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40-Year Longitudinal Caries Development in German Adolescents in the Light of New Caries Measures
(2019)
This study assessed the 40-year longitudinal caries development in German adolescents in the light of the sixth National Oral Health Survey in Children (NOHSC, 2016) employing initial DMFT (IDMFT), Significant Caries Index (SiC) and Specific Affected Caries Index (SaC). On the basis of the current NOHSC (randomized cluster selection using school list or regional community school surveys, 55,956 12-year-old sixth-graders examined by 482 calibrated community/study dentists) DMFT, SiC, a novel IDMFT including initial lesions (IT) and the recently introduced SaC were calculated and also recalculated for national and international surveys from the last 4 decades. In 2016, 78.8% of children were caries-free (DMFT = 0), 65.5% including IT lesions. The mean DMFT was 0.44 (single components: DT = 0.14, MT = 0.02, FT = 0.29, IT = 0.52) showing a clear association with the school type as marker for the socio-economic status. The mean number of affected teeth in children with DMFT >0 was 2.07 (SaC) in comparison to almost 9 teeth in the 1970s. The current care index on the tooth level was 66.3%, leaving only 7.7% of children with restorative treatment needs. Longitudinally, a continuous caries decline of more than 80%, including the risk groups (SiC/SaC), to an internationally extremely low level was observed. In conclusion, the National Oral Health Surveys reveal a continuous caries decline to a very low caries level in 12-year-old 6th-graders in Germany even if IT lesions are included (IDMFT). In spite of proportional reductions in the risk groups (SiC/SaC), the polarized caries distribution according to socio-economic parameters reveals the need for targeted preventive programmes.
In the present work high density helicon plasma discharges are created and characterized as a promising concept towards the realization of plasma wakefield accelerators to build up electric fields in the order of GV/m to accelerate electrons to energies in the TeV range with proton driving bunches. For such a concept plasma sources are needed that are able to maintain discharges with plasma densities of n_e = 7E20 m^-3 over long distances with a low variation in plasma density. Measurements at the PROMETHEUS-A device are performed for variable parameters, like magnetic induction, RF heating power and filling gas pressure. A CO2 laser interferometer, a laser induced fluorescence (LIF) diagnostic and a reaction rate model are combined to give a full picture. It is shown that in most cases the plasma density is centrally peaked with a high density region +- 5 mm from the center. The peak plasma density increases with increasing filling gas pressure, RF heating power and magnetic induction, limited by the number of neutral particles in low pressure discharges, by the transferred heating power and the increasing recombination and electron quenching rates of argon ions in high filling pressure cases. The increase in plasma density with increasing magnetic induction correlates to the direct proportionality in the helicon dispersion relation. For all investigated operational parameters the time evolution of the helicon discharge shows the same characteristics and is reliably reproducable inside the error bars. The electron temperature is determined by combining the collisional radiative model with line ratio measurements of two spontaneously emitted LIF lines. The low electron temperature regime of 1.2 eV < T_e < 1.4 eV and the electron temperature profiles are consistent with helicon wave heating via collisional power dissipation. The maximum plasma density of n_e = (6 +- 1)E20 m^-3 is measured at high RF power of P_RF = 24 kW, p_0 = 9 Pa filling gas pressure and a magnetic induction of B = 105 mT with a maximum electron temperature at 1.4 eV. At these operational parameters the plasma density peaking time and width are determined to be 270E-6 s and 50E-6 s, respectively. This shows that specific plasma density requirements for the use of a wakefield accelerator are reachable and the duration of the peak plasma density is more than sufficient for a relativistic particle to pass a 1 km long plasma cell. Additionally time-resolved LIF profile measurements for neutral and singly ionized argon were conducted to complement the previously evaluated measurements. The time resolution of the LIF diagnostic was chosen in a way to adequately represent the evolution of densities and to allow full profile measurements over one day. A resolution of 200E-6 s was chosen. The time-resolved neutral and ion metastable densities show hollow profiles with high densities at the edges over the first ms indicating higher ionization levels and increasing electron quenching rates. The metastable densities are highly determined by electron temperature, RF heating power and filling neutral gas pressure and do not reflect the neutral argon evolution. To investigate the influence of neutral depletion on the density evolution and maximum plasma density, the argon neutral and ion ground state densities are determined. Both time-resolved density profiles show a hollow profile with highest densities at the edges over a longer time interval of 3-4 ms. The penetration depths (ionization mean-free paths) indicate increased ionization of neutral argon while dissipating inwards, corresponding well to the theoretical value of lambda = 20 mm. This results in a depletion of neutrals in the center of the discharge, leading to a limitation and a fast decrease of plasma density after the neutrals are partially ionized. The shown refilling effect of neutral argon is too slow to have an important impact. At operation parameters for highest plasma density, the calculated ground states also show a fast increase in density at the end of the discharge after the RF-heating is switched off. This indicates recombination effects to these atomic states and higher ionization levels than ArII in the helicon discharge.
Understanding the fundamental mechanisms in the extracellular matrix of cells (ECM) is crucial for the development of drugs and biomaterials. Therefore, an atomistic model of the extracellular matrix is a cost-efficient way to observe influences of drugs, test the effect of mutations or misfolds in proteins or study the properties of fibril or network-forming peptides.
With this thesis, a refined molecular model of an adhesion complex is proposed that contains collagen, fibronectin and the cell receptor integrin. During the building of the model, major new insights are given for each of these proteins and a powerful protein-folding algorithm is
developed.
This thesis describes experiments with clusters stored in an electrostatic ion trap called Multi-reflection time-of-flight (MR-ToF) analyzer. These devices are established as mass separators and analyzers with high resolving powers and fast processing times. The objective was to characterize an experiment that utilizes such analyzer for cluster research, to this end a laser-ablation ion source was combined with an MR-ToF analyzer.
In the first part, an experiment scheme that combines two operating modes, namely in-trap lift operation and mirror operation, is presented and characterized for the present setup. For ion capture in-trap lift switching was employed and exit-side mirror switching for ejection with higher information content. Measurements were performed with small lead clusters to illustrate individual advantages of both techniques and the gain of combining them with focus on the ions’ ToF ejection window.
In the second part, a recently introduced method of ion separation by transversal ejection of unwanted species inside the trap was studied for the present setup. The ejection is performed by appropriate pulses of the potentials of deflector electrodes located in the trap. The various parameters affecting the selection effectivity and resolving power are illustrated with tin-cluster measurements, with resolving powers of up to several tens of thousands.
The third part presents the experiment in detail, with the construction of each component and measurements for its various performance parameters. Because the heart of the setup is the MR-ToF analyzer the characterization focuses on the trap. In addition, cluster ions were mass selected in the MR-ToF device and photodissociated. The charged fragments were stored and mass analyzed in a proof-of principle MS/MS experiment where both MS steps were performed in the MR-ToF operation mode.
For a long time the apocryphal Ladder of Jacob was accessible only in arbitrarily selected translations. Without a critical edition and a comprehensive study of the whole textual segment, scholars were unable to evaluate its significance for Early Jewish and Christian literature. Since 2015/17, with the publication of a new critical edition and German translation (accompanied by a detailed introduction, footnote commentaries and appendices with related texts), a new approach to this important but hitherto widely unknown text has been made possible. This approach verifies the different layers or strata in the text, which are: a supposed Jewish apocalypse (mid-second century), a Christian expansion of the angels speech in light of the praeparatio evangelica tradition (fourth–seventh centuries), a Jewish mystical prayer (eleventh century) and the incorporation of this narrative block into the Tolkovaja Paleja together with a series of exegetical commentaries (end of the thirteenth century). In the light of the new approach, it can be said that the Ladder of Jacob is most of all an outstanding example of mutual relations between Jewish and Christian theology.
Hyperoxia is a well-known cause of cerebral white matter injury in preterm infants with male sex being an independent and critical risk factor for poor neurodevelopmental outcome. We investigated the underlying mechanisms behind such a sex dependent difference in oligodendrocyte progenitor cells (OPCs). Our findings demonstrate that oxidative stress severely affects cellular functions related to energy metabolism, stress response, and maturation in male derived oligodendrocyte progenitor cells (OPCs) whereas the female cells remain largely unaffected. This impairment of maturation is accompanied by the downregulation of nucleoporin and nuclear lamina proteins. We identify Nup133, which regulates OPC maturation as a major target protein affected by hyperoxia in male cells and that this differential response is mediated by an inverse Nup133 regulation in the male and female cells. It also regulates mitochondrial function and oxidative stress response through its downstream target Nuclear respiratory factor 1 (Nrf1). Additionally, the presence of 17-β estradiol and higher amounts of fetal zone steroids (precursors for maternal estrogen synthesis during fetal development) confer resistance to the female cells mediated by the estrogen receptor alpha (ERα) along with Nup133. Both Nup133 and ERα regulate mitochondrial function and oxidative stress response by transcriptional regulation of Nrf1. These findings establish prominent sex based differences and the molecular mechanisms involved in differential response of OPCs towards oxidative stress and the important role of Nup133 in mediating a severe negative outcome in the male cells.
Microbial cell factories have been largely exploited for the controlled production of recombinant proteins, including industrial enzymes and biopharmaceuticals. The advent of high-throughput ‘-omics’ techniques have boosted the design of these production systems due to their valuable contribution to the field of systems metabolic engineering, a discipline integrating metabolic engineering with systems and synthetic biology. In order to thrive, the field of systems metabolic engineering needs absolute proteomics data to be generated, as proteins are the central players in the complex metabolic and adaptational networks. Due to advent of mass spectrometry-based proteomics, a substantial amount of absolute proteomic data became available in the past decade. However, membrane proteins remained inaccessible to these efforts.
Nonetheless, comparative studies targeting the membrane proteome have been quite successful in characterizing physiological processes. Hence, label-free proteomics was used in a study (Quesada-Ganuza et al, 2019 – Article I) to identify and optimize PrsA in Bacillus subtilis, for improved yield of amylase. Amylase is one of the most relevant enzymes in the biotechnological sector. By employing a label-free mass spectrometry approach targeting the membrane proteome of this bacterium, relative changes in heterologous and native levels of PrsA could be quantified. The results of this study evidenced that each PrsA shows different relative abundancies, but with no relevant impact in the yield of amylase.
Even though relative protein quantification can already provide a good visualization of the physiological changes occurring between different conditions, they are not sufficient to understand how resources are allocated in the cell under certain physiological conditions. Therefore, a global method for absolute membrane protein quantification remains the biggest requirement for systems metabolic engineering.
Hence, with this work, we successfully developed a mass spectrometry-based approach enabling the absolute quantification of membrane proteins (Antelo-Varela et al, 2019 – Article II). This study was also performed in the Gram-positive model organism Bacillus subtilis, regarded as a prolific microbial cell factory. The method developed in this work combines the comprehensiveness of shotgun proteomics with the sensitivity and accuracy of targeted mass spectrometry. Fundamental to the method is that it relies on the application of a correction and an enrichment factor to calibrate absolute membrane protein abundances derived from shotgun mass spectrometry. This has permitted, for the first time reported, the calculation of absolute membrane protein abundances in a living organism.
The newly developed approach enabled to accurately quantify ~40% of the predicted proteome of this bacterium, offering a clear visualization of the physiological rearrangements occurring upon the onset of osmotic stress. In addition, this work also provides evidence for new membrane protein stoichiometries.
Overall, this study enabled the development of a straightforward methodology long-needed in the scientific and biotechnological community and, for the first time reported, providing absolute abundances of one of the most puzzling fractions of the cell – the membrane proteome.
The next step of the work summarized here was to implement the afore described method to a biotechnological relevant strain, as absolute membrane protein abundances are essential to understand the fundamental principles of protein secretion and production stress. Hence, this work was applied in a genome-reduced B. subtilis strain, ‘midiBacillus’, expressing the major staphylococcal antigen IsaA (Antelo-Varela et al, submitted – Article III). The employed absolute membrane protein quantification methodology enabled the analysis of physiological rearrangements occurring upon the induction of heterologous protein production. This work showed that, even though IsaA was successfully secreted into the growth medium, one of the main requirements for the biotechnological sector, it was still partly accumulated in the cell membrane of this bacterium. This led to an exacerbated physiological response where membrane proteins involved in the management of secretion stress were activated. In addition, this study also showed that a rearrangement of the cell’s translocation machinery occurs upon induction of production, where a ‘game’ of in- and decrease of transporters takes place.
Anticipating the impact of genetic and environmental insults, such as the ones caused by production stress, is essential for the field of systems metabolic engineering. Thus, the highly accurate and comprehensive dataset generated during this work can be implemented in predictive mathematical models, thereby contributing in the rational design of next-generation secretion systems.
In phylogenetics, evolutionary relationships of different species are represented by phylogenetic trees.
In this thesis, we are mainly concerned with the reconstruction of ancestral sequences and the accuracy of this reconstruction given a rooted binary phylogenetic tree.
For example, we wish to estimate the DNA sequences of the ancestors given the observed DNA sequences of today living species.
In particular, we are interested in reconstructing the DNA sequence of the last common ancestor of all species under consideration. Note that this last common ancestor corresponds to the root of the tree.
There exist various methods for the reconstruction of ancestral sequences.
A widely used principle for ancestral sequence reconstruction is the principle of parsimony (Maximum Parsimony).
This principle means that the simplest explanation it the best.
Applied to the reconstruction of ancestral sequences this means that a sequence which requires the fewest evolutionary changes along the tree is reconstructed.
Thus, the number of changes is minimized, which explains the name of Maximum Parsimony.
Instead of estimating a whole DNA sequence, Maximum Parsimony considers each position in the sequence separately. Thus in the following, each sequence position is regarded separately, and we call a single position in a sequence state.
It can happen that the state of the last common ancestor is reconstructed unambiguously, for example as A. On the other hand, Maximum Parsimony might be indecisive between two DNA nucleotides, say for example A and C.
In this case, the last common ancestor will be reconstructed as {A,C}.
Therefore we consider, after an introduction and some preliminary definitions, the following question in Section 3: how many present-day species need to be in a certain state, for example A, such that the Maximum Parsimony estimate of the last common ancestor is also {A}?
The answer of this question depends on the tree topology as well as on the number of different states.
In Section 4, we provide a sufficient condition for Maximum Parsimony to recover the ancestral state at the root correctly from the observed states at the leaves.
The so-called reconstruction accuracy for the reconstruction of ancestral states is introduced in Section 5. The reconstruction accuracy is the probability that the true root state is indeed reconstructed and always takes two processes into account: on the one hand the approach to reconstruct ancestral states, and on the other hand the way how the states evolve along the edges of the tree. The latter is given by an evolutionary model.
In the present thesis, we focus on a simple symmetric model, the Neyman model.
The symmetry of the model means for example that a change from A to C is equally likely than a change from C to A.
Intuitively, one could expect that the reconstruction accuracy it the highest when all present-day species are taken into account. However, it has long been known that the reconstruction accuracy improves when some taxa are disregarded for the estimation.
Therefore, the question if there exits at least a lower bound for the reconstruction accuracy arises, i.e. if it is best to consider all today living species instead of just one for the reconstruction.
This is bad news for Maximum Parsimony as a criterion for ancestral state reconstruction, and therefore the question if there exists at least a lower bound for the reconstruction accuracy arises.
In Section 5, we start with considering ultrametric trees, which are trees where the expected number of substitutions from the root to each leaf is the same.
For such trees, we investigate a lower bound for the reconstruction accuracy, when the number of different states at the leaves of the tree is 3 or 4.
Subsequently in Section 6, in order to generalize this result, we introduce a new method for ancestral state reconstruction: the coin-toss method.
We obtain new results for the reconstruction accuracy of Maximum Parsimony by relating Maximum Parsimony to the coin-toss method.
Some of these results do not require the underlying tree to be ultrametric.
Then, in Section 7 we investigate the influence of specific tree topologies on the reconstruction accuracy of Maximum Parsimony. In particular, we consider balanced and imbalanced trees as the balance of a tree may have an influence on the reconstruction accuracy.
We end by introducing the Colless index in Section 8, an index which measures the degree of balance a rooted binary tree can have, and analyze its extremal properties.
Spinal cord injury (SCI) above mid-thoracic levels leads to autonomic dysfunction affecting both the cardiovascular system and thermoregulation. The renin-angiotensin system (RAS) which is a potent regulator of blood pressure, including its novel beneficial arm with the receptor Mas could be an interesting target in post-SCI hemodynamics. To test the hypothesis that hemodynamics, activity and diurnal patterns of those are more affected in the Mas deficient mice post-SCI we used a mouse model of SCI with complete transection of spinal cord at thoracic level 4 (T4-Tx) and performed telemetric monitoring of blood pressure (BP) and heart rate (HR). Our data revealed that hypothermia deteriorated physiological BP and HR control. Preserving normothermia by keeping mice at 30°C prevented severe hypotension and bradycardia post-SCI. Moreover, it facilitated rapid return of diurnal regulation of BP, HR and activity in wild type (WT) mice. In contrast, although Mas deficient mice had comparable reacquisition of diurnal HR rhythm, they showed delayed recovery of diurnal rhythmicity in BP and significantly lower nocturnal activity. Exposing mice with T4-Tx (kept in temperature-controlled cages) to 23°C room temperature for one hour at different time-points post-SCI, demonstrated their inability to maintain core body temperature, Mas deficient mice being significantly more impaired than WT littermates. We conclude that Mas deficient mice were more resistant to acute hypotension, delayed nocturnal recovery, lower activity and more severely impaired thermoregulation. The ambient temperature had significant effect on hemodynamics and, thus it should be taken into account when assessing cardiovascular parameters post-SCI in mice.
Summary
The susceptibility of Candida albicans biofilms to a non‐thermal plasma treatment has been investigated in terms of growth, survival and cell viability by a series of in vitro experiments. For different time periods, the C. albicans strain SC5314 was treated with a microwave‐induced plasma torch (MiniMIP). The MiniMIP treatment had a strong effect (reduction factor (RF) = 2.97 after 50 s treatment) at a distance of 3 cm between the nozzle and the superior regions of the biofilms. In addition, a viability reduction of 77% after a 20 s plasma treatment and a metabolism reduction of 90% after a 40 s plasma treatment time were observed for C. albicans. After such a treatment, the biofilms revealed an altered morphology of their cells by atomic force microscopy (AFM). Additionally, fluorescence microscopy and confocal laser scanning microscopy (CLSM) analyses of plasma‐treated biofilms showed that an inactivation of cells mainly appeared on the bottom side of the biofilms. Thus, the plasma inactivation of the overgrown surface reveals a new possibility to combat biofilms.
Introduction: Hearing and vision loss are highly prevalent in elderly adults, and thus frequently occur in conjunction with cognitive impairments. Studies have shown that hearing impairment is associated with a higher risk of dementia. However, evidence concerning the association between vision loss and dementia, as well as the co-occurrence of vision and hearing loss and dementia, has been inconclusive.
Objectives: To assess the association between: (i) either hearing or vision loss and the risk of dementia, as well as between; and (ii) the combination of both sensory impairments and the risk of dementia.
Methods: This case-control study was based on a 5-year data set that included patients aged 65 years and older who had initially been diagnosed with dementia diseases by one of 1,203 general practitioners in Germany between January 2013 and December 2017. In total, 61,354 identified dementia cases were matched to non-dementia controls, resulting in a sample size of 122,708 individuals. Hearing loss and vision loss were identified using the ICD-10 diagnoses documented in the general practitioners’ files prior to the initial dementia diagnosis. Multivariate logistic regression models were fitted to evaluate the associations between visual and/or hearing impairment and the risk of dementia and controlled for sociodemographic and clinical variables.
Results: Hearing impairment was documented in 11.2% of patients with a dementia diagnosis and 9.5% of patients without such a diagnosis. Some form of vision impairment was documented in 28.4% of patients diagnosed with dementia and 28.8% of controls. Visual impairment was not significantly associated with dementia (OR = 0.97, CI = 95% 0.97–1.02, p = 0.219). However, patients with hearing impairment were at a significantly higher risk of developing dementia (OR = 1.26, CI = 95% 1.15–1.38, p < 0.001), a finding that very likely led to the observed significant association of the combination of both visual and hearing impairments and the risk of dementia (OR = 1.14, CI = 95% 1.04–1.24, p = 0.005).
Discussion: This analysis adds important evidence that contributes to the limited body of knowledge about the association between hearing and/or vision loss and dementia. It further demonstrates that, of the two, only hearing impairment affects patients’ cognition and thus contributes to dementia risk.
Brain aging even in healthy older adults is characterized by a decline in cognitive functions including memory, learning and attention. Among others, memory is one of the major cognitive functions affected by aging. Understanding the mechanisms underlying age-related memory decline may help pave the road for novel treatment strategies. Here, we tried to elucidate the neural correlates associated with memory decline using structural and functional neuroimaging and neuromodulation with transcranial direct current stimulation (tDCS).
Over the course of three studies, we investigated 1) the influence of white matter integrity and grey matter volume on memory performance in healthy older adults, 2) the role of functional coupling within the memory network in predicting memory performance and the impact of tDCS in modulating retrieval performance in healthy older adults, 3) the effect of tDCS over the sensorimotor cortex on cognitive performance in young adults.
MRI was used to study associations of cognitive performance with white matter integrity and grey matter volume, and examine their causal relationship in the course of aging. White matter integrity was assessed by acquiring diffusion tensor imaging (DTI) and performing deterministic tractography based on constrained spherical deconvolution. Grey matter volume was estimated using fully automated segmentation. Both white matter integrity and grey matter volume were correlated with behavioral data of a verbal episodic memory task. Percentage of correct answers at retrieval was used to measure memory performance (Manuscript 1). In addition, anodal tDCS (atDCS) (1 mA, 20 min) was applied over CP5 (left temporoparietal cortex) to modulate memory formation in healthy older adults. Participants underwent resting-state fMRI before the stimulation. Functional connectivity analysis was performed to determine whether functional coupling within the memory network predicted initial memory performance, and to examine its association to tDCS-induced enhancement effect (Manuscript 2). Finally, atDCS (1 mA, 20 min) was applied over C3 (left sensorimotor cortex) to explore the effect of tDCS over the sensorimotor cortex on cognitive performance in young adults. During the stimulation, participants performed three tasks; gestural task, attentional load task and simple reaction time task (Manuscript 3).
Results showed that volumes of the left dentate gyrus (DG) and tractography-based fractional anisotropy (FA) of individual fornix pathways were positively related to memory retrieval in older adults. Brain-behavior associations were observed for correct rejections rather than hits of memory performance, indicating specificity of memory network functioning for detecting false associations. Thus, the data suggested a particular role of neural integrity that promotes successful memory retrieval in older adults. Subsequent mediation analysis showed that left DG volume mediated the effect of fornix FA on memory performance (48%), corrected for age, revealing a crucial role of hippocampal pathway microstructure in modulating memory performance in older adults (Manuscript 1). tDCS results showed that atDCS led to better retrieval performance and increasing learning curves, indicating that brain stimulation can induce plasticity of episodic memory processes in older adults. Combining tDCS and fMRI, hippocampo-temporoparietal functional connectivity was positively associated with initial memory performance in healthy older adults and was positively correlated with the magnitude of individual tDCS-induced enhancement, suggesting that individual tDCS responsiveness may be determined by intrinsic network coupling (Manuscript 2). Finally, our findings suggested that atDCS over left sensorimotor cortex reduced reaction times in the gestural-verbal integration task, specifically for incongruent pairs of gestures and verbal expressions, indicating the role of sensorimotor cortex in gestural-verbal integration in young adults (Manuscript 3).
The results of all three studies may help to elucidate age-related structural deterioration and functional coupling network underlying cognitive processes in healthy adults. Furthermore, these studies emphasized the importance of interventions like tDCS in modulating cognitive performance, specifically episodic verbal memory and gestural-verbal integration. By unveiling the specific role of brain structures and functional network coupling as well as the role of tDCS in modulating cognitive performance, our results contribute to a better understanding of brain-behavior associations, and may help to develop clinical interventional approaches, tailored for specific cognitive functions in aging.
In this thesis, we elaborate upon Bayesian changepoint analysis, whereby our focus is on three big topics: approximate sampling via MCMC, exact inference and uncertainty quantification. Besides, modeling matters are discussed in an ongoing fashion. Our findings are underpinned through several changepoint examples with a focus on a well-log drilling data.
Staphylococcus aureus is one of the commonly encountered bacteria of the human microbiome. Although mostly a seemingly harmless commensal microbe, S. aureus can act as an invasive pathogen with seriously devastating effects on its host’s health and wellbeing. A wide range of infections caused by this bacterium has been reported to affect diverse parts of the human body, including the skin, soft tissues and bones, as well as important organs like the heart, kidneys and lungs. Particularly, S. aureus is infamous for being a major causative agent of respiratory tract infections that may escalate up to necrotizing pneumonia. Due to its clinical relevance, this pathogen has been intensively studied for many years. Nonetheless, further research in this field is still needed, because of the high capacity of S. aureus to evolve drug resistance, its high genomic plasticity and adaptability and, not in the last place, the plethora of niches within the human body where it can thrive and survive. In this regard, there are still many uncertainties concerning the specific adaptations carried out by S. aureus during colonization and infection of the human body, the transition between both stages, and upon the invasion of different types of host cells. To shed more light on some of these adaptations, the research described in this thesis has employed in vitro models of infection that mimic particular conditions during the infectious process with special focus on the lung epithelium. The adaptations displayed by S. aureus were monitored using advanced proteomics. Furthermore, the analyses documented in this thesis included S. aureus strains with diverse backgrounds and epidemiology to take into account the genetic diversity encountered in this species.
Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades
(2019)
Plant-derived carbohydrates are an abundant renewable re- source. Transformation of carbohydrates into new products, in- cluding amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, bio- catalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step bio- catalysis was performed to functionalize d-galactose-contain- ing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from
Agaricus bisporus followed by the w-transaminase from Chro- mobacterium violaceum (Cvi-w-TA). Formation of 6-amino-6- deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino- 2-deoxy-6-aldo-d-galactose was confirmed by mass spectrome- try. The activity of Cvi-w-TA was highest towards 6-aldo-d-gal- actose, for which the highest yield of 6-amino-6-deoxy-d-galac- tose (67%) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6- aldo-d-galactose.
Long-term nationally representative caries data in the primary dentition are rare, but nonetheless central to assess needs in caries prevention and treatment. This study evaluated the prevalence and trends of caries levels in the primary dentition of 6- to 7-year-olds in Germany as a whole and its federal states individually. In 2016, employing a randomized cluster selection, 6- to 7-year-old first graders were included in the National German Oral Health Survey performed regularly since 1994/95. Children were examined by 482 calibrated dentists in all 17 German regions using the WHO criteria for the decayed, missing, and filled teeth (dmft) including the assessment of initial carious lesions (it). In total, 151,555 6- to 7-year-olds were examined. Caries prevalence in the primary dentition dropped from 65% in 1994 to 44% in 2016, while the mean caries experience dropped from 2.89 to 1.73 dmft (dt = 0.74, mt = 0.19, ft = 0.80). When initial lesions were included, the mean caries experience increased to idmft = 2.12 (it = 0.38). In 2016, 49.7% of the examined 6- to 7-year-olds were caries-free including initial lesions. The Care Index at the tooth level was 57.5%, and the Significant Caries Index was 4.84 dmft. Depending on the German region, the mean dmft varied considerably, ranging from 1.37 to 2.31. In conclusion, despite the overall caries decline in 6- to 7-year-olds in Germany, only minor caries reductions were observed over the last decade, with a still existing high proportion of untreated dental decay. This calls for more effective preventive and restorative efforts with focus on the primary dentition in Germany.
Changes in Interhemispheric Motor Connectivity Across the Lifespan: A Combined TMS and DTI Study
(2019)
Age-related decline in interhemispheric connectivity between motor areas has been reported with both transcranial magnetic stimulation (TMS) and diffusion tensor imaging (DTI) measurements. However, not all studies were able to confirm these findings, and previous studies did not apply structural (DTI) and functional (TMS) measurements within each individual appropriately. Here, we investigated age dependency of the ipsilateral silent period (ISP) and integrity of fibers in the corpus callosum as operationalized by fractional anisotrophy (FA), using TMS and DTI, respectively, in 20 participants between 19 and 72 years of age. We found age-dependent increase for ISP, and decrease of FA, both indicating a decrease in interhemispheric inhibition, with a negative association between FA and ISP for the dominant hemisphere (r = −0.39, p = 0.043). Our findings suggest that aging leads to decline of interhemispheric motor connectivity, as evidenced in both structural and functional parameters, which should be taken into account when interpreting disease- or medication-related changes.
The aim of the present dissertation was to investigate the biological and chemical potential of two European mushroom species: Fomitopsis betulina and Calvatia gigantea. For this purpose, different extracts of both fungi were tested for: antimicrobial, antifungal, cytotoxic, in vitro wound healing, and anti-adhesive properties. Bioassay-guided fractionation led to the isolation of bioactive compounds, altogether 20 compounds were isolated and identified. The compounds were obtained from the ethyl acetate extracts, they included triterpenes, sterols and aromatic compounds. The separated substances from both fungi were proved for biological activities, some of them showed antimicrobial and cytotoxic activities.
Due to a variety of plasma sources in terms of type of discharge, energy yield, working gas or geometric factors, it is recommended to standardize the study protocol by choosing a plasma source and easy access to rugged tumor surfaces as demonstrated by the CAP-plasma-jet. The intention of the trial shall be to optimize the plasma jet for tumor site capability and operating room implementation.
It makes sense to start clinical trials in plasma medicine with the treatment of head and neck squamous cell carcinoma patients of infected wounds and ulcerations.
CAP is able to reduce contamination of cancer ulcerations and the typical fetid odor that often accompanies head and neck cancer patients. The intention of the trial shall be to evaluate the efficiency of decontamination in head and neck cancer ulcerations in terms of pathogenic species, amount of reduction and reliability.
Standardize study protocol:
Phase I, clinical explorative single-arm, randomized, open, multicenter
Primary objective
Reduction of microbial burden of cancer ulcerations by application of CAP
Secondary objective:
Reduction of tumor following local CAP application
Inclusion:
20 Patients suffering from locally advanced oral cavity carcinoma with open tumor surfaces, treated with palliative intention and no more curative treatment options
Exclusion:
No wish for treatment, no compliance and understanding the protocol of the clinical study
Efficacy:
reduction of microbial burden; Documentation of visible changes by photography; Pathohistological and biochemical examination of specimen, taken from the tumor area and control areas
Procedure:
Plasma is applied for 1 minute per cm², spot area of 3 mm diameter distance between nozzle and tumor surface of 14 mm. 3 times/week with a break of 1 week followed by a repeated cycle for another week.
Conclusion:
The most important intention of the trial from the clinician’s point of view shall be to make CAP-treatment an effective and well-accepted addition to standard cancer therapy based upon EBM at least in palliative medicine.
Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 μM. Moreover, at 0.39 μM, nilotinib, in combination with 8 μg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations.
Determining the effect of a changing climate on tree growth will ultimately depend on our understanding of wood formation processes and how they can be affected by environmental conditions. In this context, monitoring intra-annual radial growth with high temporal resolution through point dendrometers has often been used. Another widespread approach is the microcoring method to follow xylem and phloem formation at the cellular level. Although both register the same biological process (secondary growth), given the limitations of each method, each delivers specific insights that can be combined to obtain a better picture of the process as a whole. To explore the potential of visualizing combined dendrometer and histological monitoring data and scrutinize intra-annual growth data on both dimensions (dendrometer → continuous; microcoring → discrete), we developed DevX (Dendrometer vs. Xylogenesis), a visualization application using the “Shiny” package in the R programming language. The interactive visualization allows the display of dendrometer curves and the overlay of commonly used growth model fits (Gompertz and Weibull) as well as the calculation of wood phenology estimates based on these fits (growth onset, growth cessation, and duration). Furthermore, the growth curves have interactive points to show the corresponding histological section, where the amount and development stage of the tissues at that particular time point can be observed. This allows to see the agreement of dendrometer derived phenology and the development status at the cellular level, and by this help disentangle shrinkage and swelling due to water uptake from actual radial growth. We present a case study with monitoring data for Acer pseudoplatanus L., Fagus sylvatica L., and Quercus robur L. trees growing in a mixed stand in northeastern Germany. The presented application is an example of the innovative and easy to access use of programming languages as basis for data visualization, and can be further used as a learning tool in the topic of wood formation and its ecology. Combining continuous dendrometer data with the discrete information from histological-sections provides a tool to identify active periods of wood formation from dendrometer series (calibrate) and explore monitoring datasets.
GPR68 (OGR1) belongs to the proton-sensing G protein-coupled receptors that are involved
in cellular adaptations to pH changes during tumour development. Although expression of GPR68
has been described in many tumour cell lines, little is known about its presence in human tumour
entities. We characterised the novel rabbit monoclonal anti-human GPR68 antibody 16H23L16
using various cell lines and tissue specimens. The antibody was then applied to a large series of
formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Antibody specificity
was demonstrated in a Western blot analysis of GPR68-expressing cells using specific siRNAs.
Immunocytochemical experiments revealed pH-dependent changes in subcellular localisation of the
receptor and internalisation after stimulation with lorazepam. In normal tissue, GPR68 was present in
glucagon-producing islet cells, neuroendocrine cells of the intestinal tract, gastric glands, granulocytes,
macrophages, muscle layers of arteries and arterioles, and capillaries. GPR68 was also expressed
in neuroendocrine tumours, where it may be a positive prognostic factor, in pheochromocytomas,
cervical adenocarcinomas, and endometrial cancer, as well as in paragangliomas, medullary thyroid
carcinomas, gastrointestinal stromal tumours, and pancreatic adenocarcinomas. Often, tumour
capillaries were also strongly GPR68-positive. The novel antibody 16H23L16 will be a valuable tool for
basic research and for identifying GPR68-expressing tumours during histopathological examinations.
The aim of the present study was to construct a biological age score reflecting one’s physiologic capability and aging condition with respect to tooth loss over 10 y. From the follow-up to the population-based Study of Health in Pomerania (i.e., SHIP-2), 2,049 participants were studied for their baseline biomarker measures 10 y before (i.e., in SHIP-0). Metabolic and periodontal data were regressed onto chronological age to construct a score designated as “biological age.” For either sex separately, the impact of this individualized score was used to predict tooth loss in the follow-up cohort in comparison with each participant’s chronological age. Outcome data after 10 y with respect to tooth loss, periodontitis, obesity, and inflammation were shown to be better for biologically younger subjects than as expected by their chronological age, whereas for the older subjects, data were worse. Especially for tooth loss, a striking increase was observed in subjects whose biological age at baseline appeared to be higher than their chronological age. Biological age produced significantly better tooth loss predictions than chronological age (P < 0.001). Areas under receiver operating characteristic curves for tooth loss of ≥3 teeth in men during follow-up were 0.811 and 0.745 for biological and chronological age, respectively. For women, these figures were 0.788 and 0.724. For total tooth loss, areas under the curve were 0.890 and 0.749 in men and 0.872 and 0.752 in women. Biological age combines various measures into a single score and allows identifying individuals at increased risk of tooth loss.
The purpose of this study was to compare the effect of culture on consumers’ attitudes toward Cause-Related marketing between Iran and Germany by answering the following questions:
A: What is consumer’s response concerning (1) skepticism toward CRM claim (2) attitude toward the CRM strategy, (3) attitude toward CRM brand personality, (4) attitude toward the CRM brand image and (5) CRM purchase intention (6) Warm glow? ; B: Do consumers respond differently to Cause-Related Marketing in Iran in comparison to Germany? C: Can cultural characteristics of the countries explain these differences? To answer the research questions, hypotheses were developed based on the literature which shape the research framework, in total containing 17 hypotheses. The data was gathered by questionnaire to make the research quantitative. By using convenience sampling, 564 responses were generated. The data was analysed by Structural Equation Modeling (SEM) and independent student t-test. Potential differences between Iran and Germany as well as moderation analysis are tested by critical ratio difference test as well as chi-square difference test using multiple- group analysis in AMOS. The results showed the importance of culture in applying CRM strategy. It can be said that CRM in a collectivistic culture like Iran can be successful as well as individualistic country like Germany. Although Iranian consumers were less familiar with this strategy, the benefits of CRM were similar in case of brand image and higher for purchase intention. The research found that emotions play a stronger role in Iran and it is more critical to evoke proper emotions by CRM campaign.
A successful colonization of different compartments of the human host requires multifactorial contacts between bacterial surface proteins and host factors. Extracellular matrix proteins and matricellular proteins such as thrombospondin-1 play a pivotal role as adhesive substrates to ensure a strong interaction with pathobionts like the Gram-positive Streptococcus pneumoniae and Staphylococcus aureus. The human glycoprotein thrombospondin-1 is a component of the extracellular matrix and is highly abundant in the bloodstream during bacteremia. Human platelets secrete thrombospondin-1, which is then acquired by invading pathogens to facilitate colonization and immune evasion. Gram-positive bacteria express a broad spectrum of surface-exposed proteins, some of which also recognize thrombospondin-1. This review highlights the importance of thrombospondin-1 as an adhesion substrate to facilitate colonization, and we summarize the variety of thrombospondin-1-binding proteins of S. pneumoniae and S. aureus.
Type I interferonopathies cover a phenotypically heterogeneous group of rare genetic diseases including the recently described proteasome-associated autoinflammatory syndromes (PRAAS). By definition, PRAAS are caused by inherited and/or de novo loss-of-function mutations in genes encoding proteasome subunits such as PSMB8, PSMB9, PSMB7, PSMA3, or proteasome assembly factors including POMP and PSMG2, respectively. Disruption of any of these subunits results in perturbed intracellular protein homeostasis including accumulation of ubiquitinated proteins which is accompanied by a type I interferon (IFN) signature. The observation that, similarly to pathogens, proteasome dysfunctions are potent type I IFN inducers is quite unexpected and, up to now, the underlying molecular mechanisms of this process remain largely unknown. One promising candidate for triggering type I IFN under sterile conditions is the unfolded protein response (UPR) which is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) (also referred to as ER stress). The recent observation that the UPR is engaged in subjects carrying POMP mutations strongly suggests its possible implication in the cause-and- effect relationship between proteasome impairment and interferonopathy onset. The purpose of this present review is therefore to discuss the possible role of the UPR in the pathogenesis of PRAAS. We will particularly focus on pathways initiated by the four ER-membrane proteins ATF6, PERK, IRE1-a, and TCF11/Nrf1 which undergo activation under proteasome inhibition. An overview of the current understanding of the mechanisms and potential cross-talk between the UPR and inflammatory signaling casacades is provided to convey a more integrated picture of the pathophysiology of PRAAS and shed light on potential biomarkers and therapeutic targets.
Type I interferonopathies cover a phenotypically heterogeneous group of rare genetic diseases including the recently described proteasome-associated autoinflammatory syndromes (PRAAS). By definition, PRAAS are caused by inherited and/or de novo loss-of-function mutations in genes encoding proteasome subunits such as PSMB8, PSMB9, PSMB7, PSMA3, or proteasome assembly factors including POMP and PSMG2, respectively. Disruption of any of these subunits results in perturbed intracellular protein homeostasis including accumulation of ubiquitinated proteins which is accompanied by a type I interferon (IFN) signature. The observation that, similarly to pathogens, proteasome dysfunctions are potent type I IFN inducers is quite unexpected and, up to now, the underlying molecular mechanisms of this process remain largely unknown. One promising candidate for triggering type I IFN under sterile conditions is the unfolded protein response (UPR) which is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) (also referred to as ER stress). The recent observation that the UPR is engaged in subjects carrying POMP mutations strongly suggests its possible implication in the cause-and-effect relationship between proteasome impairment and interferonopathy onset. The purpose of this present review is therefore to discuss the possible role of the UPR in the pathogenesis of PRAAS. We will particularly focus on pathways initiated by the four ER-membrane proteins ATF6, PERK, IRE1-α, and TCF11/Nrf1 which undergo activation under proteasome inhibition. An overview of the current understanding of the mechanisms and potential cross-talk between the UPR and inflammatory signaling casacades is provided to convey a more integrated picture of the pathophysiology of PRAAS and shed light on potential biomarkers and therapeutic targets.
For decades, evolutionary biologists have sought to understand the evolution of individual behaviour, physiology and ecology allowing organisms to cope to environmental change. One of the main challenges of current climate change is the unprecedent rate of temperature increase, as well as the increased occurence of extreme heat events. Interindividual response variability opens a whole new area of opportunities to understand how individual phenotypic traits are linked to individual response differences. In colour polymorphic species, colour honestly reflects an individual’s life-history strategy, and each morph may, therefore, represent an alternative life-history strategy. As such, colour polymorphic species, such as the Gouldian finch (Erythrura gouldiae), may be good models to assess how different strategies between morphs are linked to their espective responses to environmental variations. However, polymorphic species have mainly been disregarded for that purpose. In this context, the main aim of this thesis was to understand how the two morphs of the Gouldian finch respond through phenotypic plasticity to simulated heatwaves reaching thermocritical temperatures, and whether such differential responses may help to identify a ‘winner’ and a ‘loser’ morph in the light of climate change. To address these issues, we used an integrative approach including measurements of behavioural (Study 1), physiological (Study 2), and reproductive (Study 3) parameters. The novelty of our approach was to assess the immediate behavioural and physiological response variation of individuals of the two morphs longitudinally across different thermal conditions, as well as the postponed effects of this thermocritical heatwave exposure on their reproductive performance. In this study, although the behavioural responses generally did not differ between morphs or according to temperature intensity, the physiological and reproductive parameters differed in response to morph and temperature intensity. Blackheaded females, in particular, seem highly sensitive to thermocritical heatwaves, as they exhibited decreased body mass and increased oxidative damage during the thermocritical heatwaves, and advanced breeding initiation after these conditions, whereas these variables remained mostly unaffected in black-headed males and red-headed individuals. However, despite some response differences between morphs, both invested similarly in reproduction following intense heatwaves, and the offspring of both morphs were similarly affected. Based on these results, no morph therefore seems to appear more disadvantaged than the other following an intense heatwave, and red- and black-headed Gouldian finches may both be considered as climate stress ‘losers’.
Extracts from the leaves and flowers of Crataegus spp. (i.e., hawthorn species) have been traditionally used with documented preclinical and clinical activities in cardiovascular medicine. Based on reported positive effects on heart muscle after ischemic injury and the overall cardioprotective profile, the present study addressed potential contributions of Crataegus extracts to cardiopoietic differentiation from stem cells. The quantified Crataegus extract WS®1442 stimulated cardiomyogenesis from murine and human embryonic stem cells (ESCs). Mechanistically, this effect was found to be induced by promoting differentiation of cardiovascular progenitor cell populations but not by proliferation. Bioassay-guided fractionation, phytochemical and analytical profiling suggested high-molecular weight ingredients as the active principle with at least part of the activity due to oligomeric procyanidines (OPCs) with a degree of polymerization between 3 and 6 (DP3–6). Transcriptome profiling in mESCs suggested two main, plausible mechanisms: These were early, stress-associated cellular events along with the modulation of distinct developmental pathways, including the upregulation of brain-derived neurotrophic factor (BDNF) and retinoic acid as well as the inhibition of transforming growth factor β/bone morphogenetic protein (TGFβ/BMP) and fibroblast growth factor (FGF) signaling. In addition, WS®1442 stimulated angiogenesis ex vivo in Sca-1+ progenitor cells from adult mice hearts. These in vitro data provide evidence for a differentiation promoting activity of WS®1442 on distinct cardiovascular stem/progenitor cells that could be valuable for therapeutic heart regeneration after myocardial infarction. However, the in vivo relevance of this new pharmacological activity of Crataegus spp. remains to be investigated and active ingredients from bioactive fractions will have to be further characterized.
Deciphering the influence of Streptococcus pneumoniae global regulators on fitness and virulence
(2019)
Streptococcus pneumoniae (S. pneumoniae; the pneumococcus) is a Gram-positive, aerotolerant, and opportunistic bacteria, which colonizes the upper respiratory tract of human. S. pneumoniae can further migrate to other sterile parts of the body, and causes local as well as fatal infections like, pneumonia, septicaemia and meningitis. Due to incomplete amino acid pathways, pneumococci are auxotrophic for eight different amino acids including glutamine and arginine. The pneumococcus has adapted to the various host environmental conditions and a number of systems are dedicated for the transport and utilization of nutrients such as monosaccharides, amino acids and oligopeptides.
In this study the amino acid metabolism was characterised by 15N-isotopologue profiling in two different pneumococcal strains, D39 and TIGR4. Efficient uptake of a labelled amino acids mixture of 15N-labelled amino acids showed that S. pneumoniae has a preference for the amino acids transport instead of a de novo biosynthesis. It is known that glutamine (Gln) serves as main nitrogen source for S. pneumoniae. The 15N-labelled Gln used in this study demonstrated an efficient 15N-enrichment of Glu, Ala, Pro and Thr. Minor enrichment was seen for the amino acids Asp, Ile, Leu, Phe, Tyr, and Val. Remarkably, labelled Gly and Ser could be determined in strain TIGR4, whereas for strain D39 these two labelled amino acids were not detected. This confirms earlier studies with 13C-labelled glucose, which showed the biosynthesis of Ser out of Gly. Strain TIGR4 was able to grow in chemically-defined medium depleted of Gly confirming that Gly can be synthesized out of serine by the action of the enzyme serine hydroxymethyltransferase (SHMT).
The transcriptional regulator GlnR controls the Gln and Glu metabolism in S. pneumoniae. Hence, the impact of the repressor GlnR on amino acids metabolism was also studied. An increased 15N-enrichment was determined for Ala and Glu in both used pneumococcal strains, while an increased level of Pro was only measured in the isogenic glnR-mutant of non-encapsulated D39.
Arginine can also serve as nitrogen source in strain TIGR4. The arginine deiminase system metabolizes Arg into ornithine, carbamoyl phosphate and CO2 by the generation of 1 ATP and 2 mol NH3. Because of the truncation of the arcA gene strain D39 lacks arginine deiminase activity and has thus no functional ADS system. When 15N-Arg was added for growth, only in strain TIGR4, thirteen (13) labelled amino acids were detected with the highest enrichment for Ala, Glu and Thr. Genes coding for the enzymes of the arginine metabolism and for arginine uptake are regulated by the activator ArgR2 in strain TIGR4. Inactivation of ArgR2 was not accompanied by an enrichment of labelled amino acids, when the argR2-mutant was grown with 15N-labelled Arg indicative of the important role of ArgR2.
The bicistronic operon arcDT encoding the arginine/ornithine transporter ArcD and a putative peptidase ArcT belong to the peptidase family M20. The in silico comparison of structures revealed a significant homology of ArcT to PepV of L. delbrueckii and to Sapep of S. aureus known as carboxypeptidase. ArcT was heterologously expressed in E. coli and purified under reducing conditions. An enzymatic reaction was established and several dipeptides like Ala-Arg, Arg-Ala, and Ala-Asp were used as substrates. In addition, the dependency on divalent cations was analysed. Cleavage of the dipeptide Ala-Arg was detected in the presence of Mn2+ as cofactor under reducing conditions. Reduced peptidase activity was observed when Zn2+ was added. No cleavage of the tripeptide Ala-Ala-Arg could be shown indicating that ArcT acts as dipeptidase with the preference to the Arg residue at the C-terminal end.
Bacterial meningitis caused by S. pneumoniae was studied in an in vivo proteomic analysis. In a mouse meningitis model S. pneumoniae was isolated from the cerebrospinal fluid (CSF) by a filter extraction step. The MS analysis identified AliB and ComDE only from CSF isolated pneumococci indicating that these proteins are expressed under infection conditions. Mice infected with D39 wild-type and isogenic aliB, comDE and aliB-comDE double knockout mutants showed significantly less number of pleocytosis in the CSF and lower bacterial load in the blood compared to the wild-type. The results indicate that AliB and ComDE play an important role during meningitis.
Phenotypic characterization was carried out to identify differences between the wild-type and the aliB-, comDE- and aliB-comDE double mutants. Oxidative stress conditions were induced by the application of hydrogen peroxide or paraquat during growth in a chemically-defined medium similar to the CSF. No alteration in growth and survival of these mutants compared to the wild-type was observed suggesting that oxygen radicals play not an important role during the progression of meningitis. In addition, no differences of AliB expression was detected in the ComDE deficient D39. No impact of aliB and comDE-mutation on the expression of different virulence factors like pneumolysin or proteins involved in capsular biosynthesis was detected.
In vitro proteome analysis was performed to compare the wild-type to the AliB, and ComDE deficient D39 in the early and mid logarithmic growth phase. More than 70 % of theoretically expressed proteins were identified. In the aliB-mutant 33 proteins were differentally expressed in the early growth phase and 50 proteins differed during mid log growth. For the comDE mutant 24 and 11 proteins differed in expression in these two growth phases. Interestingly, high level of AliA expression was identified in all samples. The aliB-mutant had a decreased abundance of the proteins resembling an oligopeptide ABC transporter (AmiA, AmiC, AmiD, AmiE). In addition, another ABC transporter for iron transport encoded by spd_1607 to spd_ 1610 was higher expressed in the aliB-mutant. In the ComDE deficient mutant lower abundance of the Ami transporter sytem was identified. An increased abundance of proteins involved in the pyrimidine metabolism (PyrF, PyrE, PyrDb, PyrB and PyrR) was recognized only in the early growth phase of the comDE-mutant. These analyses demonstrate the marginal changes in protein synthesis during growth of S. pneumoniae. These studies demonstrated the adaptation of the proteome of S. pneumoniae to different growth conditions and the impact of regulatory proteins on the availability of carbon and nitrogen sources.
Matrix-product-state based methods, in particular the density-matrix renormalization group, are used to numerically investigate several one-dimensional systems, focusing on models with symmetry-protected topological phases that generalize the spin-1 Haldane chain. In the first part, ground state properties such as topological order parameters and the criticality at quantum phase transitions are studied.
The second part deals with dynamic properties of spin chains. Using time-dependent matrix-product-state calculations, the dynamic structure factor, and the transport properties of contacted spin chains are analyzed.
Vielfältig lässt sich die Bedeutung von Vorstrafen zeigen; jenseits ihrer gesellschaftlichen Wirkungen beschäftigen sie Straf-, Arbeits- und Zivilgerichte. Dieser Beitrag untersucht, welche Implikationen die Registrierung einer Verurteilung hat. Dabei soll aus strafrechtlicher wie auch aus grundrechtlicher Perspektive belegt werden, dass der Staat als Autor der Verurteilung alle ihre Folgen bedenken und bestimmten Konsequenzen gegebenenfalls entgegenwirken muss. Zur Einstimmung sollen fünf Fallskizzen Schlaglichter auf den Problemkreis werfen:
Determination of the Pathological Features of NPC1 Variants in a Cellular Complementation Test
(2019)
Niemann-Pick Type C (NP-C) is a rare disorder of lipid metabolism caused by mutations
within the NPC1 and NPC2 genes. NP-C is a neurovisceral disease leading to a heterogeneous,
multisystemic spectrum of symptoms in those affected. Until now, there is no investigative tool to
demonstrate the significance of single variants within the NPC genes. Hence, the aim of the study
was to establish a test that allows for an objective assessment of the pathological potential of NPC1
gene variants. Chinese hamster ovary cells defective in the NPC1 gene accumulate cholesterol in
lysosomal storage organelles. The cells were transfected with NPC1-GFP plasmid vectors carrying
distinct sequence variants. Filipin staining was used to test for complementation of the phenotype.
The known variant p.Ile1061Thr showed a significantly impaired cholesterol clearance after 12 and
24 h compared to the wild type. Among the investigated variants, p.Ser954Leu and p.Glu1273Lys
showed decelerated cholesterol clearance as well. The remaining variants p.Gln60His, p.Val494Met,
and p.Ile787Val showed a cholesterol clearance indistinguishable from wild type. Further, p.Ile1061Thr
acquired an enhanced clearance ability upon 25-hydroxycholesterol treatment. We conclude that the
variants that caused an abnormal clearance phenotype are highly likely to be of clinical relevance.
Moreover, we present a system that can be utilized to screen for new drugs.
Oils and fats from natural origin are sustainable sources for a broad range of economically relevant products in food, feed, fuel, oleochemical, and cosmetic industries. Thereby, a huge variety of lipids or lipid-derived products exist which distinguish themselves by their unique physical properties making them suitable for their individual applications. To obtain such functional lipids in an environmentally friendly manner, enzymes can be employed. In that context, lipases have been proven to be valuable biocatalysts in lipid modification, which are broadly applied in industry. Even though they have been implemented successfully in the dairy, baking, and detergent industries, there is an increasing demand for the expansion of their utilization. New technologies like protein engineering and the implementation of process development are employed in solving this task. Within the enzymes in lipid modification, lipases are the most applied catalysts and in this thesis their utilization was expanded successfully to the implementation of novel separation processes and the production of improved drug delivery matrices.
From a biopharmaceutical point of view, poor oral bioavailability of a drug is one of the greatest challenges for formulation scientists. The majority of new chemical entities (NCEs) are weakly basic drugs. Consequently, these drugs exhibit pH-dependent solubility, being higher under acidic conditions in the fasted stomach and lower under neutral conditions in the small intestine, the main site of drug absorption. For theses compounds, pH-dependent precipitation testing represents a key parameter during early development stages. In this development phase, the amount of drug available is limited, and fast and detailed investigations of simulated drug solubility are desired. Therefore, an automated small-scale in vitro transfer model, simulating drug transfer from a donor (stomach; simulated gastric fluid, SGF pH 2.0) to an acceptor (small intestine; fasted state simulated intestinal fluid, FaSSIF-phosphate pH 6.5) compartment, has been developed. In contrast to the originally published transfer model, this model allowed a detailed investigation of drug supersaturation and precipitation in a small-scale, feasible for pre-formulation purposes, through miniaturization and automation in an in-line analytical set-up. In-line drug concentration analysis in turbid samples, due to pH-dependent drug precipitation, was achieved by a pre-filtration step, the use of flow-through cuvettes and the application of UV derivative spectroscopy. Compared to the common procedure of manual sampling followed by HPLC-UV analysis for concentration determination, the supersaturation and precipitation of the model drug ketoconazole was more accurately captured by the newly developed in-line analytical set-up. In addition, the newly developed small-scale model was compared to a USP II-based transfer model, representing an established scale of the transfer model. Using a physiologically relevant simulated gastric emptying rate of 5 min half-time, supersaturation and precipitation of the model drugs ketoconazole and a new chemical entity from the research laboratories of Merck Healthcare KGaA, MSC-A, were observed to be highly comparable. Following miniaturization and automation, the developed small-scale model was used to establish eight physiologically relevant test-sets. These test-sets were used to assess the impact of gastrointestinal (GI) variability, i.e. gastric pH, gastric emptying, and GI fluid volumes, on supersaturation and precipitation of two weakly basic model compounds, ketoconazole and MSC-A. The experiments revealed that variations in all GI parameters investigated affected the in vitro supersaturation and precipitation of ketoconazole. For example, faster gastric emptying yielded higher supersaturation and faster precipitation of ketoconazole. In contrast, MSC-A supersaturation and precipitation was only affected by variability in gastric pH. Consequently, the effect of varying GI parameters was found to be drug-specific. Elevated gastric pH, as it can result from co-medication with acid-reducing drugs, resulted in lower degrees of supersaturation for both substances. For ketoconazole, this result is in agreement with the observation that the oral bioavailability of ketoconazole is lowered when proton pump inhibitors are co-administered. In addition to the physiological considerations, the small-scale model developed herein was used to establish an in vitro screening assay for precipitation inhibitors (PIs). The use of PIs represents one option of reducing the process of pH-dependent drug precipitation during simulated GI transfer. For this purpose, ketoconazole and five orally administered kinase inhibitors (i.e. pazopanib, gefitinib, lapatinib, vemurafenib, and MSC-A) were analyzed with and without the polymeric PIs HPMC, HPMCAS, PVPK17 and K30, PEG6000, and Soluplus® in the small-scale transfer model. This screening revealed that at least one effective PI could be identified for each model drug. Moreover, HPMCAS and Soluplus® were the most effective PIs. Another outcome of these studies was that gefitinib expressed highly variable amorphous precipitation which was confirmed by powder X-ray diffraction (PXRD). During the transfer model experiments, the intermediate amorphous and supersaturated state of gefitinib was stabilized using HPMCAS and Soluplus®. After the polymer investigations, the impact of the buffer species in the simulated intestinal medium on drug supersaturation and precipitation was assessed. Since luminal fluids are mainly buffered by hydrogen carbonate ions, a USP II-based transfer model equipped with the pHysio-grad® device was proposed. This allowed the use of a complex bicarbonate buffer for the preparation of FaSSIF-bicarbonate in an in vitro transfer model. Results of transfer model experiments using standard phosphate-based FaSSIF and a more physiologically relevant bicarbonate-based FaSSIF were compared. Therefore, ketoconazole, pazopanib, and lapatinib were analyzed with and without the precipitation inhibitor HPMCAS. While HPMCAS was found to be an effective precipitation inhibitor for all drugs in FaSSIF-phosphate, the effect in FaSSIF-bicarbonate was much less pronounced. Additionally, performed rat PK studies revealed that HPMCAS did not increase the exposure of any of the model compounds significantly, indicating that the transfer model employing bicarbonate-buffered FaSSIF was more predictive compared to the model using phosphate-buffered FaSSIF. The in vitro and in vivo results of these studies demonstrated that the supersaturation precipitation of poorly soluble weakly basic drugs can be significantly affected by GI variability. Furthermore, the use of the automated small-scale transfer model enabled the identification of effective precipitation inhibitors for the model drugs involved in these studies. At the same time the buffer species has been observed to be especially important to reliably predict the in vivo solubility/dissolution behavior of HPMCAS and the weakly basic model drugs.
Background: Huntington’s disease (HD) is a progressive neurodegenerative disorder. The striatum is one of the first brain regions that show detectable atrophy in HD. Previous studies using functional magnetic resonance imaging (fMRI) at 3 tesla (3 T) revealed reduced functional connectivity between striatum and motor cortex in the prodromal period of HD. Neuroanatomical and neurophysiological studies have suggested segregated corticostriatal pathways with distinct loops involving different cortical regions, which may be investigated using fMRI at an ultra-high field (7 T) with enhanced sensitivity compared to lower fields. Objectives: We performed fMRI at 7 T to assess functional connectivity between the striatum and several chosen cortical areas including the motor and prefrontal cortex, in order to better understand brain changes in the striatum-cortical pathways. Method: 13 manifest subjects (age 51 ± 13 years, cytosine-adenine-guanine [CAG] repeat 45 ± 5, Unified Huntington’s Disease Rating Scale [UHDRS] motor score 32 ± 17), 8 subjects in the close-to-onset premanifest period (age 38 ± 10 years, CAG repeat 44 ± 2, UHDRS motor score 8 ± 2), 11 subjects in the far-from-onset premanifest period (age 38 ± 11 years, CAG repeat 42 ± 2, UHDRS motor score 1 ± 2), and 16 healthy controls (age 44 ± 15 years) were studied. The functional connectivity between the striatum and several cortical areas was measured by resting state fMRI at 7 T and analyzed in all participants. Results: Compared to controls, functional connectivity between striatum and premotor area, supplementary motor area, inferior frontal as well as middle frontal regions was altered in HD (all p values <0.001). Specifically, decreased striatum-motor connectivity but increased striatum-prefrontal connectivity were found in premanifest HD subjects. Altered functional connectivity correlated consistently with genetic burden, but not with clinical scores. Conclusions: Differential changes in functional connectivity of striatum-prefrontal and striatum-motor circuits can be found in early and premanifest HD. This may imply a compensatory mechanism, where additional cortical regions are recruited to subserve functions that have been impaired due to HD pathology. Our results suggest the potential value of functional connectivity as a marker for future clinical trials in HD.
Background: Huntington’s disease (HD) is a progressive neurodegenerative disorder. The striatum is one of the first brain regions that show detectable atrophy in HD. Previous studies using functional magnetic resonance imaging (fMRI) at 3 tesla (3 T) revealed reduced functional connectivity between striatum and motor cortex in the prodromal period of HD. Neuroanatomical and neurophysiological studies have suggested segregated corticostriatal pathways with distinct loops involving different cortical regions, which may be investigated using fMRI at an ultra-high field (7 T) with enhanced sensitivity compared to lower fields. Objectives: We performed fMRI at 7 T to assess functional connectivity between the striatum and several chosen cortical areas including the motor and prefrontal cortex, in order to better understand brain changes in the striatum-cortical pathways. Method: 13 manifest subjects (age 51 ± 13 years, cytosine-adenine-guanine [CAG] repeat 45 ± 5, Unified Huntington’s Disease Rating Scale [UHDRS] motor score 32 ± 17), 8 subjects in the close-to-onset premanifest period (age 38 ± 10 years, CAG repeat 44 ± 2, UHDRS motor score 8 ± 2), 11 subjects in the far-from-onset premanifest period (age 38 ± 11 years, CAG repeat 42 ± 2, UHDRS motor score 1 ± 2), and 16 healthy controls (age 44 ± 15 years) were studied. The functional connectivity between the striatum and several cortical areas was measured by resting state fMRI at 7 T and analyzed in all participants. Results: Compared to controls, functional connectivity between striatum and premotor area, supplementary motor area, inferior frontal as well as middle frontal regions was altered in HD (all p values <0.001). Specifically, decreased striatum-motor connectivity but increased striatum-prefrontal connectivity were found in premanifest HD subjects. Altered functional connectivity correlated consistently with genetic burden, but not with clinical scores. Conclusions: Differential changes in functional connectivity of striatum-prefrontal and striatum-motor circuits can be found in early and premanifest HD. This may imply a compensatory mechanism, where additional cortical regions are recruited to subserve functions that have been impaired due to HD pathology. Our results suggest the potential value of functional connectivity as a marker for future clinical trials in HD.
Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid
(CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance
in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a
proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less
flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.
Clostridioides difficile is an intestinal human pathogen that uses the opportunity of a depleted microbiota to cause an infection. It is known, that the composition of the intestinal bile acid cocktail has a great impact on the susceptibility toward a C. difficile infection. However, the specific response of growing C. difficile cells to diverse bile acids on the molecular level has not been described yet. In this study, we recorded proteome signatures of shock and long-term (LT) stress with the four main bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). A general overlapping response to all tested bile acids could be determined particularly in shock experiments which appears plausible in the light of their common steroid structure. However, during LT stress several proteins showed an altered abundance in the presence of only a single or a few of the bile acids indicating the existence of specific adaptation mechanisms. Our results point at a differential induction of the groEL and dnaKJgrpE chaperone systems, both belonging to the class I heat shock genes. Additionally, central metabolic pathways involving butyrate fermentation and the reductive Stickland fermentation of leucine were effected, although CA caused a proteome signature different from the other three bile acids. Furthermore, quantitative proteomics revealed a loss of flagellar proteins in LT stress with LCA. The absence of flagella could be substantiated by electron microscopy which also indicated less flagellated cells in the presence of DCA and CDCA and no influence on flagella formation by CA. Our data break down the bile acid stress response of C. difficile into a general and a specific adaptation. The latter cannot simply be divided into a response to primary and secondary bile acids, but rather reflects a complex and variable adaptation process enabling C. difficile to survive and to cause an infection in the intestinal tract.
The present work focusses on the mosquito populations of two zoological gardens in Germany with the aim to better understand mosquito biology of native species and to contribute to a greater awareness of mosquito and mosquito-borne disease agent surveillance in zoos. For this purpose, data on species composition, blood meal patterns and mosquito-borne pathogens were analysed. The investigated zoological gardens differed not only in their sizes and animal stocks, but also in their surrounding environments. The 160 ha Tierpark Berlin is located in a densely populated urban area, while the 15 ha Zoological Garden Eberswalde is surrounded by forest.
To gain an overview about the mosquito fauna of both zoos, adult specimens were caught by aspirating and EVS-trapping during the 2016 season. In addition, larval stages were collected from their breeding sites located in the zoo areas. In total, 2,257 mosquitoes were sampled, belonging to 20 taxa. Seasonal differences between the zoos were documented, both in terms of species composition and the relative abundance of mosquito species collected. As the studied zoos were located in the same climatic region and both locations provided similar breeding sites, differences in species composition were attributed to the entry of mosquitoes from surrounding landscapes. Influencing factors could have been the different sizes of the zoos and variations in the potential host animal populations.
According to the vector potential of most frequently collected taxa in the Zoological Garden Eberswalde (Annulipes Group, Culiseta annulata), TAHV, USUV, WNV, filariae and avian malaria parasites appear to have the highest risk of being transmitted at this location. In the Tierpark Berlin, Aedes vexans was the most frequently collected mosquito species, suggesting a theoretical risk for the transmission of a broader spectrum of pathogens due to covered vector competences. Pathogens such as BATV, SINV, TAHV, USUV and filarial worms could be of major importance regarding transmission risk to zoo animals, as they had previously been found to circulate Germany. In addition, avian malaria parasites represent a considerable risk for susceptible exotic bird species in Berlin.
Since the blood-feeding behaviour of vector-competent mosquito species has a major influence on the transmission of a mosquito-associated pathogen, the analysis of blood meal patterns is crucial to better understand vector-pathogen cycles. Therefore, blood meals of blood-fed mosquitoes caught in 2016 and 2017 by aspirating and EVS-trapping in the Tierpark Berlin and the Zoological Garden Eberswalde were analysed. The aim was to investigate to what extent native mosquito species accept exotic zoo animals, wild native animals and humans as blood hosts. In addition, it was examined whether the collected species are generalists or specialists when selecting vertebrates for blood feeding.
A total of 405 blood-fed mosquitoes from 16 taxa were collected. The genetic analysis of blood meals identified 56 host species, which – in addition to humans – mainly originated from mammals of the zoo animal populations. In agreement with the previous study on the mosquito fauna of the Tierpark Berlin and the Zoological Garden Eberswalde, the analysis of blood meals also showed differences between the two zoos. In the smaller Zoological Garden Eberswalde, a higher number of blood-fed mosquitoes was collected than in the Tierpark Berlin, probably caused by a higher host density in Eberswalde, which may have led to an overall higher mosquito density. However, no differences between both zoos were observed with respect to the blood feeding behaviour of the analysed mosquito species: Mosquitoes of both locations were rather generalistic, although species could be grouped according their blood meals into 'amphibian', 'non-human mammal' and, ‘non-human mammal and human' feeding species. The more random selection of hosts could indicate a low probability of effective pathogen transmission by applying the 'dilution effect'. Notwithstanding, since wild animals have also been accepted as hosts, pathogen transmission by bridge vectors from one vertebrate group to another could be relevant in the sampled zoos.
Adult mosquito specimens collected in 2016 and 2017 were screened for filarial nematodes, avian Haemosporidia and mosquito-borne viruses. Dirofilaria repens was detected in a mosquito from the Zoological Garden Eberswalde. Mosquitoes from Berlin and Eberswalde were tested positive for the nematode species S. tundra. Sindbis virus was found in a mosquito pool collected in the Tierpark Berlin, while no mosquito-associated viruses were detected in specimens collected in the Zoological Garden Eberswalde. Mosquitoes from both zoos were positive for the haemosporidian parasites Haemoproteus sp. and Leucocytozoon sp., and one documentation was made for avian Plasmodium sp. in the Tierpark Berlin.
The identified pathogens have the potential to cause disease in captive and wild animals, and some of them also in humans. Most of the mosquitoes tested positive had been collected in July, suggesting a high infection risk during this month. Since most pathogen detections were made from species belonging to the Cx. pipiens complex, species of this complex seem to be most relevant in the studied zoos when it comes to mosquito-borne pathogen transmission. Although mosquitoes are no proven vectors of most of the avian malaria parasite genera found, evidence for Haemoproteus sp. and Leucozytozoon sp. demonstrated a high prevalence of avian malaria parasites in the zoos.
In summary, the results of the three studies indicate regional differences both in the mosquito species composition and in the occurrence of mosquito-borne pathogens. However, no differences were found between the mosquito communities of both zoos concerning their blood feeding behaviour, suggesting that the general behaviour of the insects is location-independent.
Several potential disease agents were found in the collected mosquitoes, although not at high abundances. Whether these pathogens were found by chance in the two zoos or whether the particular zoo environment is a hot spot of arthropod-borne pathogens cannot be determined with the studies conducted. Nonetheless, it seems clear that zoological gardens are attractive to mosquito females not only in their search for breeding sites, but also when looking for blood hosts and places for mating or resting. These advantageous conditions also attract mosquito species that have their larval habitats outside the zoological gardens, which is why elimination of breeding sites on the zoo premises alone will not necessarily keep away all mosquitoes.
A closer collaboration between zoological gardens and entomologists could be beneficial for both. Zoo officials could benefit from being able to identify potential arthropod vectors on the zoo grounds and receiving information on circulating arthropod-borne disease agents, as well as on the animal species susceptible to those. For entomologists, zoological gardens are ideal research locations, as they provide an environment with a high diversity of habitats and potential blood hosts for haematophagous arthropods in a confined space.
Studying mosquito biology will become even more significant in the future, since in a world that is getting smaller, both potential vectors and pathogens are regularly introduced into areas where they did not occur before. Therefore, it would be desirable if more studies targeting ecological as well as infectiological aspects of vector species in zoological gardens in Germany were carried out.
Species persistence in the face of rapidly progressing environmental change requires adaptive responses that allow organisms to either cope with the novel conditions in their habitat or to follow their environmental niche in space. A poleward range shift due to global warming induced habitat loss in the south has been predicted for the lesser horseshoe bat, Rhinolophus hipposideros. Theoretical as well as numerous empirical studies link range expansion success to increased dispersal and reproduction rates due to spatial sorting and r-selection resulting from low population densities at the expansion front. R. hipposideros females however are highly philopatric and the species’ life history reflects a K- rather than an r-strategy, encompassing a long life span and limited individual annual reproductive output. I therefore investigated if adaptations in these traits determining range expansion success (dispersal and reproduction) can be observed in this bat species of high conservation concern. Genetic diversity presents a critical factor for adaptive responses to global change, both for range expansion and for coping with novel environmental conditions. I hence explored the genetic diversity levels of European R. hipposideros leading edge populations and their drivers for an assessment of these populations’ evolutionary potential and the development of conservation recommendations.
Comparing range expansion traits between an expanding R. hipposideros metapopulation in Germany and a non-expanding one in France revealed that range expansion was associated with an increase in juvenile survival and fecundity, and no decrease in adult survival. These results demonstrate than an increase in reproduction and growth rates is generally possible in R. hipposideros, indicating a potential adaptation (sensu lato) to range expansion. A positive correlation between adult and juvenile survival in the expanding metapopulation suggests higher resource acquisition in the expanding metapopulation, giving rise to the question if the observed demographic changes have a genetic basis or if they are rather induced by differences in environmental conditions between the two metapopulations. Long-term range expansion success requires adaptive evolutionary changes. The relative contribution of the former and that of undirected changes resulting e.g. from differences in resource availability therefore will have to be investigated in more detail in the future to allow predictions about range expansion dynamics in R. hipposideros.
The number of individuals within a radius of approximately 60 to 90 km around a population (as a measure of connectivity) was identified as the main positive driver of the studied populations’ genetic diversity. Overall genetic diversity levels in German R. hipposideros populations were found to be reduced compared to populations in France as a legacy of demographic bottlenecks resulting from severe population declines in the mid-20th century. This finding is alarming as future range expansion can be expected to entail a further decrease in genetic diversity. The resulting loss of genetic diversity can be expected to be particularly strong in R. hipposideros due to the detected dependence of genetic diversity on connectivity, because range expansion often results in small and patchy populations.
Protecting and ideally re-installing genetic diversity in R. hipposideros leading edge populations therefore presents a conservation goal of utmost importance. To achieve this endeavour, conservation efforts should target the protection of extensive networks of well-connected populations. Geographical concentration of individuals should be avoided and populations in key locations that connect clusters must be protected particularly well to prevent populations from becoming isolated. Continuous, regular monitoring of population trends is also important for a quick registration of disturbances or threats, and the subsequent rapid development of countermeasures to preclude further demographic declines.
The reduced levels of genetic diversity in the German metapopulation precluded a reliable quantification of dispersal rates due to the reduced power of discrimination between individuals. While ongoing re-colonization and the establishment of new maternity colonies provide evidence for increased dispersal in the expanding metapopulation, evaluating the expected range expansion velocity of R. hipposideros in relation to the estimated velocity of global warming induced habitat loss will require the confirmation of the existing preliminary dispersal data by employing more genetic markers.
Drug-induced activation of integrin alpha IIb beta 3 leads to minor localized structural changes
(2019)
Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbβ3. Although the dramatic rearrangement of the overall structure of αIIbβ3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbβ3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism pectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbβ3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbβ3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.
Duckweeds include the world's smallest and fastest growing flowering plants that have the capacity to produce huge biomass with a broad range of potential applications like production of feed and food, biofuel and biogas. In order to achieve optimal and sustainable commercial system, it is necessary that suitable species and clones of duckweeds be identified and selected based on appropriate strategies. However, a high degree of reduction in their structural complexity poses serious problems in identification of closely related species of duckweeds, on a morphological basis. Use of molecular taxonomic tools is the present solution. The state of the art of molecular taxonomy of all the five genera of duckweeds (Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia) is based mainly on the techniques of fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding using sequences of plastidic DNA fragments. After more than 15 years of molecular taxonomic investigations, a certain viewpoint is now available demonstrating all five genera to be monophyletic. Also, the phenetic analyses had made huge progress in delineating the currently defined 36 species of duckweeds, although, all species cannot yet be defined with confidence. Wolffiella has turned out to be the most complicated genus as only 6 to 7 species out of the 10 can be reliably delineated. Further progress in the phylogenetic and phenetic analyses requires more advanced methods like next generation and/or whole genome sequencing. First results using the method genotyping-by-sequencing in the genus Lemna (in combination with metabolomic profiling by matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) as well as AFLP and barcoding by plastidic sequences) are more promising: The species Lemna valdiviana and Lemna yungensis were united to one species, Lemna valdiviana. This reduced the total number of Lemnaceae species to 36.
Evidence is limited regarding whether periodontal treatment improves hemoglobin A1c (HbA1c) among people with prediabetes and periodontal disease, and it is unknown whether improvement of metabolic status persists >3 mo. In an exploratory post hoc analysis of the multicenter randomized controlled trial “Antibiotika und Parodontitis” (Antibiotics and Periodontitis)—a prospective, stratified, double-blind study—we assessed whether nonsurgical periodontal treatment with or without an adjunctive systemic antibiotic treatment affects HbA1c and high-sensitivity C-reactive protein (hsCRP) levels among periodontitis patients with normal HbA1c (≤5.7%, n = 218), prediabetes (5.7% < HbA1c < 6.5%, n = 101), or unknown diabetes (HbA1c ≥ 6.5%, n = 8) over a period of 27.5 mo. Nonsurgical periodontal treatment reduced mean pocket probing depth by >1 mm in both groups. In the normal HbA1c group, HbA1c values remained unchanged at 5.0% (95% CI, 4.9% to 6.1%) during the observation period. Among periodontitis patients with prediabetes, HbA1c decreased from 5.9% (95% CI, 5.9% to 6.0%) to 5.4% (95% CI, 5.3% to 5.5%) at 15.5 mo and increased to 5.6% (95% CI, 5.4% to 5.7%) after 27.5 mo. At 27.5 mo, 46% of periodontitis patients with prediabetes had normal HbA1c levels, whereas 47.9% remained unchanged and 6.3% progressed to diabetes. Median hsCRP values were reduced in the normal HbA1c and prediabetes groups from 1.2 and 1.4 mg/L to 0.7 and 0.7 mg/L, respectively. Nonsurgical periodontal treatment may improve blood glucose values among periodontitis patients with prediabetes (ClinicalTrials.gov NCT00707369).
In this work, we theoretically investigate both aspects of charge-transferring atom-surface collisions: local-moment-type correlations and emission of secondary electrons from surfaces. Ideally, one chooses an approach that keeps as many electronic and lattice degrees of freedom at an ab-initio level as possible. In practice, however, this sophistication is hard to maintain. In this work, we do not aim to perform a description from first principles which could utilize density functional theory or quantum-chemical techniques. Instead, we keep only the most important degrees of freedom of the scattering process and use effective models for them. These are basically the Anderson-impurity model leading to time-dependent Anderson-Newns Hamiltonians and Gadzuk’s semiempirical approach to describe the projectile-target interaction from classical image shifts. In direct comparison with the description from first principles, the semiempirical approach offers a flexible basis for the modeling of a great variety of projectile-target combinations. The addition of further effective models to increase the general quality of the results is possible since the approach is very modular. The clear physical interpretation of each effective model, as well as the requirement for only a few and generally available parameters are further advantages of this approach. Rewritten in terms of Coleman’s pseudo-particle operators, the model is then numerically analyzed. This is done within a non-crossing approximation for the hybridization self-energies which are utilized by contour-ordered Green functions for each relevant electronic state of the projectile.
Climate change threatens marine ecosystems by simultaneous alterations and fluctuations in several abiotic factors like temperature, salinity and pH. Therefore, a strong ability to cope with varying environmental factors is indispensable for marine organisms. Especially, larvae of meroplanktonic species will be affected by predicted alterations in environmental conditions as planktonic larval stages are considered the most sensitive stages during life history (Anger 2001).
The European shore crab Carcinus maenas, as an ecological key species, was chosen as a model species to investigate multiple stressor effects on early life history stages of marine meroplanktonic invertebrates. The life cycle of C. maenas is biphasic consisting of five pelagic larval stages (four zoeal and one megalopal stage), followed by benthic juvenile and adult phases. The metamorphic molt from the last zoeal stage to the semi-benthic Megalopa includes dramatic changes in ecology, habitat, behavior, feeding, morphology, and physiology. During life history, zoeal stages of C. maenas are of particular interest in the course of climate change as these stages are more vulnerable than the following developmental stages to alterations in abiotic factors.
The aim of the present thesis was to develop an integrative view on effects of long-term exposure, from hatching to metamorphosis, to increased temperature and hypo-osmotic conditions on early life history stages of C. maenas. We wanted to gain insights into larval responses to climate driven environmental variables, more specifically, on how tolerance to low salinity is affected by increased temperatures.
Consequently, the present study investigated the effect of long-term exposure to twelve different sub-lethal temperature and salinity combinations in an ecological relevant range on larval development of C. maenas. In a multidisciplinary approach, larval responses in performance (survival and developmental duration) and morphology were measured. Furthermore, analysis on larval ontogeny and organogenesis created the foundation for analysis of larval response to multiple stressors in anatomy.
Results of the present thesis demonstrated that despite their different life-styles and external morphology, brachyuran larvae are smaller versions of their adults when regarding their inner organization: the adult bauplan unfolds from organ anlagen compressed into miniature organisms. In addition, they provide an overall picture of seemingly gradual organogenesis across larval development and the metamorphic molt, an insight that contrasts with the abrupt external morphological changes during metamorphosis. Gradual anatomical changes in e.g. osmoregulatory structures like gills and antennal glands allowed for ontogenetic shifts of tolerance to temperature and salinity during zoeal development and successive increase in osmo- and thermoregulatory capability. On the other hand, osmoregulatory structures as seen for adults were underdeveloped during zoeal development and therefore do not qualify for osmoregulatory function for these stages. This potentially explains the higher sensitivity of zoeae to hypo-osmotic conditions.
Early life history stages of C. maenas were affected on all response levels by the tested multiple stressors. The interaction of temperature and salinity was of antagonistic type, resulting in general reduced stress for larval stages. Nevertheless, low salinity had a strong negative impact on survival, while increased temperature caused ann acceleration of development. Furthermore, the size of zoeae of C. maenas was driven by the interaction of temperature and salinity, with extreme conditions, causing diminished growth, thus resulting in smaller larval size. On the other hand, larval shape was only slightly affected by changes of abiotic factors. Volume of the digestive gland and the heart of larvae from long-term exposure to sub-lethal temperatures and salinities showed high variability.
Larval responses were affected by the stressors intensities: moderately high temperatures lessened the negative effects of low salinities, while extreme high temperatures exceeded the ameliorating effect of temperature on stressful salinity conditions. On the other hand, the tolerance to temperature and salinity increased during larval development indicating an ontogenetic shift in response to multiple stressors with development. In addition, performance, morphology, and multiple stressor interaction showed intrapopulation variability among larvae hatched from different females, and between experimental periods.
In conclusion, this study highlighted direct effects of abiotic factors on all investigated response levels in early life history stages of the meroplanktonic larvae of the invertebrate C. maenas. High mortality rates combined with higher sensitivity confirm that planktonic early life history stages are the bottleneck during life history of this species. Nevertheless, early life history stages of C. maenas had the ability to cope with wide ranges of changing environmental factors. The antagonism between temperature and salinity on larval development offers potential for early life history stages to persist in a changing world. Furthermore, anatomical structures allow for slight eurytolerance and potentially for compensation of abiotic stress. Overall, slight increases in temperature, driven by climate change may enable larvae of C. maenas to tolerate exposure to moderately low salinities and, combinedwith intrapopulation variability, potentially allows for population persistence. Summarized, this study emphasizes the importance of testing a wide range of ecologically relevant traits in developing pelagic larvae in order to properly characterize their response to environmental change.
Changes in abundance and phenology of planktonic larvae like the zoeae of C. maenas have major potential to change a species‘ population structure significantly, and furthermore indirectly affect whole community and ecosystem structures. Therefore, this thesis may serve as a bridge to future studies in evolutionary and ecological developmental biology.
Abstract
We propose a setup enabling electron energy loss spectroscopy to determine the density of the electrons accumulated by an electropositive dielectric in contact with a plasma. It is based on a two-layer structure inserted into a recess of the wall. Consisting of a plasma-facing film made out of the dielectric of interest and a substrate layer, the structure is designed to confine the plasma-induced surplus electrons to the region of the film. The charge fluctuations they give rise to can then be read out from the backside of the substrate by near specular electron reflection. To obtain in this scattering geometry a strong charge-sensitive reflection maximum due to the surplus electrons, the film has to be most probably pre-n-doped and sufficiently thin with the mechanical stability maintained by the substrate. Taking electronegative CaO as a substrate layer we demonstrate the feasibility of the proposal by calculating the loss spectra for Al2O3, SiO2, and ZnO films. In all three cases we find a reflection maximum strongly shifting with the density of the surplus electrons and suggest to use it for charge diagnostics.
With this thesis, studies which form the bedrock for the long term goal of first wall heat load control and optimization for the advanced stellarator Wendelstein 7-X are developed, described and put into context. It is laid out how reconstruction of features of the edge magnetic field from plasma facing component heat loads is an important first step and can successfully be achieved by artificial neural networks. A detailed study of plasma facing component heat load distribution, potential overloads and overload mitigation possibilities is made in first order approximation of the impact of the main plasma dynamic effects.
Abstract
Erucic (22:1, cisΔ13) and gondoic acids (20:1, cisΔ11) are building blocks obtained from renewable sources for the oleochemical industry. Different biocatalytic strategies for the enrichment of these compounds with high recovery yields were developed in our group. Geotrichum candidum lipases (GCL) strongly discriminate against fatty acids longer than 18 carbon atoms. Thus, GCL‐I and ‐II were investigated using hydrolysis or ethanolysis reactions with Crambe and Camelina oils. Hydrolysis was also studied using fatty acid ethyl esters (FAEE) derived from the corresponding oil. Both isoforms were highly selective; however, interesting differences were observed. Although it has been reported that GCL‐I displays a higher preference toward 18 cisΔ9, which is present in the studied oils at high levels, GCL‐II showed higher enrichment values during hydrolysis independent of the substrate used. Hence, enrichments of 87% (Crambe oil) and 82% (Crambe FAEE) for erucic acid and 50% (Camelina oil) and 45% (Camelina FAEE) for gondoic acid, with recovery values between 89% and 99%, were achieved. On the contrary, the best enzyme for ethanolysis was GCL‐I (82% and 41% for erucic and gondoic acid, respectively). In this case, although GCL‐II also displayed good enrichment and recovery levels (77% and 28%, respectively), they were lower compared to the former reactions. In both ethanolysis reactions, the FAEE fraction contained between 92% and 97% of 18 unsaturated fatty acids.
Prediction models learn patterns from available data (training) and are then validated on new data (testing). Prediction modeling is increasingly common in dental research. We aimed to evaluate how different model development and validation steps affect the predictive performance of tooth loss prediction models of patients with periodontitis. Two independent cohorts (627 patients, 11,651 teeth) were followed over a mean ± SD 18.2 ± 5.6 y (Kiel cohort) and 6.6 ± 2.9 y (Greifswald cohort). Tooth loss and 10 patient- and tooth-level predictors were recorded. The impact of different model development and validation steps was evaluated: 1) model complexity (logistic regression, recursive partitioning, random forest, extreme gradient boosting), 2) sample size (full data set or 10%, 25%, or 75% of cases dropped at random), 3) prediction periods (maximum 10, 15, or 20 y or uncensored), and 4) validation schemes (internal or external by centers/time). Tooth loss was generally a rare event (880 teeth were lost). All models showed limited sensitivity but high specificity. Patients’ age and tooth loss at baseline as well as probing pocket depths showed high variable importance. More complex models (random forest, extreme gradient boosting) had no consistent advantages over simpler ones (logistic regression, recursive partitioning). Internal validation (in sample) overestimated the predictive power (area under the curve up to 0.90), while external validation (out of sample) found lower areas under the curve (range 0.62 to 0.82). Reducing the sample size decreased the predictive power, particularly for more complex models. Censoring the prediction period had only limited impact. When the model was trained in one period and tested in another, model outcomes were similar to the base case, indicating temporal validation as a valid option. No model showed higher accuracy than the no-information rate. In conclusion, none of the developed models would be useful in a clinical setting, despite high accuracy. During modeling, rigorous development and external validation should be applied and reported accordingly.
Microalgae are aquatic, unicellular, eukaryotic organisms, which perform photosynthesis. They have gained interest within the last decades not only for biofuel production due to their high amount of lipids, but also for pharmaceutical and for nutraceutical purposes. Interesting compounds are proteins, carbohydrates, or pigments, such as carotenoids. However, microalgae possess strong and rigid cell walls, which hinder a sufficient and yet, gentle extraction of those valuable compounds. Although standard extraction techniques are available, several shortcomings occur, e.g. high energy demand, use of environmentally harmful solvents or alteration of compounds due to heat or chemicals. Therefore, an alternative method is needed, which is able to address these disadvantages. Physical plasmas were thus studied to answer the question whether they are able to disintegrate the cell walls of microalgae effectively and yet, without degradation of the extractives.
First step of the thesis was to find a suitable plasma source that has an effect on the cell walls because plasma effects, such as electric fields, shockwaves, UV light emission, and the generation of reactive species can be tailored with the respective setup. It was found that spark discharges are most effective for the extraction of Chlorella vulgaris, which was chosen as model organism. All extraction yields were compared to reference methods, whereat microwave radiation was found to be the most effective reference method and were hence, applied for comparative studies.
For the next step, proteins were selected as targets to answer the question, which differences can be determined between plasms-treated and microwave-radiated proteins are observable although the extraction yields were equal. Furthermore, plasma effects, especially the effects of reactive species on the extracted proteins had to be studied. Findings indicate that heat sensitive proteins, such as photosystem-related proteins, or histones are better extractable with spark discharges than with microwave exposure and the effect of reactive species is only minor.
The last step was to determine, which plasma effect is responsible for the observed cell wall disintegration. Therefore, the tensile strength of Chlorella vulgaris was determined and compared to the shockwave pressure, which is generated from the spark channel. It was proven that the shockwave pressure exceeds by far the tensile strength of the microalgae an can be thus held responsible for mechanism for cell wall rupture.
In this thesis, it was found that spark discharges are a promising alternative for the extraction of valuable compounds from microalgae. The discharges are not only effective, but also gentle enough for sensitive compounds, such as proteins or pigments.
Despite major research interest regarding gender differences in emotion regulation, it is still not clear whether men and women differ in their basic capacity to implement specific emotion regulation strategies, as opposed to indications of the habitual use of these strategies in self-reports. Similarly, little is known on how such basic capacities relate to indices of well-being in both sexes. This study took a novel approach by investigating gender differences in the capacity for generating cognitive reappraisals in adverse situations in a sample of 67 female and 59 male students, using a maximum performance test of the inventiveness in generating reappraisals. Participants’ self-perceived efficacy in emotion regulation was additionally assessed. Analyses showed that men and women did not differ in their basic capacity to generate alternative appraisals for anxiety-eliciting scenarios, suggesting similar functional cognitive mechanisms in the implementation of this strategy. Yet, higher cognitive reappraisal capacity predicted fewer depressive daily-life experiences in men only. These findings suggest that in the case of cognitive reappraisal, benefits for well-being in women might depend on a more complex combination of basic ability, habits, and efficacy-beliefs, along with the use of other emotion regulation strategies. The results of this study may have useful implications for psychotherapy research and practice.
Introduction: Senior urology physicians represent a heterogeneous group covering various clinical priorities and career objectives. No reliable data on gender-specific variations among senior urology physicians are available concerning professional and personal aspects. Methods: The objective of this study was to analyze professional perspectives, professional and personal settings, and individual career goals. A Web-based survey containing 55 items was designed which was available for senior physicians at German urologic centers between February and April 2019. Gender-specific differences were evaluated using bootstrap-adjusted multivariate logistic regression models. Results: One hundred and ninety-two surveys were evaluable including 29 female senior physicians (15.1%). Ninety-five percent would choose urology again as their field of specialization – with no significant gender-specific difference. 81.2% of participants rate the position of senior physician as a desirable career goal (comparing sexes: p = 0.220). Based on multivariate models, male participants self-assessed themselves significantly more frequently autonomously safe performing laparoscopic, open, and endourologic surgery. Male senior physicians declared 7 times more often to run for the position of head of department/full professor. Conclusion: This first study on professional and personal aspects among senior urology physicians demonstrates gender-specific variations concerning self-assessment of surgical expertise and future career goals. The creation of well-orchestrated human resources development strategies especially adapted to the needs of female urologists seems advisable.
Introduction: Senior urology physicians represent a heterogeneous group covering various clinical priorities and career objectives. No reliable data on gender-specific variations among senior urology physicians are available concerning professional and personal aspects. Methods: The objective of this study was to analyze professional perspectives, professional and personal settings, and individual career goals. A Web-based survey containing 55 items was designed which was available for senior physicians at German urologic centers between February and April 2019. Gender-specific differences were evaluated using bootstrap-adjusted multivariate logistic regression models. Results: One hundred and ninety-two surveys were evaluable including 29 female senior physicians (15.1%). Ninety-five percent would choose urology again as their field of specialization – with no significant gender-specific difference. 81.2% of participants rate the position of senior physician as a desirable career goal (comparing sexes: p = 0.220). Based on multivariate models, male participants self-assessed themselves significantly more frequently autonomously safe performing laparoscopic, open, and endourologic surgery. Male senior physicians declared 7 times more often to run for the position of head of department/full professor. Conclusion: This first study on professional and personal aspects among senior urology physicians demonstrates gender-specific variations concerning self-assessment of surgical expertise and future career goals. The creation of well-orchestrated human resources development strategies especially adapted to the needs of female urologists seems advisable.
Genetic Regulation of Liver Metabolites and Transcripts Linking to Biochemical-Clinical Parameters
(2019)
Given the central metabolic role of the liver, hepatic metabolites and transcripts reflect the organismal physiological state. Biochemical-clinical plasma biomarkers, hepatic metabolites, transcripts, and single nucleotide polymorphism (SNP) genotypes of some 300 pigs were integrated by weighted correlation networks and genome-wide association analyses. Network-based approaches of transcriptomic and metabolomics data revealed linked of transcripts and metabolites of the pentose phosphate pathway (PPP). This finding was evidenced by using a NADP/NADPH assay and HDAC4 and G6PD transcript quantification with the latter coding for first limiting enzyme of this pathway and by RNAi knockdown experiments of HDAC4. Other transcripts including ARG2 and SLC22A7 showed link to amino acids and biomarkers. The amino acid metabolites were linked with transcripts of immune or acute phase response signaling, whereas the carbohydrate metabolites were highly enrich in cholesterol biosynthesis transcripts. Genome-wide association analyses revealed 180 metabolic quantitative trait loci (mQTL) (p < 10-4). Trans-4-hydroxy-L-proline (p = 6 × 10-9), being strongly correlated with plasma creatinine (CREA), showed strongest association with SNPs on chromosome 6 that had pleiotropic effects on PRODH2 expression as revealed by multivariate analysis. Consideration of shared marker association with biomarkers, metabolites, and transcripts revealed 144 SNPs associated with 44 metabolites and 69 transcripts that are correlated with each other, representing 176 mQTL and expression quantitative trait loci (eQTL). This is the first work to report genetic variants associated with liver metabolite and transcript levels as well as blood biochemical-clinical parameters in a healthy porcine model. The identified associations provide links between variation at the genome, transcriptome, and metabolome level molecules with clinically relevant phenotypes. This approach has the potential to detect novel biomarkers displaying individual variation and promoting predictive biology in medicine and animal breeding.
Peatlands are wetland ecosystems covering a relatively small area of the World (~3%), but at the same time storing excessive amounts of carbon for a very long time (equivalent to the four times global annual net primary production). As carbon sinks, peatlands work in spite of their slow growth, absorbing carbon dioxide (CO2) through the photosynthetic activity of the peatland plants and their low growth rates, and because high groundwater table removes oxygen from the soil and slows down the decomposition of the dead plant matter. Because of the relative lack of the oxygen in the peat, especially compared to the mineral soils, methanogen populations in the peatlands are abundant, and releasing methane (CH4), a potent greenhouse gas, to the atmosphere. Therefore, peatlands are generally at the same time significant carbon sinks and stores as well as the methane sources. The balance among the two peatland gass fluxes (CO2 and CH4) will dictate the impact of any given peatland on the global climate and primarily driven by hydrology, in the form of the groundwater table levels.
Because of the slow decomposition rates, and from radiocarbon dating of the peat as well as the subfossil records buried in it, carbon stored in peatlands is locked for a very long time (centuries to millennia). It is, therefore, crucial to gain insights into the development of peatlands and their gas balance through time. One way to get both is by studying peatland hydrology in the form of the groundwater table levels and their historical variations. Unfortunately, intensive monitoring of peatland groundwater table, when available, is an only a recent endeavor. Therefore, we need to employ proxies to reconstruct the past by leveraging the present. In statistics, proxy variables are often used when the observations of the variable of interest, are either missing or too difficult to obtain.
In this thesis, I tested whether we can use the radial growth of the Scots pines growing on peat as proxies to the peatland hydrology. To that end, I studied growth responses of the peatland Scots pines. Other proxies can and are used for the reconstructions of the groundwater table levels, but tree-growth is widely used as one of the proxies to reconstruct past environments which is at the same time annually resolved.
First, I examined the growth ecology of the peatland Scots pines by looking at their intra-annual development and trying to find relationships between it and environmental factors while at the same time comparing it with the Scots pines growing at the forest sites. I first tried with wood anatomy and found that, unfortunately, peatland Scots pines do not form enough wood cells, and consequently do not have high temporal resolution, necessary to investigate the intra-annual patterns of the radial growth. Initial results from wood anatomical investigations were interesting none-the-less, indicating that peatland Scots pines might have smaller cell features than the Scots pines from forests, but might at the same time maintain Early/Latewood ratios of those same features.
After I found that wood anatomical series were not resolved enough I decided to go with dendrometers, linear displacement sensors which constantly monitor the variations of stem radius, to get insights into the intra-annual growth patterns of the peatland Scots pines. Before using dendrometers for ecological investigations, I was involved in implementing routines commonly used in the analysis of the dendrometer signals and bringing them to R in the form of the dendrometeR package.
At one peatland complex, I installed dendrometers on ten trees in total at both peatland and forest sites and compared the pattern of the standardized signal. I inferred from the comparisons and classifications that the signal from two sites was indistinguishable for the dendrometer series shorter than five days. Furthermore, the most important environmental factor driving the radial variation at the peatland site was hydrological, daily relative humidity, indicating further that peatland hydrology might indeed be the driver behind peatland Scots pine growth.
Finally, I looked at the growth responses of peatland Scots pines from central Estonia using dendrochronological methods. Peatland hydrology, in the form of the groundwater table levels, was indeed the environmental factor with the strongest, and also stationary, correlations with the radial growth of the peatland Scots pine. That relationship indicated that peatland Scots pines are indeed possible proxies for reconstructing past levels of the peatland groundwater tables.
My study further indicated that the growth response of the peatland Scots pines was non-linear, further complicating the reconstructions of the past peatland hydrology. However, the strength of the growth response was proportional to the general hydrological regime, expressed as median groundwater table level. As the hydrological regime of the peatland does not vary considerably on the annual scales, but more on decadal it might be more appropriate to find another, independent, proxy to the hydrological regime first, and than use annually resolved radial growth of the peatland Scots pine to reconstruct past levels of the peatland groundwater table.
Helicobacter (H.) pylori is the most important cause for peptic ulcer disease and a risk factor for gastric carcinoma. How colonization with H. pylori affects the intestinal microbiota composition in humans is unknown. We investigated the association of H. pylori infection with intestinal microbiota composition in the population-based cohort Study-of-Health-in-pomerania (SHip)-tRenD. Anti-H. pylori serology and H. pylori stool antigen tests were used to determine the H. pylori infection status. the fecal microbiota composition of 212 H. pylori positive subjects and 212 matched negative control individuals was assessed using 16S rRNA gene sequencing. H. pylori infection was found to be significantly associated with fecal microbiota alterations and a general increase in fecal microbial diversity. in infected individuals, the H. pylori stool antigen load determined a larger portion of the microbial variation than age or sex. the highest H. pylori stool antigen loads were associated with a putatively harmful microbiota composition. this study demonstrates profound alterations in human fecal microbiota of H. pylori infected individuals. While the increased microbiota diversity associated with H. pylori infection as well as changes in abundance of specific genera could be considered to be beneficial, others may be associated with adverse health effects, reflecting the complex relationship between H. pylori and its human host.
The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.
AbstractThis paper takes concepts from spatial theory and globalization discourse and uses them in order to analyze the narrative function of descriptions of nature in romantic Icelandic poetry from the beginning of the 19th century and an Icelandic TV-Series from 2015. In Iceland’s romantic poetry of the early 19th century, especially in poems written by Bjarni Thorarensen, sublime nature is described as a form of guardian against foreign influences that threaten the way of living on the peripheral island. This romantic concept of Icelandic nature is closely connected to narrative patterns in the process of the Icelandic Nation-Building, as it characterizes Icelanders as simultaneously defined and protected by the harsh conditions on the island. The paper takes a comparative look at the underlying narrative concepts of nature in two of Bjarni Thorarensen’s poems and a recent Icelandic TV series, Baltasar Kormákur’s Ófærð (2015), that presents a different concept of Icelandic nature in its relation to a (threatening) global influence. The series depicts a globalized world in which crime does not only affect remote communities as an evil from the outside but as a local evil connected to forces on global scale. Nature as a narrative device in the TV series thus does not protect Icelanders from global forces, as it did in Bjarni Thorarensens poems in the early 19th century, but instead functions a catalyst that reveals the evil from the outside and the evil from within.
Phenolics and its derivatives are aromatic compounds with a wide range of industrial applications. Gallic acid, protocatechuic acid, catechol or pyrogallol are only a few examples of industrially relevant aromatics. The production of bulk fine chemicals primarily for chemical and pharmaceutical industry has put a strong emphasis on optimizing manufacturing conditions. Commercial production of many chemicals is still based on organic chemical synthesis using petroleum derivatives as starting material. Since these processes are considered environmentally unfriendly and posing an irresponsible strain on limited fossil resources, much attention is paid to the development of new microbial factories for the bioproduction of industrially relevant chemicals using renewable sources or organic pollutants as starting material. Arxula adeninivoras is a non-conventional yeast possessing attractive properties for industrial application such as thermo- and osmotolerance. Another major advantage of this organism is its broad substrate spectrum with tannin at the forefront. The present project is dedicated to the study of the tannic acid degradation pathway in A. adeninivorans. Two genes encoding enzymes annotated as gallic acid decarboxylase (AGDC1) and catechol-1,2-dioxygenase (ACDO1) have been selected and investigated. Both enzymes were characterized and their function in tannin catabolism analyzed.
Hepeviruses are small viruses with a RNA-genome of positive polarity that form the family Hepeviridae. The family includes two genera: members of the genus Piscihepevirus were detected in fish species and members of the genus Orthohepevirus were found in different mammal and bird species. The genus Orthohepevirus contains four different species, namely Orthohepevirus A, B, C and D. The species Orthohepevirus A contains five human pathogenic genotypes, with three of them being zoonotic. The species Orthohepevirus C contains mammal-associated pathogens, which were identified in rats and carnivores. The human pathogenic genotypes are responsible for a self-limiting acute hepatitis in humans, which could become chronically in immunocompromised individuals. The main route of transmission is the consumption of undercooked meat and direct contact with HEV-positive excreta or blood. In Germany, hepatitis E is a notifiable disease since 2001 with an increased number of cases per year. Rats are the reservoir of rat-associated HEV (ratHEV), but also the zoonotic HEV-3 genotype was detected in rats. The European rabbit (Oryctolagus cuniculus) was identified as a reservoir host of a subgenotype of human pathogenic HEV-3 (HEV-3ra).
For the development of small mammal animal models, the objective of this study was to evaluate different small mammal populations for novel hepeviruses and to study the presence of HEV and sequence divergence of ratHEV and rabbitHEV in rat and rabbit populations from Europe.
Approximately 3000 rodents from Germany and the Czech Republic were screened by broad spectrum HEV-RT-PCR. As a result, 13 common voles (Microtus arvalis) and one bank vole (Myodes glareolus) were detected to be HEV-RNA positive. Comparison of the obtained sequences, complete genome determination and phylogenetic analysis indicated the finding of a novel common vole-associated HEV (cvHEV), which shows a high sequence divergence towards other members of the species Orthohepevirus C, but shares a high sequence similarity to a HEV-genome derived from a kestrel (Falco tinnunculus). The finding of cvHEV-RNA in a bank vole might be caused by a spillover infection. The cvHEV genome shares the hepevirus-typical open reading frames, but also has unique cvHEV-specific attributes in its genome.
The investigation of 420 Norway rats (Rattus norvegicus) and 88 Black rats (Rattus rattus) identified HEV-RNA in Norway rats from eight of nine and Black rats from two of four European countries. In a single Norway rat from Belgium, a HEV-3-strain with high sequence similarities to rabbitHEV (HEV-3ra), was detected. The investigation of zoo animals revealed a ratHEV spillover infection in a Syrian brown bear (Ursus arctos syriacus). This infection was most likely caused by ratHEV-infected free-living, wild rats from the same zoo.
Investigation of wild rabbit populations trapped in and around Frankfurt am Main, Germany, showed anti-HEV antibodies (34.7%) and rabbitHEV-RNA (25%). A high sequence similarity of rabbitHEV in the animals trapped at the urban site was observed, whereas a high sequence divergence was seen for the animals trapped at the rural trapping sites.
In conclusion, hepeviruses are widespread among different small mammal populations in Europe. The broad geographical distribution of these hepeviruses should be taken into account in further public health risk assessments. Further investigations are needed to characterize the presence of cvHEV in more detail, especially by taking the population dynamics of common voles into account. The detected HEV-strains could be taken as basis for the establishment of novel HEV-animal models, which might replace the so far used swine and non-human primate models.
Immunogenicity and protectivity of surface-localized lipoproteins of Streptococcus pneumoniae
(2019)
Steptococcus pneumoniae (pneumococcus) represents a common colonizer of the human upper respiratory tract (URT). However, under certain conditions, for example following viral infections, or in indiciduals with a weakened immune system, including young children, elderly and immunocompromised persons, it can cause a wide range of life-threatening diseases, such as pneumonia, meningitis or sepsis. Based on the polysaccharide capsule that surrounds the bacterium, pneumococci are classified into so far 98 different serotypes. Prevention of S. pneumoniae infections was achieved by the development of pneumococcal polysaccharide-based (PPSV) vaccines. However, these vaccines have important limitations, including high manufacturing costs and restricted serotype coverage facilitating replacement by non-vaccine serotypes. Aiming for the development of a serotype-independent vaccine, the potential of surface-exposed and highly conserved pneumococcal lipoproteins was evaluated for being targeted as a future protein-based vaccine. Therefore, selected lipoproteins were examined i) for their surface abundance and accessibility, ii) for their presence in clinically relevant S. pneumoniae strains, and iii) for their immunogenicity. Finally, based on these initial screenings, the most promising candidates were selected to analyze their protective efficacy in a moude model of colonization. DacB and PnrA were identified as highly abundant lipoproteins on the pneumococcal surface. They showed to be immunogenic both during natural infection using convalescent patient sera and when given to mice as a subunit vaccine formulation. Following intranasal immunization and challenge of mice with two heterologous S. pneumoniae strains, both proteins reduced the pneumococcal load in the nasopharynx. The protection correlated with increased production of IL-17A indicative for a Th17-mediated immunity, which is strongly suggested to play a critical role in preventing pneumococcal colonization and infection. Lipoproteins are triggering innate receptors on antigen-presenting cells, thereby linking innate with adaptive immune responses. Therefore, lipidated proteins were evaluated for their potential to be used as an adjuvant for vaccination. Lipidation clearly enhanced humoral immune responses to DacB and PnrA without the need of an additional adjuvant. However, an additional adjuvant was required to confer protection against pneumococcal colonization. In conclusion, Lipoproteins are interesting candidates for future protein-based vaccine strategies because they are highly conserved, abundant and immunogenic. PnrA and DacB were identified as potential candidates, since they induced protection against pneumococcal colonization, which in turn may lead to a decline in infections and transmission.
Neuroblastoma (NB) is an aggressive, poorly immunogenic tumor in childhood. Therapy for high-risk NB remains challenging. Immunotherapy with anti-GD 2 antibody ch14.18/CHO effectively prolongs the survival of NB patients.
Killer-immunoglobulin-like receptor (KIR)/human leucocyte antigen (HLA) mismatch and Fc gamma receptor (FCGR) polymorphisms are reported to affect antibody-dependent cellular cytotoxicity (ADCC) induced by monoclonal antibodies. To determine whether FCGR polymorphisms and KIR/HLA mismatch are associated with the survival following ch14.18-based immunotherapy, genotyping methods that allow for genotype determination of FCGR2A, -3A, -3B, KIR2DL1, 2DL2, 2DL3, and 3DL1 have been established and applied to the analysis of 53 NB patients treated with ch14.18/CHO.
High-affinity polymorphisms of FCGR2A (H131) and FCGR3A (V158) were associated with improved survival. Importantly, patients displaying both the FCGR3A-V158 and FCGR2A-H131 alleles exhibited significantly improved event-free survival. No association was found between KIR/HLA genotypes or FCGR3B alleles and patients’ survival in our patient cohort.
In conclusion, impact of FCGR2A and -3A genotypes in response to ch14.18/CHO immunotherapy in combination with IL2 was demonstrated. FCGR2A and -3A might therefore provide a prognostic marker when conducting ch14.18/CHO-based immunotherapy.
The advances in high-throughput sequencing technologies have revolutionized the possibilities for pathogen identification in cases of unknown disease origin. Diagnostic metagenomics allows the unbiased and simultaneous detection of almost all nucleic acids in a clinical sample, with the potential to provide pivotal insights into otherwise undeterminable causes of human or animal disease.
In this thesis, possibilities, pitfalls and the suitability of Ion Torrent and Illumina sequencing platforms for comprehensive use in diagnostic metagenomics were assessed and optimized procedures developed. Clinical field samples, undiagnosable by standard diagnostics, were taken as real-life examples for the investigations. The results show that cross-contamination due to index swapping and run-to-run-carryover constitute a major issue on Illumina platforms, severely compromising the correct interpretation of results for clinical specimens. In contrast, Ion Torrent platforms did not display any form of cross-contamination, however, the commercial library preparation method is less efficient. Combining the advantages of both platforms, customized Y adapters, facilitating highly efficient library preparation, were developed for Ion Torrent sequencing and applied in further experiments. The obstacles of strongly degraded RNA in formalin-fixed paraffin-embedded samples were identified and the workflow adapted to meet the requirements of smaller fragments. Additionally, it was shown that adequate sampling is a very important step, if not the most important step, in the workflow, as well as subsequent validation of the obtained results in terms of causation. The achievements in this study allow other researchers the application of a sensitive and optimized diagnostic metagenomics workflow.
Furthermore, the investigations on the clinical samples resulted in the discovery of a novel respirovirus with putative zoonotic potential, the first description of Borna disease virus 1 in human organ transplant recipients, and the discovery of a very distantly related novel ovine picornavirus. These discoveries build a basis for further research and expand the knowledge regarding new and emerging viruses.
Abstract
Proteome analyses are often hampered by the low amount of available starting material like a low bacterial cell number obtained from in vivo settings. Here, the single pot solid‐phase enhanced sample preparation (SP3) protocol is adapted and combined with effective cell disruption using detergents for the proteome analysis of bacteria available in limited numbers only. Using this optimized protocol, identification of peptides and proteins for different Gram‐positive and Gram‐negative species can be dramatically increased and, reliable quantification can also be ensured. This adapted method is compared to already established strain‐specific sample processing protocols for Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila. The highest species‐specific increase in identifications is observed using the adapted method with L. pneumophila samples by increasing protein and peptide identifications up to 300% and 620%, respectively. This increase is accompanied by an improvement in reproducibility of protein quantification and data completeness between replicates. Thus, this protocol is of interest for performing comprehensive proteomics analyses of low bacterial cell numbers from different settings ranging from infection assays to environmental samples.
Objectives: Performing proper toothbrushing is a complicated process for children. Therefore, the aim of this study was to investigate the effect of a smartphone app for improving manual toothbrushing via a gravitation sensor. Methods: In this prospective, controlled, single-blinded, randomized clinical trial, 49 children (mean age 5.1 ± 0.6 years, 27 female) were randomly assigned to test (n = 26) and control (n = 23) groups. All children were provided with manual toothbrushes with an integrated gravitation sensor and they received oral health instructions. Only the children of the test group got an additional smartphone app to visualize and reward proper brushing in form and time. At baseline and recalls after 6 and 12 weeks, plaque and gingival indices (QHI, PBI) were recorded for analysis between the two groups. Results: At baseline, there were no significant differences between the test and control group regarding plaque and gingival indices (QHI: 2.36 ± 0.7 and 2.42 ± 0.8; p = 0.94; PBI: 0.42 ± 0.2 and 0.47 ± 0.3; p = 0.59). At the 6- and 12-week recalls, the test group showed statistically significantly better oral health indices than the controls (6-week recall, QHI: 0.8 ±0.5 and 1.88 ± 0.9; p < 0.001; PBI: 0.08 ± 0.1 and 0.26 ± 0.2; p < 0.001; 12-week recall, QHI: 0.44 ± 0.5 and 1.49 ± 0.7; p < 0.001; PBI: 0.05 ± 0.18 and 0.21 ± 0.1; p < 0.001). Conclusion: The results highlight the enormous possibilities of a toothbrushing application via the smartphone, at least for medium-term oral hygiene improvement in preschool children and even after excluding the app. The long-term effect should also be investigated to exclude the expected novelty effect.