@phdthesis{Rosenbaum2016,
  author    = {Rosenbaum, Corinna},
  title     = {The role of enteric glial cells under inflammatory conditions of the intestine},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-138946},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The enteric nervous system (ENS) innervates the gastrointestinal (GI) tract and controls central aspects of GI physiology including contractility of the intestinal musculature, glandular secretion and intestinal blood flow. The ENS is composed of neurons that conduct electrical signals and of enteric glial cells (EGCs). EGCs resemble central nervous system (CNS) astrocytes in their morphology and in the expression of shared markers such as the intermediate filament protein glial fibrillary acidic protein (GFAP). They are strategically located at the interface of ENS neurons and their effector cells to modulate intestinal motility, epithelial barrier stability and inflammatory processes. The specific contributions of EGCs to the maintenance of intestinal homeostasis are subject of current research. From a clinical point of view EGC involvement in pathophysiological processes such as intestinal inflammation is highly relevant. Like CNS astrocytes ECGs can acquire a reactive, tissue-protective phenotype in response to intestinal injury. In patients with chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, alterations in the EGC network are well known, particularly a differential expression of GFAP, which is a hallmark of reactive gliosis in the CNS. With increasing recognition of the role of EGCs in intestinal health and disease comes the need to study the glial population in its complexity. The overall aim of this thesis was to comprehensively study EGCs with focus on the reactive GFAP-expressing subpopulation under inflammatory conditions in vivo and in vitro. In a first step, a novel in vivo rat model of acute systemic inflammation mimicking sepsis was employed to investigate rapidly occuring responses of EGCs to inflammation. This study revealed that within a short time frame of a few hours, EGCs responded to the inflammation with an upregulation of Gfap gene expression. This inflammation-induced upregulation was confined to the myenteric plexus and varied in intensity along the intestinal rostro-caudal axis. This highly responsive myenteric GFAP-expressing EGC population was further characterized in vivo andin vitro using a transgenic mouse model (hGFAP-eGFP mice). Primary purified murine GFAP-EGC cultures in vitro were established and it was assessed how the transcriptomic and proteomic profiles of these cells change upon inflammatory stimulation. Here, myenteric GFAP-EGCs were found to undergo a shift in gene expression profile that predominantly affects expression of genes associated with inflammatory responses. Further, a secretion of inflammatory mediators was validated on protein level. The GFAP+ subpopulation is hence an active participant in inflammatory pathophysiology. In an acute murine IBD model in vivo, GFAP-EGCs were found to express components of the major histocompatibility complex (MHC) class II in inflamed tissue, which also indicates a crosstalk of EGCs with the innate and the adaptive lamina propria immune system in acute inflammation. Taken together, this work advances our knowledge on EGC (patho-)physiology by identifying and characterizing an EGC subpopulation rapidly responsive to inflammation. This study further provides the transcriptomic profile of this population in vivo and in vitro, which can be used to identify targets for therapeutic intervention. Due to the modulating influence of EGCs on the intestinal microenvironment, the study further underlines the importance of integrating EGCs into in vitro test systems that aim to model intestinal tissues in vitro and presents an outlook on a potential strategy.},
  subject      = {Darmwandnervensystem},
  language  = {en}
}
@phdthesis{Costea2016,
  author    = {Costea, Paul Igor},
  title     = {Stratification and variation of the human gut microbiota},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139649},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The microbial communities that live inside the human gastrointestinal tract -the human gut microbiome- are important for host health and wellbeing. Characterizing this new "organ", made up of as many cells as the human body itself, has recently become possible through technological advances. Metagenomics, the high-throughput sequencing of DNA directly from microbial communities, enables us to take genomic snapshots of thousands of microbes living together in this complex ecosystem, without the need for isolating and growing them. Quantifying the composition of the human gut microbiome allows us to investigate its properties and connect it to host physiology and disease. The wealth of such connections was unexpected and is probably still underestimated. Due to the fact that most of our dietary as well as medicinal intake affects the microbiome and that the microbiome itself interacts with our immune system through a multitude of pathways, many mechanisms have been proposed to explain the observed correlations, though most have yet to be understood in depth. An obvious prerequisite to characterizing the microbiome and its interactions with the host is the accurate quantification of its composition, i.e. determining which microbes are present and in what numbers they occur. Historically, standard practices have existed for sample handling, DNA extraction and data analysis for many years. However, these were generally developed for single microbe cultures and it is not always feasible to implement them in large scale metagenomic studies. Partly because of this and partly because of the excitement that new technology brings about, the first metagenomic studies each took the liberty to define their own approach and protocols. From early meta-analysis of these studies it became clear that the differences in sample handling, as well as differences in computational approaches, made comparisons across studies very difficult. This restricts our ability to cross-validate findings of individual studies and to pool samples from larger cohorts. To address the pressing need for standardization, we undertook an extensive comparison of 21 different DNA extraction methods as well as a series of other sample manipulations that affect quantification. We developed a number of criteria for determining the measurement quality in the absence of a mock community and used these to propose best practices for sampling, DNA extraction and library preparation. If these were to be accepted as standards in the field, it would greatly improve comparability across studies, which would dramatically increase the power of our inferences and our ability to draw general conclusions about the microbiome. Most metagenomics studies involve comparisons between microbial communities, for example between fecal samples from cases and controls. A multitude of approaches have been proposed to calculate community dissimilarities (beta diversity) and they are often combined with various preprocessing techniques. Direct metagenomics quantification usually counts sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to technology-inherent differences in sampling depth, normalizing counts is necessary, for instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by subsampling. To derive a single value for community (dis-)similarity, multiple distance measures have been proposed. Although it is theoretically difficult to benchmark these approaches, we developed a biologically motivated framework in which distance measures can be evaluated. This highlights the importance of data transformations and their impact on the measured distances. Building on our experience with accurate abundance estimation and data preprocessing techniques, we can now try and understand some of the basic properties of microbial communities. In 2011, it was proposed that the space of genus level variation of the human gut microbial community is structured into three basic types, termed enterotypes. These were described in a multi-country cohort, so as to be independent of geography, age and other host properties. Operationally defined through a clustering approach, they are "densely populated areas in a multidimensional space of community composition"(source) and were proposed as a general stratifier for the human population. Later studies that applied this concept to other datasets raised concerns about the optimum number of clusters and robustness of the clustering approach. This heralded a long standing debate about the existence of structure and the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in the context of the vastly increased amounts of available data. We propose a refined framework in which the different types should be thought of as weak attractors in compositional space and we try to implement an approach to determining which attractor a sample is closest to. To this end, we train a classifier on a reference dataset to assign membership to new samples. This way, enterotypes assignment is no longer dataset dependent and effects due to biased sampling are minimized. Using a model in which we assume the existence of three enterotypes characterized by the same driver genera, as originally postulated, we show the relevance of this stratification and propose it to be used in a clinical setting as a potential marker for disease development. Moreover, we believe that these attractors underline different rules of community assembly and we recommend they be accounted for when analyzing gut microbiome samples. While enterotypes describe structure in the community at genus level, metagenomic sequencing can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis methodology for this level of resolution has only recently been developed and little exploration has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the landscape of genomic variation seems highly structured even beyond species resolution, indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several cases, these subspecies exhibit geo-stratification, with some subspecies only found in the Chinese population. Generally however, they present only minor dispersion limitations and are seen across most of our study populations. Within one individual, one subspecies is commonly found to dominate and only rarely are several subspecies observed to co-occur in the same ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable over periods of more than three years. When interrogating their functional properties we find many differences, with specific ones appearing relevant to the host. For example, we identify a subspecies of E. rectale that is lacking the flagellum operon and find its presence to be significantly associated with lower body mass index and lower insulin resistance of their hosts; it also correlates with higher microbial community diversity. These associations could not be seen at the species level (where multiple subspecies are convoluted), which illustrates the importance of this increased resolution for a more comprehensive understanding of microbial interactions within the microbiome and with the host. Taken together, our results provide a rigorous basis for performing comparative metagenomics of the human gut, encompassing recommendations for both experimental sample processing and computational analysis. We furthermore refine the concept of community stratification into enterotypes, develop a reference-based approach for enterotype assignment and provide compelling evidence for their relevance. Lastly, by harnessing the full resolution of metagenomics, we discover a highly structured genomic variation landscape below the microbial species level and identify common subspecies of the human gut microbiome. By developing these high-precision metagenomics analysis tools, we thus hope to contribute to a greatly improved understanding of the properties and dynamics of the human gut microbiome.},
  subject      = {Mensch},
  language  = {en}
}
@phdthesis{Ulrich2016,
  author    = {Ulrich, Natalie},
  title     = {Processing of Near Outcomes and Outcome Sequences in Gambling: Implications for the Biopsychological Basis of Problem Gambling},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139612},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Gambling is a popular activity in Germany, with 40\% of a representative sample reporting having gambled at least once in the past year (Bundeszentrale f{\"u}r gesundheitliche Aufkl{\"a}rung, 2014). While the majority of gamblers show harmless gambling behavior, a subset develops serious problems due to their gambling, affecting their psychological well-being, social life and work. According to recent estimates, up to 0.8\% of the German population are affected by such pathological gambling. People in general and pathological gamblers in particular show several cognitive distortions, that is, misconceptions about the chances of winning and skill involvement, in gambling. The current work aimed at elucidating the biopsychological basis of two such kinds of cognitive distortions, the illusion of control and the gambler's and hot hand fallacies, and their modulation by gambling problems. Therefore, four studies were conducted assessing the processing of near outcomes (used as a proxy for the illusion of control) and outcome sequences (used as a proxy for the gambler's and hot hand fallacies) in samples of varying degrees of gambling problems, using a multimethod approach. The first study analyzed the processing and evaluation of near outcomes as well as choice behavior in a wheel of fortune paradigm using electroencephalography (EEG). To assess the influence of gambling problems, a group of problem gamblers was compared to a group of controls. The results showed that there were no differences in the processing of near outcomes between the two groups. Near compared to full outcomes elicited smaller P300 amplitudes. Furthermore, at a trend level, the choice behavior of participants showed signs of a pattern opposite to the gambler's fallacy, with longer runs of an outcome color leading to increased probabilities of choosing this color again on the subsequent trial. Finally, problem gamblers showed smaller feedback-related negativity (FRN) amplitudes relative to controls. The second study also targeted the processing of near outcomes in a wheel of fortune paradigm, this time using functional magnetic resonance imaging and a group of participants with varying degrees of gambling problems. The results showed increased activity in the bilateral superior parietal cortex following near compared to full outcomes. The third study examined the peripheral physiology reactions to near outcomes in the wheel of fortune. Heart period and skin conductance were measured while participants with varying degrees of gambling problems played on the wheel of fortune. Near compared to full outcomes led to increased heart period duration shortly after the outcome. Furthermore, heart period reactions and skin conductance responses (SCRs) were modulated by gambling problems. Participants with high relative to low levels of gambling problems showed increased SCRs to near outcomes and similar heart period reactions to near outcomes and full wins. The fourth study analyzed choice behavior and sequence effects in the processing of outcomes in a coin toss paradigm using EEG in a group of problem gamblers and controls. Again, problem gamblers showed generally smaller FRN amplitudes compared to controls. There were no differences between groups in the processing of outcome sequences. The break of an outcome streak led to increased power in the theta frequency band. Furthermore, the P300 amplitude was increased after a sequence of previous wins. Finally, problem gamblers compared to controls showed a trend of switching the outcome symbol relative to the previous outcome symbol more often. In sum, the results point towards differences in the processing of near compared to full outcomes in brain areas and measures implicated in attentional and salience processes. The processing of outcome sequences involves processes of salience attribution and violation of expectations. Furthermore, problem gamblers seem to process near outcomes as more win-like compared to controls. The results and their implications for problem gambling as well as further possible lines of research are discussed.},
  subject      = {Spielsucht},
  language  = {en}
}
@phdthesis{Asmus2016,
  author    = {Asmus, Elisabeth},
  title     = {Mode of Action of Adjuvants for Foliar Application},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-138159},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Adjuvants are compounds added to an agrochemical spray formulation to improve or modify the action of an active ingredient (AI) or the physico-chemical characteristics of the spray liquid. Adjuvants can have more than only one distinct mode of action (MoA) during the foliar spray application process and they are generally known to be the best tools to improve agrochemical formulations. The main objective for this work was to elucidate the basic MoA of adjuvants by uncoupling different aspects of the spray application. Laboratory experiments, beginning from retention and spreading characteristics, followed by humectant effects concerning the spray deposit on the leaf surface and ultimately the cuticular penetration of an AI, were figured out to evaluate overall in vivo effects of adjuvants which were also obtained in a greenhouse spray test. For this comprehensive study, the surfactant classes of non-ionic sorbitan esters (Span), polysorbates (Tween) and oleyl alcohol polyglycol ether (Genapol O) were generally considered because of their common promoting potential in agrochemical formulations and their structural diversity. The reduction of interfacial tension is one of the most crucial physico-chemical properties of surfactants. The dynamic surface tension (DST) was monitored to characterise the surface tension lowering behaviour which is known to influence the droplet formation and retention characteristics. The DST is a function of time and the critical time frame of droplet impact might be at about 100 ms. None of the selected surfactants were found to lower the surface tension sufficiently during this short timeframe (chapter I). At ca. 100 ms, Tween 20 resulted in the lowest DST value. When surfactant monomers are fully saturated at the droplet-air-interface, an equilibrium surface tension (STeq) value can be determined which may be used to predict spreading or run-off effects. The majority of selected surfactants resulted in a narrow distribution of STeq values, ranging between 30 and 45 mN m- 1. Nevertheless, all surfactants were able to decrease the surface tension considerably compared to pure water (72 mN m- 1). The influence of different surfactants on the wetting process was evaluated by studying time-dependent static contact angles on different surfaces and the droplet spread area on Triticum aestivum leaves after water evaporation. The spreading potential was observed to be better for Spans than for Tweens. Especially Span 20 showed maximum spreading results. To transfer laboratory findings to spray application, related to field conditions, retention and leaf coverage was measured quantitatively on wheat leaves by using a variable track sprayer. Since the retention process involves short time dynamics, it is well-known that the spray retention on a plant surface is not correlated to STeq but to DST values. The relationship between DST at ca. 100 ms and results from the track sprayer showed increasing retention results with decreasing DST, whereas at DST values below ca. 60 mN m- 1 no further retention improvement could be observed. Under field conditions, water evaporates from the droplet within a few seconds to minutes after droplet deposition on the leaf surface. Since precipitation of the AI must essentially being avoided by holding the AI in solution, so-called humectants are used as tank-mix adjuvants. The ability of pure surfactants to absorb water from the surrounding atmosphere was investigated comprehensively by analysing water sorption isotherms (chapter II). These isotherms showed an exponential shape with a steep water sorption increase starting at 60\% to 70\% RH. Water sorption was low for Spans and much more distinct for the polyethoxylated surfactants (Tweens and Genapol O series). The relationship between the water sorption behaviour and the molecular structure of surfactants was considered as the so-called humectant activity. With an increasing ethylene oxide (EO) content, the humectant activity increased concerning the particular class of Genapol O. However, it could be shown that the moisture absorption across all classes of selected surfactants correlates rather better with their hydrophilic-lipophilic balance values with the EO content. All aboveground organs of plants are covered by the cuticular membrane which is therefore the first rate limiting barrier for AI uptake. In vitro penetration experiments through an astomatous model cuticle were performed to study the effects of adjuvants on the penetration of the lipophilic herbicide Pinoxaden (PXD) (chapter III). In order to understand the influence of different adjuvant MoA like humectancy, experiments were performed under three different humidity levels. No explicit relationship could be found between humidity levels and the PXD penetration which might be explained by the fact that humidity effects would rather affect hydrophilic AIs than lipophilic ones. Especially for Tween 20, it became obvious that a complex balance between multiple MoA like spreading, humectancy and plasticising effects have to be considered. Greenhouse trials, focussing the adjuvant impact on in vivo action of PXD, were evaluated on five different grass-weed species (chapter III). Since agrochemical spray application and its following action on living plants also includes translocation processes in planta and species dependent physiological effects, this investigation may help to simulate the situation on the field. Even though the absolute weed damage was different, depending both on plant species and also on PXD rates, adjuvant effects in greenhouse experiments displayed the same ranking as in cuticular penetration studies: Tween 20 > Tween 80 > Span 20 ≥ Span 80. Thus, the present work shows for the first time that findings obtained in laboratory experiments can be successfully transferred to spray application studies on living plants concerning adjuvant MoA. A comparative analysis, using radar charts, could demonstrate systematic derivations from structural similarities of adjuvants to their MoA (summarising discussion and outlook). Exemplarily, Tween 20 and Tween 80 cover a wide range of selected variables by having no outstanding MoA improving one distinct process during foliar application, compared to non-ethoxylated Span 20 and Span 80 which primarily revealed a surface active action. Most adjuvants used in this study represent polydisperse mixtures bearing a complex distribution of EO and aliphatic chains. From this study it seems alike that adjuvants having a wide EO distribution offer broader potential than adjuvants with a small EO distribution. It might be a speculation that due to this broad distribution of single molecules, all bearing their individual specific physico-chemical nature, a wide range of properties concerning their MoA is covered.},
  subject      = {Adjuvans},
  language  = {en}
}
@phdthesis{Yadav2016,
  author    = {Yadav, Preeti},
  title     = {Studying Neuronal Cytoskeleton Defects and Synaptic Defects in Mouse Model of Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-138093},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Amyotrophic lateral sclerosis and spinal muscular atrophy are the two most common motoneuron diseases. Both are characterized by destabilization of axon terminals, axon degeneration and alterations in neuronal cytoskeleton. Accumulation of neurofilaments has been observed in several neurodegenerative diseases but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, I show that increased neurofilament expression in motor nerves of pmn mutant mice causes disturbed microtubule dynamics. Depletion of neurofilament by Nefl knockout increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Depletion of neurofilament increases stathmin-Stat3 interaction and stabilizes the microtubules. Consequently, the axonal maintenance is improved and the pmn mutant mice survive longer. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation is a prominent feature. Next, using Smn-/-;SMN2 mouse as a model, the molecular mechanism behind synapse loss in SMA is studied. SMA is characterized by degeneration of lower α-motoneurons in spinal cord; however, how reduction of ubiquitously expressed SMN leads to MN-specific degeneration remains unclear. SMN is involved in pre-mRNA splicing (Pellizzoni, Kataoka et al. 1998) and its deficiency in SMA affects the splicing machinery. Neuromuscular junction denervation precedes neurodegeneration in SMA. However, there is no evidence of a link between aberrant splicing of transcripts downstream of Smn and reduced presynaptic axon excitability observed in SMA. In this study, we observed that expression and splicing of Nrxn2, that encodes a presynaptic protein is affected in the SMA mouse and that Nrxn2 could be a candidate that relates aberrant splicing to synaptic motoneuron defects in SMA.},
  subject      = {Neurofilament},
  language  = {en}
}
@phdthesis{Bankoglu2016,
  author    = {Bankoglu, Ezgi Eyl{\"u}l},
  title     = {Oxidative status and genomic damage in an obesity model},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137566},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Several cohort studies showed that obesity increases the risk of chronic disease such as T2DM, hypertension and non-alcoholic fatty liver disease and various types of cancer. Different factors were described that might be involving in these diseases in obesity. Some of these suggested factors were chronic infection, elevated free fatty acids, increased ROS formation, mitochondrial dysfunction and raised NAPDH oxidase activity. Obesity is a multifactorial disease and it is very hard to distinguish between all of these factors. In this study, we wanted to focus on the association between obesity, oxidative stress and genomic damage in kidney, liver and colon, which are the most relevant organs for cancer risk according to the cohort studies. Our findings indicated elevated oxidative stress in kidney, liver and colon together with elevated lipid, RNA and DNA oxidation in the whole body. Additionally, we were able to show increased DNA damage in kidney, liver and colon. Since obesity has become an epidemic all over the world, possible therapeutic applications such as life style changes (diet and sport), pharmacological supplements and various type of surgeries are increasing. As a second question, we focused on the effect of weight loss, which is supplied either by Roux-en-Y gastric bypass surgery or by caloric restriction designed in a way to provide the same extent of weight loss, on oxidative stress and genomic damage. Our results indicated that weight loss either by gastric bypass surgery or by caloric restriction led to reduced oxidative stress and genomic damage in kidney, liver and colon. We could not find any difference between the weight loss methods, except the DNA oxidation and repair marker urinary 8-oxodG, which was still elevated after RYGB, but not after caloric restriction. It is known that hyperinsulinemia and in the long term T2DM are among the biggest concerns in obese individuals. Since we know the mutagenic potential of elevated insulin levels from previous data in our working group, the correlation between the highly mutagenic DNA DBSs marker, γ-H2AX and the plasma insulin level was tested and the findings indicated a positive correlation. In order to demonstrate the association between insulin-related oxidative stress and genomic damage, we used in vitro and in vivo models with Pten deficiency. In this part of study, the work was focused on liver. Pten is a known negative regulator of the PI3K/Akt pathway, which is responsible for the elevated NADPH oxidase activity and mitochondrial dysfunction through elevated insulin levels. Pten inhibition or deficiency were used to sensitize the system to insulin. Non-transformed immortalized human hepatocytes were used to show the mutagenic potential of elevated insulin and these in vitro data revealed once more the link between insulin signaling, elevated oxidative stress and genomic damage. Since the metabolic function of the liver is not only due to the extent of the hepatic insulin response but is also affected by systemic interactions, a whole-body Pten haplodeficient mouse model with an additional Pten+/-/Akt2-/- group was utilized for in vivo investigation of insulin-mediated toxicity. Our findings in this model suggested that Pten deficiency alone can cause an increase in oxidative stress. HFD alone was sufficient to increase the expression of HO-1 and genomic damage significantly. Moreover, the combination (whole-body Pten haplodeficient mice fed with HFD) showed significantly elevated oxidative stress and genomic damage in mouse liver. However, Akt2 knockout could only reduce the oxidative stress and DNA damage in high fat diet fed mice significantly. All these findings demonstrated that obesity can induce oxidative stress and genomic damage. Elevated insulin levels are associated with obesity-mediated oxidative stress and genomic damage. However, the underlying mechanisms are surely multifaceted and complicated. For example, Pten as oncogene might also induce other mechanisms besides the elevation of the PI3K/Akt pathway activity. In conclusion, it is clear that oxidative stress and DNA damage are linked to obesity and that weight loss can reduce these two factors. Since DNA-damage is associated with an elevated cancer risk, it might be logical to use an antioxidant therapy in obese individuals to reduce the side effects and oxidative stress dependent mutagenicity and cancer risk in these individuals. However, much more research will be needed to support this idea experimentally.},
  subject      = {{\"U}bergewicht},
  language  = {en}
}
@phdthesis{Eck2016,
  author    = {Eck, Saskia},
  title     = {The impact of thermogenetic depolarizations of specific clock neurons on Drosophila melanogaster's circadian clock},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137118},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms' behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly's circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock's interneuronal communication. Drosophila melanogaster's circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly's locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature. Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs). The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs \& l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day. To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner. However, the molecular link between clock neuron depolarization and shifts in the molecular clock's cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock. Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.},
  subject      = {Chronobiologie},
  language  = {en}
}
@phdthesis{VasquezOspina2016,
  author    = {Vasquez Ospina, Juan Jose},
  title     = {Development of tools for the study of gene regulation in Trypanosoma brucei},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133996},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The protozoan parasite Trypanosoma brucei is the causal agent of sleeping sickness and besides its epidemiological importance it has been used as model organism for the study of many aspects of cellular and molecular biology especially the post-transcriptional control of gene expression. Several studies in the last 30 years have shown the importance of mRNA processing and stability for gene regulation. In T. brucei genes are unusually arranged in polycistronic transcription units (PTUs) and a coupled process of trans-splicing and polyadenylation produces the mature mRNAs. Both processes, mRNA processing and stability, cannot completely explain the control of gene expression in the different life cycle stages analyzed in T. brucei so far. In recent years, the relevance of expression regulation at the level of translation has become evident in other eukaryotes. Therefore, in the first part of my thesis I studied the impact of translational regulation by means of a genome-wide ribosome profiling approach. My data suggest that translational efficiencies vary between life cycle stages of the parasite as well as between genes within one life cycle stage. Furthermore, using ribosome profiling I was able to identify many new putative un-annotated coding sequences and to evaluate the coding potential of upstream open reading frames (uORF). Comparing my results with previously published proteomic and RNA interference (RNAi) target sequencing (RIT-seq) datasets allowed me to validate some of the new coding sequences and to evaluate their relevance for the fitness of the parasite. In the second part of my thesis I used the transcriptomic and translatomic profiles obtained from the ribosome profiling analysis for the identification of putative non-coding RNAs (ncRNAs). These results led to the analysis of the coding potential in the regions upstream and downstream of the expressed variant surface glycoprotein (VSG), which is outlined in the third part of the results section. The region upstream of the VSG, the co-transposed region (CTR), has been implicated in an increase of the in situ switching rate upon its deletion. The ribosome profiling results indicated moderate transcription but not translation in this region. These results raised the possibility that the CTR may be transcribed into ncRNA. Therefore, in the third part of my thesis, I performed a primary characterization of the CTR-derived transcripts based on northern blotting and RACE. The results suggested the presence of a unique transcript species of about 1,200 nucleotides (nt) and polyadenylated at the 3'-end of the sequence. The deletion of the CTR sequence promoting and increase of the in situ switching rates was performed around 20 years ago by means of inserting reporter genes. With the recent development of endonuclease-based tools for genome editing, it is now possible to delete sequences in a marker-free way. In the fourth part of my thesis, I show the results on the implementation of the highly efficient genome-editing CRISPR-Cas9 system in T. brucei using episomes. As a proof of principle, I inserted the sequence coding for the enhanced green fluorescent protein (eGFP) at the end of the SCD6 coding sequence (CDS). Fluorescent cells were observed as early as two days after transfection. Therefore, after the successful set up of the CRISPR-Cas9 system it will be possible to modify genomic regions with more relevance for the biology of the parasite, such as the substitution of codons present in gene tandem arrays. The implementation of ribosome profiling in T. brucei opens the opportunity for the study of translational regulation in a genome-wide scale, the re-annotation of the currently available genome, the search for new putative coding sequences, the detection of putative ncRNAs, the evaluation of the coding potential in uORFs and the role of unstranslated regions (UTRs) in the regulation of translation. In turn, the implementation of the CRISPR-Cas9 system offers the possibility to manipulate the genome of the parasite at a nucleotide resolution and without the need of including resistant makers. The CRISPR-Cas9 system is a powerful tool for editing ncRNAs, UTRs, multicopy gene families and CDSs keeping their endogenous UTRs. Moreover, the system can be used for the modification of both alleles after just one round of transfection and of codons coding for amino acids carrying post-translational modifications (PTMs) among other possibilities.    },
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{Schuster2016,
  author    = {Schuster, Ann-Christin},
  title     = {Chemical and functional analyses of the plant cuticle as leaf transpiration barrier},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133475},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Cuticles cover all above-ground primary plant organs and are lipoid in nature consisting of a cutin matrix with cuticular waxes embedded within or deposited on its surface. The foremost function of the plant cuticle is the limitation of transpirational water loss into the surrounding atmosphere. Transpiration of water vapour from plants differs between stomatal and cuticular transpiration. Stomatal closure minimises the stomatal water loss and the remaining, much lower water transpiration occurs through the plant cuticle. Temperature influence on the transpiration barrier properties of intact leaves is not yet known, despite the importance of the cuticular transpiration especially under drought and heat conditions. The present study focuses on the temperature-dependent minimum water permeability of whole leaves, in comparison to the temperature effect on the cuticular permeance of isolated, astomatous cuticles (Chapter I - III). The minimum water permeability was determined gravimetrically from leaf drying curves and represents the cuticular water permeability of intact, stomatous leaves under conditions of complete stomatal closure. The temperature effect on the transpiration barrier of the desert plant Rhazya stricta and the Mediterranean sclerophyll Nerium oleander exposed a continuous increase of minimum water permeabilities with an increase in temperature. In contrast to other published studies, no abrupt and steep increase of the water permeability at high temperatures was detected. This steep increase indicates structural changes of the barrier properties of isolated cuticular membranes with a drastic decrease of efficiency. A stabilising impact of the cell wall on the plant cuticle of intact leaves was proposed. This steadying effect was confirmed with different experimental approaches measuring the cuticular water permeability of Prunus laurocerasus intact leaves. Physiological analysis of water transport on isolated, astomatous leaf cuticles indicated a drastic decline of the barrier properties at elevated temperatures for Prunus laurocerasus but not for Nerium oleander. Cuticular components were quantitatively and qualitatively analysed by gas chromatography with a flame ionisation detector and a mass spectrometric detector, respectively. A high accumulation of pentacyclic triterpenoids as cuticular wax components in relation to the cutin monomer coverage was detected for Nerium oleander and for Rhazya stricta leaves, too. Accordingly, reinforcing of the cutin matrix by triterpenoids was proposed to improve the mechanical strength and to reduce the extensibility of plant cuticles. Thus, structural changes of the cuticular barrier properties were potentially suppressed at elevated temperatures. The function of the cuticular wax amount and/or wax composition and its relation with the cuticular water permeability remains to be elucidated. In the second part of this work the cuticular wax quantity and quality as well as its impact on the transpiration barrier properties was analysed in order to deduce a potential relation between chemistry and function of plant cuticles (Chapter IV - V). Chemical analyses of the cuticular wax components of a wide range of plant species, including one tropical (Vanilla planifolia), temperate (Juglans regia, Plantago lanceolata), Mediterranean (Nerium oleander, Olea europaea) and one desert (Rhazya stricta) plant species, were conducted. The cuticular wax compositions of nine characteristic plant species from xeric limestone sites naturally located in Franconia (Southern Germany) were determined for the first time. The corresponding minimum or cuticular water permeabilities of both stomatous and astomatous leaf surfaces were measured to detect a potential relationship between the cuticular wax amount, wax composition and the cuticular barrier properties. It was demonstrated that abundant cuticular wax amounts did not constitute more efficient transpiration barriers. However, 55\% of the cuticular barrier function can be attributed to the very-long-chain aliphatic wax coverages. These new findings provide evidence that the acyclic wax constituents play a pivotal role establishing efficient transpiration barriers. Additionally, these findings strengthen the hypothesis that cyclic components, such as pentacyclic triterpenoids, do not hinder the water diffusion through plant cuticles as effectively as acyclic constituents. For the first time a relationship between the cuticular wax composition and the transpiration barrier properties of a wide range of plant species proved insights into the potential relation between chemistry and function of plant cuticles.},
  subject      = {Kutikula},
  language  = {en}
}