
In this work, studies with respect to the exhaust problem were performed
in the stellarator experiment Wendelstein 7-X with different target concepts and different magnetic field geometries. Different infrared cameras were used to study the heat flux from the plasma onto the PFC. In the first publication, the limiter set-up was used with a simpler magnetic topology in the plasma edge. The radial fall-off of the parallel heat flux for inboard limiters in W7-X shows, similar to inboard limiters in tokamaks, two different radial fall-off lengths, a short (narrow) one, characterizing the near-SOL, and a long (broad) characterizing the far-SOL. For the far-SOL, the heating power and connection length have been identified as the main scaling parameters, while for the near-SOL, the electron temperature close to the LCFS has been identified as the main scaling parameter. The two fall-off lengths differ by a factor 10, and the found scalings for both regimes differ from known models and experimental scalings in tokamaks. A turbulent-driven feature was discussed in the publication as a possible explanation for the behavior of the fall-off length in W7-X.
The gained information and data have been further used to support many
other publications, covering the symmetry of the heat loads, the
energy balance of the machine, and seeding experiments.
The heat exhaust in W7-X with an island divertor was studied in the second
and third publication. Definitions of parameters such as peaking factor and
wetted area were applied for the heterogeneous heat flux pattern on the
W7-X divertor. It was shown that the island divertor concept is capable
of spreading out the heat efficiently, resulting in large wetted areas of up to 1.5 m2. The reached values for the wetted area are comparable to the ones of the larger tokamak JET but with a much smaller ratio of wetted
area to the area of the last closed flux surface. Furthermore, a positive
scaling of the wetted area with the power in the SOL was observed. This
scaling is beneficial for future reactors but needs further investigation of the involved transport processes. The peaking factor (discussed in the second publication) describes how concentrated the heat load is within the region of the strike line. It was shown that this factor is decreasing for increasing densities without affecting the wetted area. The present work paves the way for further analysis of the transport processes of the heat flux towards the island divertor of Wendelstein 7-X.
Species have to cope with climate change either by migration or by adaptation and acclimatisation. Especially for long-living tree species with a low seed dispersal capacity (e.g. European beech, hereafter called beech), the in situ responses through genetic adaptation and phenotypic plasticity play an important role for their persistence. Beech, the dominant climax tree species in Central Europe, shows a high drought sensitivity and its distribution range is expected to shift northwards. On the other hand, projected northward shifts need to be taken with caution, as some studies suggest a sensitivity of beech to frost events in winter and spring. However, studies on the growth performance of cold-marginal beech populations are still rare. Previous studies on beech populations found local adaptation to drought and phenotypic plasticity in fitness-related traits as well as phenological traits. However, studies on the regeneration of beech under natural conditions are yet missing, although germination and establishment of young trees are a very first selective bottleneck and are crucial for tree population persistence and for successful range shifts.
This PhD-thesis aimed to identify the potential of plasticity and local adaptation in the important early life-history traits germination, establishment after the 1st year, and survival after the 2nd year in a reciprocal transplantation experiment at 11 sites across and even beyond the distribution range of beech (Manuscript 1). Moreover, this thesis investigated the climate sensitivity and the adaptation potential of beech populations by conducting dendroecological studies along a large climatic gradient across the distribution range (Manuscript 2) and along a strong winter temperature gradient towards the cold distribution margin in Poland (Manuscript 3). In addition, the impact of local climatic singularities was studied in a local study at the southern margin (Manuscript 4).
Warm and dry conditions limited natural regeneration, which was indicated by very low survival of young trees, even though germination rates increased with increasing temperature (Manuscript 1). This was also the case in parts of the distribution centre due to the hot and dry conditions in 2018. Although the transplantation experiment revealed high plasticity in the early life-history traits, this plasticity might thus not buffer against climate change under dry conditions. Local adaptation was not detected for any of these traits along the climatic gradient. In contrast, the results of the dendroecological study across the gradient (Manuscript 2) hint towards an adaptation potential of adult trees to drought at the southern margin. Thus, adult trees seemed to be adapted to drought at the southern margin, whereas tree growth in the distribution centre was sensitive to drought. These results indicate that parts of the centre may become ecologically marginal with increasing drought frequency in times of climate change. Interestingly, Manuscript 4 shows that beech growth was positively influenced by frequent fog immersion at the southern distribution margin in north-eastern Spain. This study underlines the importance of local climatic singularities, as they may allow marginal populations to grow in climate refugia in an otherwise unfavourable climate.
At the cold distribution margin, the study in Manuscript 1 found a remarkably higher survival of young trees in Sweden than in Poland. Moreover, the dendroecological studies revealed that beech was hampered by both drought at the cold-dry margin (Manuscript 2) and by winter cold at the cold-wet margin in Poland (Manuscript 3). All these results highlight the importance to study climate sensitivity of adult trees and the response of early life-history traits at the cold margin with a more differentiated view comparing cold-dry against the cold-wet populations and growing conditions. However, the high plasticity of the early life-history traits may allow for an increasing germination rate with climate warming at the northern margin and may thus facilitate natural regeneration there. In contrast, the dendroecological studies suggest that adult trees at the cold distribution margin may suffer either from drought or from winter cold and that the risk for spring frost may increase. Thus, the often-predicted compensation of dry-marginal population decline by a northward range expansion should be discussed more critically.
In conclusion, my PhD thesis provides new knowledge about the potential of natural regeneration and about climate sensitivity of adult trees across the distribution range of beech. Moreover, it underlines the importance to study both the young tree stages as well as adult trees to assess the performance and vulnerability of tree species under climate change, as both showed differences in their response to changing environmental conditions.
Lipoproteins of Staphylococcus aureus represent a major class of surface proteins, which are anchored to the outer leaflet of the cell membrane. Although they play a key role in the immune response and virulence, the majority of lipoproteins in this organism is still of unknown function. The aim of our study was to investigate the function of so far poorly or uncharacterized lipoproteins in S. aureus strain Newman. To this end, an integrated bioinformatical approach was applied to define the pan-lipoproteome of 123 completely sequenced S. aureus strains. In total, this analysis predicted 192 different potential lipoproteins, with a core lipoproteome of 39 and a variable lipoproteome of 153 lipoproteins. Out of those 192 lipoproteins, 141 are so far functionally uncharacterized. Primarily focusing on members of the core-lipoproteome with unknown or poorly characterized function, 24 lipoproteins or co-encoded neighbor proteins were selected for further characterization. Of those 24 proteins, 20 S. aureus markerless deletion mutants were constructed (S. aureus delta l01 - delta l20) and screened for an altered growth behavior under various conditions. Here, three mutants showed a temperature-sensitive phenotype, two mutants formed aggregates in the TSB of the manufacturer Merck (TSBMerck), and four mutants showed reduced growth under osmotic stress with 8% NaCl. An altered aggregation behavior was observed for four mutants in the presence of Triton X-100 and for eleven mutants in the presence of SDS. Furthermore, ten mutants revealed an impaired biofilm formation capacity as well as reduced hemolytic activity. Interestingly, S. aureus deletion mutants delta l14 (delta NWMN_1435) and delta l16 (delta NWMN_0646) showed an altered phenotype under nearly all tested growth and stress conditions. Most strikingly, both deletion mutants demonstrated dramatic defects in cell morphology and cell division during the transient growth phase in TSBMerck and were therefore selected for further detailed characterization. Electron microscopy imaging of the two mutants revealed an irregular cell shape, increased cell size, multiple displaced division septa, and incomplete separation of daughter cells resulting in the formation of cell aggregates in TSBMerck. Complementarily, microarray-based transcriptome analysis and whole-genome sequencing of S. aureus delta l14 and delta l16 suppressor mutants strongly point to a functional association of both lipoproteins with cell envelope- or cell division-related processes. Specifically, multiple hints suggest a functional connection of both lipoproteins with lipo- or wall teichoic acids. Of note, the phenotypes of S. aureus delta l14 and delta l16 are conditional and appear under some, but not all growth conditions. Thus, it is conceivable that the function of L14 and L16 is modulated by metabolic processes, or that the proteins might be part of a “backup system” becoming important only under certain conditions. Collectively, we propose that L14 and L16 fulfill a basic role in cell envelope- or cell division-related processes under specific growth conditions. Particularly, the activity of L14 and L16 might be necessary for the function or localization of lipo- or wall teichoic acids, and thus, might be linked to the regulation of autolysins. In conclusion, this study reveals important insights into the function of two so far uncharacterized but highly conserved lipoproteins in S. aureus.
The global prevalence of kidney diseases has been steadily rising over the last decades. Today, around 10% of the world population suffers from relevant chronic kidney disease. Podocytes are highly specialized and terminally differentiated cells residing in the filtering units of the kidneys, the so-called glomeruli. With their interdigitating foot-processes, these cells are a crucial part of the renal filtration barrier. As podocytes are post-mitotic, injury or loss of these cells results in an impairment of the filtration barrier with subsequent loss of global kidney function. Therefore, the question whether a relevant amount of podocytes can be regenerated and if this regeneration can be influenced is crucial for future therapeutic developments. As in vivo microscopic imaging of podocytes in higher animals like mice or rats is rather challenging, larval zebrafish have been applied as an animal model for podocyte development and kidney filtration. 48 hours post fertilization, zebrafish larvae develop a single filtering glomerulus with a similar morphology and molecular construction to that in mammals. For evaluation of podocyte morphology and filtration, we used transgenic zebrafish strains in which podocytes were labeled with fluorescence proteins. Additionally, podocytes expressed the bacterial enzyme nitroreductase fused to the fluorescence protein mCherry. In this model, application of the antibiotic metronidazole leads to podocyte-specific cell death. Through cross-breeding we established strains that additionally express an eGFP-labeled protein in the blood plasma. Using in vivo two-photon microscopy, we could image podocyte-loss induced impairments of the glomerular filtration barrier. Additionally, we tracked characteristic morphological changes of podocyte morphology including podocyte foot process effacement, development of sub-podocyte pseudocysts and finally detachment of whole cells from the glomerular basement membrane. These changes have been before described histologically in different animal models as well as in patient biopsies. Using the in vivo microscopy approach, we could clearly describe the temporal sequence of these alterations. Finally, we also tracked individual, non-detached podocytes over up to 24 hours, and found that these cells were non-migratory. These results show that early podocyte-regeneration through immigration of intra- or extraglomerular cells is unlikely within the first 24 hours of acute glomerular injury.
Background: Depressive disorders are highly prevalent and disabling diseases. Epidemiological studies have shown that they often co-occur with addictive behaviors, which in part might be explained by common risk factors. Rumination might be such a risk factor. Comorbidity can have substantial adverse effects for those affected. Thus, combined treatment approaches are needed. These should not be restricted to individuals with clinical disorders. In light of an apparent treatment gap, new treatment approaches that provide widespread access to evidence-based treatments need to be explored. In recent years, e-health interventions received a lot of attention. With their potential to be widely disseminated, they might be suitable to provide population-based intervention approaches. Developing population-based interventions might present special challenges to intervention developers, for example, in terms of intervention design or the selection of samples to preliminary test interventions. This thesis explored the application of e-health interventions in the treatment and prevention of depressive symptoms and addictive behaviors. Its first aim was to provide an overview on publicly accessible evidence-based e-health interventions for the treatment and prevention of depressive symptoms (study 1). The second aim was to test the feasibility, acceptability and potential effectiveness of a newly developed computer-based expert system intervention simultaneously targeting hazardous alcohol consumption and depressive symptoms and to investigate the importance of the sample selection when preliminary testing interventions (study 2). The third aim was to further investigate rumination with its subfactors brooding and reflection as a common cause of depression and addictive behaviors and thus as a potential target for combined interventions by analyzing its associations with symptoms of pathological gambling (SPGs; study 3).
Methods: This thesis provides a summary of different working steps in the process of developing and testing a computer-based intervention for health care patients (HCPs) with comorbid hazardous alcohol consumption patterns and depressive symptoms. In study 1, a systematic literature search was conducted to identify evidence-based e-health interventions for depressive symptoms. Interventions were considered for further inspection if studies provided evidence for at least small intervention effects and if the interventions were accessible to at least selected groups of individuals. For study 2, 2773 consecutive HCPs were screened for hazardous drinking and depressive symptoms. Of the 41 HCPs who were offered to participate in the study, 27 (65.9%) consented. To investigate the importance of the sample selection when preliminary testing interventions, HCPs were compared to media recruited volunteers (MVs). Over a period of 6 months, study participants received 6 individualized counselling letters and weekly short messages. Pre-post data were analyzed for 30 participants (15 HCPs, 15 MVs). Intervention acceptability was assessed in post-intervention interviews conducted with 32 study participants. In study 3, cross-sectional data of 506 (80.4% male) individuals aged 14 to 64 years with a history of gambling problems were analyzed. Associations between the rumination subfactors and SPGs across different levels of problem gambling severity were investigated by means of sequential quantile regression.
Results: In study 1, 37 publicly accessible evidence-based e-health interventions for depressive symptoms were identified. Most interventions (81.1%) were available in English. For the German language area, only 3 interventions were identified. In study 2, HCPs and MVs reduced regular binge drinking (HCPs: p = 0.016; MVs: p = 0.031) and depressiveness (HCPs: p = 0.020; MVs: p < 0.001). MVs further reduced average daily consumption (p = 0.034). Both subsamples rated the intervention positive. Compared to HCPs, MVs rated the alcohol module more favorably (p = 0.012). Intervention usage was higher in MVs than in HCPs (p = 0.013). Study 3 showed that at the median, ruminative brooding was positively associated with the severity of problem gambling after controlling for covariates (p = 0.005). Along the distribution of problem gambling severity, findings did hold for all but the lowest severity level. Ruminative reflection was not associated with problem gambling severity at the median (p = 0.347).
Conclusions: E-health interventions show great potential in the treatment and prevention of depressive symptoms and addictive behaviors. However, more research is needed to clarify how to make the most of this potential. Important questions that remain to be answered include, for example, how to best provide e-health interventions to those in need or how to design interventions in order to maximize their reach and thus their public health impact. This thesis showed that 1) publicly accessible evidence-based e-health interventions for depressive symptoms were available. However, the supply in the German language area was low. 2) The computer-based expert system intervention targeting hazardous alcohol consumption and depressive symptoms was technically and logistically feasible, acceptable, and may have the potential to reduce hazardous drinking and depressive symptoms in different populations, including populations unselected in terms of their motivation to change. To avoid biased conclusions about the potential of interventions, intervention developers should preliminary test interventions on intended target populations. 3) Rumination might be important in the development and maintenance of addictive behaviors. With its relations to depression and addictive behaviors, it should be considered as a target for future combined interventions.
This paper is a literary analysis of Sebastian Barry’s six novels “The Whereabouts of Eneas McNulty” (1998), “Annie Dunne” (2002), “A Long Long Way” (2005), “The Secret Scripture” (2008), “On Canaan’s Side” (2011) and “The Temporary Gentleman” (2014) and his two plays “The Steward of Christendom” (1995) and “Our Lady of Sligo” (1998). The analysis focuses on Barry’s presentation of (Irish) history, (Irish) nationalism, and personal and national identity in order to ascertain to what extent Barry can be labelled an Irish historical revisionist and compares Barry’s texts to revisionist fiction by other modern Irish authors to delineate Barry’s vision of a – generally more inclusive - Irish identity.
The active screen plasma nitrocarburizing (ASPNC) technology is a state-of-the-art plasma-assisted heat treatment for improving surface hardness and wear resistance of metallic workpieces based on thermochemical diffusion. In comparison to conventional plasma nitrocarburizing, the use of an active screen (AS) improves thermal homogeinity at the workload and reduces soot formation. Further it can serve as a chemical source for the plasma processes, e.g. by use of an AS made of carbon-fibre reinforced carbon. This compilation of studies investigates the plasma-chemical composition of industrial- and laboratory-scale ASPNC plasmas, predominantly using in-situ laser absorption spectroscopy with lead-salt tuneable diode lasers, external-cavity quantum cascade lasers, and a frequency comb. In this way the temperatures and concentrations of the dominant stable molecular species HCN, NH3, CH4, C2H2, and CO, as well as of less prevelant species, were recorded as functions of e.g. the pressure, the applied plasma power, the total feed gas flow and its composition. Additionally, the diagnostics were applied to a chemically similar plasma-assisted process for diamond deposition.
Resulting from this thesis are new insights into the practical application of an AS made of CFC, the plasma-chemistry involving hydrogen, nitrogen, and carbon, and the particular role of CO as an indicator for reactor contamination. The effect of the feed gas composition on the resulting nitrogen- and carbon-expanded austenite layers was proven by combination of in-situ laser absorption spectroscopy with post-treatment surface diagnostics. Furthermore this work marks the first use of frequency comb spectroscopy with sub-nominally resolved Michelson interferometry for investigation of a low-pressure molecular discharge. This way the rotational bands of multiple species were simultaneously measured, resulting in temperature information at a precision hitherto not reached in the field of nitrocarburizing plasmas.
In this thesis, I present work motivated, in part, by a series of upcoming laboratory experiments (APEX), which seeks to uncover some of the inner workings of turbulence and stability in electron- positron plasmas in closed field-line systems. I present the results of several distinct, but connected, problems addressing the theory of electron-positron plasmas.
This work is partitioned into several parts, which loosely correspond to different particulars of the APEX experiment and the different theoretical physics problems which reside within.
I begin with the derivation of a kinetic theory for plasmas which are optically thin to cyclotron emission, as indeed, experimental pair plasmas are expected to be. The results of this section include: (1) the derivation of a general kinetic theory of cyclotron radiation in electron-ion plasmas; (2) a calculation showing that cyclotron emission results in strongly anisotropic distribution functions on the radiation timescale; (3) calculation of the evolution of the distribution function under collisional scattering which, in the absence of any radiation terms, acts to drive the plasma towards a Maxwellian; (4) generalisation of this theory to more exotic geometries; (5) specialisation of this theory to pair plasmas of experimental interest; and (6) presentation of the applications and the limitations of this theory.
The second project is primarily concerned with non-neutral plasmas. We begin with gyrokinetic theory and a novel extension of this theoretical framework to plasmas with arbitrary degree of neutrality in straight field-line geometry. I go on to discuss the gyrokinetic stability theory of such plasmas in this simplified geometry. I conclude this project with a discussion of some further
nuances in the theory of singly-charged non-neutral plasmas, this time concerning global features. Namely, I declare an interest in the equilibria such plasmas might be able to attain.
The final project pertains to plasmas which are in state of Maxwellian equilibrium i.e., electron- positron plasmas with sufficiently large number densities of each species to attain a stationary quasineutral plasma. To this end, I present gyrokinetic flux-tube simulations of electron-positron plasmas in complex, and experimentally relevant, magnetic geometries on the road towards a study of gyrokinetic turbulence. The results of this work include: (1) the first simulations of electron- positron plasmas in a stellarator and ring-dipole geometry; (2) an analytic theory of trapped particle modes in electron-positron plasmas, a result which can also be verified numerically; and (3) extension of several important theoretical results in electron-positron plasmas to experimentally relevant geometries. The culmination of this project is the roadmap ahead towards demonstration of the so-called “inward pinch” effect in electron-positron plasmas in a magnetic Z-pinch.
Over thousands of years, peatlands around the world have accumulated carbon (C) stocks of global importance. Drainage for agriculture, forestry and peat extraction has transformed many peatlands from long-term sinks into strong sources of carbon dioxide (CO2). Peat extraction is worldwide responsible for about ten percent of drained peatlands and is mainly carried out in northern countries and Eastern Europe. In Belarus, 0.3 Mha of peatlands are drained for peat extraction, which is twelve percent of the country's peatland area. From 2006 to 2013, 21,333 ha of this area have been rewetted to protect these peatlands from fire and further degradation, reduce their greenhouse gas (GHG) emissions, turn them back into C sinks and promote biodiversity. A further 260,000 ha are no longer used for peat extraction and their rewetting would be a great benefit for nature conservation and climate protection.
Rewetting of abandoned peat extraction areas usually leads to inundation of large areas where not adapted plants die and new species establish, depending on water level and nutrient conditions. Beavers, of which there are many in Belarus, also play an important role in the rewetting of peatlands. They dam up ditches in drained and rewetted peatlands, thus contributing to water level increases and vegetation changes. The aim of this PhD thesis was to investigate the impact of inundation on vegetation and GHG emissions in formerly extracted fens in Belarus, to determine the role of water level in this process, and to study whether such fens develop back into C sinks with an almost neutral GHG balance within one or two decades after rewetting (Papers II and III). Also the potential of beaver activities for peatland restoration was assessed (Paper III).
Two very different fens, rewetted after peat extraction, were chosen as study areas. The first one, Giel'cykaŭ Kašyl, is a former flood mire and was rewetted with water from the Jasiel'da River in 1985. During the study period 2010–2012 this site was a shallow lake (~ 1 m deep) dominated by very productive, tall reed. Shallower areas along the edges had a partly floating vegetation cover of cattail (Typha latifolia, T. angustifolia) and sedges (Carex elata, C. vesicaria). The second fen, Barcianicha, is fed by groundwater. Rewetting from 1995 onwards resulted in water levels at or slightly above surface and a lower nutrient availability compared to Giel'cykaŭ Kašyl'. This was reflected in the establishment of mesotrophic communities of Eriophorum angustifolium and Carex rostrata. Phragmites australis stands, which were also dominant here, were shorter and less productive than in Giel'cykaŭ Kašyl'. The southern area of Barcianicha was not used for peat extraction and has not been rewetted. Until 2009 vegetation of this part was characterized by forbs (Urtica dioica) and wet meadows (Agrostis stolonifera). From autumn 2009, a beaver dam in the main drainage ditch caused flooding of these areas and led to diverging vegetation development depending on water levels.
Within the framework of this doctoral thesis annual fluxes of CO2, methane (CH4) and nitrous oxide (N2O) and the development of water levels and vegetation were monitored for two years at nine sites and evaluated (Papers II and III). Three of the sites, respectively, were located (a) in Giel’cykaŭ Kašyl’, flooded in 1985, (b) in the central area of Barcianicha, which was rewetted in 1995, and (c) in the southern part of Barcianicha, which was flooded by beavers end of 2009. GHG measurements were carried out with manual chambers from August 2010 to September 2012. Annual net CO2 exchange rates (NEE) were modeled based on light response curves of gross primary production (GPP) and on temperature response curves of ecosystem respiration (Reco), which were determined every third to fourth week by alternating measurements with transparent (cooled) and opaque chambers (both with fan) along the daily amplitude of photosynthetically active radiation (PAR) and temperature. Annual CH4 emissions were calculated mainly based on the temperature response of CH4 fluxes over the course of the year, based on biweekly (in summer) to monthly (in winter) repeated single measurements with opaque chambers (without fan). This was done, although all longer rewetted sites were dominated by aerenchymatic plants whose gas transport during the vegetation period may change over the course of the day and can be influenced by shading. This might apply to the six longer rewetted sites, two of which were dominated by Phragmites australis, and the others by Typha latifolia, Carex elata, Carex rostrata or Eriophorum angustifolium. For these six sites therefore studies on the daily course of CH4 release and the influence of chamber shade were conducted, covering 8–24 hours and lasting at least from sunrise to afternoon. Also the extent to which flux rates were affected by a lack of chamber headspace mixing by fans was investigated in the mentioned studies (Papers I and II).
The daytime course of CH4 emissions showed a pronounced dynamic for Phragmites australis in both fens, with minimum release during the night and maximum during the day (Paper I). The other sites in contrast did not show a significant diurnal CH4 flux dynamic (Paper II). Lack of headspace mixing by fans as compared to chambers with fan resulted in a slight underestimation of CH4 emissions at very high chambers (220 and 250 cm), as used for Phragmites australis in Giel'cykaŭ Kašyl', while there was no difference at lower chambers (≤185 cm), as used for the other sites. Opaque chambers resulted for sites dominated by Typha latifolia and Carex elata in significantly (1.2 times and 1.1 times, respectively) lower CH4 fluxes compared to transparent chambers. For the other sites, opaque chambers did not significantly reduce CH4 emissions. This result was unexpected, especially for Phragmites australis, as PAR out of all parameters tested had the strongest influence on CH4 emissions from both reed sites, and clouds directly led to reduction of their emissions. Presumably the gas flow in the reed shoots located within opaque chambers was maintained by shoots outside the chamber that were connected to the enclosed shoots by rhizomes (Paper I). The investigations showed that single measurements between 9 a.m. and 6 p.m. with opaque chambers without fan, as performed for the determination of annual CH4 fluxes, resulted for Carex rostrata and Eriophorum angustifolium in estimates similar to the daily mean, but for Phragmites australis in estimates that were rather above the daily mean. Annual CH4 fluxes from Phragmites australis could therefore be slightly overestimated. CH4 fluxes from Typha latifolia and Carex elata during the vegetation period were corrected by a factor of 1.2, although darkness inside of opaque chambers matters only at day, not at night. Daily and annual CH4 fluxes from these sites have been therefore most likely slightly overestimated, too.
Water saturation and the establishment of adapted vegetation were the most important conditions for the restoration of C sinks (gaseous CO2 and CH4 fluxes) in the investigated peatlands. The only site with falling water levels in summer and thus temporarily aerated peat was the beaver flooded forbs (Urtica dioica) site at Barcianicha. This site was a very strong CO2 emitter and the only significant N2O source of the entire study (Paper III). All other sites were permanently wet, had much lower CO2 emissions or were even net C sinks (Papers II and III). Establishment of adapted vegetation depended on inundation depth and time since rewetting. For example, within one year the meadow site in Barcianicha shallowly flooded by beaver was colonized by Carex rostrata and other adapted helophytes and developed into a CO2 sink, while the deeper flooded site at the same meadow initially attracted only Chara and some individuals of Alisma plantago aquatica and remained a moderate CO2 source. However, the results of the longer rewetted sites show, that also deeply (~ 1 m) flooded fen areas can become densely populated with mire plants in the course of 25 years and develop into net C sinks. Highest annual C uptake in both fens was achieved by the reed sites. Eriophorum angustifolium and Carex rostrata in mesotrophic Barcianicha were smaller C sinks. Typha latifolia and Carex elata in the eutrophic Giel'cykaŭ Kašyl', on the other hand, released CO2, presumably because the high and fluctuating water levels imposed stress to the plants, and because the large supply of nutrients and dead plant material allowed for strong heterotrophic respiration (Paper II). The simultaneously high CH4 emissions made Typha latifolia and Carex elata major sources of GHG. CH4 emissions from Phragmites australis in Giel'cykaŭ Kašyl' were even higher, but due to extremely high CO2 uptake the site was only a small net GHG source. CH4 emissions in Barcianicha were much lower and comparable to undisturbed sedge fens. The difference between Giel'cykaŭ Kašyl' and Barcianicha was mainly due to the different nutrient supply and the related productivity of the plants. Important conclusions are that stable inundation is an appropriate measure for restoration of the C sink of formerly extracted fens, but nutrient input with water needs to be stopped or reduced in order to decrease CH4 production. If this is not possible, establishment of Phragmites australis and other strong C sinks could help to compensate for the climate impact of high CH4 emissions from eutrophic sites.
The effect of the beaver dam on the development of the southern part of Barcianicha depended not only on the initial situation but mainly on the water level. Under optimal conditions, it led to the rapid establishment of adapted mire plants, the restoration of a C sink and a significant reduction of GHG emissions. However, this situation in the shallowly flooded meadow was achieved by chance. In comparison to planned rewetting measures, which aim to raise the water level evenly over the entire peatland, beavers dam ditches in order to improve their immediate habitat, thus influencing water levels only up to a certain distance, but rarely over the entire peatland. Nevertheless, beaver activity is of high value both for mire conservation projects, where existing dams are supplemented by beaver dams, and for abandoned, drained peatlands, like former peat extraction areas in Belarus, many of which at least partially have been rewetted by beavers.
Twisted topological K-theory is a twisted version of topological K-theory in the sense of twisted generalized cohomology theories. It was pioneered by Donavan and Karoubi in 1970 where they used bundles of central simple graded algebras to model twists of K-theory. By the end of the last century physicists realised that D-brane charges in the field of string theory may be studied in terms of twisted K-theory. This rekindled interest in the topic lead to a wave of new models for the twists and new ways to realize the respective twisted K-theory groups. The state-of-the-art models today use bundles of projective unitary operators on separable Hilbert spaces as twists and K-groups are modeled by homotopy classes of sections of certain bundles of Fredholm operators. From a physics perspective these treatments are not optimal yet: they are intrinsically infinite-dimensional and these models do not immediately allow the inclusion of differential data like forms and connections.
In this thesis we introduce the 2-stack of k-algebra gerbes. Objects, 1-morphisms and 2-morphisms consist of finite-dimensional geometric data simultaneously generalizing bundle gerbes and bundles of central simple graded k-algebras for k either the field of real numbers or the field of complex numbers. We construct an explicit isomorphism from equivalence classes of k-algebra gerbes over a space X to the full set of twists of real K-theory and complex K-theory respectively. Further, we model relative twisted K-groups for compact spaces X and closed subspaces Y twisted by algebra gerbes. These groups are modeled directly in terms of 1-morphisms and 2-morphisms of algebra gerbes over X. We exhibit a relation to the K-groups introduced by Donavan and Karoubi and we translate their fundamental isomorphism -- an isomorphism relating K-groups over Thom spaces with K-groups twisted by Clifford algebra bundles -- to the new setting. With the help of this fundamental isomorphism we construct an explicit Thom isomorphism and explicit pushforward homomorphisms for smooth maps between compact manifolds, without requiring these maps to be K-oriented. Further -- in order to treat K-groups for non-torsion twists -- we implement a geometric cocycle model, inspired by a related geometric cycle model developed by Baum and Douglas for K-homology in 1982, and construct an assembly map for this model.
In agricultural grasslands, management practice highly determines reproductive success for ground-nesting bird species. The most effective conservation measure is the delay of first mowing dates until broods fledge or bird friendly mowing. Late mowing often implies economical losses for farmers and may increase land use abandonment, which will, in turn, cause habitat deterioration. Thus, grassland bird conservation involves the challenge to protect broods against land use and to promote an appropriate management to sustain habitat quality at the same time. Because of their late and extended breeding season Corncrakes Crex crex are in particular vulnerable to frequent mowing which increases nest destruction, chick mortality and habitat loss.
This thesis aims to gain knowledge on favourable habitat characteristics and brood protection in relation with grassland management to derive implications for the conservation of Corncrake breeding sites in floodplain meadows. Study area is the Lower Oder Valley National Park in northeastern Germany that holds a Corncrake population of 50 to 250 calling males. The study covered two study periods, before (1998-2000) and after (2012-2015) the implementation of new Corncrake conservation measures allowing inferences on the effects of different timing and intensity of mowing for brood protection and habitat conservation.
Breeding was only confirmed on meadows with high forb cover, low sedge cover, low litter heights and a close location to ditches. Radio-tracked females preferred areas with high cover of forbs (> 30%) and a distinct relief heterogeneity, which was associated with increased vegetation diversity. Vegetation characteristics on sites with day calling activity of males showed more similarity with breeding sites than with sites only used for nocturnal calling, supporting the assumption that diurnal calling indicates the occurrence of females. Favoured vegetation structure was best provided by mowing in the preceding year. Low-intensity grazing was less effective in reducing litter and sedge cover, especially when conducted late in the season. In the absence of management, meadows rapidly overgrow and dense litter accumulates from dead plant material in eutrophic floodplains, which increases walking resistance for Corncrakes and may impede prey accessibility. Plant species richness and forb cover declined after land use cessation. Male Corncrakes abandoned calling sites on meadows unmanaged for longer periods.
Besides the availability of suitable nesting sites, food supply and nest predation risk are also related to vegetation structure and may indirectly influence the habitat quality. Faecal samples of Corncrakes consisted mainly of beetles and their larvae, followed by snails, spiders and earthworms. Invertebrate biomass, sampled with pitfall-traps, was twice as high, the numbers of large ground beetles even five times higher on previously unmanaged than on managed meadows. Invertebrate abundance was highest in the first and second years after land use abandonment, but strongly decreased afterwards to a similar level like under annual management. Therefore, unmown refuge strips for Corncrake protection and alternating mowing also enhance invertebrate prey resources in floodplain meadows.
Mammals caused the majority of all observed artificial ground nest predations. Nest predation risk was higher on previously unmanaged than on managed sites. Unmanaged meadows probably attract mammalian predators, because they provide a more favourable vegetation structure for foraging and harbour high numbers of small rodents, increasing also the risk of incidental nest predations. These findings suggest that an annual removal of vegetation, if conducted late in the season to protect grassland birds may reduce predation risk of ground nests in the subsequent year.
Whereas during 1998-2000 half of the study area was managed by the end of July, land use was delayed on meadows occupied by Corncrakes until at least 15 July or 15 August during 2012-2015. On meadows mown between 15 July and 15 August refuge strips were applied. The majority of Corncrake broods were started in the second half of May and mowing postponement until 15 August allows 80% of chicks to fledge without disturbance in the study area. In 65% of broods chicks reach independence (> 14 days old) until 15 July and can be protected by Corncrake friendly mowing because then they are large enough to successfully escape during mowing. Both adults and chicks survived in 10 m wide refuge strips. Because most birds tried to leave the unmown block for the first time when it was up to 30 m wide and only 15 to 30 m wide strips served as temporary habitat for unfledged chicks from mowing to departure, 10 m should be considered as the absolute minimum width for refuge strips.
The strong reduction of land use especially during July should have allowed more chicks to survive until fledging in 2012-2015 than 1998-2000. Besides the protection of nests and higher chick survival, the decline of mowing intensity increased the extent of habitat available for second breeding attempts. In 2012-2015, broods were initiated until late July in the study area. Male Corncrakes showed continuous arrival and departure during the breeding season. Similar departure rates were estimated by a multi-state occupancy model and for radio-tracked males in the same study area and periods, which both left their home ranges spontaneous and due to the impact of mowing. Compared to 1998-2000, total departure of males during June and July was reduced by 50% in 2012-2015, when more calling sites were protected from mowing. Although male Corncrakes show high intra-seasonal dispersal due to their sequential polygamous breeding system, postponed land use should have increased mating opportunities and re-nesting at first breeding sites.
Therefore, future directions of Corncrake conservation in eutrophic floodplains should address the increase of annual late mowing to protect broods and maintain favourable habitat conditions by creating a more flexible mowing regime adjusted to actual occurrence of Corncrakes. This requires expert advice to farmers based on an intense monitoring of calling Corncrakes. Repeated nocturnal surveys during May and June are highly recommended because low detection probability in combination with constant departure substantially underestimated the number of males present. Additionally, diurnal calling activity could improve the identification of breeding sites and timing could be used to estimate chick age in July to select sites for Corncrake friendly mowing. Because currently late mowing dates are unattractive for farmers conservation actions should along with financial compensations for mowing after 15 August promote the utilization of late-cut grass with poor nutrient quality for combustion. Energy production could provide an alternative income for farmers operating in conservation areas with delayed land use dates and will increase their acceptance of Corncrake protection measures.
Polysaccharide is a major constituent of the total organic carbon that is generated by photosynthetic eukaryotes. In the marine realm, where approximately half of annual global carbon fixation occurs, algae can produce large amounts of polysaccharide during bloom events. Phytoplankton blooms are frequently seasonal phenomena, and spring blooms in particular have been a focus of study as they are predictable in space and time. This makes them much more amenable model systems in which to explore the processes that occur as organic carbon is recycled.
It is assumed that the bulk of the polysaccharides algae produce serve one of two primary functions - namely acting as an energy storage molecule, or they serve as structural polymers in the cell walls. Other polysaccharides may also have protective functions as exudates. Regardless of function in algae, the polysaccharides are a valuable energy source for heterotrophic bacteria. The combination of abundance and predictable or semi-predictable structure of the polysaccharides has led to proliferation of variations on a particular sequestration and degradation strategy among the Bacteroidetes and Gammaproteobacteria that is frequently characterised as being ‘selfish’. The strategy is based on uptake of poly- and especially oligosaccharides into the periplasm via the use of TonB-dependent transporters. Once in the periplasmic space, oligomers can be further degraded to monomers that can then be transported into the cytosol. This mechanism is beneficial to the cell as it needn’t then lose the nutritive benefit of the polysaccharide to other cells, which may or may not have manufactured their own degradative carbohydrate active enzymes (CAZymes).
The research articles that make up this thesis are thus based around attempts to find and elucidate the polysaccharide preferences of heterotrophic bacteria that become abundant following phytoplankton blooms.The first article is a study into the abundance of TonB-dependent transporter proteins in metaproteomes and metagenomes across a single spring phytoplankton bloom at the long term research station at Helgoland. This investigation identifies transporters for laminarin and alpha-glucans, the two most abundant glucose-based storage polysaccharides, are the most abundant predicted polysaccharide transporting TonB-dependent transporters during the bloom. However, as the bloom progressed, and particularly following a doubling of bacterial cell numbers, the proportion of predicted polysaccharide transporters dedicated to laminarin and alpha-glucan transport declined relative to transporters for less readily degraded mannose-, xylose-, and fucose-containing polysaccharides. The inference is that this change is an active response to the availability of the different polysaccharides, or their relative attractiveness as growth substrates during the period.
The second article is an in-depth look at one of the most abundant Bacteroidetes clades, which was previously unnamed, and has not to date been cultivated. The most abundant species in this clade grows rapidly and often peaks earlier than other heterotrophic clades. It was found to be limited in predicted polysaccharide consumption capability, having only PULs for predicted laminarin degradation. It is also detectable in many locations at higher latitudes where phytoplankton blooms are expected to occur, indicating this is a globally successful consumer of algal organic matter, and may have an outsize significance for global laminarin degradation given its high abundance.
The third article is a more holistic study of phytoplankton bloom associated Gammaproteobacteria, which have otherwise been rather ignored compared to the Bacteroidetes. Gammaproteobacteria overlap with Bacteroidetes to some extent in being clear consumers of laminarin, but fewer of them are clearly capable of consuming the more complex cell-wall derived polysaccharides. Some may, however, be producers of alginate, an otherwise mysteriously popular polysaccharide with Bacteroidetes, given that it is not known to be produced by bloom forming microalgae.
The fourth article then goes into detail on the PUL content of Bacteroidetes, based on metagenomic data. It finds five substrates, alpha- and beta-glucans, xylose and mannose rich polysaccharides, and alginate, are the most frequent predicted polysaccharide substrates for Bacteroidetes PULs among populations responding to the Helgoland spring blooms.
This thesis thus summarises multiple metagenomic and metaproteomic investigations into the polysaccharide consumption capabilities of marine heterotrophic bacteria. These bacteria have a profound impact on the overall carbon cycle in coastal regions, and are critical for understanding how changes in atmospheric carbon concentrations impact carbon turnover and storage in the world's oceans.
Bats spend half of their life at roosting sites. Hence, exploring for potential roosts is an essential task for their survival, especially for those species which switch roosts regularly, such as several temperate bat species. However, localizing new roosts is a difficult task due to bats’ sensory limitations (e.g., vision, echolocation range). To compensate such constrains, it has been hypothesized that bats rely on cognitive processes like associative learning, spatial memory, social information use and memory retention for an efficient roost localization. However, no previous study has assessed these cognitive skills under natural conditions.
The aim of my thesis was to assess how individually RFID-marked, free-ranging bats use different cognitive processes when localizing suitable day roosts. For this purpose, I used a pairwise roost-quality (suitable vs. unsuitable) choice experiment with automatic monitoring and assessed bats’ cognitive processes according to different cues available. Cues were echo-reflective (spectral signature of boxes), spatial (position of the box within the experimental pair) and social (presence of conspecific at roosts), each one linked to a different cognitive process.
I found that Bechstein’s bats (Myotis bechsteinii) used associative learning to discriminate between suitable and unsuitable newly placed boxes according to their echo-reflective cues. However, when individuals returned to known suitable roosts, they relied more on spatial memory to localize them. This was evidenced by the higher proportion of visits to the unsuitable boxes after swapping box positions within the same experimental pairs. When social cues were available, bats discovered a higher number of suitable roosts and re-localized previously occupied roosts more accurately. Taken together, Bechstein’s bats used multiple cognitive processes and prioritized one process over another depending on the relevance of the cues and search context.
Memory retention of the learned association was analyzed one year later, after the bats had returned to their breeding sites from their hibernacula. I found no evidence that individuals remembered the association between roosts’ suitability and their respective echo-reflective cue. The lack of memory retention could be attributed to hibernation or the duration of the period that the bats spent away from their summer habitat without the opportunity to reinforce the association contingencies. Nevertheless, bats quickly relearned the same association in a short period of time. This emphasizes the high behavioral flexibility of the bats.
Given the ability of Bechstein’s bats to quickly learn to discriminate roosts based on their external echo-reflective cue via associative learning, I investigated whether the use of echo-reflective cues improves box detectability and further occupancy. This was also assessed in free-ranging Natterer’s (Myotis nattereri) bats and the brown long-eared bats (Plecotus auritus). I found that the use of echo-reflective cues did not improve the detectability and occupancy of newly placed boxes despite the previous experience of the colonies with such cues. There were differences among species in the number of discovered boxes, visits and roosting days. These differences could be related to the species-specific explorative behavior and roost-switching behavior. Box supplementations programs aimed to conserve or relocate bat colonies should consider these behaviors to increase their likelihood of success even when bat colonies are used to roosting in artificial shelters.
My research underlined the importance of evaluating multiple cues under natural conditions to understand how natural selection has shaped the cognitive process used for localizing resources. Cognitive field studies are logistically challenging given the number of factors to control. However, automatic monitoring techniques like the one used in this study give the possibility to deepen the understanding of the cognitive ecology of animals. I finally discuss two venues of further research to understand the spread of information within colony members about novel roosts and the recruitment dynamic to novel roosts.
Abstract
In this work we investigated immunological mechanisms involved in the onset of PE, a multifactorial pregnancy related disease of global importance. The clinical symptoms range from de novo hypertension, renal and hepatic damage, to IUGR and convulsions (eclampsia). An imbalance between vasospastic and vasodilatory mediators, leading to generalized endothelial dysfunction, is most probable responsible for the onset of the disorder. Autoimmune reactions provoked by the semi-allogen fetus have also been postulated as a possible cause. Preterm delivery is the only curative therapie available.
Our focus was on a subset of B lymphocytes, the CD19+ CD5+ B1-a B cells. These cells belong to the innate immune system and produce natural polyreactive (and possibly autoreactive) antibodies such as AT1-AA but also different cytokines. In the context of PE it has been reported that B1-a B cells in the peripheral blood are augmented. Female sex hormones, pregnancy associated hCG and its isoform h-HCG modulate the immune functions in pregnancy and thus may be involved in the development of PE. Cytokine production patterns of B1-a B cells and the impact of female sex hormones were analyzed.
For our experiments an established mouse model for immunological pregnancy loss (CBA/J x DBA/2J model) was used. Aditionally, isolated human peripheral blood B1-a B cells were used.
In the mouse model we could demonstrate that in vivo transferred B1-a B cells induced deposits in the mothers’ kidneys, correlating with renal damage. Secretion patterns of the cytokines IL10, IFN-γ, TNF-α and IL17 in disturbed pregnancies were altered as measured by FACS or MBAA respectively. Our data revealed an increased expression of anti-inflammatory IL10 in normal pregnant mice.
The activation levels of human and murine B1-a B cells (as recorded by CD69 and CD86 expression) were influenced by female sex hormones in a dose-dependent manner. In humans, recombinant h-HCG had a strong capacity to activate B1-a B cells. PG exerted a comparable effect on murine B1-a B cells.
We provide further evidence for a possible autoimmune component in the pathogenesis of PE. B1-a B cell involvement might include AA secretion as well as cytokine production. H-HCG emerges as a potentially important factor for human B1-a B cell activation in vitro.
Introduction: Multiparametric MRI (mpMRI) and MRI targeted biopsies (MRtb) are a new standard in prostate cancer (PCa) screening and diagnosis. Guidelines already include this approach for patients at risk. We aimed to gather information from German urologists about their knowledge, routine use, and attitude toward mpMRI and consecutive biopsy methods. Materials and Methods: An anonymous online questionnaire was sent via Survey Monkey to the members of the German Society of Urology (DGU). Statistical analyses were performed using SPSS version 25.0. Results: 496 members with a median age of 48.6 years (±11.7) participated in the survey. The majority rated mpMRI of the prostate as a very useful diagnostic tool (72.7%). MRtb of the prostate was considered as very advantageous (71.5%). MpMRI was used by 95.9%, and 83.2% also recommended MRtb predominantly in clinical institutions. For targeted biopsy, MRI-ultrasound fusion biopsy was clearly favored (75.8%). MpMRI was mostly used in patients with previously negative biopsy (90.9%) and in patients under active surveillance (60.9%). Arguments against the use of prostate mpMRI are costs (84.9%) and/or lack of sufficient radiological infrastructure (17.4%). Conclusion: Our data illustrate the meanwhile high acceptance and clinical use of the prostate mpMRI and MRtb in Germany.
The genus Sphagnum (L.) belongs to the Bryophyte plant division and includes 150 to 400 species. As all mosses Sphagnum has no roots and can hardly regulate its water uptake. As long as enough water is available Sphagnum can grow nearly unlimited while the lower, older parts die off and may accumulate as peat. Single Sphagnum species are able to build up an acrotelm as a hydrological self-regulating mechanism of a bog, a type of intact peatland (mire) only fed by precipitation. Because Sphagnum dominates nearly half of the peatlands in the world, it is one of the globally most important peat formers.
Sphagnum biomass is an important raw material for many valuable products, but in a much larger scale Sphagnum is used in its fossil state – as Sphagnum peat. With a consumption of c. 40 million m³ per year globally, Sphagnum peat is the predominant raw material for horticultural growing media. To get Sphagnum biomass it is currently collected from wild populations, to get Sphagnum peat it is extracted from bogs.
By far, more peatlands (including bogs) are subjects to drainage for agri- and silvicultural use since centuries, which harms their ecosystem services, including their typical biodiversity, carbon storage capacity, water regulation function and palaeo-environmental archive. In Europe, c. 25 % of all peatlands are used for agriculture, in Germany more than 80 %. Globally drained peatlands cover 0.4 % of land surface but produce 5 % of all anthropogenic greenhouse gas emissions.
Sphagnum farming aims to cultivate Sphagnum biomass on rewetted degraded bogs as a new agricultural crop. Sphagnum farming is paludiculture and contributes to the protection of bogs and their peat by conserving the peat body through rewetting and by offering a climate-friendly alternative to fossil peat in horticulture. Next to climate change mitigation, Sphagnum farming has benefits for nutrient retention and biodiversity conservation.
This thesis contributes to the development of Sphagnum farming by studying the conditions under which Sphagnum may reach maximal growth. Under (semi)controlled glasshouse conditions, we tested the effects of different water regimes and fertilisation levels on the productivity of various Sphagnum species. On a 1260 m² large irrigated field on cut-over bog in Lower Saxony (Germany) we studied length increase, biomass productivity and tissue nutrient content of Sphagnum over a period of 10 years. Finally, we reviewed all scientific literature and practical experiences with respect to Sphagnum farming worldwide as a first step towards a science-based implementation manual.
The main conclusions of our studies are:
1. It is possible to cultivate Sphagnum on rewetted cut-over bog and on rewetted former bog grassland.
2. The rapid establishment of a closed, highly productive Sphagnum lawn requires the deployment of a loose, >1(–5) cm thick Sphagnum layer (80–100 m³ of Sphagnum founder material per hectare) at the start of the growing season (when long frost periods are no longer probable) and adequate water supply.
3. Water table management must be very precise until a dense, well-growing Sphagnum lawn has established. For highest yields the water table should rise with Sphagnum growth and be kept a few centimetres below the Sphagnum capitula. Water supply via open irrigation ditches seems to function better than via subsurface irrigation pipes.
4. Fertilisation does not increase Sphagnum productivity on sites with high atmospheric nitrogen deposition and irrigation with phosphate-rich surface water from the agricultural surroundings. To avoid growth reduction a balanced stoichiometry is important.
5. From all studied species, Sphagnum fallax has the highest productivity. Its fast decomposition and low water holding capacity, however, may make this species less suitable for use in horticultural substrates.
6. Vascular plant cover on Sphagnum production fields can be kept low (<50 % cover) by regular mowing. Higher covers retard Sphagnum growth and reduce its quality for growing media.
7. Pathogenic fungi occurred far more in the glasshouse than in the field and have to be controlled for highest Sphagnum yields. We found Sphagnum vitality and growth rate to be stimulated by high water levels, where Sphagnum is less vulnerable to fungal or algal infection despite high nutrient loads.
8. The rate of Sphagnum biomass accumulation may remain constant over at least 4–5 years after establishing a Sphagnum production field with sufficient water supply. At dry conditions Sphagnum biomass accumulation is lower as a result of lower biomass productivity and higher decomposition rates.
Organic cation transporter OCT1 is strongly expressed in the sinusoidal membrane of hepatocytes. OCT1 mediates the uptake of weakly basic and cationic compounds from the blood into the liver and may thereby facilitate the first step in hepatic metabolism or excretion of many cationic drugs. OCT1 is a polyspecific transporter and has a very broad spectrum of structurally highly diverse ligands (substrates and inhibitors). The exact transport mechanism and the amino acids involved in polyspecific ligand binding of OCT1 are poorly understood.
The aim of this work was to utilize the polyspecificity to better understand the structure-function relationships of OCT1 and to gain first insights into potential mechanisms conferring the polyspecificity. We followed two strategies, analyzing the effects of variability in both ligand and transporter structure on OCT1 function. The effects of ligand structure were analyzed by comparing uptake and inhibitory potencies of structurally similar drugs of the group of opioids. The effects of transporter structure were analyzed by comparing the effects of variability caused by naturally occurring genetic variants or artificial mutations on OCT1 uptake and inhibition of several substrates. Most importantly, the effects of interspecies variability in transporter structure were analyzed by comparing uptake kinetics between human and mouse OCT1 orthologs. To this end, we used stably or transiently transfected HEK293 cells overexpressing OCT1 and different chimeric and mutant variants thereof.
Focusing on OCT1 ligands, we compared the uptake and inhibitory potencies of structurally similar opioids. Only minor changes of the ligand structure strongly affected the interaction with OCT1. The presence of the ether linkage between C4 and C5 of the morphinan ring was associated with reduced OCT1 inhibitory potencies, while passive membrane permeability was the major negative determinant of OCT1-mediated uptake among structurally highly similar morphinan opioids. Only minor structural changes strongly increased the inhibitory potency by 28-fold from the lowest IC50 of 2004 µM for oxycodone to 72 µM for morphine. Additional removal of the ether linkage between C4-C5 increased the inhibitory potency by a total of 313-fold to the lowest IC50 of 6 µM for dextrorphan. Consequently, our data demonstrates that despite its polyspecificity, OCT1-mediated uptake and inhibition of this uptake is still somewhat very specific.
Focusing on OCT1 protein structure, we first analyzed the effects of variability caused by naturally occurring genetic variants on OCT1 uptake and inhibition. OCT1 transport was strongly affected by OCT1 genetic variants and these effects were often substrate-specific. Correlation of these effects revealed several substrates that were similarly affected by the variants and may therefore be suggested to share similar or overlapping binding sites in OCT1. In addition, the effects of the genetic variants OCT1*2 and OCT1*3 on different substrates correlated well which may suggest that the structural variability caused by these two variants similarly affects substrate uptake. OCT1 genetic variants also affected the inhibition of OCT1, with both substrate and genotype-specific differences. Ranitidine inhibited the uptake of several substrates, among them the clinically relevant drugs metformin and morphine. Moreover, the inhibition was more potent (about 2-fold) on the uptake mediated by the common genetic variant OCT1*2 than on the uptake mediated by the reference OCT1*1.
Second, we analyzed the effects of artificial mutations of key amino acids. Tyr222 and Asp475 in rat OCT1 had strongly substrate-specific and also species-specific effects on both OCT1-mediated uptake and inhibition. Mutation of these amino acids strongly decreased OCT1-mediated uptake, which further underscored an important role especially of Asp475. Interestingly, despite a proposed essential role of this amino acid, we observed Asp475-independent transport. This transport was observed in mouse, but not in human OCT1 and was substrate-specific. TMH10 was identified to be involved in determining the Asp475-independent uptake of mouse OCT1.
Finally and most importantly, we analyzed the effects of sequence differences between human and mouse OCT1 on the transport kinetics of several OCT1 substrates. The transport kinetics differed strongly between human and mouse OCT1 orthologs. These differences were substrate-specific and affected both the affinity (KM) and capacity (vmax) of transport. Human OCT1 had an 8-fold higher capacity of trospium transport, while mouse OCT1 had an 8-fold higher capacity of fenoterol transport. Furthermore, mouse OCT1 had a 5-fold higher affinity for metformin transport compared to human OCT1. The difference between Phe32 in human and Leu32 in mouse OCT1 in TMH1 was identified to confer a higher capacity of transport by human compared to mouse OCT1, while the difference between Cys36 in human and Tyr36 in mouse OCT1 in TMH1 was identified to confer a higher capacity of transport by mouse compared to human OCT1. Furthermore, Leu155 in human OCT1, corresponding to Val156 in mouse OCT1 in TMH2, in concert with TMH3 were identified to confer the differences in affinity for metformin transport between the species.
It may be speculated that ligand binding in OCT1 involves a core binding region that includes Asp474/475 and that polyspecific ligand binding is enabled by providing further binding partners (different amino acids) in more peripheral regions that different ligands can selectively interact with. This mechanism may also be a first step in explaining the substrate-specific effects of genetic variants with clinical relevance. Based on our findings, these “polyspecificity regions” may include TMH1, TMH2, and TMH3. Further analyses are warranted to characterize and narrow down these regions to unravel the structure-function relationships and with that the polyspecificity of OCT1.
To summarize, variability in both ligand and transporter structure strongly affected OCT1 function and we were able to identify ligand structures that affect inhibitory potency and protein structures that confer species-specific differences in OCT1 transport. This work emphasizes again the complexity of OCT1 transport and structure-function relationships. We also showed that, in spite of the difficulties for experimental analysis and data interpretation that arise from the polyspecific nature of OCT1, polyspecificity can also be used as a tool to better understand the structure-function relationships of this transporter.
Global climate change is omnipresent all over the world and is affecting and challenging organisms in various ways. Species either have to adapt to the changing environmental conditions or move to new habitats in order to avoid extinction.
Possible ways for an organism to react can be dispersal, phenotypic plasticity, genetic adaptation or a combination of these factors. Among the various consequences of climate change, especially changes in temperature affect plenty of species. In ectotherms, the body temperature and associated mechanisms are strongly dependent on environmental conditions.
The aim of this work was to investigate the mechanisms underlying adaptation to thermal variation and heat stress in the widespread butterfly species <i>Pieris napi<i>.
Focusing on indicators of individual condition, including morphology, physiology and life history traits, the purpose was to specify whether the species’ responses to temperature variation have a plastic or genetic basis. In the first experiment, phenotypic variation along a latitudinal and altitudinal cline was investigated. Yellow reflectance of wings was negatively correlated with wing melanisation, providing evidence for a trade-off between a sexually selected trait (yellow color) and thermoregulation (black color). Body size decreased with increasing latitude and led to the assumption that warmer conditions are more beneficial for <i>P. napi<i> than cooler ones. An increased flight performance at higher altitudes but not latitudes may
indicate stronger challenges for flight activity in high-altitude environments.
The second experiment focused on clinal variation and plasticity in morphology, physiology and life history in F1-generation individuals reared in captivity at different temperatures. It could be shown that individuals from cooler environments were less heat-tolerant, had a longer development but were nevertheless smaller, and had more melanised wings. These differences were genetically-based. Furthermore, it could be shown that a higher developmental temperature speeded up development, reduced body size, potential metabolic activity, and wing melanisation but increased heat tolerance, documenting plastic responses.
In a third experiment, we examined physiological responses to heat stress. A transcriptome analysis revealed an upregulation in molecular chaperones under hot conditions, whereas antioxidant responses and oxidative damage remained unaffected. The antioxidant glutathione (GSH) though was reduced under both cold and hot conditions. Interestingly, Swedish individuals were characterized by higher levels of GSH, lower early fecundity, and lower larval growth rates compared with German or Italian populations, suggesting a ‘pace-of-life’ syndrome. Thus, the individuals from warmer regions show the opposite pattern with a lower investment into maintenance but a faster lifestyle.
In summary, we found clinal variation in body size, growth rates and concomitant development time, wing aspect ratio, wing melanisation and heat tolerance. The effects of high developmental temperature very likely reflect adaptive phenotypic plasticity. When speeding up development; heat tolerance is increasing while body size, potential metabolic activity and wing melanisation are decreasing. Overall, body size of <i>P. napi<i> individuals decreased from south to north while the melanisation of the wings increased. Furthermore, we found a connection between increased wing melanisation and decreased yellow reflectance, most likely caused by a trade-off between the two. We could confirm that <i>P. napi<i> individuals from warmer environments were more heat-tolerant and larger than individuals from colder environments. Due to increasing temperatures and heat waves becoming more frequent in the future, being able to cope with such conditions will be advantageous. As warmer conditions had positive effects on individual development, <i>P. napi<i> may benefit from global warming, but its association with moist habitats suggests negative consequences of climate change. We could also reveal pronounced plastic and genetic responses in <i>P. napi<i>, which may indicate high adaptive capacities. Thus, increasing temperature may not be too problematic for the species, as it seems to be rather well equipped to deal with such challenges. However, as climate change entails changes in precipitation / humidity along with temperature changes, such issues need further investigation.
Reactive species play an essential role in orchestrating wound healing responses. They act as secondary messengers and drive redox-signaling pathways that are involved in the hemostatic, inflammatory, proliferative and remodeling phases of wound healing. Cold plasma produces a profusion of short- and long-lived redox species that promotes wound healing, however, until today, the knowledge of CAP mediated wound healing remained scarce. In this thesis, CAP mediated wound healing mechanism and their effect on extracellular matrix and adhesion molecules have been investigated. To this end, a keratinocyte cell line (HaCaT), skin fibroblast cell line (GM Fbs) and an in vitro coculture model including both HaCaT and GM Fbs at a 2:1 ratio, were employed to investigate the cross talk between these two skin cell types.
We examined the impact of CAP on extracellular matrix proteins and cell adhesion molecules in GM Fbs and observed a significant impact of cold plasma treatment on the expression level of collagen moieties, cell adhesion molecule like integrin, cadherin, versican, MMPs as well as extracellular matrix proteins.
Moreover, scratch assays with monocultures of HaCaT, GM Fbs and coculture of these two cell types were performed. We detected that, CAP accelerated the migratory capability of HaCaT cells cocultured with fibroblasts. In fact, compared to HaCaT monoculture, a significant acceleration on cell migration was observed in coculture upon CAP treatment. NAC, a potent antioxidant could abrogate this CAP-stimulated cell migration in coculture, further pointing towards the importance of well-orchestrated reactive species in wound healing. To better understand this CAP-mediated effect on cell migration, we examined the signaling pathways involved in tissue homeostasis and regeneration. We checked the HIPPO signaling pathway and observed an upregulation of several signaling molecules at transcriptional level in GM Fbs upon CAP treatment.
YAP is the central nuclear executer of HIPPO signaling pathway. YAP was upregulated in both HaCaT cells and GM Fbs. The major downstream effectors of the HIPPO signaling pathway (CTGF and Cyr61) were also upregulated in dermal fibroblasts at both transcriptional and protein level. However, administration of antioxidant NAC inhibited CAP-mediated wound healing and abrogated the gene expression of the HIPPO downstream effectors. These results confirm that the upregulation of YAP-CTGF-CYR61 axis is due to CAP-generated redox species. In HaCaT cells, both CTGF and Cyr61 was minimally transcribed. Even though CTGF was rarely detected in HaCaT cells on the protein level,Cyr61 remained undetected. This again shows the importance of the cross talk between fibroblasts and keratinocytes.
The coculture with the inclusion of fibroblasts showed an accelerated migration rate, compared to HaCaT monoculture which specifies a cross talk between these two cell types. Thus, monoculture of HaCaT cells were incubated with CAP-treated and untreated fibroblast conditioned medium. Interestingly, we observed that HaCaT cells exhibited an improved cell migration rate when incubated with CAP-treated fibroblast-conditioned media compared to that observed after incubation with untreated media. Upon investigation, an induction of CTGF and Cyr61 secretion was observed upon CAP treatment in the fibroblast-conditioned media. Furthermore, exposure to recombinant CTGF and Cyr61 could also significantly improve HaCaT cell migration which confirms that CAP mediated accelerated cell migration is due to activation of YAP-CTGF-Cyr61 axis.
In conclusion, this study revealed a completely new mechanical insight of CAP mediated wound healing. Along with several other ECM molecules, CAP activates a regenerative signaling pathway i.e., HIPPO signaling pathway in dermal fibroblasts at the onset of wound healing. Dermal fibroblasts drive a paracrine interaction by secreting CTGF and Cyr61 in close vicinity of wound, resulting in accelerated keratinocyte migration and wound healing in coculture.
Abstract
Relatively little is known about how plant–soil feedbacks (PSFs) may affect plant growth in field conditions where factors such as herbivory may be important. Using a potted experiment in a grassland, we measured PSFs with and without aboveground insect herbivory for 20 plant species. We then compared PSF values to plant landscape abundance. Aboveground herbivory had a large negative effect on PSF values. For 15 of 20 species, PSFs were more negative with herbivory than without. This occurred because plant biomass on “home” soils was smaller with herbivory than without. PSF values with herbivory were correlated with plant landscape abundance, whereas PSF values without herbivory were not. Shoot nitrogen concentrations suggested that plants create soils that increase nitrogen uptake, but that greater shoot nitrogen values increase herbivory and that the net effect of positive PSF and greater aboveground herbivory is less aboveground biomass. Results provided clear evidence that PSFs alone have limited power in explaining species abundances and that herbivory has stronger effects on plant biomass and growth on the landscape. Our results provide a potential explanation for observed differences between greenhouse and field PSF experiments and suggest that PSF experiments need to consider important biotic interactions, like aboveground herbivory, particularly when the goal of PSF research is to understand plant growth in field conditions.
Objectives: To examine the association between third molars and orofacial pain. We hypothesized that impacted third molars are a cause of orofacial pain.
Methods: Magnetic resonance images of 1808 participants from two populationbased cohorts from Northeastern Germany were analysed to define the status of third molars according to the Pell and Gregory classification. A self-reported questionnaire and a clinical dental examination were used to detect chronic and acute complaints of orofacial pain, masticatory muscle pain, migraine and other types of headache. Logistic regression models were used to analyse the associations between third molar status and orofacial pain.
Results: Individuals with impacted third molars in the maxilla had a higher chance of chronic orofacial pain than those with erupted third molars (odds ratio 2.19; 95% CI 1.19-4.02). No such association was detected for third molars in the lower jaw. Third molars were not associated with masticatory muscle pain, migraine or other types of headache.
Conclusions: Impacted maxillary third molars might be a cause of chronic orofacial pain. Thus, physicians should consider the eruption/impaction status of third molars in their decision-making process when treating patients who complain of orofacial pain.
I/R injury occurs during stroke and TBI. It represents a complex pathological event including several processes that can lead to cell membrane disruption, cellular dysfunction and death. The reintroduction of blood flow after the ischemic event may cause detrimental injury to the brain beyond the harm caused by ischemia itself and, therefore, represents a clinical challenge. This so-called I/R injury damages cells in a variety of ways including poration of cell membranes. Hence, methods to improve the endogenous membrane resealing capacity are crucial to prevent neuronal injury.
In the present work, treatment during reoxygenation with the probably most studied CCMS, P188, was investigated in an in-vitro simulation of stroke and TBI in primary isolated cortical mouse neurons. P188 offers a unique hydrophilic/lipophilic character that has been reported to protect different cells and tissues in various experimental settings against I/R and mechanical injury by sealing membranes. The aim of this study was to establish an in-vitro stroke and TBI model and further investigate if P188 directly interacts with neurons after compression and H/R (simulated I/R) injury, when administered at the start of reoxygenation.
The outcome of this treatment was evaluated in regard to cell number/viability, mitochondrial viability, membrane damage by LDH release and FM1-43 incorporation as well as activation of apoptosis by Caspase 3. It could be demonstrated that 5 hours hypoxia ± compression with 2 hours reoxygenation appear to be a suitable model for testing novel treatments. Compared to normoxic cells not exposed to compression, cell number and mitochondrial viability decreased, whereas membrane injury by LDH per total/FM1-43 dye incorporation and Caspase 3 activity increased in cells exposed to hypoxic conditions ± compression followed by reoxygenation.
However, it could not be shown that P188 is capable to protect isolated neurons from H/R and/or compression injury when administered purely as a postconditioning agent. It therefore seems likely that P188 does not directly affect isolated neurons. Yet, it may be able to provide neuronal protection in a different experimental setting.
In conclusion, this work contributes a new model of simulated stroke and TBI in-vitro. In addition, further knowledge about the impact of P188 on injured neurons can be gained. The extent to which the in-vitro results can be transferred to in-vivo mechanisms is yet unclear and offers opportunities for further investigations.
National oral health survey on refugees in Germany 2016/2017: caries and subsequent complications
(2020)
Objectives To assess oral health, caries prevalence, and subsequent complications among recently arrived refugees in Germany and to ompare these findings with the German resident population. Methods This multicenter cross-sectional study recruited 544 refugees aged 3–75+ years; they were examined at ten registration institutions in four federal states in Germany by two calibrated dentists. The refugees were screened for caries (dmft/DMFT) and its complications pufa/PUFA); this data was compared to the resident population via the presentative national oral health surveys). Results The deciduous dentition of the 3-year-old refugees had a mean dmft value of 2.62 ± 3.6 compared with 0.48 dmft in the German resident population, and caries increased to 5.22 ± 3.4 for 6–7-year-olds (Germany: 1.73 dmft). Few refugee children had naturally healthy teeth (7% in 6–7-year-olds, Germany: 56%). In the permanent dentition, the gap in caries prevalence between refugees and the German population decreased with age (35–44-year-olds: 10.55 ± 7.1 DMFT; Germany: 11.2), but refugees exhibited more caries defects (35–44-year-olds DT = 3.13 ± 3.0; Germany: 0.5). German residents had more restorations (35–44-year-olds FT = 4.21 ± 4.6). Regarding complications, the 6–7-year-olds exhibited the highest pufa index (0.86 ± 1.4) which decreased in adolescence (13–17-year-olds, 0.18 ± 0.6) and increased in adults (45–64-year-olds, 0.45 ± 0.8). Conclusion The refugees had high caries experience, often untreated caries teeth and more complications compared with the German resident population, especially in children. Closing this gap by extending preventive systems to the refugees would decrease future treatment needs. Clinical relevance European countries should be prepared for the higher dental treatment needs in recent refugees, especially in
children.
For an Organisation for Caries Research/European Federation of Conservative Dentistry consensus, this systematic review is aimed to assess the question of how to manage the caries process in the case of early childhood caries (ECC). Medline via PubMed was searched systematically regarding management of ECC. First priority was existing systematic reviews or randomized clinical trials otherwise cohort studies dealing with management of ECC, primarily with carious anterior teeth. After data extraction, the potential risk of bias was estimated depending on the study types, and the level of evidence was evaluated. Regarding management of ECC, results are presented for silver diamine fluoride (SDF, n = 5), nonoperative caries management (NOCM, n = 10), and restorative approaches (RA, n = 8) separately, as different kinds of studies with different levels of evidence were found for the different aspects in the management of ECC. The 5 systematic reviews on SDF showed a high potential for arrest of ECC on a high level of evidence. In NOCM, a low level of evidence for a moderate effect of fluoride varnish in arresting or remineralizing, especially non-cavitated lesions, was assessed. For RA in carious anterior upper primary teeth, a low level of evidence was found for higher failure rates of glass ionomer cement and composite fillings than composite strip crowns even if placed under general anaesthesia and especially compared to other crowns (stainless steel and zirconia). In conclusions, ECC may be managed successfully with nonoperative (SDF, regular fluoride application) and moderately well with operative approaches, but the decision is affected by many other variables such as pulpal involvement, the child’s cooperation, or a general anaesthesia setting.
Barrier corona (BC) arrangements are employed in different plasma-based applications such as material surface and exhaust gas treatments. However, a comprehensive study about the discharge behavior and properties in such strongly asymmetric arrangements is still missing. This dissertation is devoted to the detailed investigation of single microdischarges (MDs) in a sinusoidally driven BC discharge in air at atmospheric pressure. The discharge arrangement consist of a sharp metal pin and a dielectric-covered hemispherical electrode. It is the first study of volume BC discharges, in which phasially-resolved spatio-temporal development of the MDs are recorded using a multi-dimensional time-correlated single photon counting (TC-SPC) technique. The morphology of the MDs is recorded using an ICCD camera. A voltage probe and a current probe are employed to measure the applied voltage and current pulses. Furthermore, phase-resolved current measurements and statistical studies of current pulse amplitudes are realized using an oscilloscope.
Due to the asymmetric geometry and material of the electrodes, discharge behavior in the two polarities of the applied sinusoidal voltage is significantly different. For the voltage amplitude being applied, mostly two MDs appear in the anodic pin half-cycles. It is observed that the breakdown mechanism in both MDs is a positive streamer starting near the anode, similar to the single MDs in symmetric dielectric barrier discharges (DBDs). However, the second MDs have different properties, such as longer duration of the bulk plasma and broader current pulses. It is considered that the differences are mainly due to the positive surface charges deposited by the first MDs on the dielectric. It is proposed, for the first time, that the current pulse derivative maximum corresponds to the arrival of the streamer head at the cathode surface. This is used to synchronize the spatio-temporal development of the MDs with their current pulses. The accuracy of the synchronization is limited to the rise-time of the current probe (350 ps). In each cathodic pin half-cycle, only one major MD appears. The appearance and amplitude of the MDs are more erratic compared to the anodic pin polarity. The TC-SPC recordings show that the MDs appearing at low applied voltages have a similar spatio-temporal development to the MDs of the anodic pin polarity. On the other hand, at high applied voltages a development similar to transient sparks, i.e. a double-streamer starting near the tip of the pin (cathode), is observed. The statistical study shows that in DBD-like MDs the current pulse amplitude is not dependent on the appearance phase (or applied voltage), but this is not the case for the transient sparks.
Since BC reactors are also used for air cleaning, a set of experiments is done with 35 ppm toluene additive. It is observed that adding toluene results in 500~V lower breakdown voltage. Hence, the discharge in the presence of toluene is operated under over-voltage condition, resulting in stronger MDs in the anodic pin, and earlier-appearing as well as weaker MDs in the cathodic pin half-cycles.
The results of this dissertation about the spatio-temporal development and statistical behavior of the single MDs are foreseen to be employed in the study and optimization of plasma reactors, such as "Stacked DBD Reactor," which are developed for exhaust gas and material surface treatment. Furthermore, the results are a benchmark for the study of a novel discharge arrangement with a rotating dielectric electrode.
The achievement and monitoring of a good environmental status on continental shelf seas requires
the use of acoustic remote sensing techniques due to their range. The interpretation of acoustic signals
for the identification of benthic communities, however, is still in its infancy. In this thesis, the results
of two field campaigns conducted in a sandy environment off the shore of Sylt Island (North Sea)
utilizing ship- and lander-based acoustic and optical remote sensing techniques are discussed. The
objective of the thesis is a better knowledge of the impact of the polychaete Lanice conchilega on
physical seafloor properties, especially roughness at a cm to mm scale, which is relevant for
understanding acoustic scatter. The results show a clear impact of L. conchilega on roughness even in
sparse populations of less than 2% coverage. However, these sparsely populated areas could not be
reliably identified with acoustic data; a denser population of L. conchilega provided a clearer signal for
the acoustic remote sensing methods. The results are promising regarding the broader use of acoustic
remote sensing techniques for environmental monitoring in selected habitats, although the
determination of minimum population thresholds that can be identified will require further studies.
High resolution palaeo-ecological analysis of an Arctic ice-wedge polygon mire (Kytalyk, NE Siberia)
(2020)
Ice-wedge polygon mires are typical features of the Artic and therefore especially affected by climate change. They show, caused by soil-ice action, an amazing regular polygonal structure in meter dimension of higher and lower elevated dry and wet parts, and to this microtopography adapted vegetation. Polygon mires play, analogous to other mires, an important role in carbon sequestration, water balance, wildlife habitat and archive value with local to global significance. By storing enormous amounts of the global soil carbon polygon mires are crucial for our climate. Despite this relevance by covering large areas, polygon mires are comparatively poorly scientifically investigated and understood. It is still difficult to make forecasts on how polygon mires will develop under a changing climate in the Arctic, especially because internal factors and self-organisation complicate the understanding of their functioning. Therefore the investigation of modern and past polygon mires is necessary. This dissertation presents high resolution palaeo-ecological studies of a Northeast Siberian model polygon: ice-wedge polygon Lhc11 located in the Indigirka Lowlands at the scientific station Kytalyk. During field work in July 2011 the study site, covering an area of 26 × 21 m was divided into 546 plots, in which vegetation composition and microtopographical elevation characteristics were assessed and surface samples were collected. For palaeoecological analysis a 105.5 cm long peat section was excavated from the same site. Cluster analysis revealed five plant communities, which are clearly separated with respect to ground surface height, frost surface height and coverages of open water and vegetation, confirming the pattern already identified in other studies of Arctic ice-wedge polygons. The correct recognition of these patterns is crucial in palaeoecological studies in order to reconstruct landscape elements and their dynamics. This recognition requires insight in the short-distance relationships between surface elevation/wetness, vegetation and pollen deposition. The applied pollen-vegetation reference study shows that in general modern pollen deposition in polygon Lhc11 corresponds well with actual vegetation, allowing accurate reconstruction of local site conditions from fossil palynomorph sequences, including the reconstruction of the dynamics of closely spaced microtopographical elements. We conducted an evaluation of common palaeo proxies to compare their wetness reconstruction potential. The analysed proxies macrofossils, pollen, testate amoebae, geochemistry and sediment properties show similar wetness trends. Macrofossils provided the most detailed wetness reconstruction, spanning several wetness classes from very dry to wet, because they could be identified to genus or species level. However, as the proxies sometimes show contradictory results, a multi-proxy approach is preferable over a single proxy interpretation as it allows the reconstruction of environmental development in a broader palaeoecological context. For a better understanding of polygon dynamics and former greenhouse gas fluxes, more detailed and better quantified palaeo-microtopographical information is required. Therefore we developed a new transfer approach for modelling past Ground Surface Heights (GSH) in polygon mires from plant fossils. Based on the composition of modern vegetation we constructed two sets of potential fossil types (plant macrofossils and pollen), an extensive and a more restricted one. We applied Canonical Correspondence Analysis to model the relationships between potential fossil types and measured GSH. Both models show a strong relationship between modelled and measured GSH values and a high accuracy in prediction. Finally, we used the models to predict GSH values for Holocene peat samples. We found a fair correspondence with expert-based multi-proxy reconstruction of wetness conditions, even though only a minor part of the encountered fossils were represented in the GSH models, illustrating the robustness of the approach. The method can thus be used to reconstruct palaeoenvironmental conditions in a more objective way and can serve as a template for further palaeoecological studies. The 4000 years lasting history of the Lhc11 polygon site started with the establishment of a low-centre polygon in a drained thermokarst lake basin. Polygon Lhc11 formed part of a low-centre polygon for about 2000 years, experiencing enormous environmental influences discernible by incidence of silt, charred detritus, change of fossils composition and strongly declined peat accumulation rates and finally developed into a mature and degradation stage, into a low-high-centre polygon, currently characterized by high elevation differences. In the context of less studied but large-scale polygon mire occurrence, the high-resolution analysed ice-wedge polygon Lhc11 delivers insights into state and dynamics of a representative Siberian polygon site, in terms of modern and past vegetation and elevation characteristics. Furthermore the present study provides facilities for palaeoecological polygon studies including a new quantitative elevation modelling approach and provides valuable datasets for future research, e.g. greenhouse gas emissions and therefore contributes to a better understanding of these climate relevant ecosystems.
A common task in natural sciences is to
describe, characterize, and infer relations between discrete
objects. A set of relations E on a set of objects V can
naturally be expressed as a graph G = (V, E). It is
therefore often convenient to formalize problems in natural
sciences as graph theoretical problems.
In this thesis we will examine a number of problems found in
life sciences in particular, and show how to use graph theoretical
concepts to formalize and solve the presented problems. The
content of the thesis is a collection of papers all
solving separate problems that are relevant to biology
or biochemistry.
The first paper examines problems found in self-assembling
protein design. Designing polypeptides, composed of concatenated
coiled coil units, to fold into polyhedra turns out
to be intimately related to the concept of 1-face embeddings in
graph topology. We show that 1-face embeddings can be
canonicalized in linear time and present algorithms to enumerate
pairwise non-isomorphic 1-face embeddings in orientable surfaces.
The second and third paper examine problems found in evolutionary
biology. In particular, they focus on
inferring gene and species trees directly from sequence data
without any a priori knowledge of the trees topology. The second
paper characterize when gene trees can be inferred from
estimates of orthology, paralogy and xenology relations when only
partial information is available. Using this characterization an
algorithm is presented that constructs a gene tree consistent
with the estimates in polynomial time, if one exists. The
shown algorithm is used to experimentally show that gene trees
can be accurately inferred even in the case that only 20$\%$ of
the relations are known. The third paper explores how to
reconcile a gene tree with a species tree in a biologically
feasible way, when the events of the gene tree are known.
Biologically feasible reconciliations are characterized using
only the topology of the gene and species tree. Using this
characterization an algorithm is shown that constructs a
biologically feasible reconciliation in polynomial time, if one
exists.
The fourth and fifth paper are concerned with with the analysis
of automatically generated reaction networks. The fourth paper
introduces an algorithm to predict thermodynamic properties of
compounds in a chemistry. The algorithm is based on
the well known group contribution methods and will automatically
infer functional groups based on common structural motifs found
in a set of sampled compounds. It is shown experimentally that
the algorithm can be used to accurately
predict a variety of molecular properties such as normal boiling
point, Gibbs free energy, and the minimum free energy of RNA
secondary structures. The fifth and final paper presents a
framework to track atoms through reaction networks generated by a
graph grammar. Using concepts found in semigroup theory, the
paper defines the characteristic monoid of a reaction network. It
goes on to show how natural subsystems of a reaction network organically
emerge from the right Cayley graph of said monoid. The
applicability of the framework is proven by applying it to the
design of isotopic labeling experiments as well as to the
analysis of the TCA cycle.
Periodontitis is one of the most prevalent oral diseases worldwide caused by multifactorial interactions between host and oral bacteria. Altered cellular metabolism of host and microbes releases a number of intermediary end-products known as metabolites. Recently, there is an increasing interest in identifying metabolites from oral fluids like saliva to widen the understanding of the complex pathogenesis of periodontitis. It is believed, that some metabolites might serve as indicators toward early detection and screening of periodontitis and perhaps even for monitoring its prognosis in the future. Because contemporary periodontal screening methods are deficient, there is an urgent need for novel approaches in periodontal screening procedures. To this end we associated oral parameters (clinical attachment level, periodontal probing depth, supragingival plaque, supragingival calculus, number of missing teeth, and removable denture) with a large set of salivary metabolites (n=383) obtained by mass spectrometry among a subsample (n=909) of non-diabetic participants of the Study of Health in Pomerania (SHIP-Trend-0). Linear regression analyses were performed in age-stratified groups and adjusted for potential confounders. A multifaceted image of associated metabolites (n=107) with considerable differences according to age groups was revealed. In the young (20-39 years) and middle-aged groups (40-59 years), we found metabolites predominantly associated with periodontal variables; whereas among the older subjects (60 + years), tooth loss was strongly associated with metabolite levels. Metabolites associated with periodontal variables were clearly linked to tissue destruction, host- defence mechanisms and bacterial metabolism. Across all age groups, the bacterial metabolite phenylacetate was significantly associated with periodontal variables. Our results revealed alterations of the salivary metabolome in association with age and oral health status. Among our comprehensive panel of metabolites, periodontitis was significantly associated with the bacterial metabolite phenylacetate, a promising substance for further biomarker research.
Purpose: To assess the comparative efficacy and the long-term cost-utility of alternative minimally invasive glaucoma surgeries (MIGSs) when combined with cataract surgery in patients with primary open-angle glaucoma (POAG).
Methods: Treatment effects, as measured by the 1-year reduction in intraocular pressure (IOP), were estimated with an adjusted indirect treatment comparison. Evidence from randomized clinical trials was identified for four different MIGS methods. A disease-transition model was developed by capturing clinically relevant POAG stages and the expected natural disease evolution. Outcomes of the disease-transition model were the comparative utility [quality-adjusted life years (QALYs)], cost and cost-utility of included strategies in a lifetime horizon.
Results: Estimated 1-year IOP reductions were: cataract surgery - 2.05 mmHg (95% CI - 3.38; - 0.72), one trabecular micro-bypass stent - 3.15 mmHg (95% CI - 5.66; - 0.64), two trabecular micro-bypass stents - 4.85 mmHg (95% CI - 7.71; - 1.99) and intracanalicular scaffold - 2.25 mmHg (95% CI - 4.87; 0.37). Discounted outcomes from the disease-transition model appraised the strategy of two trabecular micro-bypass stents with cataract surgery in the moderate POAG stage as the one providing the greatest added value, with 10,955€ per additional QALY. Improved outcomes were seen when assessing MIGS in the moderate POAG stage.
Conclusions: When indirectly comparing alternative MIGS methods combined with cataract surgery, the option of two trabecular micro-bypass stents showed both a superior efficacy and long-term cost-utility from a German perspective. Moreover, outcomes of the disease-transition model suggest POAG patients to beneficiate the most from an earlier intervention in the moderate stage contrary to waiting until an advanced disease is present.
Bats belong to the most gregarious and diverse mammals with highly complex social behaviors. Despite extensive research on their ecology and social behavior in some bat species, gained insights are restricted to only few of the more than 1300 species. In the recent past, bats have also become a central topic of a different branch of research: Since the 1990s bats came to the fore of virologists and immunologists due to the bats’ apparent importance as reservoir hosts and vectors of several (mostly tropical) diseases. While this research is focused mainly on emerging infectious diseases linked to bats, and their zoonotic potential, little has been invested regarding the link between disease transmission and bat social systems.
In my work, I aim at filling this gap by merging automated daily roosting observations, social network analysis, and a virological screening in Natterer’s bats (Myotis nattereri). In a collaborative approach, my co-workers and I analyzed the social structure of individually marked Natterer’s bats, their astrovirus detection rate and transmission pathways within their colony, as well as roosting interactions between different co-occurring con- and heterospecific bat colonies.
We discovered Natterer’s bats to display a very divergent social network structure that contradicts the findings of previous studies on large fission-fusion groups. Contrary to the modular social network structure found in e.g. primates or other bats species, the social network of Natterer’s bats consists of only one highly interconnected community. Moreover, although the close proximity between bat hosts in the colony should strongly promote direct transmission, we found indications that astrovirus infections follow at least partly an indirect transmission pathway via contaminated roost use. Lastly, our results prove that co-occurring con- and heterospecific bat colonies, e.g. as in this study Natterer’s bats, brown long-eared bats and Bechstein’s bats, can influence each other in their roost use by avoiding conspecific roosts and by being attracted towards those of heterospecifics. This holds implication for the transmission of parasites and pathogens within and between different colonies with opportunities for spillovers. To conclude, this multidisciplinary study led to valuable insights in the hitherto hidden mechanisms within and among bat colonies.
Abstract
Background
Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin–glycoprotein‐complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para‐clinical parameters in the early disease phase in the well‐established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long‐term pre‐clinical study in mdx mice.
Methods
IgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology.
Results
IgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all).
Conclusions
The present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long‐term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.
Abstract
Objectives
In this study, the pharmacological properties of six spirocyclic piperidines 1–6 showing very high σ1 receptor affinity (Ki = 0.2–16 nm) were investigated.
Methods
In vitro receptor binding studies, retinal ganglion assay and in vivo capsaicin assay were used to determine the affinity, selectivity and activity. Influence on human tumour cell growth (cell lines A427, LCLC‐103H, 5637 and DAN‐G) was determined in different assays. The effect on the ergosterol and cholesterol biosynthesis was determined by GLC/MS analysis.
Key findings
Receptor binding studies demonstrated high selectivity for the σ1 receptor. The increased Ca2+ influx mediated by 2 and the analgesic activity of 1, 4, 5 and 6 confirm σ1 receptor antagonistic activity. Inhibition of human tumour cell growth further supports the σ1 antagonistic effects. Treatment of A427 tumour cells with 2 led to cell detachment and cell degradation. Whereas the ergosterol biosynthesis was not affected, the sterol C14‐reductase, a key enzyme in the cholesterol biosynthesis, was weakly inhibited.
Conclusions
Due to the high selectivity, off‐target effects are not expected. The antiallodynic activity underlines the clinical potential of the spirocyclic piperidines for the treatment of neuropathic pain. Due to the antiproliferative activity, the spirocyclic σ1 antagonists represent promising antitumour agents.
Abstract
Background
A growing proportion of older people in Germany receive long‐term care from informal and professional caregivers at home. Their personal assessment of the individual care situation is scarcely considered.
Aim
This study aimed to explore the subjective views of care recipients, informal and professional caregivers on the adequacy of care provision in long‐term home care arrangements.
Design and Methods
Qualitative semi‐structured face‐to‐face interviews were conducted with ten care recipients, ten professional caregivers and eight informal caregivers to capture their perspectives on the adequacy of the care received and delivered. Qualitative content analysis was applied using MAXQDA software.
Results
All groups highlighted that they perceive an underprovision of care, even though their explanations differed. The underprovision was mainly described regarding the quality rather than quantity of services. It occurs especially in interpersonal relationships and social inclusion, where the gap between the self‐perceived current situation and the desires of those affected is most prominent. The ambivalent impact of home care on social participation becomes apparent. Perceptions of an overprovision of care range from the view that it appears mainly with respect to informal care to the statement that it is currently non‐existent or generally impossible. Misprovision of care is experienced as serious whenever the interviewees face the challenge of preserving existing abilities or regaining certain skills.
Abstract
Because isoenzymes of the experimentally and therapeutically extremely relevant sirtuin family show high similarity, addressing the unique selectivity pocket of sirtuin 2 is a promising strategy towards selective inhibitors. An unrelated approach towards selective inhibition of isoenzymes with varied tissue distribution is targeted drug delivery or spatiotemporal activation by photochemical activation. Azologization of two nicotinamide‐mimicking lead structures was undertaken to combine both approaches and yielded a set of 33 azobenzenes and azopyridines that have been evaluated for their photochemical behaviour and bioactivity. For some compounds, inhibitory activity reached the sub‐micromolar range in their thermodynamically favoured E form and could be decreased by photoisomerization to the metastable Z form. Besides, derivatization with long‐chain fatty acids yielded potent sirtuin 2 inhibitors, featuring another intriguing aspect of azo‐based photoswitches. In these compounds, switching to the Z isomer increased aqueous solubility and thereby enhanced biological activity by up to a factor of 21. The biological activity of two compounds was confirmed by hyperacetylation of sirtuin specific histone proteins in a cell‐based activity assay.
Obesity and diabetes have reached epidemic proportions and have emerged as massive public health problems globally. The etiology of both obesity and diabetes are related, multifactorial, highly complex, and involves interplay of genetic, environmental, socio-economic and physiological factors, which calls for a more extensive research in understanding the risk factors and biological pathways. Hence, this dissertation contributed in part to understanding the role of iron markers in the development of type 2 diabetes mellitus and the role of intrauterine hyperglycemia in influencing the risk of offspring obesity along with investigating potential pathways.
In the first part of my dissertation, the associations of iron markers (ferritin and transferrin) with type 2 diabetes mellitus and metabolic syndrome were investigated using the population-based Study of Health in Pomerania. The present analyses were based on 3,232 participants aged 20-81 years with a follow-up time of nearly 11 years. The results suggest that serum ferritin concentrations were associated with a higher prevalence of type 2 diabetes mellitus and metabolic syndrome in the total population as well as in men. However, the effects of serum ferritin on incident type 2 diabetes mellitus were observed only in women, while the effects on incident metabolic syndrome were seen in the total population. Serum ferritin is also known to reflect systemic inflammation or hepatic dysfunction in addition to increased iron stores. Hence, upon further analyses, the associations were found to be attenuated after adjustment for hepatic enzymes but not after adjustment for inflammation. Transferrin was not associated with any of the outcomes. Thus, our study provides evidence for a link between the iron marker ferritin and type 2 diabetes mellitus and metabolic syndrome, although the association seemed to vary by sex. Moreover, hepatic dysfunction seems likely to be in the pathway between ferritin and type 2 diabetes mellitus and metabolic syndrome.
In the second part of my dissertation, the association between maternal hyperglycemia and the risk of offspring overweight and obesity were investigated using three different cohorts: TEDDY, TEENDIAB and BABYDIAB/BABYDIET. The present analyses were based on a total of 8,103 children who were followed until 6 years of age in TEDDY study and until 18 years of age in TEENDIAB and BABYDIAB/BABYDIET studies. The dissertation revealed that maternal hyperglycemia in general may be associated with increased risk for childhood overweight and obesity, and that the association gets stronger as children grow older, with the risk being clearly evident at late childhood and adolescence. Moreover, this dissertation adds that this association can be driven by different pathways based on the type of maternal diabetes to which the offspring was exposed. The association of maternal gestational diabetes mellitus with offspring overweight can be largely explained by the confounding influence of maternal BMI, whereas the association of maternal type 1 diabetes mellitus with offspring overweight can be substantially explained by birthweight in all three studies. In our attempt to understand biological pathways at a cellular level, we found that the offspring metabolome was unlikely to be in the causal pathway between maternal type 1 diabetes mellitus and overweight, because this association could not be explained by any of the potentially relevant metabolites.
To conclude, this dissertation acknowledges the fact that prevention and early intervention of obesity and diabetes is of paramount importance to lessen the impact of these public health problems. Thus, our findings of the role of ferritin in increasing the risk of type 2 diabetes mellitus/ metabolic syndrome and the role of intrauterine hyperglycemia in increasing the risk of offspring overweight helped to identify particular risk groups who may need closer attention with respect to prevention of obesity and diabetes.
Does really no one care? Analyzing the public engagement of communication scientists on Twitter
(2020)
Recent publications question the public visibility of communication science as a discipline and its relevance for the broader society. To address this issue, we analyze the public engagement of communication scientists by using the example of their Twitter activity. We theoretically distinguish eight types of engagement and explore their empirical prevalence. The results show that a large share of communication is between peers, fulfilling social networking functions. Nevertheless, more than a quarter of the tweets are on political and social topics. In this way, communication scientists bring society into their scholarly community and thus act as bridge builders. They also reach diverse publics outside of science, such as followers from the field of economics. Our study thus highlights the diversity of connections between science and society and can offer a starting point to further research other fields of public engagement and the impact of the discipline on the public discourse.
Experience in the construction of optimized stellarators shows the coil system is a significant challenge. The precision necessary allow the generation of accurate flux surfaces in recent experiments affected both cost and schedule negatively. Moreover, recent experiments at Wendelstein 7-X have shown that small field corrections were necessary for the operation of specific desired magnetic configurations. Therefore, robust magnetic configurations in terms of coil geometry and assembly tolerances have a high potential to facilitate swifter and less expensive construction of future, optimized stellarators. We present a new coil optimization technique that is designed to seek out coil configurations that are resilient against 3D coil displacements. This stochastic version of stellarator coil optimization uses the sampling average approach to incorporate an iterative perturbation analysis into the optimization routine. The result is a robust magnetic configuration that simultaneously reproduces the target magnetic field more accurately and leads to a better fusion performing coil configuration.
Tree growth in northern and upper treeline ecotones of the circumpolar boreal forest is
generally limited by temperature, i.e., trees grow generally more under warm, and less under
cold climatic conditions. Based on the assumption that this relationship between tree growth
and climate is linear and stable through time, dendroclimatologists use tree rings as natural
archives to reconstruct past temperature conditions. Such tree-ring based reconstructions,
together with other natural archives (e.g., ice cores and pollen), constitute our understanding of
past climatic conditions that reach beyond modern instrumental records.
However, a steadily increasing amount of studies reports a recent reduction or loss of the
summer temperature signal for several species and sites of the boreal forest. Such a reduction
of temperature sensitivity results in temporally unstable climate-tree growth relationships,
which challenges the work of dendroclimatologists by potentially leading to miscalibrations of
past climatic conditions. On the upside, this shift in the trees’ climate sensitivity might point to
a shift in tree growth-limiting factors and thus serve as an early indicator of climate change
impacts. There is evidence that this recent reduction in temperature sensitivity might be caused
by the observed strong temperature increase at high latitudes, and thus temperature-induced
drought stress. Other potential drivers and amplifiers of this phenomenon are differing microsite
conditions (dry vs. wet soils) and factors inherent to trees, like genetic properties or age
effects.
In this PhD thesis, I systematically assessed the effects of frequently discussed drivers of
unstable climate-tree growth relationships (climate change, micro-site effects, genetical
predisposition) on two representative species of the boreal forest, white spruce in North
America and Scots pine in Eurasia, across various temporal and spatial scales. I used classical
(tree-ring width) and more novel (wood density, quantitative wood anatomy)
dendrochronological proxies to unravel the effects from annual to sub-monthly resolution.
More precisely, in chapter I, white spruce clones were compared to non-clones at two treeline
sites in Alaska to test whether their growth patterns differ, and whether white spruce clones are
generally suitable for dendroclimatic assessments. Clonal reproduction is frequent at treeline
due to harsh conditions, but might lead to competition among individuals due to the close
proximity among each other, which in turn might obscure their climatic signal. Second, I tested
the effect of warmer and drier climatic conditions on the summer temperature signal of Scots
pine in Eurasia (chapter II) and on the growing season moisture signal of white spruce in North
America (chapter III), respectively. Temperature-induced drought stress is expected to be the
most important driver of unstable climate-growth relationships in the boreal forest. I included
several sites across latitudinal (50-150 km) and longitudinal (1,000-2,200 km) gradients to
cover large parts of the species’ distribution ranges. Since Scots pine covers a wide range of
ecological habitats, I additionally tested the effect of dry and wet micro-site conditions on the
summer temperature signal of Scots pine in chapter II. Finally, in chapter IV, a systematic
literature review was carried out in order to investigate the distribution of unstable climategrowth
relationships in global tree-ring studies, and the usage of such series in climate
reconstructions. Furthermore, the scientific impact of these potentially inaccurate climate
reconstructions was assessed.
In this PhD project, warmer and drier climatic conditions led to temporally unstable climate
signals in both Scots pine (chapter II) and white spruce (chapter III), as expected. Unstable
climate-growth relationships were found for all tested tree-ring proxies and at all sites in North
America, and at most sites in Eurasia. Micro-site effects (chapter II) and clonal growth
(chapter I) had no significant effect on the climate sensitivity and high-frequency variability
of the tested species, but affected absolute growth. The review (chapter IV) revealed that the
phenomenon of unstable climate-growth relationships is globally widespread, and occurs
independent of tree species, geographic location, and tree-ring and climate proxies. While
reconstructions inferred from these unstable relationships are frequent and respective papers
have a high impact, the tree-ring community seems to increasingly recognize the challenge of
unstable climate-growth relationships.
With these findings, this PhD project helped to shed more light on the frequency, underlying
drivers, and the impact of unstable climate-growth relationships in boreal forest trees, as well
as underlying reaction processes in trees. Above all, this PhD project suggests that the loss of
climate sensitivity is caused by a change of growth limiting factors: temperature limitation
seems to be suspended in warmer and drier years for Scots pine in Eurasia, and moisture
limitation first arises under warm/dry conditions for white spruce in North America. Due to
plastic growth responses in trees, the general assumption in dendroclimatology – that climategrowth
relationships are stable through time – seems to be incompatible with the principle of
limiting factors (one factors is always most growth limiting).
To improve the validity of future climate reconstructions, statistical approaches considering
synchronously or changing climatic limiting factors need to be promoted, along with attempts
to select the best responding trees from a dataset. Furthermore, a better understanding of nonclimatic
factors potentially affecting tree growth (e.g., age, disturbance, soil parameters) is
needed. A growth reduction of mature and dominant white spruce trees sampled in this PhD
project seems likely under future warming conditions, with series of wood cells being valuable
early indicators of climate change effects in white spruce. However, inferences cannot be
extended to the entire stand due to the applied sample design. Projected climate warming will
probably lead to a further reduction of the summer temperature signal in trees of the northern
boreal forest, while wider consequences for forest growth and productivity are unclear.
This thesis contains studies on a special class of topological insulators, so called anomalous Floquet topological insulators, which exclusively occur in periodically driven systems. At the boundary of an anomalous Floquet topological insulator, topologically protected transport occurs even though all of the Floquet bands are topologically trivial. This is in stark contrast to ordinary topological insulators of both static and Floquet type, where the topological invariants of the bulk bands completely determine the chiral boundary states via the bulk-boundary correspondence. In anomalous Floquet topological insulators, the boundary states are instead characterized by bulk invariants that account for the full dynamical evolution of the Floquet system.
Here, we explore the interplay between topology, symmetry, and non-Hermiticity in two-dimensional anomalous Floquet topological insulators. The central results of this exploration are (i) new expressions for the topological invariants of symmetry-protected anomalous Floquet topological phases which can be efficiently computed numerically, (ii) the construction of a universal driving protocol for symmetry-protected anomalous Floquet topological phases and its experimental implementation in photonic waveguide lattices, (iii) the discovery of non-Hermitian boundary state engineering which provides unprecedented possibilities to control and manipulate the topological transport of anomalous Floquet topological insulators.
Symbiotic interactions are a key element of biological systems. One powerful strategy to gain insight into these interactions, and into biological systems in general, is the analysis of proteins expressed in situ using metaproteomics. In this thesis, host-microbe interactions in two mutualistic associations between chemosynthetic sulfur-oxidizing endosymbionts and marine invertebrates, the deep-sea tubeworm Riftia pachyptila and the shallow-water clam Codakia orbicularis, were studied by adapted and optimized metaproteomics methods.
The Riftia symbiosis, which inhabits hydrothermal vents in the deep sea, and in which the host completely depends on its symbiont for nutrition, has fascinated researchers for about four decades. Yet, the interaction mechanisms between both partners have been understudied so far. Additionally, while different aspects of the host’s biology have been described, a comprehensive analysis has been lacking. Moreover, although only one symbiont 16S rRNA phylotype is present in Riftia, the symbiont population of the same host expresses proteins of various redundant or opposed metabolic pathways at the same time. As the symbionts also exhibit a wide variety in size and shape, symbionts of different size might have dissimilar physiological functions, which remained as of now to be elucidated. In this thesis, we addressed both, the host-symbiont interaction mechanisms, and physiological roles of symbiont subpopulations. A comprehensive Riftia host and symbiont protein database was generated as prerequisite for metaproteomics studies by de novo sequencing the host’s transcriptome and combining it with existing symbiont protein databases. This database was then used for metaproteomics comparisons of symbiont-containing and symbiont-free Riftia tissues, to gain insights into host-symbiont interactions on the protein level. The impact of energy availability on host-symbiont interactions was studied by comparing specimens with stored sulfur (i.e., high energy availability) with specimens in which sulfur storages were depleted. We employed optimized liquid chromatography peptide separation to increase metaproteome coverage. With this analysis, we identified proteins and mechanisms likely involved in maintaining the symbiosis, under varying environmental conditions. We unraveled key interaction mechanisms, i.e.: (i) the host likely digests its symbionts using abundant digestive enzymes, and, at the same time, (ii) a considerable part of the worm’s proteome is involved in creating stable internal conditions, thus maintaining the symbiont population. Furthermore, (iii) the symbionts probably employ eukaryote-like proteins to communicate with the host. (iv) Under conditions of restricted energy availability, the host apparently increases digestion pressure on the symbiotic population to sustain itself.
Riftia symbionts of different size apparently have dissimilar metabolic roles, as revealed in this thesis. We enriched symbionts of different sizes using gradient centrifugation. These enrichments were subjected to protein extraction using a protocol optimized for the small sample amount available. Metaproteomics analysis included a gel-based workflow and evaluation of the complex dataset with machine learning techniques. Based on our metaproteomics study, we propose that Riftia symbionts of different cell size correspond to dissimilar physiological differentiation stages. Smaller cells are apparently engaged in cell differentiation and host interactions. Larger cells, on the other hand, seem to be more involved in synthesis of various organic compounds. Supposedly, in large symbionts endoreduplication cycles lead to polyploidy. Our results indicate that the Riftia symbiont employs a large part of its metabolic repertoire at the same time in the stable host environment.
The symbiont of the shallow-water clam Codakia orbicularis, which, like the Riftia symbiont, relies on reduced sulfur compounds as energy source and fixes inorganic carbon, is, unexpectedly, also able to fix atmospheric nitrogen, as shown by metaproteomic, genomic and biochemical analysis. Potentially, this benefits the host, as Codakia digests its symbiont and might thus supplement its diet with organic nitrogen fixed by the symbionts in addition to organic carbon in its nitrogen-poor seagrass habitat.
Herpesviruses are enveloped DNA viruses which are dependent on two fusion steps for efficient replication in the host cell. First, they have to fuse their envelope with the cellular plasma membrane or with the vesicle membrane after endocytic uptake to enter the host cell and second, they have to export the newly generated nucleocapsids from the site of assembly to the cytoplasm by fusion of the primary virion envelope with the outer nuclear membrane (ONM). The main goal of this project was to provide a better understanding of how herpesvirus capsids exit the nucleus. On the one hand this thesis aimed at finding cellular proteins involved in nuclear egress (Paper I), while on the other the focus was on further characterization of the viral nuclear egress complex (NEC, Paper II) and its interaction with the capsid (Paper III).
It is the hallmark of viruses, including herpesviruses, to hijack host cell proteins for their efficient replication. Some of those interactions are well characterized, while others might not yet have been discovered. In the last step of the nuclear egress, where the primary virion membrane fuses with the ONM, most likely a cellular machinery is involved. The presented work focused on Torsin, the only known AAA+ ATPase localizing in the endoplasmic reticulum and the perinuclear space (PNS). For this, the effect of overexpression of WT and mutant proteins, as well as CRISPR/Cas9 generated knock-out cell lines, on PrV replication was analyzed. Neither single overexpression nor single knockouts of TorA or TorB had any significant effects on virus titers. However, infection of TorA/B double knockout cells revealed reduced viral titers and an accumulation of primary virions in the PNS at early infection times, indicating a delay in nuclear egress.
The process of nuclear egress has been intensively investigated without revealing all its details. To address some of the missing aspects we generated monoclonal antibodies (mAbs) against the NEC and its components (pUL31 and pUL34) for a better visualization of the process in transfected as well as infected cells. These mAbs provide a useful tool for future analyses.
The publication of the NEC crystal structure formed the basis for intensive research on the molecular details of the NEC formation and its interaction with the nucleocapsid. Recently, our lab showed that lysine (K) at position 242 in the membrane-distal part of pUL31 is crucial for incorporation of the nucleocapsid into budding vesicles. Replacing K by alanine (A) resulted in accumulations of vesicles in the PNS, while mature capsids were not incorporated. To test whether this is due to electrostatic interference or structural restrictions we substituted K242 by different aa to determine the requirements for nucleocapsid uptake into the nascent primary particles. To analyze whether the defect of pUL31-K242A can be compensated by second-site mutations, PrV-UL31-K242A was passaged and mutations in revertants were analyzed. Different mutations have been identified compensating for the K242A defect. A considerable number of mutations indicates that the NEC is much more flexible than previously thought. Further, we gained information that the K at position 242 is not directly involved in capsid interaction, while it is more likely involved in rearrangements within the NEC coat.
In fixed orthodontic treatments debonding of brackets during treatment is an unpleasant occurrence for the clinician and the patients and resultes in an increase in treatment costs and duration. For Damon Q brackets recycling would considered as an economic saving option which could be done with using of in office methods such as the sandblasting.
A sample of sixty sound bovine first upper central incisers, were collected, cleaned, and mounted in acrylic blocks for shear bond strength testing.
The total sample was equally divided into two main groups. Each group had 30 teeth and 30 brackets.
The first group had 30 teeth bonded with metal Damon Q brackets, the second group had 30 teeth bonded with metal Mini-Mono® brackets.The study included bonding and rebonding experiments. Therefore the same brackets with their same teeth were used in bonding and in the rebonding experiments. The bonding and the rebonding procedures were done with using 3M Unitek etching, Grengloo adhasive, and Ortho solo bonding. In addition the rebonding procedure was done after cleaning the teeth and recycling their brackets with sandblasting. All specimens were recycled 5000 times for the bonding and rebonding experiments.
The first and second debonding forces were done in Newton using a Zwick Roell machine.
After that SBS and SRS were computed in MPa. Furthermore all the teeth, after each debonding, were examined under a digital scanning microscope VHX-5000, 50X magnifying, to performe the ARA and ARI.
The collected data was statistically analyzed for descriptive statistics as well as significance of differences among the different bracket types, and their ARI scores, in the bonding and rebonding experiments.
The results showed that SRS was significantly higher than SBS of both types of the brackets, and Damon Q brackets had higher SBS, and SRS than Mini-Mono® brackets, and there was no correlation between SBS, SRS and their ARI, ARA.
Recent climate change and its consequences for living organisms constitute one of the greatest problems of our century. Global warming entails an increase in mean temperature and the frequencies of extreme weather events. Those changes in environmental conditions affect both plants and animals. Because of their inability to escape from unsuitable environments, plants have evolved a wide spectrum of molecular programs to protect themselves against changing conditions. Responding on altered environmental conditions will change plants chemical composition and therefore also affect plants interaction with other species (e.g., predator-prey or symbiotic relationships). For instance, changes in the chemical composition of plants may influence the survival of associated herbivores. In other words, these herbivores will be affected indirectly by climate change due to changes in the suitability / quality of their food. The aim of this doctoral thesis was to discover the effects of climate change within the relationship of the butterfly Pieris napi and its host plant (Sinapis alba used here as host plant), including individual conditions (e.g. chemical compositions of plants; morphology, physiology of the butterfly) and behavior of female butterflies and larvae. In the first experiment, the influence of simulated climate change on the chemical composition of the plant Sinapis alba was investigated. The second experiment aimed to examine the influence of changes in plant composition on the butterfly P. napi. Glucosinolates (secondary compound of plants) are known to have an important effect on the preference and performance of herbivores. Therefore, in the third experiment, the impact of glucosinolates on the preference and performance of P. napi was investigated in order to see if these plant compounds had the most important influence on this butterfly. Furthermore, in the fourth experiment, it was explored whether there is a latitudinal gradient within the species´ responses to changes in its host plant. The fifth and last experiment aimed to examine, if there are general principles across species regarding indirect effects of climate change.
Climate change, simulated by different combinations of temperature and water regimes, had an effect on the plant chemistry. The combination of temperature and water availability changed plant composition substantially. Especially the amount of carbon and glucosinolates (here above all sinalbin) in S. alba plants varies between the different treatments and therefore between the different combinations of temperature and water regimes. Regarding glucosinolates, elevated temperatures increased their concentration in leaves, whereas water deficit in combination with higher temperature reversed this pattern. For carbon content, all plants, except those of the control group, showed a decreased amount of total carbon. However, simulated heat waves had no effect on plants, leading to the assumption that the plants were able to recover from heat stress sufficiently during the control phases. Changes in plant composition affected both larvae and females of the butterfly P. napi. Therefore, changed host-plant chemistry alters the plant quality for this herbivore, meaning that plants of different treatments represent different plant qualities defined by their composition. Females of P. napi may be able to differentiate between plant qualities and even show a direct preference. Therefore, glucosinolates seem to act as oviposition stimulants. However, preferring another plant quality with lower amount of glucosinolates suggest that females of this butterfly species were attracted by more than high levels of glucosinolates alone. Larvae fed with different plant qualities performed differently, indicated by smaller wings (lighter bodies) and prolonged development when fed with plants contained higher amount of the glucosinolate sinalbin. It can be assumed that a higher amount of sinalbin decreases the quality of the host plant and therefore lead to these responses. Probably larvae need to shift their resources from growth to detoxification and therewith survival. Furthermore, drought conditions during plant growth seem to reduce the overall negative effects of higher temperatures, lead to an increase of host plant quality. Larvae seem to benefit from feeding on these “double-stressed” plants. Comparison between the results of the preference and performance tests suggests that there might be a mismatch between female preference and larval performance. It seems that the stimulating effect of high concentration of glucosinolates, in this case sinalbin, misdirects females´ decision to less suitable host plants, meaning that the advantage of less competition for larvae come at costs through detoxification. Using Brassica napus plants with genetically fixed glucosinolate levels, it could be demonstrate that there must be other plant components influencing females´ oviposition behavior been seen in the choice experiment with S. alba. The comparison of German and Italian populations to changes in host-plant quality showed fewer differences between countries as expected. However, German and Italian individuals differed in their reaction to altered plant quality, at least in developmental time and larval growth rate. It seems that Italian larvae benefitted from plants grown under higher temperatures, whereas drought-stressed plants affected them negatively. German individuals in contrast seem to benefit only from water stress during plant growth. With regard to the sexes of P. napi, it seems that females respond differently than males to changes in plant quality. Furthermore, the results of the performance test on Bicyclus anynana showed that there might be some general principles for the respond of butterflies to changes of its host plant. B. anynana responded in a similar way to different host plant qualities as P. napi did, meaning that plants grown under higher temperatures and drought conditions seem to be beneficial for the larval performance.
In summary, these findings may have important implications for the indirect effects of climate change on this butterfly in natural environments. First, climate change seems to have an impact on the chemical composition of plants. Second, changes in plants caused by increasing temperature and droughts seem to influence the preference and performance of this butterfly. However, there are differences between populations, which seem to be induced by former adaptation. And third, there might be some general principles for the respond of butterflies to changes in their host plants. This thesis focuses only on possible indirect effects of climate change. However, there are direct effects, which may alter the responses of herbivores to changes in their host plant as well. Therefore, further investigations in this linkage and in other plant-herbivore relationships will be necessary to explore how climate change may alter the relationship between herbivores and their hosts.
The importance of ion propulsion devices as an option for in-space propulsion of space
crafts and satellites continues to grow. They are more efficient than conventional chemi-
cal thrusters, which rely on burning their propellant, by ionizing the propellant gas in a
discharge channel and emitting the heavy ions at very high velocities. The ion emission
region of a thruster is called the plume and extends several meters axially and radially
downstream from the exit of a thruster. This region is particularly important for the effi-
ciency of a thruster, because it determines energy and angular distribution of the emitted
ions. It also determines the interaction with the carrier space craft by defining the electric
potential shape and the fluxes and energies of the emitted high energy ions, which are the
key parameters for sputter erosion of satellite components such as solar panels. Developing
new ion thrusters is expensive because of the high number of prototypes and testing cycles
required. Numerical modeling can help to reduce the costs in thruster development, but
the vastly differing length and time scales of the system, particularly the large differences of
scales between the discharge chamber and the plume, make a simulation challenging. Often
both regions are considered to be decoupled and are treated with different models to make
their simulation technically feasible. The coupling between channel and plume plasmas and
its influence on each other is disregarded, because there is no interaction between the two
regions. Therefore, this thesis investigates the physical effects which arise from this cou-
pling as well as models suitable for an integrated simulation of the whole coupled problem
of channel and plume plasmas. For this purpose the High Efficiency Multistage Plasma
Thruster (HEMP-T) ion thruster is considered.
For the discharge channel plasma, a fully kinetic model is required and the Particle-in-Cell
(PIC) method is applied. The PIC method requires very high spatial and temporal resolu-
tions which makes it computationally costly. As a result, only the discharge channel and the
near-field plume close to the channel exit can be simulated. In the channel, the results show
that electrons are magnetized and follow the magnetic field lines. The orientation of the
magnetic field there is mostly parallel to the symmetry axis and the channel walls which re-
sults in a high parallel electron transport and leads to a flat electric potential and a reduced
plasma-wall sheath. Only at the magnetic cusps, which are characteristic of HEMP-Ts the
electrons are guided towards the wall, with ions following due to quasineutrality, where a
classical plasma-wall sheath develops. The ion-wall contact is thus limited to the cusp re-
gion. The small radial drop of the potential towards the wall gives rather low energies of
ions impinging at the wall and minimizes erosion in the HEMP-T.
In the near-field plume, which extends from the thruster exit plane to some centimeters
downstream, the ion emission characteristics is defined. The ratio of radial and axial elec-
tric field components in this region determines the ion emission angle which should be
minimized for maximum thruster efficiency. The plasma discharge in the channel produces
high plasma densities and the subsequent drop from plasma to vacuum potential occurs
further downstream for higher densities. This increases the ratio of radial and axial electric
field components because the plasma expands radially outside of the confinement from the
dielectric discharge channel walls. The potential structure in the near-field plume impacts
also the supply of electrons for the channel discharge because the electrons enter the channel
from the plume. An effect which arises from this coupling is the breathing mode oscilla-
tion. It is an oscillation which is observed in all plasma quantities and is located near the
thruster exit. The oscillation frequency measured in the simulation is in good agreement
with a predator-prey estimate which validates this ansatz. However, the electron tempera-
ture, assumed constant in the predator-prey model, correlates inversely with the oscillation,
i.e. it is minimal at the current maximum and vice versa, which contributes to the observed
oscillations. Because of the oscillation of the plasma number density, the potential drop also
oscillates in the exit region and thus the ratio of radial to axial electric field components,
which results in the oscillation of the mean ion emission angle.
Regarding suitable models for a combined simulation of channel and plume plasmas, the
PIC model for channel and near-field plume is explicitly coupled to a hybrid fluid-PIC
model for the plume. The latter treats the electrons as a fluid, hence increasing the effective
spatial and temporal resolutions which can be applied in the plume simulations at the cost
of reduced accuracy of the electron model. Plasma densities decrease by two orders of
magnitude two meters downstream from the channel exit. The explicitly coupled kinetic
and hybrid PIC models are well suited for the computation of a HEMP-T and its plume
expansion, but they disregard the coupling of channel and plume plasmas for which other
methods are necessary. For this purpose a new approach is presented with a proof-of-
principle validation. The limited spatial resolution in the plume can be overcome with the
mesh-coarsening method, which increases the resolution in regions of low plasma density
without numerical artifacts. Sub-cycling for the electrons in the plume can then be used
to increase the temporal resolution in the plume. The combination of both methods, called
the sub-cycling mesh-coarsening (SMC) algorithm in the scope of this work, promises high
savings in computational cost which can make a combined simulation of plume and channel
plasmas feasible.
Given a manifold with a string structure, we construct a spinor bundle on its loop space. Our construction is in analogy with the usual construction of a spinor bundle on a spin manifold, but necessarily makes use of tools from infinite dimensional geometry. We equip this spinor bundle on loop space with an action of a bundle of Clifford algebras. Given two smooth loops in our string manifold that share a segment, we can construct a third loop by deleting this segment. If this third loop is smooth, then we say that the original pair of loops is a pair of compatible loops. It is well-known that this operation of fusing compatible loops is important if one wants to understand the geometry of a manifold through its loop space. In this work, we explain in detail how the spinor bundle on loop space behaves with respect to fusion of compatible loops. To wit, we construct a family of fusion isomorphisms indexed by pairs of compatible loops in our string manifold. Each of these fusion isomorphisms is an isomorphism from the relative tensor product of the fibres of the spinor bundle over its index pair of compatible loops to the fibre over the loop that is the result of fusing the index pair. The construction of a spinor bundle on loop space equipped with a fusion product as above was proposed by Stolz and Teichner with the goal of studying the Dirac operator on loop space". Our construction combines facets of the theory of bimodules for von Neumann algebras, infinite dimensional manifolds, and Lie groups and their representations. We moreover place our spinor bundle on loop space in the context of bundle gerbes and bundle gerbe modules.
Morphological changes of the complex 3-D architecture of podocytes as well as the loss of these post-mitotic cells often result in severe kidney disease. Since currently, there are no curative drugs, we focused on the identification of non-invasive biomarkers, allowing an early detection of the onset of such diseases. Therefore, we analyzed the cellular- and the cell-free fractions of urine samples from patients suffering from chronic kidney disease (CKD), especially for injury markers as well as for exosome-derived miRNAs.
We identified the mRNA of the neuronal protein brain-derived neurotrophic factor (BDNF) in the cellular fraction of 120 CKD patients and found that the expression was highly correlated with the mRNA expression of the kidney injury marker molecule 1 (KIM-1). Furthermore, we found that both were correlated with the mRNA expression of the podocyte-specific gene Nephrin (NPHS1), suggesting that podocytes are very likely the cellular source.
Beside this, we observed that BDNF is upregulated in biopsies of diabetic patients and seems to be involved in the differentiation of podocytes. Immunofluorescence staining clearly showed that BDNF is localized in the cell body and major processes of podocytes within the glomerulus. Knockdown experiments in zebrafish larvae, a well-established animal model to study kidney function, showed the importance of BDNF on kidney function, morphology and filtration in vivo.
Additionally, we analyzed circulating exosomal microRNAs (miRs) isolated from the cell-free urine fraction. After the optimization of a column-based isolation protocol for exosomes, we identified miR-16 from a pre-selected set of candidates as a suitable endogenous reference gene for data normalization. Subsequently, we analyzed the exosomal levels of miR-21, miR-30a-5p and miR-92a in urine samples of 41 CKD patients and 5 healthy controls. We found significantly enhanced levels of miR-21 in CKD patients that were also negatively correlated with the eGFR, suggesting a negative influence on kidney function. MiR-21 was also highly upregulated in de-differentiated glomeruli and in kidneys of nephrotoxic serum- (NTS-) treated mice as an in vivo kidney injury model.
To summarize, we identified two promising new and non-invasive biomarkers for CKD in the urine of patients which may also have a functional relevance on kidney function.
Being the victim of traumatizing events has consequences that can lead to wellknown mental disorders, such as depression. However, newest studies show that these events do not only affect the victims’ behavior, but also the expression levels of specific genes in their blood and in their brain. Latest research discovered little pieces of RNA in the cells that were long thought to be genetic junk. Nevertheless, these so-called miRNAs can regulate the expression of multiple genes, thus modulating metabolism and cell functioning. The aim of this study was to see if childhood traumatization led to a set of differentially expressed miRNA profiles in the peripheral blood. For this, we used subjects from the SHIP trend cohort, who had previously answered various questionnaires, among them the Childhood Trauma Questionnaire and the Patients Health Questionnaire-9 and analyzed the miRNAs in their blood to find out whether there was an association between the score and the dysregulation of certain miRNAs. Furthermore, we selected 5 different independent variables: PHQ-trend, CTQ score, as well as its subscales Abuse and Neglect, and Major Depressive Disorder lifetime prevalence. The analyses showed a set of up- or downregulated miRNAs in the blood. In a second step, we tried to replicate our results comparing them to results in the literature. Some of the significantly dysregulated miRNAs had previously been described as key players in the pathogenesis of MDD, a few even displaying similar results to ours. The next step was to see if the significant miRNAs had common target genes and if these had been described in the literature as having an influence on MDD, showing positive results. One last step was to see if there were also common biological pathways that were modulated by the differentially expressed miRNA. This analysis did not show promising results since there were almost no brain pathways among the results. For future studies, it will be necessary to validate our results using a clinical sample, such as GANI_MED, where the prevalence of childhood traumatization, as well as MDD, is much higher. By doing this, new possibilities of trauma treatment through modulation of epigenetic pathways could arise. If childhood traumatization leads to a set of dysregulated miRNAs that can end in a positive diagnosis of MDD in adulthood, what effects could have a targeted miRNA therapy on the pathogenesis of these psychiatric disorders?
Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.
This thesis describes recent developments in multi-reflection time-of-flight mass spectrometry (MR-ToF MS) with ions exhibiting large masses and mass differences at an MR-ToF setup at the University of Greifswald. A series of in-trap manipulation techniques to selectively retain or eject ion bunches of multiple species with disparate mass-to-charge ratios is investigated. These highlight the possibility to correct long-term flight-time drifts using a reference ion species far away in mass from the species of interest and also the ability to use such a pair to perform single-reference precision mass determinations. In both cases, the results obtained with disparate-mass ion pairs are comparable to those known from operation with isobaric species.
In addition, an in-trap photoexcitation technique is developed and applied to study the dissociation behavior of atomic bismuth clusters (systems of some number of bismuth atoms). Compared to previous works by other groups, the probed cluster-size range is expanded for both ion polarities, resulting in a more comprehensive picture of the underlying dissociation pathways. The known significance of neutral-tetramer breakoff is confirmed, however, evidence is also found for the loss of larger neutral fragments.
Lastly, the principle of tandem high-resolution MR-ToF MS is introduced. This new method allows the study of the change in dissociation behavior of the cationic bismuth octamer resulting from substituting one of its atoms for lead. It is found that the lead-doping opens new preferential fragmentation pathways that outstrip the dominant tetramer breakoff for this specific precursor cluster size. As a first proof-of-principle experiment, the case of the cationic octamer shows that tandem MR-ToF MS is well-suited for the investigation of compound clusters.
Background: Physical inactivity is one of the main risk factors for cardiovascular disease,
which remains a major cause of death in Germany and around the globe. Thus, investigating
prevalences, population trends, high-risk groups, and intervention effects of physical activity
(PA) and sedentary time (ST) is highly relevant to public health. To receive reliable data, a
key issue in research is to apply an appropriate study design including the carefully
considered use of assessments. Otherwise, bias to PA and ST data may be introduced. The
present thesis investigates three often overlooked issues related to the impact of measurement
on PA and ST research data. The first aim was to examine whether mere measurement alters
PA and ST over the course of twelve months (study 1). The second aim was to identify
potential socio-demographic and cardiometabolic moderators of the mere-measurement effect
(study 2). The third aim was to present design, protocol, and preliminary results of an interim
analysis of a randomized controlled trial (RCT) aiming to test whether a video demonstration
of PA intensity levels reduces the lack of agreement between self-reported and objectively
measured PA (study 3).
Methods: Studies 1 and 2 were based on data of a trial to test the feasibility of a brief tailored
letter intervention to increase PA and to reduce ST during leisure time. Among a sample of
subjects with no history of myocardial infarction, stroke, or vascular interventions, a number
of 175 individuals aged 40 to 65 years participated in the study. At baseline, participants
received standardized measurement of blood pressure and waist circumference, blood sample
taking, and seven-day accelerometry. At baseline and after one, six, and twelve months,
participants completed the International Physical Activity Questionnaire (IPAQ). A random
subsample received a brief tailored letter intervention at months one, three, and four. A
number of 153 participants were included in study 1 using all available data across 12 months.
Changes in PA and ST were analyzed using latent growth modeling. For study 2, baseline and
one-month follow-up data of 175 participants were used. Dependence of one-month changes
in PA and ST on socio-demographic and cardiometabolic variables was analyzed using linear
regression models. In study 3, individuals aged between 40 and 75 years were recruited at a
shopping mall in Greifswald, Germany. Participants received seven-day accelerometry and
were invited to the cardiovascular examination center of the University Medicine Greifswald.
After random allocation to experimental and control group, they completed the selfadministered
IPAQ – Short Form via tablet-computer. The experimental group additionally
received a video demonstration of PA intensity levels before answering the questionnaire. A number of 131 participants were analyzed to receive preliminary results of an interim analysis
in order to verify the presumptions made for the a priori power calculation and to decide on
early stopping of the study. The difference between the study groups in the agreement
between self-reported and accelerometer-based PA was analyzed using a two-sample t-test.
Results: In study 1, results revealed no change in leisure-time PA, an increase in transportrelated
PA (p = .023), and a tendency towards a reduction of ST (p = .060) between baseline
and one-month assessment. Further, ST decreased between six and twelve months (p = .037).
Time trends of the intervention group did not differ significantly from those of the
assessment-only group. Results of study 2 revealed that men increased transport-related PA
more than women (p = .031) and men with higher triglycerides increased transport-related PA
less than men with lower triglycerides (p = .043). Men with higher systolic blood pressure
reduced ST more than those with lower systolic blood pressure (p = .028). However, this
linear association ceased to exist at a level of approximately 145 mmHg. A similar
relationship was found for glycated hemoglobin and ST in men. In study 3, preliminary
results of the interim analysis revealed a lower formal mean difference in the video group (M
= 21.8 min/day, SD = 108.9) compared to the control group (M = 41.0 min/day, SD = 117.4,
t(129) = 0.97, p = .166). The p-value lay between the significance (p < .010) and futility (p >
.269) boundaries of the test simulations.
Conclusions: Results of the present thesis have three implications for considering the impact
of PA and ST assessments in cardiovascular research. First, mere-measurement effects within
a feasibility trial were found in transport-related PA and ST suggesting to interfere with
potential intervention effects. Thus, measurement effects should be considered when planning
studies and interventions and when interpreting outcomes. Second, male sex and more
favorable triglycerides levels in men were associated with a higher increase of transportrelated
PA whereas worse health in men was associated with a higher reduction of ST. Thus,
using the mere-measurement effect for prevention purposes may require researchers and
practitioners to tailor PA and ST intervention components to individuals’ health condition.
Third, the design and protocol of the RCT seems appropriate to test the effect of a novel video
on the gap between self-reported and accelerometer-based PA. Preliminary results point to the
efficacy of the video.
Infections with Helicobacter pylori are a global challenge that affects both developed and developing countries. This infection is currently treated using multiple antimicrobials that are mostly absorbed after oral administration and subsequently secreted into the gastric lumen. The eradication rates from the different therapeutic regimens, however, are declining nowadays, primarily due to high antibiotic resistance and possibly the mode of drug delivery. H. pylori is commonly found adhering to epithelial cells, and therefore, intragastric drug delivery may be a more direct treatment option. In this work, we developed a new strategy for the local eradication of H. pylori within the stomach.
Initial in vitro experiments revealed that penicillin G shows promising antibiotic activity against resistant strains of H. pylori with MIC values of 0.125 µg/mL. To provide luminal concentrations above the MIC for an extended time, we decided to follow two different formulation strategies: effervescent granules and HPMC-based hydrogel matrix tablets. Among the granule formulations, only one batch was stable and demonstrated excellent performance with respect to drug content, effervescent action, and drug release. It was therefore selected for further in vitro studies. All matrix tablets showed the desired tablet quality requirements and drug release was scalable in vitro by the HPMC concentration.
In order to quantify PGS in various formulations and media, an HPLC method was developed and validated. Due to the stability concerns, the degradation behavior of PGS was studied at different pH. PGS was found to be unstable at acidic pH values, but its stability was higher at more neutral pH values. Sufficient stability was exhibited at pH values above pH 4.5. Due to the instability of PGS in acidic media, alkalizers were added to the matrix tablets to prevent the degradation of the drug within the tablet. Among the alkalizers tested, NaHCO3 showed the most promising results as it significantly enhanced the stability within the matrix and also the concentration of PGS in the dissolution media. The stabilizing effect was caused mainly by the modulation of the microenvironmental pH rather than a pH change in the dissolution media. As a result, these matrix tablets were selected for further in vitro characterization.
In order to guide formulation development, a flow-through model (FTM), which was able to simulate various physiological conditions of the gastric environment, was developed and applied. In contrast to compendial dissolution methods, the FTM allowed studying the effect of gastric secretion, mixing and emptying on the gastric concentration of the drug in vitro. It could be shown that the granules generated a high initial concentration, which decreased over time. On the contrary, the matrix tablets did not provide such a profile due to the absence of pressure events in the model. Further investigations of the matrix tablets in a dissolution stress test device revealed faster drug release if pressure events of physiological relevance are simulated.
In the last part of this thesis, the two formulation concepts were compared in vivo by using the salivary tracer technique. For this purpose, caffeine was used as a model drug. The in vivo investigations suggested that granules administered in a fed state demonstrated longer gastric retention than in a fasted state. In a fed state, effervescent granules provided longer gastric retention of caffeine in comparison to the matrix tablets. Interestingly, the administration of the granules together with 240 mL of tap water provided an even better gastric retention of caffeine than the smaller volume (20 mL). Additional MRI investigations after 4 h of tablets’ intake revealed that the matrix tablets were already disintegrated in vivo.
In conclusion, effervescent granules dosed after food are expected to better maintain intragastric drug concentration over an extended period compared to matrix tablets. Moreover, the carbon dioxide generated after disintegration supports the mixing of the drug with the chyme and thus, provides a uniform distribution of the drug. By this, bacterial sanctuary sites within the stomach can be avoided. The major challenge could be the stability of PGS in acidic media. This problem could be addressed via concomitant administration of PPIs. H2 blockers could also be recommended to address nocturnal acid-breakthrough during the mid-night. In combination with an acid-reducing agent, PGS granule formulations alone or part of the treatment regimens could enable the local eradication of H. pylori directly within the stomach.
Oral administration of drugs is the most common, convenient, safest and economical route of drug administration. There is lack of established tools to study the function of transporters in the intestinal absorption of drugs. Because of its favorable physico-chemical, pharmacokinetic and pharmacodynamic characteristics, trospium could be potentially used as a probe substrate to study the function of drug transporters. Therefore, this study was conducted to examine the suitability of trospium chloride as a probe drug to study the function of multidrug transporters in the human body. To this end, two randomized, controlled, four-period, cross-over pharmacokinetic drug interaction studies of oral and intravenous trospium with co-medication of oral clarithromycin or ranitidine were performed in 24 healthy subjects to mechanistically characterize the role of P-gp, OATP1A2, OCT1, OCT2, MATE1 and MATE2-K in the absorption and disposition of trospium. The contribution of the drug transporters in the absorption and disposition of trospium were examined in isolated systems using in vitro uptake and inhibition assays in transporter transfected human cell lines.
OCT1 (Vmax = 0.8 ± 0.1 nmol/min × mg) is a high capacity transporter of trospium compared to OCT2 (Vmax = 0.04 ± 0.01 nmol/min × mg). But the OCT2 (Km = 0.5 ± 0.1 µM) transporter demonstrated a high affinity in the transport of trospium compared to OCT1 (Km = 17.4 ± 2.1 µM). OCT1 genetic alleles *2, *3, *4 and *7 resulted in significant loss of activity and the alleles *5 and *6 caused complete loss of uptake of trospium. The common OCT2 genetic allele Ser270 caused slight but significant increase in activity of OCT2.
Ranitidine inhibits OCT1 (IC50 = 186 ± 25 µM), MATE1 (IC50 = 134 ± 37 µM) and MATE2-K (IC50 = 35 ± 11 µM)-mediated uptake of trospium in vitro. But it is a weak inhibitor of OCT2 transporter (IC50 = 482 ± 105 µM). Using FDA and EMA in vitro to in vivo extrapolation models, ranitidine was predicted to have a potential inhibition effect on intestinal OCT1 ([I]2/IC50 ~40), renal MATE1 ([I]1/IC50 ~0.02) and MATE2-K ([I]1/IC50 ~0.1) transporters in vivo. Clarithromycin was predicted to cause DDI by inhibiting P-gp-mediated efflux of trospium at the intestine ([I]2/IC50 of ~310) and hepatocytes ([I]3/IC50 ~1). Therefore, co-medication of oral clarithromycin was expected to result in an increase in oral absorption and hepatic clearance of trospium but not changes in distribution volume.
In healthy subjects, oral trospium is slowly (MAT ~10 h) and poorly (F ~10 %) absorbed from the jejunum and cecum/ascending colon, widely distributed into the body (Vss = 5 - 6 l/kg) and slowly eliminated (t1/2 = 9 - 10 h) majorly via renal glomerular filtration and tubular secretion (CLR ~500 ml/min). After co-medication of clarithromycin (inhibitor of P-gp), on the contrary to our IVIVE prediction, we found a non-expected but significant expansion of the shallow and deep distribution spaces for trospium by ~27 %. A single dose administration of trospium with co-medication of ranitidine (inhibitor of OCT1) resulted in no effect on the intestinal absorption of trospium. But the renal clearance of trospium decreased slightly (15 %) but significantly.
Intravenously administered trospium (2 mg TC) might be a suitable probe drug to evaluate the effects of a P-gp inhibitor on distribution of a drug. Oral trospium chloride can be selected for DDI studies with new chemical entities (NCE) with predicted inhibitory potential on OCT1 and P-gp and which are available after oral absorption along the small intestine and in the cecum/ascending colon. Another kind of application of trospium chloride might be pharmacogenomics studies in subjects with functionally relevant polymorphisms of P-gp and OCT1 or in patients with suspected transport failure due to intestinal diseases. The function of the efflux transporters MATE1 and MATE2-K in the PTC of the kidneys can be well assessed with the probe drug trospium by measuring its renal clearance.
To enable control of African swine fever (ASF) in Eastern and Southern Africa, prototype live vaccine candidates were generated by targeted gene deletions from a Kenyan genotype IX ASF virus (ASFV). It was attempted to delete known nonessential genes involved in virulence (encoding TK, dUTPase, CD2v, 9GL), possibly essential genes (p12, pA104R, ribonucleotide reductase), and genes with widely unknown functions (pK145R). Isolation of the desired virus recombinants by plaque assays or limiting dilutions on a wild boar lung cell line (WSL-HP) was facilitated by substitutive reporter gene insertions encoding fluorescent proteins (GFP, DsRed), or the human membrane protein CD4. The latter protein permitted enrichment of recombinant virus particles by magnetic activated cell sorting (MACS). The isolated ASFV recombinants were characterized by PCR and sequencing of the mutated genome parts, and replication kinetics and virus spread in cell culture were investigated. Deletion of TK, CD2v, or pK145R had no detectable effect on in vitro growth of ASFV Kenya. Interestingly, virus mutants lacking the DNA binding protein pA104R which has been considered to be essential for DNA replication, also exhibited almost wild type-like growth properties.
In contrast, ASFV mutants lacking ribonucleotide reductase or p12 could not be purified to homogeneity on WSL-HP cells, indicating these proteins are essential for virus replication in cell culture. Therefore, trans-complementing cells lines stably expressing ASFV p12 have been prepared which can now be used for mutant virus purification. If this approach is successful the resulting defective mutant ASFV Kenya-p12 might be suitable as a safe “disabled in second cycle” (DISC) live vaccine in swine.
In a novel approach to improve reverse genetics of ASFV the CRISPR/Cas9 cell line WSL-gRp30 (Hübner et al., 2018a) was co-transfected with genomic DNA of ASFV-KenyaCD2vDsRed, sgRNA plasmids targeting K145R or 9GL, and GFP-expressing recombination plasmids for homology-directed repair. For booting up of the noninfectious virus genome the cells were infected with phylogenetically distant helper virus (genotype II ASFV Armenia, 84% identity) which was selectively inhibited on the used cell line. The desired double-fluorescent double-deletion mutants could be isolated after few plaque purification steps on selective WSL-gRp30 cells. Next generation sequence (NGS) analyses of reconstituted ASFV Kenya genomes showed that no unwanted recombination with the helper virus occurred, indicating that the method might be also suitable for booting of synthetic ASFV genomes cloned and mutagenized in E. coli or yeast.
The modified CRISPR/Cas9 system of S. pyogenes might be also usable for generation of ASFV resistant pigs. To evaluate this alternative control measure WSL cell clones stably expressing Cas9 nuclease and single or multiple sgRNAs against essential ASFV proteins were prepared and tested for their susceptibility to infection. Strain specific sgRNAs targeting the p30 gene of ASFV Kenya or Armenia selectively inhibited the respective viruses, and a p12 gene-specific sgRNA abrogated replication of both genotypes almost completely. Interestingly, coexpression of four ASFV-specific sgRNAs did not enhance virus inhibition, but might help to reduce the frequency of escape mutants which were occasionally isolated from the single sgRNA-expressing cells, and exhibited silent base substitutions or in-frame deletions within the target genes. First attempts to express the in vitro tested CRISPR/Cas9 constructs in transgenic pigs are in progress.
CRISPR/Cas9 supported rescue of a defective BAC clone of pseudorabies virus (PrV) vaccine strain Bartha (Hübner et al., 2018b) was used to develop putative vectored vaccines against ASFV. In the present study expression cassettes for the codon-optimized p12 and p54 genes of ASFV were successfully inserted into the PrV genome. The insertions did not significantly affect PrV recombination in cell culture, and the transgenes were expressed at similar levels as in ASFV-infected cells. It has to be tested whether coinfection with vector constructs for these and other immunogenic ASFV proteins is able to protect pigs against a lethal challenge.
For characterization of the generated ASFV mutants and PrV vector constructs, monospecific antisera against several ASFV gene products (p11.5, p12, p54, pK145R, p285L) were prepared by immunization of rabbits with bacterial GST fusion proteins. The anti-p12 serum showed only weak and strain-specific reactions with the ASFV Kenya protein, but was nevertheless useful for identification of p12-expressing PrV recombinants and WSL cell lines. All other sera showed satisfying reactions in Western blot and mostly immunofluorescence analyses, and allowed i.a. precise localization of the pK145R and p285L proteins in ASFV-infected cells and virions (Hübner et al., 2019).
Cardiovascular diseases are the most common cause of death in industrial nations. The basis of these diseases is a dysfunction in the interaction between the cells the heart is composed of. The main types of cells making up the human heart are cardiomyocytes that build the myocardium and provide the contraction properties, endothelial cells that delimit the blood flowing through the inner chambers and coronary arteries from the myocardial tissue, and fibroblasts, which build the connective tissue. A common process in the development of cardiovascular diseases is the formation of fibrosis due to injury of the endothelium and subsequent infiltration of the cardiac tissue by immune cells, and inflammatory agents like cytokines. Cytokines exert different functions in cardiac cells. Tumor necrosis factor α (TNFα) is an inducer of apoptosis. Transforming growth factor ß (TGFß) is known for activation of proliferation. Other cytokines like C-X-C motif chemokine 11 (CXCL11), interleukin-6 (IL-6), or brain-derived neurotrophic factor (BDNF) have not yet been investigated or their impact on such cells is unknown. Eventually, however, fibrotic scar tissue arises from the transition from fibroblasts to myofibroblasts leading to a stiffening of the cardiac muscle and impaired pump function. In order to prevent the occurrence of these events the balance of proliferation, migration, and differentiation of cardiac cells needs to be controlled very delicately.
The mechanisms controlling these interactions are still not well understood, which is why this work aimed at the elucidation of molecular mechanisms within the three main cell types that might play a role in the regulation of cardiac function. A proteomic approach using mass spectrometry was used to identify alterations in protein levels that could provide hints about the involved pathways and find new players as candidates for more detailed investigation. Initially, the proteomic composition of HL-1 cardiomyocytes, L929 fibroblasts, and human umbilical vein endothelial cells (HUVECs) that were cultivated in standard growth conditions without stress was investigated. Half of the total protein intensity was made up by only 42 to 53 proteins, depending on the cell type. More than a third of all proteins were identified in all three cell types, which may be proteins performing common cell functions. Indeed, the proteins displaying the highest abundance seem to be predominantly involved in such common cellular functions as the regulation of glucose metabolism or the cytoskeleton. More specific functions like heart development and muscle contraction were found enriched in cardiomyocytes as were mitochondrial proteins. The proportion of proteins with extracellular localization and function was higher in fibroblasts and endothelial cells.
Secondly, the impact of cytokines on the proliferative behavior and the proteomic composition of cardiomyocytes and fibroblasts was analyzed. HL-1 cardiomyocytes and L929 fibroblasts were treated with different concentrations of cytokines with a cytotoxic, proliferative, or yet unknown effect on these cells. While HL-1 cells exhibited no macroscopic reaction to any of the cytokines used, cytotoxic/growth inhibitory (TNFα, CXCL11) and proliferative (TGFß, IL6, BDNF) effects were observed for L929 cells. The latter also showed CXCL11-induced upregulated EIF2 signaling, pointing to a higher need of protein synthesis.
The third aim was the examination of proteome adaptations in endothelial cells due to different kinds of stress, as these cells are the first line of defense against inflammatory agents or injury and therefore prone to wounding. The role of the growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounding and starvation was another object of this study as they are known for their angiogenic and cell survival supporting properties. Additionally, the impact of the cellular sex on the response to stress and growth factors was examined, because a person’s sex plays an important role in susceptibility, risk factors, and outcome of cardiovascular diseases. This has mainly been attributed to the different hormone levels, especially the higher levels of estrogen in premenopausal women, which exerts cardioprotective properties, but also genetic background was reported to play an important role. Only few studies that examined the molecular properties of HUVECs considered the cellular sex and if so, the genetic bias of unrelated samples was not taken into account. This is why Lorenz and colleagues at the Charité in Berlin collected HUVECs from newborn twins of opposite sex, cultivated them without stress in standard growth medium, exposed them to wounding and serum starvation, and investigated the impact of the growth factors and the sex on migrational behavior and metabolic issues. The current work focused on the alterations of not only the intra- but also the extracellular proteome, because paracrine signaling is crucial for intercellular communication in order to cope with stress. General differences between male and female cells were observed for proteins encoded on the X chromosome with higher levels in females (DDX3X, UBA1, EIF1AX, RPS4X, HDHD1), except for one protein with higher levels in male cells (G6PD). A Y-chromosomal protein was, for the first time, identified in endothelial cells (DDX3Y). Wounding, starvation, and growth factor treatment led to alterations and sex-specific different levels in an unexpectedly high number of proteins, with VEGF showing a stronger impact than bFGF. Many proteins with alterations observed without taking the sex into account, were actually only changed in male or female cells. Some proteins were regulated in opposite directions, or growth factors inhibited their secretion in a sex-specific way by unknown mechanisms. Tissue factor pathway inhibitor 2 (TFPI2) should be emphasized as a protein with sex-specific differences, especially in the extracellular space and with increased levels after starvation and VEGF treatment. These observations suggest a temporal lack in TFPI2 synthesis and secretion in male cells, which might explain the enhanced adaptation of females to wounding.
The results of this work lay the basis for future investigation by providing a database of intra- and extracellular proteome changes due to different environmental circumstances. It strongly suggests the investigation of male and female HUVECs, and other cells, separately to avoid the impact of the sex observed in this work. Essentially, the observations suggest a number of candidate proteins for more detailed investigations of endothelial and cardiovascular diseases.
New World arenaviruses represent an important group of zoonotic pathogens that pose a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell responses to infection, or how they contribute to virulence; however, TCRV strongly induces caspase-dependent apoptosis (i.e. non-inflammatory programmed cell death) in infected cells, whereas JUNV does not.
In order to better understand the connection between apoptosis and pathogenesis, we sought to unravel the regulation of pro- and anti-apoptotic signaling in response to arenavirus infection. We demonstrated that apoptosis induced by TCRV proceeds over the mitochondrial-regulated intrinsic pathway and involves activation of p53 (accumulation and phosphorylation), activation of the pro-apoptotic BH3-only factors Puma and Noxa (accumulation), as well as inactivation of another pro-apoptotic factor called Bad (phosphorylation). The regulation of these factors in response to TCRV infection is accompanied by other classical hallmarks of intrinsic apoptosis, such as disorganization of the mitochondrial network, cytochrome c release, PS flipping, caspase cleavage and nuclear condensation. The involvement of the BH3-only factors as key players in regulating TCRV-induced apoptosis could also be validated in knockout cells, which showed either suppressed or increased apoptosis depending on the respective activation (i.e. Puma and Noxa) or inactivation (i.e. Bad) status of the respective BH3 protein. Interestingly, while JUNV does not trigger late stages of apoptosis induction (i.e. caspase activation, nuclear condensation and cell death), we could show that it activates similar upstream pro-apoptotic signaling events including activation of p53, Puma and Noxa. This supports the current hypothesis that JUNV actively evades the induction of apoptosis through the involvement of a mechanism targeting late steps in the apoptotic cascade. Specifically, this model proposes that intrinsic activation is suppressed at the level of caspase activation by JUNV NP, which serves as an alternative substrate for caspase cleavage.
Additionally, in order to identify viral factors associated with the induction of apoptosis, a full genome sequencing of TCRV was performed and contributed to the validation and correction of substantial errors reported in existing sequences for TCRV. With the help of this sequence, correct expression plasmids containing the viral genes for NP, GP and Z were constructed and tested for their ability to induce apoptosis in vitro. This revealed that both TCRV and JUNV Z are triggers for apoptosis, which further supports our finding that JUNV also induces activation of pro-apoptotic factors. Again, consistent with a model where JUNV NP blocks caspase activation directly, co-expression of JUNV Z and NP abrogated caspase activation, while simultaneous expression of TCRV NP and Z still resulted in cell death.
Finally, identification of the specific apoptotic factors involved in regulating TCRV-induced apoptosis (i.e. Bad, Puma and Noxa) and the generation of the respective knockout cell lines allowed us to investigate what influence apoptosis induction has on virus infection. Interestingly, knockout of these factors showed no direct impact on virus growth in Vero cells. However, TCRV particles produced in cells with the individual pro-apoptotic (i.e. Puma and Noxa) or anti-apoptotic (i.e. Bad) factors knocked out showed altered infectivity in primary human monocytes and macrophages, which represent important target cells for arenaviruses. Since TCRV particles that originate from the different knockout cells would be expected to contain different amounts of PS in their envelope (depending on the level of apoptosis taking place), this suggests a role of apoptosis in facilitating PS-receptor-mediated entry and/or PS-receptor signaling through downstream kinases, either of which could be contributing to successful infection in professional phagocytic cells. In particular, phosphorylation of some of the identified factors involved in regulating TCRV-induced apoptosis indicates the involvement of upstream kinases from diverse signaling pathways, some of which also play a role in regulating cytokine production – another host cell reaction that differs significantly between TCRV- and JUNV-infected monocytes and macrophages. As such, these findings represent an exciting basis for a possible connection between apoptotic responses and the regulation of pro- and anti-inflammatory cytokine responses via their associated upstream signaling processes and provide a starting point for future studies that will help us to better understand how these processes contribute to arenavirus pathogenicity.
The benefit of regular physical activity and exercise training for the prevention of cardiovascular and metabolic diseases is undisputed. Many molecular mechanisms mediating exercise effects have been deciphered. Personalised exercise prescription can help patients in achieving their individual greatest benefit from an exercise-based cardiovascular rehabilitation programme. Yet, we still struggle to provide truly personalised exercise prescriptions to our patients. In this position paper, we address novel basic and translational research concepts that can help us understand the principles underlying the inter-individual differences in the response to exercise, and identify early on who would most likely benefit from which exercise intervention. This includes hereditary, non-hereditary and sex-specific concepts. Recent insights have helped us to take on a more holistic view, integrating exercise-mediated molecular mechanisms with those influenced by metabolism and immunity. Unfortunately, while the outline is recognisable, many details are still lacking to turn the understanding of a concept into a roadmap ready to be used in clinical routine. This position paper therefore also investigates perspectives on how the advent of ‘big data’ and the use of animal models could help unravel inter-individual responses to exercise parameters and thus influence hypothesis-building for translational research in exercise-based cardiovascular rehabilitation.
Abstract
The frequency of mechanical circulatory support (MCS) device application has increased in recent years. Besides implantation in the emergency setting, such as circulatory arrest, MCS is also increasingly used electively to ensure hemodynamic stability in high‐risk patients, for example, during percutaneous coronary interventions (PCI), valve interventions or off‐pump coronary bypass surgery. Lifebridge (Zoll Medical GmbH, Germany) is a compact percutaneous MCS device widely used in daily clinical routine. The present study aimed to investigate the indications, feasibility, and outcomes after use of Lifebridge in cardiac interventions, evaluating a large‐scale multicenter database. A total of 60 tertiary cardiovascular centers were questioned regarding application and short‐term outcomes after the use of the Lifebridge system (n = 160 patients). Out of these 60 centers, eight consented to participate in the study (n = 39 patients), where detailed data were collected using standardized questionnaires. Demographic and clinical characteristics of the patient population, procedural as well as follow‐up data were recorded and analyzed. In 60 interrogated centers, Lifebridge was used in 74% of emergency cases and 26% in the setting of planned interventions. The subcohort interrogated in detail displayed the same distribution of application scenarios, while the main cardiovascular procedure was high‐risk PCI (82%). All patients were successfully weaned from the device and 92% (n = 36) of the patients studied in detail survived after 30 days. As assessed 30 days after insertion of the device, bleeding requiring red blood cell (RBC) transfusion constituted the main complication, occurring in 49% of cases. In our analysis of clinical data, the use of Lifebridge in cardiac intervention was shown to be feasible. Further prospective studies are warranted to identify patients who benefit from hemodynamic MCS support despite the increased rate of RBC transfusion due to challenges in access sites during cardiovascular procedures.
The thesis investigates the occurrence of the Early Modern European witch-hunt within the distinctively diverse society of the Grand Duchy of Lithuania. Positioned at the intersection of Latin and post-Byzantine cultures, along with Western and Eastern Christianity, this region lay on the frontlines of the Reformation and Counter-Reformation. The research aims to analyze the specific characteristics of the witch-hunt in this area, considering it a case study of social and cultural interaction within a borderland. It focuses particularly on identifying the distinctive aspects of the Lithuanian witch-hunt, examining the social and cultural roots of the witch trials, and exploring their relationship to the broader social and cultural developments of the period.
Central to this study is a detailed examination of the witch trials and an analysis of court materials. The thesis posits a socio-cultural interpretation of witch persecutions, arguing that they were culturally influenced manifestations of social tensions, enacted through legal mechanisms. The emergence of new Early Modern challenges, such as the social consequences of agrarian reform, the expansion of manorialism, and serfdom, led to novel tensions, conflicts, and responses, including accusations of witchcraft. The importation of authoritative foreign ideas about witchcraft reinvigorated, facilitated, and shaped pre-existing moderate indigenous beliefs, a process facilitated by religious struggles and Catholic post-Tridentine confessionalization. The Lithuanian legal system provided an environment conducive to an intensive witch-hunt, with witchcraft being a secular grave felony tried in highly decentralized and poorly supervised courts. However, this potential was largely unrealized. The study argues that the cultural diversity of the society played a major role in inhibiting the spread of Western witchcraft discourse, thereby limiting the extent of the witch-hunt in the Grand Duchy of Lithuania.
Summary
Streptococcus pneumoniae (the pneumococcus), a bacterium belonging to the normal flora in the human respiratory tract, continues to be an important pathogen due to its contribution to morbidity and mortality among children, the elderly, and immunocompromised persons. Global estimates of pneumococcal deaths among children declined by 51% between 2000 and 2015. This achievement was mainly due to the introduction of pneumococcal conjugate vaccines (PCVs) in countries with the highest pneumococcal burden. Since May 2012, children in Ghana have been receiving PCV vaccination as part of routine immunization. The continuous monitoring of the pneumococcus after PCV introduction is essential to understand the changing epidemiology of the pathogen in the population.
This study therefore, aims to determine the (1) prevalence, serotypes, and sequence types of pneumococcal isolates, (2) antibiotic susceptibility patterns and the genetic basis for the antibiotic resistance among these pneumococcal isolates, and (3) prevalence of selected virulence genes that have been identified as potential vaccine candidates. Nasopharyngeal swabs were obtained from vaccinated children under five years of age in Cape Coast, Ghana. Six years after PCV implementation, we provide data on the epidemiology of pneumococcal strains circulating among children in Cape Coast Ghana. Standard microbiological and molecular techniques were used to identify and characterize the pneumococcal strains.
Overall, pneumococcal carriage prevalence was 29.4% (151/513). All participating children were fully vaccinated. Of the 26 different serotypes identified, the top five PCV13 serotypes (VT) were 6B, 23F, 19F, 3, 6A and non-PCV13 vaccine serotypes (NVT) were 23B, 13, 11A, 15B, and 34. PCV13 coverage was 38.4%, however, more than half of the isolates were NVT with a coverage rate of 61.6%. The isolates were highly susceptible to levofloxacin, ceftriaxone, vancomycin, and erythromycin. However, marked resistance to cotrimoxazole and tetracycline was observed. The reduction in penicillin resistance (35.8%) as compared to pre-vaccination data (45% - 63%) suggests an attributable effect from PCV13 vaccination. However, penicillin resistance was also detected in some NVT serotypes. Overall, 28.5% of the isolates resistant to three or more different classes of antibiotics were classified as multidrug-resistant (MDR). To analyze the genetic basis for resistance to penicillin, erythromycin and tetracycline, pbp2b, ermB, mefA, and tetM genes were amplified.
Thirty-eight (70%) out of the 54 penicillin-resistant isolates contained the pbp2b resistance gene. Out of the 11 erythromycin-resistant isolates, 7 (63.6) and 4 (36.4%) were positive for the ermB and mefA genes, respectively. The tetM gene was detected in 85 (98.8%) of the 86 tetracycline resistance isolates.
To determine the extent to which potential protein-based vaccines could be protective in Ghanaian children, we sought to determine the prevalence of selected virulence genes among the isolates. The lytA, pavB, and cpsA genes were present in all the carrier isolates. However, psrP, pcpA, pilus islet (PI) PI-1, and PI-2 were present in 62.7%, 87.5%, 11.8%, and 6.5% of the strains, respectively. The psrP and pcpA virulence genes were evenly distributed among all the serotypes. However, the pilus islets were detected in only seven serotypes namely 19F, 6B, 9V, 6A, 13, 11A, and 23B. Five serotype 19F isolates possessed both PI-1 and PI-2. Furthermore, the pilus islets were associated with multidrug resistance.
The predominant NVT serotype 23B and isolates resistant to ≥ 4 antibiotics were analysed by multilocus sequence typing (MLST). Nine known sequence types (STs) and 10 novel STs were identified. Seven out of the 10 new STs belonged to serotype 23B, while the remaining 3 were VTs 6B and 19F. A capsular switch was identified among isolates of ST802, which comprised of both serotype 23F and 19F. The majority of serotype 23B strains belonged to ST172. The ST172 is associated with serotype 23F and a single locus variant (SLV) of internationally disseminated clone ST338 (Colombia23F-26). Consequently, ST172 was characterised with marked antibiotic resistance and with traits of capsular switching. One serotype 6B strain was identified as a SLV of ST273 (Greece6B-22) while two serotype 9V strains belonged to the internationally disseminated clone ST156 (Spain9V-3).
In conclusion, this study showed a marginal decline in overall pneumococcal carriage prevalence, persistence of VTs despite the increase in NVTs, and the occurrence of serotype replacement and capsular switching. In addition, sequence types related to internationally disseminated clones are circulating in Ghana. With the high pcpA and psrP coverage detected,including these genes in protein-based vaccines could provide adequate protection for Ghanaian Children.
Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.
Overall, the present thesis provides tools for virus characterization. Importantly, the application of the developed tools contributed to the fundamental knowledge of selected, veterinary relevant viruses in terms of their underlying biology and virus-host interaction.
By using in vitro models and full-genome sequencing, important new findings were gained that contributed to the deeper understanding of the selected viruses. Results show that in vitro models can be successfully modified to enable study of specific host factors that are important for viral entry. By genetically modifying a bovine cell line using CRISPR/CAS9 technology , a stable cell culture model was established that is now available to the research community, to study the virus-host interaction of pestiviruses. The model was further used to elucidate the adaptability of bovine viral diarrhea the virus and impact on infectivity and growth. By using deep sequencing, genetic changes that occurred during the adaption process of bovine viral diarrhea virus were identified and linked to the phenotype, allowing the characterization of genetic regions important for virus binding to the host cell.
Whole-genome analysis using deep-sequencing was further used to characterize circulating rabbit haemorrhagic disease virus (RHDV) strains from Germany. The study provides more than 50 full genomes of RHDV strains sampled between 2013 and 2020. Since the virus family is drastically under sampled, in particular in central Europe, these sequences represent a very valuable addition to the field. The investigation led further to the discovery of a novel recombinant virus strain in hares, that is likely still circulating today. This finding is of special interest, since it is the first detection of a recombination event between the genogroups RHDV and European brown hare syndrome virus (EBHSV) of Lagoviruses. It highlights the importance of full genome virus surveillance and the potential risk of virus variants that might evade diagnostic detection.
Serological assay were used to study the persistency of antibodies developed during a natural infection with Schmallenberg virus. It could be shown that these antibodies are long lasting and therefore, re-emergence of this virus in Europe is likely favoured by introduction of naïve animals into a herd and not by decreasing antibody-titers over time.
Overall, the discoveries described in this thesis underline the importance of adequate tools for virus characterization and they give valuable answers to fundamental questions regarding the biology of the different viruses.
Over the last decades, thyroid hormone metabolites (THMs) received marked attention as it has been demonstrated that they are bioactive compounds. Their concentrations were determined by immunoassay or mass-spectrometry methods. Among those metabolites, 3,5-diiodothyronine (3,5-T2), occurs at low nanomolar concentrations in human serum, but might reach tissue concentrations similar to those of T4 and T3, at least based on data from rodent models. However, the immunoassay-based measurements in human sera revealed remarkable variations depending on antibodies used in the assays and thus need to be interpreted with caution. In clinical experimental approaches in euthyroid volunteers and hypothyroid patients using the immunoassay as the analytical tool no evidence of formation of 3,5-T2 from its putative precursors T4 or T3 was found, nor was any support found for the assumption that 3,5-T2 might represent a direct precursor for serum 3-T1-AM generated by combined deiodination and decarboxylation from 3,5-T2, as previously documented for mouse intestinal mucosa. We hypothesized that lowered endogenous production of 3,5-T2 in patients requiring T4 replacement therapy after thyroidectomy or for treatment of autoimmune thyroid disease, compared to production of 3,5-T2 in individuals with intact thyroid glands might contribute to the discontent seen in a subset of patients with this therapeutic regimen. So far, our observations do not support this assumption. However, the unexpected association between high serum 3,5-T2 and elevated urinary concentrations of metabolites related to coffee consumption requires further studies for an explanation. Elevated 3,5-T2 serum concentrations were found in several situations including impaired renal function, chronic dialysis, sepsis, non-survival in the ICU as well as post-operative atrial fibrillation (POAF) in studies using a monoclonal antibody-based chemoluminescence immunoassay. Pilot analysis of human sera using LC-linear-ion-trap-mass-spectrometry yielded 3,5-T2 concentrations below the limit of quantification in the majority of cases, thus the divergent results of both methods need to be reconciliated by further studies. Although positive anti-steatotic effects have been observed in rodent models, use of 3,5-T2 as a muscle anabolic, slimming or fitness drug, easily obtained without medical prescription, must be advised against, considering its potency in suppressing the HPT axis and causing adverse cardiac side effects. 3,5-T2 escapes regular detection by commercially available clinical routine assays used for thyroid function tests, which may be seriously disrupted in individuals self-administering 3,5-T2 obtained over-the counter or from other sources.
Age-related deterioration in white and gray matter is linked to cognitive deficits. Reduced microstructure of the fornix, the major efferent pathway of the hippocampus, and volume of the dentate gyrus (DG), may cause age-associated memory decline. However, the linkage between these anatomical determinants and memory retrieval in healthy aging are poorly understood. In 30 older adults, we acquired diffusion tensor and T1-weighted images for individual deterministic tractography and volume estimation. A memory task, administered outside of the scanner to assess retrieval of learned associations, required discrimination of previously acquired picture-word pairs. The results showed that fornix fractional anisotropy (FA) and left DG volumes were related to successful retrieval. These brain-behavior associations were observed for correct rejections, but not hits, indicating specificity of memory network functioning for detecting false associations. Mediation analyses showed that left DG volume mediated the effect of fornix FA on memory (48%), but not vice versa. These findings suggest that reduced microstructure induces volume loss and thus negatively affects retrieval of learned associations, complementing evidence of a pivotal role of the fornix in healthy aging. Our study offers a neurobehavioral model to explain variability in memory retrieval in older adults, an important prerequisite for the development of interventions to counteract cognitive decline.
Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
Staphylococcus aureussuperantigens (SAgs) are among the most potent T cell mitogensknown.They stimulate large fractions of T cells by cross-linking their T cell receptor withmajor histocompatibility complex class-II molecules on antigen presenting cells, resulting in Tcell proliferation and massive cytokine release. To date, 26 different SAgs have been described in thespeciesS. aureus; they comprise the toxic shock syndrome toxin (TSST-1), as well as 25 staphylococcalenterotoxins (SEs) or enterotoxin-like proteins (SEls). SAgs can cause staphylococcal food poisoningand toxic shock syndrome and contribute to the clinical symptoms of staphylococcal infection. Inaddition, there is growing evidence that SAgs are involved in allergic diseases. This review providesan overview on recent epidemiological data on the involvement ofS. aureusSAgs and anti-SAg-IgEin allergy, demonstrating that being sensitized to SEs—in contrast to inhalant allergens—is associatedwith a severe disease course in patients with chronic airway inflammation. The mechanisms by whichSAgs trigger or amplify allergic immune responses, however, are not yet fully understood. Here, wediscuss known and hypothetical pathways by which SAgs can drive an atopic disease
Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.
Differences in salinity are boundaries that act as barriers for the dispersal of most aquatic organisms. This creates distinctive biota in freshwater and brackish water (mesohaline) environments. To test how saline boundaries influence the diversity and composition of host-associated microbiota, we analyzed the microbiome within the digestive tract of Theodoxus fluviatilis, an organism able to cross the freshwater and mesohaline boundary. Alpha-diversity measures of the microbiome in freshwater and brackish water were not significantly different. However, the composition of the bacterial community within freshwater T. fluviatilis differed significantly compared with mesohaline T. fluviatilis and typical bacteria could be determined for the freshwater and the mesohaline digestive tract microbiome. An artificial increase in salinity surrounding these freshwater snails resulted in a strong change in the bacterial community and typical marine bacteria became more pronounced in the digestive tract microbiome of freshwater T. fluviatilis. However, the composition of the digestive tract microbiome in freshwater snails did not converge to that found within mesohaline snails. Within mesohaline snails, no cardinal change was found after either an increase or decrease in salinity. In all samples, Pseudomonas, Pirellula, Flavobacterium, Limnohabitans, and Acinetobacter were among the most abundant bacteria. These bacterial genera were largely unaffected by changes in environmental conditions. As permanent residents in T. fluviatilis, they may support the digestion of the algal food in the digestive tract. Our results show that freshwater and mesohaline water host-associated microbiomes respond differently to changes in salinity. Therefore, the salinization of coastal freshwater environments due to a rise in sea level can influence the gut microbiome and its functions with currently unknown consequences for, e.g., nutritional physiology of the host.
Streptococcus pneumoniaeinfections lead to high morbidity and mortality rates worldwide.Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but havea limited range of protection, which encourages the development of a broadly protective protein-basedalternative. We and others have shown that immunization with pneumococcal lipoproteins that lackthe lipid anchor protects against colonization. Since immunity againstS. pneumoniaeis mediatedthrough Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects ofa lipid modification on the induced immune responses in either intranasally or subcutaneouslyvaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrAstrongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhancedantibody concentrations and different induction kinetics. The induced humoral immune responsewas modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-typeimmunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderatebut consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins,indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus,protein lipidation represents a promising approach for the development of a serotype-independentpneumococcal vaccine.
Transcriptional Basis for Differential Thermosensitivity of Seedlings of Various Tomato Genotypes
(2020)
Over the last years, there has been a resurge in the interest to study the relationship between interoception and emotion. By now, it is well established that interoception contributes to the experience of emotions. However, it may also be possible that interoception contributes to the regulation of emotions. To test this possibility, we studied the relationship between interoception and emotion regulation in a sample of healthy individuals (n = 84). We used a similar heartbeat detection task and a similar self-report questionnaire for the assessment of interoceptive accuracy and emotion regulation as in previous studies. In contrast to previous studies, we differentiated between male and female individuals in our analyses and controlled our analyses for individual characteristics that may affect the relationship between interoceptive accuracy and emotion regulation. We found sex-differences in interoceptive accuracy and emotion regulation that amounted to a sex-specific relationship between interoceptive accuracy and emotion regulation: Whereas interoceptive accuracy was related to reappraisal but not to suppression in male individuals, interoceptive accuracy was unrelated to reappraisal and suppression in female individuals. These findings indicate that the relationship between interoception and emotion regulation is far more complex than has been suggested by previous findings. However, these findings nonetheless support the view that interoception is essential for both, the regulation and experience of emotions.
The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (Fpassive) of cardiomyocytes. Protein kinase D (PKD) enzymes play crucial roles in regulating myocardial contraction, hypertrophy, and remodeling. PKD phosphorylates myofilament proteins, but it is not known whether the giant protein titin is also a PKD substrate. Here, we aimed to determine whether PKD phosphorylates titin and thereby modulates cardiomyocyte Fpassive in normal and failing myocardium. The phosphorylation of titin was assessed in cardiomyocyte-specific PKD knock-out mice (cKO) and human hearts using immunoblotting with a phosphoserine/threonine and a phosphosite-specific titin antibody. PKD-dependent site-specific titin phosphorylation in vivo was quantified by mass spectrometry using stable isotope labeling by amino acids in cell culture (SILAC) of SILAC-labeled mouse heart protein lysates that were mixed with lysates isolated from hearts of either wild-type control (WT) or cKO mice. Fpassive of single permeabilized cardiomyocytes was recorded before and after PKD and HSP27 administration. All-titin phosphorylation was reduced in cKO compared to WT hearts. Multiple conserved PKD-dependent phosphosites were identified within the Z-disk, A-band and M-band regions of titin by quantitative mass spectrometry, and many PKD-dependent phosphosites detected in the elastic titin I-band region were significantly decreased in cKO. Analysis of titin site-specific phosphorylation showed unaltered or upregulated phosphorylation in cKO compared to matched WT hearts. Fpassive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered Fpassive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher Fpassive compared to control hearts and significantly lower Fpassive after PKD treatment. In addition, we found higher phosphorylation at CaMKII-dependent titin sites in HCM compared to control hearts. Expression and phosphorylation of HSP27, a substrate of PKD, were elevated in HCM hearts, which was associated with increased PKD expression and phosphorylation. The relocalization of HSP27 in HCM away from the sarcomeric Z-disk and I-band suggested that HSP27 failed to exert its protective action on titin extensibility. This protection could, however, be restored by administration of HSP27, which significantly reduced Fpassive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.
The aim of this follow-up study was, to compare the effects of mechanical periodontal therapy with or without adjunctive amoxicillin and metronidazole on the subgingival microbiome of smokers with periodontitis using 16S rDNA amplicon next generation sequencing. Fifty-four periodontitis patients that smoke received either non-surgical periodontal therapy with adjunctive amoxicillin and metronidazole (n = 27) or with placebos (n = 27). Subgingival plaque samples were taken before and two months after therapy. Bacterial genomic DNA was isolated and the V4 hypervariable region of the bacterial 16S rRNA genes was amplified. Up to 96 libraries were normalized and pooled for Illumina MiSeq paired-end sequencing with almost fully overlapping 250 base pairs reads. Exact ribosomal sequence variants (RSVs) were inferred with DADA2. Microbial diversity and changes on the genus and RSV level were analyzed with non-parametric tests and a negative binomial regression model, respectively. Before therapy, the demographic, clinical, and microbial parameters were not significantly different between the placebo and antibiotic groups. Two months after the therapy, clinical parameters improved and there was a significantly increased dissimilarity of microbiomes between the two groups. In the antibiotic group, there was a significant reduction of genera classified as Porphyromonas, Tannerella, and Treponema, and 22 other genera also decreased significantly, while Selenomonas, Capnocytophaga, Actinomycetes, and five other genera significantly increased. In the placebo group, however, there was not a significant decrease in periodontal pathogens after therapy and only five other genera decreased, while Veillonella and nine other genera increased. We conclude that in periodontitis patients who smoke, microbial shifts occurred two months after periodontal therapy with either antibiotics or placebo, but genera including periodontal pathogens decreased significantly only with adjunctive antibiotics.
Drained peatlands are significant sources of the greenhouse gas (GHG) carbon dioxide.Rewetting is a proven strategy used to protect carbon stocks; however, it can lead to increasedemissions of the potent GHG methane. The response to rewetting of soil microbiomes as drivers ofthese processes is poorly understood, as are the biotic and abiotic factors that control communitycomposition. We analyzed the pro- and eukaryotic microbiomes of three contrasting pairs ofminerotrophic fens subject to decade-long drainage and subsequent long-term rewetting. Abiotic soilproperties including moisture, dissolved organic matter, methane fluxes, and ecosystem respirationrates were also determined. The composition of the microbiomes was fen-type-specific, but allrewetted sites showed higher abundances of anaerobic taxa compared to drained sites. Based onmulti-variate statistics and network analyses, we identified soil moisture as a major driver ofcommunity composition. Furthermore, salinity drove the separation between coastal and freshwaterfen communities. Methanogens were more than 10-fold more abundant in rewetted than in drainedsites, while their abundance was lowest in the coastal fen, likely due to competition with sulfatereducers. The microbiome compositions were reflected in methane fluxes from the sites. Our resultsshed light on the factors that structure fen microbiomes via environmental filtering.
Glutathione (GSH) was initially identified and characterized for its redox properties andlater for its contributions to detoxification reactions. Over the past decade, however, the essentialcontributions of glutathione to cellular iron metabolism have come more and more into focus. GSH isindispensable in mitochondrial iron-sulfur (FeS) cluster biosynthesis, primarily by co-ligating FeSclusters as a cofactor of the CGFS-type (class II) glutaredoxins (Grxs). GSH is required for the exportof the yet to be defined FeS precursor from the mitochondria to the cytosol. In the cytosol, it is anessential cofactor, again of the multi-domain CGFS-type Grxs, master players in cellular iron and FeStrafficking. In this review, we summarize the recent advances and progress in this field. The mosturgent open questions are discussed, such as the role of GSH in the export of FeS precursors frommitochondria, the physiological roles of the CGFS-type Grx interactions with BolA-like proteins andthe cluster transfer between Grxs and recipient proteins