@phdthesis{Spohn1999, author = {Spohn, Gunther}, title = {The transcriptional control of virulence gene expression in Helicobacter pylori}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-2334}, school = {Universit{\"a}t W{\"u}rzburg}, year = {1999}, abstract = {The Gram-negative, spiral-shaped, microaerophilic bacterium Helicobacter pylori is the causative agent of various disorders of the upper gastrointestinal tract, such as chronic superficial gastritis, chronic active gastritis, peptic ulceration and adenocarcinoma. Although many of the bacterial factors associated with disease development have been analysed in some detail in the recent years, very few studies have focused so far on the mechanisms that regulate expression of these factors at the molecular level. In an attempt to obtain an overview of the basic mechanisms of virulence gene expression in H. pylori, three important virulence factors of this pathogen, representative of different pathogenic mechanisms and different phases of the infectious process, are investigated in detail in the present thesis regarding their transcriptional regulation. As an essential factor for the early phase of infection, including the colonisation of the gastric mucosa, the flagella are analysed; the chaperones including the putative adhesion factors GroEL and DnaK are investigated as representatives of the phase of adherence to the gastric epithelium and persistence in the mucus layer; and finally the cytotoxin associated antigen CagA is analysed as representative of the cag pathogenicity island, which is supposed to account for the phenomena of chronic inflammation and tissue damage observed in the later phases of infection. RNA analyses and in vitro transcription demonstrate that a single promoter regulates expression of cagA, while two promoters are responsible for expression of the upstream divergently transcribed cagB gene. All three promoters are shown to be recognised by RNA polymerase containing the vegetative sigma factor sigma 80. Promoter deletion analyses establish that full activation of the cagA promoter requires sequences up to -70 and binding of the C-terminal portion of the alpha subunit of RNA polymerase to an UP-like element located between -40 and -60, while full activation of the major cagB promoter requires sequences upstream of -96 which overlap with the cagA promoter. These data suggest that the promoters of the pathogenicity island represent a class of minimum promoters, that ensure a basic level of transcription, while full activation requires regulatory elements or structural DNA binding proteins that provide a suitable DNA context. Regarding flagellar biosynthesis, a master transcriptional factor is identified that regulates expression of a series of flagellar basal body and hook genes in concert with the alternative sigma factor sigma 54. Evidence is provided that this regulator, designated FlgR (for flagellar regulatory protein), is necessary for motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR is shown to act as a repressor of transcription of the sigma 28-regulated promoter of the flaA gene, while changes in DNA topology are shown to affect transcription of the sigma 54-regulated flaB promoter. These data indicate that the regulatory network that governs flagellar gene expression in H. pylori shows similarities to the systems of both Salmonella spp. and Caulobacter crescentus. In contrast to the flagellar genes which are regulated by three different sigma factors, the three operons encoding the major chaperones of H. pylori are shown to be transcribed by RNA polymerase containing the vegetative sigma factor sigma 80. Expression of these operons is shown to be regulated negatively by the transcriptional repressor HspR, a homologue of a repressor protein of Streptomyces spp., known to be involved in negative regulation of heat shock genes. In vitro studies with purified recombinant HspR establish that the protein represses transcription by binding to large DNA regions centered around the transcription initiation site in the case of one promoter, and around -85 and -120 in the case of the the other two promoters. In contrast to the situation in Streptomyces, where transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible with thermal stimuli. Transcription of two of the three chaperone encoding operons is induced by osmotic shock, while transcription of the third operon, although HspR-dependent, is not affected by salt treatment. Taken together, the analyses carried out indicate that H. pylori has reduced its repertoire of specific regulatory proteins to a basic level that may ensure coordinate regulation of those factors that are necessary during the initial phase of infection including the passage through the gastric lumen and the colonisation of the gastric mucosa. The importance of DNA topology and/or context for transcription of many virulence gene promoters may on the other hand indicate, that a sophisticated global regulatory network is present in H. pylori, which influences transcription of specific subsets of virulence genes in response to changes in the microenvironment.}, subject = {Helicobacter-pylori-Infektion}, language = {en} } @phdthesis{Ye2004, author = {Ye, Fang}, title = {The role of DNA supercoiling in the coordinated regulation of gene expression in Helicobacter pylori}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-9878}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {Summary Mechanisms of global gene regulation in bacteria are not well characterized yet. Changes in global or local supercoiling of chromosomal DNA are thought to play a role in global gene silencing and gene activation. In Helicobacter pylori, a bacterium with few dedicated transcriptional regulators, the structure of some promoters indicates a dependency on DNA topology. For example, the promoter of the major flagellar subunit gene flaA ({\´o}28-dependent) has a shorter spacing of 13 nucleotides (nt) in comparison to the consensus promoter (15 nt). Supercoiling changes might be a mechanism of gene-specific and global transcriptional regulation in this bacterium. The aim of this study was to elucidate, if changes in global supercoiling have an influence on global gene regulation in H. pylori, and on the temporal regulation of the flagellar biosynthesis pathway in this organism. In the present work, global DNA supercoiling in H. pylori was visualized for the first time, by determining the supercoiling state of plasmids under different growth conditions. Using this method, we showed that cellular supercoiling was clearly growth phase-dependent in H. pylori. Coinciding with increased supercoiling during the growth phases, transcription of the flaA gene was increased, while the transcription of a second {\´o}28-dependent gene with regular promoter spacing (HP0472) was reduced, supporting the hypothesis that growth phase-dependency of promoters might be mediated by changes of DNA topology. Supercoiling in H. pylori could be influenced in a reproducible fashion by inhibition of gyrase using novobiocin, which led to DNA relaxation and to a concomitant decrease of flaA transcript levels. Promoter spacer mutagenesis of the flaA promoter was performed. With flaA promoters of increased or reduced length, transcription of flaA was reduced, less susceptible to supercoiling changes, and, under specific conditions, inverted as compared to the wild type promoter. Transcriptional interdependence between the coupled topA-flaB genes and flaA was found by analysis of the flaA promoter mutants. Chromosomally linked gyrA-flgR, and topA-flaB genes were all dependent on supercoiling and coregulated with each other. Comprehensive transcript profiling (DNA microarrays) of wildtype H. pylori with and without novobiocin treatment identified a number of genes (10\% of total genes), including flagellin, virulence and housekeeping genes, which were strongly dependent on and appeared to be synchronized by supercoiling changes (transcriptional up- or downregulation). These findings indicate a tightly coupled temporal regulation of flagellar biogenesis and metabolism in H. pylori, dependent on global supercoiling. A specific group of genes was also regulated in H. pylori by overexpression of Topoisomerase I, as detected by genome-wide analysis (DNA microarray). The DNA-bending protein HU is thought to be responsible for influencing the negative supercoiling of DNA, through its ability to wrap DNA. HU is encoded by the hup single gene in H. pylori, and constitutively expressed during the whole growth curve. An H. pylori hup mutant was constructed. H. pylori cells lacking HU protein were viable, but exhibited a severe growth defect. Our data indicate that the lack of HU dramatically changes global DNA supercoiling, indicating an important function of HU in chromosome structuring in H. pylori. Transcriptome analyses were performed and demonstrated that a total of 66 genes were differentially transcribed upon hup deletion, which include virulence genes and many other cell functions. The data indicate that HU might act as further important global regulator in H. pylori. Increased gene expression of heat shock proteins and a decreased transcription of the urease gene cluster may indicate a co-ordinated response of H. pylori to changes of environmental conditions in its specific ecological niche, mediated by HU. After the whole genomic sequences of H. pylori strains 26695 and J99 were published, two ORFs (HP0116 and HP0440) were presumptively annotated as topoisomerase I orthologs. HP0116 is the functional H. pylori topoisomerase I (TopA). HP0440 (topA2) was found in only few (5 of 43) strains. Western blot analysis indicated that TopA2 is antigenically different from TopA. TopA2 is transcribed in H. pylori, but the protein must be functionally different from TopA, since it is lacking one functionally essential zinc finger motif, and was not able to functionally complement a TopA-deficient E. coli. Like topA, topA2 was also transcribed in a growth phase-dependent manner. We did not find a function of TopA2 in DNA structuring or topology, but, in the present study, we were able for the first time to establish a unique function for TopA2 in global gene regulation, by comprehensive transcriptome analysis (DNA microarray). Transcriptome analysis showed that a total of 46 genes were differentially regulated upon topA2 deletion, which included flagellar genes and urease genes. These results suggest that TopA2 might act as a novel important regulator of both flagellar biosynthesis and urease in H. pylori.}, subject = {Helicobacter pylori}, language = {en} } @phdthesis{Wedel2018, author = {Wedel, Carolin}, title = {The impact of DNA sequence and chromatin on transcription in \(Trypanosoma\) \(brucei\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173438}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {For cellular viability, transcription is a fundamental process. Hereby, the DNA plays the most elemental and highly versatile role. It has long been known that promoters contain conserved and often well-defined motifs, which dictate the site of transcription initiation by providing binding sites for regulatory proteins. However, research within the last decade revealed that it is promoters lacking conserved promoter motifs and transcribing constitutively expressed genes that constitute the majority of promoters in eukaryotes. While the process of transcription initiation is well studied, whether defined DNA sequence motifs are required for the transcription of constitutively expressed genes in eukaryotes remains unknown. In the highly divergent protozoan parasite Trypanosoma brucei, most of the proteincoding genes are organized in large polycistronic transcription units. The genes within one polycistronic transcription unit are generally unrelated and transcribed by a common transcription start site for which no RNA polymerase II promoter motifs have been identified so far. Thus, it is assumed that transcription initiation is not regulated but how transcription is initiated in T. brucei is not known. This study aimed to investigate the requirement of DNA sequence motifs and chromatin structures for transcription initiation in an organism lacking transcriptional regulation. To this end, I performed a systematic analysis to investigate the dependence of transcription initiation on the DNA sequence. I was able to identify GT-rich promoter elements required for directional transcription initiation and targeted deposition of the histone variant H2A.Z, a conserved component during transcription initiation. Furthermore, nucleosome positioning data in this work provide evidence that sites of transcription initiation are rather characterized by broad regions of open and more accessible chromatin than narrow nucleosome depleted regions as it is the case in other eukaryotes. These findings highlight the importance of chromatin during transcription initiation. Polycistronic RNA in T. brucei is separated by adding an independently transcribed miniexon during trans-splicing. The data in this work suggest that nucleosome occupancy plays an important role during RNA maturation by slowing down the progressing polymerase and thereby facilitating the choice of the proper splice site during trans-splicing. Overall, this work investigated the role of the DNA sequence during transcription initiation and nucleosome positioning in a highly divergent eukaryote. Furthermore, the findings shed light on the conservation of the requirement of DNA motifs during transcription initiation and the regulatory potential of chromatin during RNA maturation. The findings improve the understanding of gene expression regulation in T. brucei, a eukaryotic parasite lacking transcriptional Regulation.}, subject = {Transkription}, language = {en} } @phdthesis{Subota2011, author = {Subota, Ines}, title = {Switches in trypanosome differentiation: ALBA proteins acting on post-transcriptional mRNA control}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85707}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Trypanosoma brucei is a digenetic eukaryotic parasite that develops in different tissues of a mammalian host and a tsetse fly. It is responsible for sleeping sickness in sub-saharan Africa. The parasite cycle involves more than nine developmental stages that can be clearly distinguished by their general morphology, their metabolism and the relative positioning of their DNA-containing organelles. During their development, trypanosomes remain exclusively extracellular and encounter changing environments with different physico-chemical properties (nutritional availability, viscosity, temperature, etc.). It has been proposed that trypanosomes use their flagellum as a sensing organelle, in agreement with the established role of structurally-related cilia in metazoa and ciliates. Recognition of environmental triggers is presumed to be at the initiation of differentiation events, leading to the parasite stage that is the best suited to the new environment. These changes are achieved by the modification of gene expression programmes, mostly underlying post-transcriptional control of mRNA transcripts. We first demonstrate that the RNA-binding proteins ALBA3/4 are involved in specific differentiation processes during the parasite development in the fly. They are cytosolic and expressed throughout the parasite cycle with the exception of the stages found in the tsetse fly proventriculus, as shown by both immunofluorescence and live cell analysis upon endogenous tagging with YFP. Knock-down of both proteins in the developmental stage preceding these forms leads to striking modifications: cell elongation, cell cycle arrest and relocalization of the nucleus in a posterior position, all typical of processes acting in parasites found in the proventriculus region. When ALBA3 is over-expressed from an exogenous copy during infection, it interferes with the relocalization of the nucleus in proventricular parasites. This is not observed for ALBA4 over-expression that does not visibly impede differentiation. Both ALBA3/4 proteins react to starvation conditions by accumulating in cytoplasmic stress granules together with DHH1, a recognized RNA-binding protein. ALBA3/4 proteins also partially colocalize with granules formed by polyA+ RNA in these conditions. We propose that ALBA are involved in trypanosome differentiation processes where they control a subset of developmentally regulated transcripts. These processes involving ALBA3/4 are likely to result from the specific activation of sensing pathways. In the second part of the thesis, we identify novel flagellar proteins that could act in sensing mechanisms. Several protein candidates were selected from a proteomic analysis of intact flagella performed in the host laboratory. This work validates their flagellar localization with high success (85\% of the proteins examined) and defines multiple different patterns of protein distribution in the flagellum. Two proteins are analyzed during development, one of them showing down-regulation in proventricular stages. The functional analysis of one novel flagellar membrane protein reveals its rapid dynamics within the flagellum but does not yield a visible phenotype in culture. This is coherent with sensory function that might not be needed in stable culture conditions, but could be required in natural conditions during development. In conclusion, this work adds new pieces to the puzzle of identifying molecular switches involved in developmental mRNA control and environmental sensing in trypanosome stages in the tsetse fly.}, subject = {Trypanosoma brucei}, language = {en} } @phdthesis{Hofmann2013, author = {Hofmann, Sebastian}, title = {Studies on the function and regulation of CD84, GPVI and Orai2 in genetically modified mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87949}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Platelet activation and aggregation at sites of vascular injury are essential processes to limit blood loss but they also contribute to arterial thrombosis, which can lead to myocardial infarction and stroke. Stable thrombus formation requires a series of events involving platelet receptors which contribute to adhesion, activation and aggregation of platelets. Regulation of receptor expression by (metallo-)proteinases has been described for several platelet receptors, but the molecular mechanisms are ill-defined. The signaling lymphocyte activation molecule (SLAM) family member CD84 is expressed in immune cells and platelets, however its role in platelet physiology was unclear. In this thesis, CD84 deficient mice were generated and analyzed. In well established in vitro and in vivo assays testing platelet function and thrombus formation, CD84 deficient mice displayed phenotypes indistinguishable from wild-type controls. It was concluded that CD84 in platelets does not function as modulator of thrombus formation, but rather has other functions. In line with this, in the second part of this thesis, a novel regulation mechanism for platelet CD84 was discovered and elucidated. Upon platelet activation, the N-terminus of CD84 was found to be cleaved exclusively by the a disintegrin and metalloproteinase 10 (ADAM10), whereas the intracellular part was cleaved by calpain. In addition, regulation of the platelet activating collagen receptor glycoprotein VI (GPVI) was studied and it was shown that GPVI is in contrast to CD84 differentially regulated by ADAM10 and ADAM17. A novel role of CD84 under pathophysiological conditions was revealed as CD84 deficient mice were protected from ischemic stroke in the model of transient middle cerebral artery occlusion and this protection was based on the lack of CD84 in T cells. Ca2+ is an essential second messenger that facilitates activation of platelets and diverse functions in different eukaryotic cell types. Store-operated Ca2+ entry (SOCE) represents the major mechanism leading to rise in intracellular Ca2+ concentration in non-excitable cells. The Ca2+ sensor STIM1 (stromal interaction molecule 1) and the SOC channel subunit protein Orai1 are established mediators of SOCE in platelets. STIM2 is the major STIM isoform in neurons, but the role of the SOC channel subunit protein Orai2 in platelets and neurons has remained elusive. In the third part of this thesis, Orai2 deficient mice were generated and analyzed. Orai2 was dispensable for platelet function, however, Orai2 deficient mice were protected from ischemic neurodegeneration and this phenotype was attributed to defective SOCE in neurons.}, subject = {Thrombozyt}, language = {en} } @phdthesis{Kriegebaum2009, author = {Kriegebaum, Claudia}, title = {Spatio-temporal Expression Patterns of the Serotonin Synthesis Enzymes TPH1 and TPH2 and Effects of Acute Stress}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-40839}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotions and stress and point to their potential relevance to the etiology and pathogenesis of various neuropsychiatric disorders. However, the differential expression pattern of the two isoforms TPH1 and TPH2 which encode two forms of the rate-limiting enzyme of 5-HT synthesis is controversial. Here, a comprehensive spatio-temporal analysis clarifies TPH1 and TPH2 expression during pre- and postnatal development of the mouse brain and in adult human brain as well as in peripheral organs including the pineal gland. Four different methods (real time PCR, in situ hybridization, immunohistochemistry and Western blot analysis) were performed to systematically control for tissue-, species- and isoform-specific expression on both the pre- and posttranslational level. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibres in the deep pineal gland and in the gastrointestinal tract. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. Also under conditions like stress and clearing the tissue from blood cells, no changes in expression levels were detectable. Furthermore, the reuptake of 5-HT into the presynaptic neuron by the serotonin transporter (SERT) is the major mechanism terminating the neurotransmitter signal. Thus, mice with a deletion in the Sert gene (Sert KO mice) provide an adequate model for human affective disorders to study lifelong modified 5-HT homeostasis in interaction with stressful life events. To further explore the role of TPH isoforms, Tph1 and Tph2 expression was studied in the raphe nuclei of Sert deficient mice under normal conditions as well as following exposure to acute immobilization stress. Interestingly, no statistically significant changes in expression were detected. Moreover, in comparison to Tph2, no relevant Tph1 expression was detected in the brain independent from genotype, gender and treatment confirming expression in data from native animals. Raphe neurons of a brain-specific Tph2 conditional knockout (cKO) model were completely devoid of Tph2-positive neurons and consequently 5-HT in the brain, with no compensatory activation of Tph1 expression. In addition, a time-specific Tph2 inducible (i) KO mouse provides a brain-specific knockdown model during adult life, resulting in a highly reduced number of Tph2-positive cells and 5-HT in the brain. Intriguingly, expression studies detected no obvious alteration in expression of 5-HT system-associated genes in these brain-specific Tph2 knockout and knockdown models. The findings on the one hand confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan and on the other hand indicate that neither developmental nor adult Tph2-dependent 5-HT synthesis is required for normal formation of the serotonergic system, although Tph1 does not compensate for the lack of 5-HT in the brain of Tph2 KO models. A further aim of this thesis was to investigate the expression of the neuropeptide oxytocin, which is primarily produced in the hypothalamus and released for instance in response to stimulation of 5-HT and selective serotonin reuptake inhibitors (SSRIs). Oxytocin acts as a neuromodulator within the central nervous system (CNS) and is critically involved in mediating pain modulation, anxiolytic-like effects and decrease of stress response, thereby reducing the risk for emotional disorders. In this study, the expression levels of oxytocin in different brain regions of interest (cortex, hippocampus, amygdala, hypothalamus and raphe nuclei) from female and male wildtype (WT) and Sert KO mice with or without exposure to acute immobilization stress were investigated. Results showed significantly higher expression levels of oxytocin in brain regions which are involved in the regulation of emotional stimuli (amygdala and hippocampus) of stressed male WT mice, whereas male Sert KO as well as female WT and Sert KO mice lack these stress-induced changes. These findings are in accordance with the hypothesis of oxytocin being necessary for protection against stress, depressive mood and anxiety but suggest gender-dependent differences. The lack of altered oxytocin expression in Sert KO mice also indicates a modulation of the oxytocin response by the serotonergic system and provides novel research perspectives with respect to altered response of Sert KO mice to stress and anxiety inducing stimuli.}, subject = {Serotonin}, language = {en} } @phdthesis{Post2014, author = {Post, Antonia}, title = {Snap25 heterozygous knockout mice as a potential model for attention deficit/hyperactivity disorder (ADHD)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122899}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {SNAP25 (Synaptosomal-Associated Protein of 25 kDa; part of the SNARE complex) is involved in the docking and fusion of synaptic vesicles in presynaptic neurons necessary for the regulation of neurotransmitter release, as well as in axonal growth and synaptic plasticity. In humans, different single nucleotide polymorphisms of SNAP25 have repeatedly been associated with attention deficit/hyperactivity disorder (ADHD). Thus, in this study heterozygous Snap25 knockout mice were investigated as a model of ADHD. Heterozygous (+/-) Snap25 knockout mice as well as their wild-type (+/+) littermates were reared under control conditions or underwent a Maternal Separation (MS) procedure. Starting at the age of 2 months, mice were tested for locomotor activity in a repeated long-term Open Field (OF) task, for attention deficits and impulsive behavior in the 5 Choice Serial Reaction Time Task (5CSRTT), for anxiety-like behavior in the Light-Dark Box (LDB) and for depression-like behavior in the Porsolt Forced Swim Test (FST). The brains of these mice were subsequently tested for the expression of several ADHD related genes in a quantitative Real-Time PCR (qRT-PCR) study. Another group of female mice (+/+; +/-) underwent a one hour OF test after oral administration of 45 mg/kg Methylphenidate (MPH) or placebo. To find an optimized dosage for this MPH challenge, a pilot study was performed. Wild-type C57BL/6 mice were tested in a long-term OF with several dosages of MPH both intraperitoneally (i.p.) and orally. The brains of these animals were afterwards investigated for neurotransmitter concentrations. In this pilot study the dosages of MPH that were similarly behaviorally effective without causing symptoms of overdosing were 7.5-15 mg/kg intraperitoneally and 30-60 mg/kg orally. However, even though it was possible to find intraperitoneal and oral doses that correlate behaviorally, the neurochemistry was mostly different. In the study on Snap25-deficient mice, unstressed controls showed a hyperactive phenotype in the second of two long-term OF sessions (60 min) spaced three weeks apart. Considering all groups, there was a significant interaction of stress and genotype in the second session, with animals subjected to MS being overall hyperactive with no genotype differences. In the training phase of the 5CSRTT only effects of stress were found, with MS animals finding and consuming fewer rewards. In the single test trial, several genotype effects became apparent, with tendencies for the number of correct nose pokes and the number of rewards eaten, and a significant effect for the number of rewards eaten directly after the correct response. In all of these variables +/- mice performed worse than their wild-type littermates. In the LDB +/- mice entered the lit compartment of the arena earlier than the controls, thus showing attenuated anxiety-like behavior. Regarding depressive-like behavior in the FST, male +/- mice spent significantly less time struggling than male +/+ mice. In the gene expression study, +/- mice had lower expression levels of Maoa and Comt, and higher expression levels of Nos1 than wild-types. Finally, the locomotor activity response to MPH was exaggerated in +/- mice as compared to controls. Heterozygous Snap25 knockout mice show some of the behavioral characteristics of ADHD, as for example a mild hyperactivity in a familiar environment, difficulties in the correct execution of a given task and even some behavior that can be interpreted as delay aversion. Additionally, expression levels of three ADHD related genes were changed in these animals. Although the exaggerated locomotor activity response to MPH is not to be expected of an ADHD model, the difference in the response between +/+ and +/- mice nonetheless implicates a potential dysfunction of the brain dopaminergic system.}, subject = {Aufmerksamkeitsdefizit-Syndrom}, language = {en} } @phdthesis{BakariSoale2024, author = {Bakari Soale, Majeed}, title = {Regulation of the Variant Surface Glycoprotein (VSG) Expression and Characterisation of the Nucleolar DExD/H box Protein Hel66 in \(Trypanosoma\) \(brucei\)}, doi = {10.25972/OPUS-25809}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-258090}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {The variant surface glycoprotein (VSG) of African trypanosomes plays an essential role in protecting the parasites from host immune factors. These trypanosomes undergo antigenic variation resulting in the expression of a single VSG isoform out of a repertoire of around 2000 genes. The molecular mechanism central to the expression and regulation of the VSG is however not fully understood. Gene expression in trypanosomes is unusual due to the absence of typical RNA polymerase II promoters and the polycistronic transcription of genes. The regulation of gene expression is therefore mainly post-transcriptional. Regulatory sequences, mostly present in the 3´ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 100 \% conserved 16mer motif within the 3´ UTR has been shown to modulate the stability of VSG transcripts and hence their expression. As a stability-associated sequence element, the absence of nucleotide substitutions in the motif is however unusual. It was therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts. In this study, it was demonstrated that the 100 \% conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. It was further shown that the intact motif in the active VSG 3´ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Crosstalk between the VSG and procyclin genes during differentiation to the insect vector stage is also unaffected in cells with a mutated 16mer motif. Ectopic overexpression of a second VSG however requires the intact motif to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The 16mer motif therefore plays a dual role in VSG in situ switching and stability of VSG transcripts. The additional role of the 16mer in the essential process of antigenic variation appears to be the driving force for the 100 \% conservation of this RNA motif. A screen aimed at identifying candidate RNA-binding proteins interacting with the 16mer motif, led to the identification of a DExD/H box protein, Hel66. Although the protein did not appear to have a direct link to the 16mer regulation of VSG expression, the DExD/H family of proteins are important players in the process of ribosome biogenesis. This process is relatively understudied in trypanosomes and so this candidate was singled out for detailed characterisation, given that the 16mer story had reached a natural end point. Ribosome biogenesis is a major cellular process in eukaryotes involving ribosomal RNA, ribosomal proteins and several non-ribosomal trans-acting protein factors. The DExD/H box proteins are the most important trans-acting protein factors involved in the biosynthesis of ribosomes. Several DExD/H box proteins have been directly implicated in this process in yeast. In trypanosomes, very few of this family of proteins have been characterised and therefore little is known about the specific roles they play in RNA metabolism. Here, it was shown that Hel66 is involved in rRNA processing during ribosome biogenesis. Hel66 localises to the nucleolus and depleting the protein led to a severe growth defect. Loss of the protein also resulted in a reduced rate of global translation and accumulation of rRNA processing intermediates of both the small and large ribosomal subunits. Hel66 is therefore an essential nucleolar DExD/H protein involved in rRNA processing during ribosome biogenesis. As very few protein factors involved in the processing of rRNAs have been described in trypanosomes, this finding represents an important platform for future investigation of this topic.}, subject = {Trypanosoma brucei}, language = {en} } @phdthesis{Zhang2014, author = {Zhang, Yi}, title = {Regulation of Agrobacterial Oncogene Expression in Host Plants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-102578}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Virulent Agrobacterium tumefaciens strains transfer and integrate a DNA region of the tumor-inducing (Ti) plasmid, the T-DNA, into the plant genome and thereby cause crown gall disease. The most essential genes required for crown gall development are the T-DNA-encoded oncogenes, IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) for auxin, and Ipt (isopentenyl transferase) for cytokinin biosynthesis. When these oncogenes are expressed in the host cell, the levels of auxin and cytokinin increase and cause cell proliferation. The aim of this study was to unravel the molecular mechanisms, which regulate expression of the agrobacterial oncogenes in plant cells. Transcripts of the three oncogenes were expressed in Arabidopsis thaliana crown galls induced by A. tumefaciens strain C58 and the intergenic regions (IGRs) between their coding sequences (CDS) were proven to have promoter activity in plant cells. These promoters possess eukaryotic sequence structures and contain cis-regulatory elements for the binding of plant transcription factors. The high-throughput protoplast transactivation (PTA) system was used and identified the Arabidopsis thaliana transcription factors WRKY18, WRKY40, WRKY60 and ARF5 to activate the Ipt oncogene promoter. No transcription factor promoted the activity of the IaaH and IaaM promoters, despite the fact that the sequences contained binding elements for type B ARR transcription factors. Likewise, the treatment of Arabidopsis mesophyll protoplasts with cytokinin (trans-zeatin) and auxin (1-NAA) exerted no positive effect on IaaH and IaaM promoter activity. In contrast, the Ipt promoter strongly responded to a treatment with auxin and only modestly to cytokinin. The three Arabidopsis WRKYs play a role in crown gall development as the wrky mutants developed smaller crown galls than wild-type plants. The WRKY40 and WRKY60 genes responded very quickly to pathogen infection, two and four hours post infection, respectively. Transcription of the WRKY18 gene was induced upon buffer infiltration, which implicates a response to wounding. The three WRKY proteins interacted with ARF5 and with each other in the plant nucleus, but only WRKY40 together with ARF5 increased activation of the Ipt promoter. Moreover, ARF5 activated the Ipt promoter in an auxin-dependent manner. The severe developmental phenotype of the arf5 mutant prevented studies on crown gall development, nevertheless, the reduced crown gall growth on the transport inhibitor response 1 (TIR1) tir1 mutant, lacking the auxin sensor, suggested that auxin signaling is required for optimal crown gall development. In conclusion, A. tumefaciens recruits the pathogen defense related WRKY40 pathway to activate Ipt expression in T-DNA-transformed plant cells. IaaH and IaaM gene expression seems not to be controlled by transcriptional activators, but the increasing auxin levels are signaled via ARF5. The auxin-depended activation of ARF5 boosts expression of the Ipt gene in combination with WRKY40 to increase cytokinin levels and induce crown gall development.}, subject = {Agrobacterium tumefaciens}, language = {en} } @phdthesis{Zheng2012, author = {Zheng, Peilin}, title = {Ptpn22 silencing in the NOD model of type 1 diabetes indicates the human susceptibility allele of PTPN22 is a gain-of-function variant}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73869}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {PTPN22 encodes the lymphoid tyrosine phosphatase Lyp that can dephosphorylate Lck, ZAP-70 and Fyn to attenuate TCR signaling. A single-nucleotide polymorphism (C1858T) causes a substitution from arginine (R) to tryptophan (W) at 620 residue (R620W). Lyp-620W has been confirmed as a susceptible allele in multiple autoimmune diseases, including type 1 diabetes (T1D). Several independent studies proposed that the disease-associated allele is a gain-of-function variant. However, a recent report found that in human cells and a knockin mouse containing the R620W homolog that Ptpn22 protein degradation is accelerated, indicating Lyp-620W is a loss-of-function variant. Whether Lyp R620W is a gain- or loss-of-function variant remains controversial. To resolve this issue, we generated two lines (P2 and P4) of nonobese diabetic (NOD) mice in which Ptpn22 can be inducibly silenced by RNAi. We found long term silencing of Ptpn22 increased spleen cellularity and regulatory T (Treg) cell numbers, replicating the effect of gene deletion reported in the knockout (KO) B6 mice. Notably, Ptpn22 silencing also increased the reactivity and apoptotic behavior of B lymphocytes, which is consistent with the reduced reactivity and apoptosis of human B cells carrying the alleged gain-of-function PTPN22 allele. Furthermore, loss of Ptpn22 protected P2 KD mice from spontaneous and Cyclophosphamide (CY) induced diabetes. Our data support the notion that Lyp-620W is a gain-of-function variant. Moreover, Lyp may be a valuable target for the treatment of autoimmune diseases.}, subject = {Diabetes mellitus}, language = {en} } @phdthesis{Brede2013, author = {Brede, Christian}, title = {Peripheral alloantigen expression directs the organ specific T cell infiltration after hematopoietic cell transplantation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85365}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {In acute graft-versus-host disease (GVHD) alloreactive donor T cells selectively damage skin, liver, and the gastrointestinal tract while other organs are rarely affected. The mechanism of this selective target tissue infiltration is not well understood. We investigated the importance of alloantigen expression for the selective organ manifestation by examining spatiotemporal changes of cellular and molecular events after allogeneic hematopoietic cell transplantation (allo-HCT). To accomplish this we established a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized protocols for antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following allo-HCT. In addition, we proofed that LSFM is suitable to map individual T cell subsets after HCT and detected rare cellular events. We employed this versatile technique to study the role of alloantigen expression for the selective organ manifestation after allo-HCT. Therefore, we used a T cell receptor (TCR) transgenic mouse model of GVHD that targets a single peptide antigen and thereby mimics a major histocompatibility complex (MHC)-matched single antigen mismatched (miHAg-mismatched) HCT. We transplanted TCR transgenic (OT-I) T cells into myeloablatively conditioned hosts that either express the peptide antigen ovalbumin ubiquitously (βa-Ova) or selectively in the pancreas (RIP-mOva), an organ that is normally not affected by acute GVHD. Of note, at day+6 after HCT we observed that OT-I T cell infiltration occurred in an alloantigen dependent manner. In βa-Ova recipients, where antigen was ubiquitously expressed, OT-I T cells infiltrated all organs and were not restricted to gastrointestinal tract, liver, and skin. In RIP-mOva recipients, where cognate antigen was only expressed in the pancreas, OT-I T cells selectively infiltrated this organ that is usually spared in acute GVHD. In conditioned RIP-mOva the transfer of 100 OT-I T cells sufficed to effectively infiltrate and destroy pancreatic islets resulting in 100\% mortality. By employing intact tissue LSFM in RIP-mOva recipients, we identified very low numbers of initial islet infiltrating T cells on day+4 after HCT followed by a massive T cell migration to the pancreas within the following 24 hours. This suggested an effective mechanism of effector T cell recruitment to the tissue of alloantigen expression after initial antigen specific T cell encounter. In chimeras that either expressed the model antigen ovalbumin selectively in hematopoietic or in parenchymal cells only, transplanted OT-I T cells infiltrated target tissues irrespective of which compartment expressed the alloantigen. As IFN-γ could be detected in the serum of transplanted ovalbumin expressing recipients (βa-Ova, βa-Ova-chimeras and RIP-mOva) at day+6 after HCT, we hypothesized that this cytokine may be functionally involved in antigen specific OT-I T cell mediated pathology. In vitro activated OT-I T cells responded with the production of IFN-γ upon antigen re-encounter suggesting that IFN-γ might be relevant in the alloantigen dependent organ infiltration of antigen specific CD8+ T cell infiltration after HCT. Based on these data we propose that alloantigen expression plays an important role in organ specific T cell infiltration during acute GVHD and that initial alloreactive T cells recognizing the cognate antigen propagate a vicious cycle of enhanced T cell recruitment that subsequently culminates in the exacerbation of tissue restricted GVHD.}, subject = {Alloantigen}, language = {en} } @phdthesis{Jessen2021, author = {Jessen, Christina}, title = {NRF2 links antioxidant and immune-relevant features in melanoma}, doi = {10.25972/OPUS-23349}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-233495}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The transcription factor NRF2 is considered as the master regulator of cytoprotective and ROS-detoxifying gene expression. Due to their vulnerability to accumulating reactive oxygen species, melanomas are dependent on an efficient oxidative stress response, but to what extent melanomas rely on NRF2 is only scarcely investigated so far. In tumor entities harboring activating mutations of NRF2, such as lung adenocarcinoma, NRF2 activation is closely connected to therapy resistance. In melanoma, activating mutations are rare and triggers and effectors of NRF2 are less well characterized. This work revealed that NRF2 is activated by oncogenic signaling, cytokines and pro-oxidant triggers, released cell-autonomously or by the tumor microenvironment. Moreover, silencing of NRF2 significantly reduced melanoma cell proliferation and repressed well-known NRF2 target genes, indicating basal transcriptional activity of NRF2 in melanoma. Transcriptomic analysis showed a large set of deregulated gene sets, besides the well-known antioxidant effectors. NRF2 suppressed the activity of MITF, a marker for the melanocyte lineage, and induced expression of epidermal growth factor receptor (EGFR), thereby stabilizing the dedifferentiated melanoma phenotype and limiting pigmentation markers and melanoma-associated antigens. In general, the dedifferentiated melanoma phenotype is associated with a reduced tumor immunogenicity. Furthermore, stress-inducible cyclooxygenase 2 (COX2) expression, a crucial immune-modulating gene, was regulated by NRF2 in an ATF4-dependent manner. Only in presence of both transcription factors was COX2 robustly induced by H2O2 or TNFα. COX2 catalyzes the first step of the prostaglandin E2 (PGE2) synthesis, which was described to be associated with tumor immune evasion and reduction of the innate immune response. In accordance with these potentially immune-suppressive features, immunocompetent mice injected with NRF2 knockout melanoma cells had a strikingly longer tumor-free survival compared to NRF2-proficient cells. In line with the in vitro data, NRF2-deficient tumors showed suppression of COX2 and induction of MITF. Furthermore, transcriptomic analyses of available tumors revealed a strong induction of genes belonging to the innate immune response, such as RSAD2 and IFIH1. The expression of these genes strongly correlated with immune evasion parameters in human melanoma datasets and NRF2 activation or PGE2 supplementation limited the innate immune response in vitro. In summary, the stress dependent NRF2 activation stabilizes the dedifferentiated melanoma phenotype and facilitates the synthesis of PGE2. As a result, NRF2 reduces gene expression of the innate immune response and promotes the generation of an immune-cold tumor microenvironment. Therefore, NRF2 not only elevated the ROS resilience, but also strongly contributed to tumor growth, maintenance, and immune control in cutaneous melanoma.}, subject = {Melanom}, language = {en} } @phdthesis{Friedrich2009, author = {Friedrich, Torben}, title = {New statistical Methods of Genome-Scale Data Analysis in Life Science - Applications to enterobacterial Diagnostics, Meta-Analysis of Arabidopsis thaliana Gene Expression and functional Sequence Annotation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39858}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.}, subject = {Genomik}, language = {en} } @phdthesis{Sommerlandt2017, author = {Sommerlandt, Frank M. J.}, title = {Mechanisms of visual memory formation in bees: About immediate early genes and synaptic plasticity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136997}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Animals form perceptual associations through processes of learning, and retain that information through mechanisms of memory. Honeybees and bumblebees are classic models for insect perception and learning, and despite their small brains with about one million neurons, they are organized in highly social colonies and possess an astonishing rich behavioral repertoire including navigation, communication and cognition. Honeybees are able to harvest hundreds of morphologically divergent flower types in a quick and efficient manner to gain nutrition and, back in the hive, communicate discovered food sources to nest mates. To accomplish such complex tasks, bees must be equipped with diverse sensory organs receptive to stimuli of different modalities and must be able to associatively learn and memorize the acquired information. Particularly color vision plays a prominent role, e.g. in navigation along landmarks and when bees identify inflorescences by their color signals. Once acquired, bees are known to retain visual information for days or even months. Numerous studies on visual perception and color vision have been conducted in the past decades and largely revealed the information processing pathways in the brain. In contrast, there are no data available on how the brain may change in the course of color learning experience and whether pathways differ for coarse and fine color learning. Although long-term memory (LTM) storage is assumed to generally include reorganization of the neuronal network, to date it is unclear where in the bee brain such changes occur in the course of color learning and whether visual memories are stored in one particular site or decentrally distributed over different brain domains. The present dissertation research aimed to dissect the visual memory trace in bees that is beyond mere stimulus processing and therefore two different approaches were elaborated: first, the application of immediate early genes (IEG) as genetic markers for neuronal activation to localize early processes underlying the formation of a stable LTM. Second, the analysis of late consequences of memory formation, including synaptic reorganization in central brain areas and dependencies of color discrimination complexity. Immediate early genes (IEG) are a group of rapidly and transiently expressed genes that are induced by various types of cellular stimulation. A great number of different IEGs are routinely used as markers for the localization of neuronal activation in vertebrate brains. The present dissertation research was dedicated to establish this approach for application in bees, with focus on the candidate genes Amjra and Amegr, which are orthologous to the two common vertebrate IEGs c-jun and egr-1. First the general requirement of gene transcription for visual LTM formation was proved. Bumblebees were trained in associative proboscis extension response (PER) conditioning to monochromatic light and subsequently injected with an inhibitor of gene transcription. Memory retention tests at different intervals revealed that gene transcription is not required for the formation of a mid-term memory, but for stable LTM. Next, the appliance of the candidate genes was validated. Honeybees were exposed to stimulation with either alarm pheromone or a light pulse, followed by qPCR analysis of gene expression. Both genes differed in their expression response to sensory exposure: Amjra was upregulated in all analyzed brain parts (antennal lobes, optic lobes and mushroom bodies, MB), independent from stimulus modality, suggesting the gene as a genetic marker for unspecific general arousal. In contrast, Amegr was not significantly affected by mere sensory exposure. Therefore, the relevance of associative learning on Amegr expression was assessed. Honeybees were trained in visual PER conditioning followed by a qPCR-based analysis of the expression of all three Amegr isoforms at different intervals after conditioning. No learning-dependent alteration of gene expression was observed. However, the presence of AmEgr protein in virtually all cerebral cell nuclei was validated by immunofluorescence staining. The most prominent immune-reactivity was detected in MB calyx neurons. Analysis of task-dependent neuronal correlates underlying visual long-term memory was conducted in free-flying honeybees confronted with either absolute conditioning to one of two perceptually similar colors or differential conditioning with both colors. Subsequent presentation of the two colors in non-rewarded discrimination tests revealed that only bees trained with differential conditioning preferred the previously learned color. In contrast, bees of the absolute conditioning group chose randomly among color stimuli. To investigate whether the observed difference in memory acquisition is also reflected at the level of synaptic microcircuits, so called microglomeruli (MG), within the visual domains of the MB calyces, MG distribution was quantified by whole-mount immunostaining three days following conditioning. Although learning-dependent differences in neuroarchitecture were absent, a significant correlation between learning performance and MG density was observed. Taken together, this dissertation research provides fundamental work on the potential use of IEGs as markers for neuronal activation and promotes future research approaches combining behaviorally relevant color learning tests in bees with examination of the neuroarchitecture to pave the way for unraveling the visual memory trace.}, subject = {Biene}, language = {en} } @phdthesis{Wu2006, author = {Wu, Rongxue}, title = {Integrins and SPARC : potential implications for cardiac remodeling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-17531}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Der enorme Umbau des Herzgewebes, wie man ihn nach Druck{\"u}berlastung des Ventrikels oder MyokardInfarkt beobachten kann, gilt als eine der kausalen Ursachen des Herzversagens. Die Ver{\"a}nderungen in der Architektur des Herzens beeinflussen die mechanischen Eigenschaften des Herzmuskels, begr{\"u}ndet sind sie jedoch in Anpassungsprozessen auf der zellul{\"a}ren Ebene vor allem in einer Modulation der Expression bestimmter Gene. Gemeinsam mit Integrinen, den Transmembran-Rezeptoren, welche die extrazellul{\"a}re Umgebung mit dem intrazellul{\"a}ren Zytoskelett verbinden, geh{\"o}ren Proteine der extrazellul{\"a}ren Matrix (ECM) und matrizellul{\"a}re Proteine zu den Schl{\"u}sselkomponenten, die den Umbauprozess im Herzen steuern. Aus diesen Gr{\"u}nden hatte diese Doktorarbeit zum Ziel, die Rolle der Integrine f{\"u}r die Regulation der Genexpression und die Leistungsf{\"a}higkeit des Herzmuskels w{\"a}hrend der durch Druck{\"u}berlastung oder myokardialen Infarkt (MI) hervorgerufenen Wundheilungsprozesse zu analysieren. Um die Beteiligung von Integrin Beta 1 zu untersuchen, wurde ein experimentelles Modell der Druck{\"u}berlastung im Mausherzen (aortic banding; Konstriktion der Aorta; AB) eingesetzt, wobei M{\"a}use mit einer konditionalen, Herz-spezifischen Deletion des Integrin Beta 1 Gens untersucht wurden. Ein besonderes Augenmerk wurde dabei auf die physiologischen Unterschiede und eine ver{\"a}nderte Genexpression im gestressten Herzen in An- oder Abwesenheit von Integrin Beta 1 gelegt. Interessanterweise wurden die M{\"a}use, welche eine Kombination aus Integrin knock-out Allel und dem Kardiomyozyten-spezifischen konditionalen knock-out Allel von Integrin Beta 1 aufwiesen im normalen Mendelschen Verh{\"a}ltnis geboren und wuchsen normal auf. Obwohl diese Tiere immer noch geringe Mengen von Integrin Beta 1 in ihrem Herzen aufwiesen (exprimiert von nicht-Myozyten), besaßen diese M{\"a}use eine ver{\"a}nderte Herzfunktion und waren sehr sensitiv gegen{\"u}ber AB. Im Gegensatz zu der kompensatorischen hypertrophischen Reaktion, die in Wildtyp M{\"a}usen zu beobachten war, zeigte sich in den Integrin Beta 1-defizienten Mausherzen kein Gewebeumbau. Auch die erh{\"o}hte Expression von verschiedenen ECM Proteinen, insbesondere die verst{\"a}rkte Expression des matrizellul{\"a}ren Proteins SPARC, unterblieb nach AB in den Integrin Beta 1-defizienten Tieren. Interessanterweise konnte auch eine transiente Erh{\"o}hung der SPARC mRNA w{\"a}hrend der Umbauprozesse im Herzen in Folge von myokardialem Infarkt (MI) mittels cDNA Makroarrays festgestellt werden. In der Tat fanden sich gr{\"o}ßere Mengen von SPARC bereits 2 Tage (~2,5-fach erh{\"o}ht), 7 Tage (~4-fach erh{\"o}ht) und 1 Monat (~2-fach erh{\"o}ht) nach MI, w{\"a}hrend ein spezifischer Inhibitor der Integrin alpha v Untereinheit diese Hochregulation von SPARC in vivo verhinderte. Immunfluoreszenz Untersuchungen von Herzgewebe verdeutlichten, dass sich die erh{\"o}hte Expression von SPARC auf das Infarktareal beschr{\"a}nkte, dass die Expression von SPARC nach einer anf{\"a}nglichen Erh{\"o}hung im Verlauf von 1 Monaten wieder auf das Anfangsniveau zur{\"u}ckging und dass die verst{\"a}rkte Expression von der Einwanderung von Fibroblasten in das isch{\"a}mische Herzgewebe begleitet war. In vitro stimulierten die Wachstumsfaktoren TGF-Beta 1 und PDGF-BB die Expression von SPARC durch Fibroblasten. Wie sich an Hand von ELISA und Western Blot Untersuchungen feststellen ließ, war die Inhibition von Integrin Beta v nicht in der Lage, die durch TGF-Beta 1 oder PDGF induzierte Sekretion von SPARC zu beeinflussen. Jedoch zeigte sich, dass Vitronektin, ein Ligand von Integrin alpha v, sowohl die Sekretion von TGF-Beta 1 als auch von PDGF-BB durch Kardiomyozyten induzierte und diese Reaktion wurde durch den Integrin alpha v Inhibitor komplett unterdr{\"u}ckt. In funktioneller Hinsicht wirkte SPARC auf die durch ECM Proteine induzierte Migration von Fibroblasten ein, so dass man davon ausgehen kann, dass die lokale Freisetzung von SPARC nach myokardialem Infarkt zur Wundheilung im Herzen beitr{\"a}gt. Zusammenfassend l{\"a}ßt die Kombination der in vivo und in vitro erhobenen experimentellen Daten den Schluss zu, dass mehrere Integrin Untereinheiten eine entscheidende Rolle w{\"a}hrend der Gewebeumbildung im Herzen spielen. Integrin-abh{\"a}ngige Genexpressionsereignisse wie beispielsweise die erh{\"o}hte Expression von SPARC nach MI sind entscheidend an der Koordination der Wundheilung beteiligt. Diese Prozesse scheinen auf einer komplexen Wechselwirkung und Kommunikation zwischen verschiedenen Zelltypen wie Kardiomyozyten und Fibroblasten zu beruhen, um lokal begrenzt eine Heilung und Vernarbung des verletzten Gewebes zu regulieren. Die Aufkl{\"a}rung des fein abgestimmten Wechselspiels zwischen Integrinen matrizellul{\"a}ren Proteinen wie SPARC und Wachstumsfaktoren wird sicherlich zu einem besseren und klinisch nutzbarem Verst{\"a}ndnis der molekularen Mechanismen des Gewebeumbaus im Herzen beitragen.}, subject = {Integrine}, language = {en} } @phdthesis{Merker2014, author = {Merker, S{\"o}ren}, title = {Genome-wide screenings in attention-deficit/hyperactivity disorder (ADHD): investigation of novel candidate genes SLC2A3 and LPHN3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-100129}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Attention-deficit/hyperactivity disorder (ADHD) is a highly prevalent childhood-onset neurodevelopmental disorder that involves a substantial risk of persisting into adolescence and adulthood. A number of genome-wide screening studies in ADHD have been conducted in recent years, giving rise to the discovery of several variants at distinct chromosomal loci, thus emphasising the genetically complex and polygenic nature of this disorder. Accordingly, promising novel candidate genes have emerged, such as the gene encoding the glucose transporter isoform 3 (SLC2A3) and the gene encoding the latrophilin isoform 3 (LPHN3). In this thesis, both genes were investigated in form of two separated projects. The first focused on SLC2A3 polymorphisms associated with ADHD and their potential physiological impact. For this purpose, gene expression analyses in peripheral cell models were performed as well as functional EEG measurements in humans. The second project concerned the murine gene Lphn3 including the goal of developing a mouse line containing a genetically modified Lphn3 with conditional knockout potential. In this respect, a specific DNA vector was applied to target the Lphn3 gene locus in murine embryonic stem (ES) cells as a prerequisite for the generation of appropriate chimeric mice. The results of the first project showed that SLC2A3 duplication carriers displayed increased SLC2A3 mRNA expression in peripheral blood cells and significantly altered event-related potentials (ERPs) during tests of cognitive response control and working memory, possibly involving changes in prefrontal brain activity and memory processing. Interestingly, ADHD patients with the rs12842 T-allele, located within and tagging the SLC2A3 gene, also exhibited remarkable effects during these EEG measurements. However, such effects reflected a reversed pattern to the aforementioned SLC2A3 duplication carriers with ADHD, thus indicative of an opposed molecular mechanism. Besides, it emerged that the impact of the aforementioned SLC2A3 variants on different EEG parameters was generally much more pronounced in the group of ADHD patients than the healthy control group, implying a considerable interaction effect. Concerning the second project, preliminary results were gathered including the successful targeting of Lphn3 in murine ES cells as well as the production of highly chimeric, phenotypically unremarkable and mostly fertile mouse chimeras. While germline transmission of the modified Lphn3 allele has not yet occurred, there are still several newborn chimeric mice that will be tested in the near future. In conclusion, the findings suggest that SLC2A3 variants associated with ADHD are accompanied by transcriptional and functional changes in humans. Future research will help to elucidate the molecular network and neurobiological basis involved in these effects and apparently contributing to the complex clinical picture of ADHD. Moreover, given the increasing number of publications concerning latrophilins in recent years and the multitude of research opportunities provided by a conditional knockout of Lphn3 in mice, the establishment of a respective mouse line, which currently is in progress, constitutes a promising approach for the investigation of this gene and its role in ADHD.}, subject = {Genexpression}, language = {en} } @phdthesis{Kiser2019, author = {Kiser, Dominik Pascal}, title = {Gene x Environment Interactions in Cdh13-deficient Mice: CDH13 as a Factor for Adaptation to the Environment}, doi = {10.25972/OPUS-17959}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179591}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Neurodevelopmental disorders, including attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are disorders of mostly unknown etiopathogenesis, for which both genetic and environmental influences are expected to contribute to the phenotype observed in patients. Changes at all levels of brain function, from network connectivity between brain areas, over neuronal survival, synaptic connectivity and axonal growth, down to molecular changes and epigenetic modifications are suspected to play a key roles in these diseases, resulting in life-long behavioural changes. Genome-wide association as well as copy-number variation studies have linked cadherin-13 (CDH13) as a novel genetic risk factor to neuropsychiatric and neurodevelopmental disorders. CDH13 is highly expressed during embryonic brain development, as well as in the adult brain, where it is present in regions including the hippocampus, striatum and thalamus (among others) and is upregulated in response to chronic stress exposure. It is however unclear how CDH13 interacts with environmentally relevant cues, including stressful triggers, in the formation of long-lasting behavioural and molecular changes. It is currently unknown how the environment influences CDH13 and which long term changes in behaviour and gene expression are caused by their interaction. This work therefore investigates the interaction between CDH13 deficiency and neonatal maternal separation (MS) in mice with the aim to elucidate the function of CDH13 and its role in the response to early-life stress (ELS). For this purpose, mixed litters of wild-type (Cdh13+/+), heterozygous (Cdh13+/-) and homozygous knockout (Cdh13-/-) mice were maternally separated from postnatal day 1 (PN1) to postnatal day 14 (PN14) for 3 hours each day (180MS; PN1-PN14). In a first series of experiments, these mice were subjected to a battery of behavioural tests starting at 8 weeks of age in order to assess motor activity, memory functions as well as measures of anxiety. Subsequently, expression of RNA in various brain regions was measured using quantitativ real-time polymerase chain reaction (qRT-PCR). A second cohort of mice was exposed to the same MS procedure, but was not behaviourally tested, to assess molecular changes in hippocampus using RNA sequencing. Behavioural analysis revealed that MS had an overall anxiolytic-like effect, with mice after MS spending more time in the open arms of the elevated-plus-maze (EPM) and the light compartment in the light-dark box (LDB). As a notable exception, Cdh13-/- mice did not show an increase of time spent in the light compartment after MS compared to Cdh13+/+ and Cdh13+/- MS mice. During the Barnes-maze learning task, mice of most groups showed a similar ability in learning the location of the escape hole, both in terms of primary latency and primary errors. Cdh13-/- control (CTRL) mice however committed more primary errors than Cdh13-/- MS mice. In the contextual fear conditioning (cFC) test, Cdh13-/- mice showed more freezing responses during the extinction recall, indicating a reduced extinction of fear memory. In the step-down test, an impulsivity task, Cdh13-/- mice had a tendency to wait longer before stepping down from the platform, indicative of more hesitant behaviour. In the same animals, qRT-PCR of several brain areas revealed changes in the GABAergic and glutamatergic systems, while also highlighting changes in the gatekeeper enzyme Glykogensynthase-Kinase 3 (Gsk3a), both in relation to Cdh13 deficiency and MS. Results from the RNA sequencing study and subsequent gene-set enrichment analysis revealed changes in adhesion and developmental genes due to Cdh13 deficiency, while also highlighting a strong link between CDH13 and endoplasmatic reticulum function. In addition, some results suggest that MS increased pro-survival pathways, while a gene x environment analysis showed alterations in apoptotic pathways and migration, as well as immune factors and membrane metabolism. An analysis of the overlap between gene and environment, as well as their interaction, highlighted an effect on cell adhesion factors, underscoring their importance for adaptation to the environment. Overall, the stress model resulted in increased stress resilience in Cdh13+/+ and Cdh13+/- mice, a change absent in Cdh13-/- mice, suggesting a role of CDH13 during programming and adaptation to early-life experiences, that can results in long-lasting consequences on brain functions and associated behaviours. These changes were also visible in the RNA sequencing, where key pathways for cell-cell adhesion, neuronal survival and cell-stress adaptation were altered. In conclusion, these findings further highlight the role of CDH13 during brain development, while also shedding light on its function in the adaptation and response during (early life) environmental challenges.}, subject = {Cadherine}, language = {en} } @phdthesis{Nietzer2010, author = {Nietzer, Sarah}, title = {Gene and environment interactions in serotonin transporter knockout mice - how stress influences gene expression and neuronal morphology}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54391}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Serotonin (5-HT) is an important modulator of many physiological, behavioural and developmental processes and it plays an important role in stress coping reactions. Anxiety disorders and depression are stress-related disorders and they are associated with a malfunction of the 5-HT system, in which the 5-HT transporter (5-HTT) plays an important role. 5-Htt knockout (KO) mice represent an artificially hyperserotonergic environment, show an increased anxiety-like behaviour and seem to be a good model to investigate the role of the 5-HT system concerning stress reactions and anxiety disorders. As synaptic proteins (SPs) seem to be involved in stress reactions, the effect of acute immobilization stress on the expression of the three SPs Synaptotagmin (Syt) I, Syt IV and Syntaxin (Stx) 1A was studied in the 5-Htt KO mouse model as well as the expression of the two immediate early genes (IEGs) FBJ osteosarcoma oncogene (c-Fos) and fos-like antigen 2 (Fra-2). Additionally, the expression of the corticotrophin releasing hormone (CRH) and its two receptors CRHR1 and CRHR2 was investigated as part of the hypothalamic-pituitary-adrenal (HPA) stress system. Based on gender- and genotype-dependent differences in corticosterone levels, expression differences in the brain were investigated by performing a quantitative real time-PCR study using primer pairs specific for these SPs and for the IEGs c-Fos and Fra-2 in five different brain regions in 5-Htt KO and 5-Htt wild-type (WT) mice. Mainly gender-dependent differences could be found and weaker stress effects on the expression of SPs could be demonstrated. Regarding the expression of IEGs, stress-, gender- and genotype-dependent differences were found mainly in the hypothalamus. Also in the hypothalamus, gender effects were found concerning the expression of CRH and its both receptors. Additionally, in a second study, male 5-Htt WT and male 5-Htt deficient mice were subjected to a resident-intruder-paradigm which stresses the animals through a loser experience. The morphological changes of neurons were subsequently analyzed in Golgi-Cox-stained sections of limbic brain areas in stressed and unstressed animals of both genotypes using the computer-based microscopy system Neurolucida (Microbrightfield, Inc.). While no differences concerning dendritic length, branching patterns and spine density were found in the hippocampus and no differences concerning dendritic length and branching patterns could be shown in the cingulate cortex (CG), pyramidal neurons in the infralimbic cortex (IL) of stressed 5-Htt WT mice displayed longer dendrites compared to unstressed 5-Htt WT mice. The results indicate that, although in this model drastic alterations of neuronal morphology are absent, subtle changes can be found in specific brain areas involved in stress- and anxiety-related behaviour which may represent neural substrates underlying behavioural phenomena.}, subject = {Serotonin}, language = {en} } @phdthesis{Busold2006, author = {Busold, Christian}, title = {Facilitating functional interpretation of microarray data by integration of gene annotations in Correspondence Analysis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-21150}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {DNS-Chips ('Microarrays') haben sich zu einer der Standardmethoden zur Erstellung von genomweiten Expressionsstudien entwickelt. Mittlerweile wurden dazu eine Vielzahl von Methoden zur Identifizierung von differentiell regulierten Genen ver{\"o}ffentlicht. Ungeachtet dessen stellt die abschliessende funktionelle Interpretation der Ergebnisse einen der Engp{\"a}sse in der Analyse von Chip-Daten dar. Die Mehrzahl der Analysemethoden stellt die signifikant regulierten Gene in Listen dar, aus denen in einem weiteren Schritt gemeinsame funktionelle Eigenschaften abgeleitet werden m{\"u}ssen. Dies stellt nicht nur eine arbeitsintensive Arbeit dar, die mit steigender Anzahl an experimentellen Konditionen immer weniger praktikabel wird, sondern ist auch fehleranf{\"a}llig, da diese Auswertung im allgemeinen auf dem visuellen Vergleich von Listen beruht. In der vorliegenden Arbeit wurden Methoden f{\"u}r eine rechnergest{\"u}tzte Auswertung von funktionellen Geneigenschaften entwickelt und validiert. Hierzu wurde die 'Gene Ontology' als Quelle f{\"u}r die Annotationsdaten ausgew{\"a}hlt, da hier die Daten in einem Format gespeichert sind, das sowohl eine leichte menschliche Interaktion sowie die statistische Analyse der Annotationen erm{\"o}glicht. Diese Genannotation wurden als Zusatzinformationen in die Korrespondenzanalyse integriert, welches eine simultane Darstellung von Genen, Hybridisierungen und funktionellen Kategorien in einer Grafik erm{\"o}glicht. Aufgrund der st{\"a}ndig wachsenden Anzahl an verf{\"u}gbaren Annotationen und der Tatsache, daß zwischen den meisten experimentellen Bedingungen nur wenige funktionelle Prozesse differentiell reguliert sind, wurden Filter entwickelt, die die Anzahl der dargestellten Annotationen auf eine im gegebenen experimentellen Kontext relevante Gruppe reduzieren. Die Anwendbarkeit der Visualisierung und der Filter wurde auf Datens{\"a}tzen unterschiedlicher Komplexit{\"a}t getestet: beginnend mit dem gut verstandenen Glukosestoffwechsel im Modellorganismus S. cerevisiae, bis hin zum Vergleich unterschiedlicher Tumortypen im Menschen. In beiden F{\"a}llen generierte die Methode gut zu interpretierende Grafiken, in denen die funktionellen Hauptunterschiede durch die dargestellten Annotationen gut beschrieben werden [90]. W{\"a}hrend die Integration von Annotationsdaten wie GO die funktionelle Interpretation vereinfacht, fehlt die M{\"o}glichkeit zur Identifikation einzelner relevanter Schl{\"u}sselgene. Um eine solche Analyse zu erm{\"o}glichen, wurden Daten zum Vorkommen von Transskriptionsfaktorbindestellen in den 5'-Bereichen von Genen integriert. Auch diese Methode wurde an Datens{\"a}tzen von S. cerevisiae und vergleichenden Studien von humanen Krebszelllinien validiert.In beiden F{\"a}llen konnten Transkriptionsfaktoren identifiziert werden, die f{\"u}r die beobachteten transkriptionellen Unterschiede von entscheidender Bedeutung sind [206]. Zusammenfassend, erm{\"o}glicht die Integration von Zusatzinformationen in die Korrespondenzanalyse eine simultane Visualisierung von Genen, Hybridisierungen und Annotationsdaten in einer einzigen, gut zu interpretierenden Grafik. Dies erlaubt auch in komplexen experimentellen Bedingungen eine intuitive Identifizierung von relevanten Annotationen. Der hier vorgestellte Ansatz, ist nicht auf die gezeigten Datenstrukturen beschr{\"a}nkt, sondern kann auf die Mehrzahl der verf{\"u}gbaren Annotationsdaten angewendet werden.}, subject = {Microarray}, language = {en} } @phdthesis{Jager2002, author = {Jager, Tertia ¬de¬}, title = {Estrogen action in the myocardium}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-1182573}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2002}, abstract = {Cardiovascular disease is the leading cause of mortality in both men and women in the Western world. Earlier observations have pointed out that pre-menopausal women have a lower risk of developing cardiovascular disease than age-matched men, with an increase in risk after the onset of menopause. This observation has directed the attention to estrogen as a potential protective factor in the heart. So far the focus of research and clinical studies has been the vascular system, leaving the current knowledge on the role of estrogen in the myocardium itself rather scarce. Functional estrogen receptor-alpha as well as -beta have recently been identified in the myocardium, making the myocardium an estrogen target organ. The focus of this thesis was 1) to investigate the role of estrogen and estrogen receptors in modulating myocardial gene expression both in vivo in an animal model for cardiac hypertrophy (spontaneously hypertensive rats; SHR), as well as in vitro in isolated neonatal cardiomyocytes, 2) to investigate the mechanisms of the rapid induction of an estrogen target gene, the early growth response gene-1 (Egr-1) and 3) to initiate the search for novel estrogen target genes in the myocardium. 1) The effects of estrogen on the expression of one of the major myocardial specific contractile proteins, the alpha-myosin heavy chain (alpha-MHC) have been investigated. In ovarectomised animals treated either with 17beta-estradiol alone or in combination with a specific estrogen receptor antagonist, ICI 182780, it was shown that both alpha-MHC mRNA and protein were upregulated by estrogen in an estrogen receptor specific manner. The in vivo results were confirmed in vitro in isolated neonatal cardiomyocytes which showed that estrogen has a direct action on the myocardium potent enough to upregulate the expression of alpha-MHC. Furthermore it was shown that the alpha-MHC promoter is induced by estrogen in an estrogen receptor-dependent manner and first investigations into the mechanisms involved in this upregulation identified Egr-1 as a potential transcription factor which, upon induction by estrogen, drives the expression of the alpha-MHC promoter. 2) Previously it was shown that Egr-1 is rapidly induced by estrogen in an estrogen receptor-dependent manner which was mediated via 5 serum response elements (SREs) in the promoter region and surprisingly not via the estrogen response elements (EREs). In this study it was shown that estrogen-treatment of cardiomyocytes resulted in the recruitment of serum response factor (SRF), or an antigenically related protein, to the SREs in the Egr-1 promoter, which was specifically inhibited by the estrogen receptor antagonist ICI 182780. Transfection experiments showed that estrogen induced a heterologous promoter consisting only of 5 tandem repeats of the c-fos SRE in an ER-dependent manner, which identified SREs as promoter elements able to confer an estrogen response to target genes. 3) Potentially new target genes regulated by estrogen in vivo were analysed using hearts of ovarectomised animals as well as ovarectomised animals treated with estrogen. Analyses of cDNA microarray filters containing 1250 known genes identified 24 genes that were modified by estrogen in vivo. Among these genes, some might have potentially important functions in the heart and further analyses of these genes will create a more global picture of the role and function of estrogen in the myocardium. Taken together, the results showed that estrogen does have a direct action on the myocardium both by regulating the expression of myocardial specific genes in vivo, as well as exerting rapid non-nuclear effects in cardiac myocytes. It was shown that SREs in the promoter region of genes can confer an estrogen response to genes identifying SREs as important elements in regulation of genes by estrogen. Furthermore, 24 potentially new estrogen targets were identified in the myocardium, contributing to the general understanding of estrogen action in the myocardium.}, subject = {Herzmuskel}, language = {en} } @phdthesis{Hondke2014, author = {Hondke, Sylvia}, title = {Elucidation of WISP3 function in human mesenchymal stem cells and chondrocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109641}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {WISP3 is a member of the CCN family which comprises six members found in the 1990's: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes. WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis. Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells.}, subject = {Knorpelzelle}, language = {en} } @phdthesis{Simann2015, author = {Simann, Meike}, title = {Aufkl{\"a}rung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von prim{\"a}ren humanen Knochenmark-Stroma-Zellen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119322}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach. This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching. Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect. The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature. Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.}, subject = {Mesenchymzelle}, language = {en} } @phdthesis{Li2009, author = {Li, Naixin}, title = {Dorso-ventral Differentiation and Specification of the Mesencephalon in Early Chick Embryos}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-32950}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {The chick midbrain is subdivided into functionally distinct ventral and dorsal domains, tegmentum and optic tectum. In the mature tectum, neurons are organized in layers, while they form discrete nuclei in the tegmentum. An interesting characteristic of the embryonic brain is the development of a large optic tectum, of which the growth becomes obvious at embryonic day 3 (E3). Dorsoventral (DV) specification of the early midbrain should thus play a crucial role for the organization of the neuronal circuitry in optic tectum and tegmentum. In the first part of my thesis, I investigated regional commitment and establishment of cellular differences along the midbrain DV axis. I examined the commitment of gene expression patterns in isolated ventral and dorsal tissue in vivo and in vitro, and studied their cell mixing properties. Explant cultures, and grafting of dorsal midbrain into a ventral environment or vice versa, revealed a gradual increase in the autonomy of region-specific gene regulation between, which was accompanied by a gradual increase in differential adhesive properties from E2 to E3, once the DV axis polarity was fixed. These events happened at a time-point when the majority of midbrain cells are not yet differentiated. Long-term transplantation (6 - 9 days) using quail cells from ventral midbrain as grafts showed the same result. Hence, the results suggest that progressive specification of the midbrain DV axis is accompanied by progressively reduced cell mixing between dorsal and ventral precursors, leading to a partial regionalization of midbrain tissue into autonomous units of precursor cell populations. In the second part I investigated the genes that might be involved in regulating the growth of the tectum. In particular, I focused on the role of Pax7 transcription factor, a paired domain protein. The results suggested that Pax7 was involved in regulating the medial-lateral extension of the tectum. Over expression of Pax7 in dorsal midbrain led to an enlarged tectum accompanied by a raise in cell division, while Pax7 knockdown by shrank caused a reduction in tectum. The overall pattern of neuronal differentiation was not disturbed by an up or down regulation of Pax7. Pax7 also positively regulated Pax3, another pair-ruled gene expressed dorsally. These results suggest that Pax7 very likely together with Pax3 could facilitate or maintain neural cell proliferation in the midbrain at early stages and that a regulation of the size in that region does not influence the neuronal patterning of the developmental field. I further checked the expression and function of a GFPase Rab 23, that was suggested to be involved in the DV patterning in mouse neural tube as a negative regulator of Shh signaling. Overexpression of Rab23 indicated that it facilitated the expression of Pax7 and Pax3 in the neural tube and suppressed ventral genes like Nkx6.1 cell autonomously, however, it did not disturb neuronal patterning. Interestingly, a thorough expression study of Rab 23 during chick early development revealed that Rab23 is already expressed very early and asymmetrically during gastrulation, suggesting a possible role of Rab23 on the left-right determination of Hensen's node. In combination with the result that Rab23 is expressed in the notochord early in development, I assume that both Rab23 and Shh exist in all neural progenitor cells initially, and when their expression patterns separate gradually the neural cells adopt a ventral or dorsal fate according to their location along the dorsoventral axis. The avian embryo is a classic system used widely to investigate questions of vertebrate development. The easy and cheap accessibility of the embryo for in ovo or ex ovo experiments all around the year make it an ideal animal model to work with. The only recently developed method of over expressing genes in specific cells or regions in the chick embryo by electroporation enabled me to study different ways of gene suppression using this way of gene transfection. Thus, I compared the effect of long-hairpin and short hairpin dsRNA in different vectors and antisense morpholino oligonucleotides. The results revealed that all hairpin dsRNA constructs did reduce gene and protein expression often accompanied by morphological changes. Most efficiently were shRNAi constructs cloned into a siRNA-specific vector - pSilencer 1.0-U6. Gene silencing was already well observed 36 hours after transfection. In comparison antisense morpholino oligonucleotides did not show such big gene reduction as the shRNA in pSilencer. Taken together, this methodical research proposes that the shRNA in the pSilencer vector was a good and effective tool to reduce gene and protein expression locally.}, subject = {Differenzierung}, language = {en} } @phdthesis{Engelmann2008, author = {Engelmann, Julia Cath{\´e}rine}, title = {DNA microarrays: applications and novel approaches for analysis and interpretation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-29747}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {In der vorliegenden Dissertation wird die Entwicklung eines phylogenetischen DNA Microarrays, die Analyse von mehreren Microarray-Genexpressionsdatens{\"a}tzen und neue Ans{\"a}tze f{\"u}r die Datenanalyse und Interpretation der Ergebnisse vorgestellt. Die Entwicklung und Analyse der Daten eines phylogenetischen DNA Microarrays wird in der ersten Publikation dargestellt. Ich konnte zeigen, dass die Spezies-Detektion mit phylogenetischen Microarrays durch die Datenanalyse mit einem linearen Regressionsansatz signifikant verbessert werden kann. Standard-Methoden haben bislang nur Signalintensit{\"a}ten betrachtet und eine Spezies als an- oder abwesend bezeichnet, wenn die Signalintensit{\"a}t ihres Messpunktes oberhalb eines willk{\"u}rlich gesetzten Schwellenwertes lag. Dieses Verfahren ist allerdings aufgrund von Kreuz-Hybridisierungen nicht auf sehr nah verwandte Spezies mit hoher Sequenzidentit{\"a}t anwendbar. Durch die Modellierung des Hybridisierungs und Kreuz-Hybridisierungsverhaltens mit einem linearen Regressionsmodell konnte ich zeigen, dass Spezies mit einer Sequenz{\"a}hnlichkeit von 97\% im Markergen immer noch unterschieden werden k{\"o}nnen. Ein weiterer Vorteil der Modellierung ist, dass auch Mischungen verschiedener Spezies zuverl{\"a}ssig vorhergesagt werden k{\"o}nnen. Theoretisch sind auch quantitative Vorhersagen mit diesem Modell m{\"o}glich. Um die großen Datenmengen, die in {\"o}ffentlichen Microarray-Datenbanken abgelegt sind besser nutzen zu k{\"o}nnen, bieten sich Meta-Analysen an. In der zweiten Publikation wird eine explorative Meta-Analyse auf Arabidopsis thaliana-Datens{\"a}tzen vorgestellt. Mit der Analyse verschiedener Datens{\"a}tze, die den Einfluss von Pflanzenhormonen, Pathogenen oder verschiedenen Mutationen auf die Genexpression untersucht haben, konnten die Datens{\"a}tze anhand ihrer Genexpressionsprofile in drei große Gruppen eingeordnet werden: Experimente mit Indol-3-Essigs{\"a}ure (IAA), mit Pathogenen und andere Experimente. Gene, die charakteristisch f{\"u}r die Gruppe der IAA-Datens{\"a}tze beziehungsweise f{\"u}r die Gruppe der Pathogen-Datens{\"a}tze sind, wurden n{\"a}her betrachtet. Diese Gene hatten Funktionen, die bereits mit Pathogenbefall bzw. dem Einfluss von IAA in Verbindung gebracht wurden. Außerdem wurden Hypothesen {\"u}ber die Funktionen von bislang nicht annotierten Genen aufgestellt. In dieser Arbeit werden auch Prim{\"a}ranalysen von einzelnen Arabidopsis thaliana Genexpressions-Datens{\"a}tzen vorgestellt. In der dritten Publikation wird ein Experiment beschrieben, das durchgef{\"u}hrt wurde um herauszufinden ob Mikrowellen-Strahlung einen Einfluss auf die Genexpression einer Zellkultur hat. Dazu wurden explorative Analysemethoden angewendet. Es wurden geringe aber signifikante Ver{\"a}nderungen in einer sehr kleinen Anzahl von Genen beobachtet, die experimentell best{\"a}tigt werden konnten. Die Funktionen der regulierten Gene und eine Meta-Analyse mit {\"o}ffentlich zug{\"a}nglichen Datens{\"a}tzen einer Datenbank deuten darauf hin, dass die pflanzliche Zellkultur die Strahlung als eine Art Energiequelle {\"a}hnlich dem Licht wahrnimmt. Des weiteren wird in der vierten Publikation die funktionelle Analyse eines Arabidopsis thaliana Genexpressionsdatensatzes beschrieben. Die Analyse der Genexpressions eines pflanzlichen Tumores zeigte, dass er seinen Stoffwechsel von aerob und auxotroph auf anaerob und heterotroph umstellt. Gene der Photosynthese werden im Tumorgewebe reprimiert, Gene des Aminos{\"a}ure- und Fettstoffwechsels, der Zellwand und Transportkan{\"a}le werden so reguliert, dass Wachstum und Entwicklung des Tumors gef{\"o}rdert werden. In der f{\"u}nften Publikation in dieser Arbeit wird GEPAT (Genome Expression Pathway Analysis Tool) beschrieben. Es besteht aus einer Internet- Anwendung und einer Datenbank, die das einfache Hochladen von Datens{\"a}tzen in die Datenbank und viele M{\"o}glichkeiten der Datenanalyse und die Integration anderer Datentypen erlaubt. In den folgenden zwei Publikationen (Publikation 6 und Publikation 7) wird GEPAT auf humane Microarray-Datens{\"a}tze angewendet um Genexpressionsdaten mit weiteren Datentypen zu verkn{\"u}pfen. Genexpressionsdaten und Daten aus vergleichender Genom-Hybridisierung (CGH) von prim{\"a}ren Tumoren von 71 Mantel-Zell-Lymphom (MCL) Patienten erm{\"o}glichte die Ermittlung eines Pr{\"a}diktors, der die Vorhersage der {\"U}berlebensdauer von Patienten gegen{\"u}ber herk{\"o}mmlichen Methoden verbessert. Die Analyse der CGH Daten zeigte, dass auch diese f{\"u}r die Vorhersage der {\"U}berlebensdauer geeignet sind. F{\"u}r den Datensatz von Patienten mit großzellig diffusem B-Zell-Lymphom DLBCL konnte aus den Genexpressionsdaten ebenfalls ein neuer Pr{\"a}diktor vorgeschlagen werden. Mit den zwischen lang und kurz {\"u}berlebenden Patienten differentiell exprimierten Genen der MCL Patienten und mit den Genen, die zwischen den beiden Untergruppen von DLBCL reguliert sind, wurden Interaktionsnetzwerke gebildet. Diese zeigen, dass bei beiden Krebstypen Gene des Zellzyklus und der Proliferation zwischen Patienten mit kurzer und langer {\"U}berlebensdauer unterschiedlich reguliert sind.}, subject = {Microarray}, language = {en} } @phdthesis{Sauer2019, author = {Sauer, Markus}, title = {DHX36 function in RNA G-quadruplex-mediated posttranscriptional gene regulation}, doi = {10.25972/OPUS-18395}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-183954}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The expression of genetic information into proteins is a key aspect of life. The efficient and exact regulation of this process is essential for the cell to produce the correct amounts of these effector molecules to a given situation. For this purpose, eukaryotic cells have developed many different levels of transcriptional and posttranscriptional gene regulation. These mechanisms themselves heavily rely on interactions of proteins with associated nucleic acids. In the case of posttranscriptional gene regulation an orchestrated interplay between RNA-binding proteins, messenger RNAs (mRNA), and non-coding RNAs is compulsory to achieve this important function. A pivotal factor hereby are RNA secondary structures. One of the most stable and diverse representatives is the G-quadruplex structure (G4) implicated in many cellular mechanisms, such as mRNA processing and translation. In protein biosynthesis, G4s often act as obstacles but can also assist in this process. However, their presence has to be tightly regulated, a task which is often fulfilled by helicases. One of the best characterized G4-resolving factors is the DEAH-box protein DHX36. The in vitro function of this helicase is extensively described and individual reports aimed to address diverse cellular functions as well. Nevertheless, a comprehensive and systems-wide study on the function of this specific helicase was missing, so far. The here-presented doctoral thesis provides a detailed view on the global cellular function of DHX36. The binding sites of this helicase were defined in a transcriptome-wide manner, a consensus binding motif was deviated, and RNA targets as well as the effect this helicase exerts on them were examined. In human embryonic kidney cells, DHX36 is a mainly cytoplasmic protein preferentially binding to G-rich and G4-forming sequence motifs on more than 4,500 mRNAs. Loss of DHX36 leads to increased target mRNA levels whereas ribosome occupancy on and protein output of these transcripts are reduced. Furthermore, DHX36 knockout leads to higher RNA G4 levels and concomitant stress reactions in the cell. I hypothesize that, upon loss of this helicase, translationally-incompetent structured DHX36 target mRNAs, prone to localize in stress granules, accumulate in the cell. The cell reacts with basal stress to avoid cytotoxic effects produced by these mis-regulated and structured transcripts.}, subject = {RNS}, language = {en} } @phdthesis{WeinstockgebPattschull2019, author = {Weinstock [geb. Pattschull], Grit}, title = {Crosstalk between the MMB complex and YAP in transcriptional regulation of cell cycle genes}, doi = {10.25972/OPUS-17086}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170866}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The Myb-MuvB (MMB) multiprotein complex is a master regulator of cell cycle-dependent gene expression. Target genes of MMB are expressed at elevated levels in several different cancer types and are included in the chromosomal instability (CIN) signature of lung, brain, and breast tumors. This doctoral thesis showed that the complete loss of the MMB core subunit LIN9 leads to strong proliferation defects and nuclear abnormalities in primary lung adenocarcinoma cells. Transcriptome profiling and genome-wide DNA-binding analyses of MMB in lung adenocarcinoma cells revealed that MMB drives the expression of genes linked to cell cycle progression, mitosis, and chromosome segregation by direct binding to promoters of these genes. Unexpectedly, a previously unknown overlap between MMB-dependent genes and several signatures of YAP-regulated genes was identified. YAP is a transcriptional co-activator acting downstream of the Hippo signaling pathway, which is deregulated in many tumor types. Here, MMB and YAP were found to physically interact and co-regulate a set of mitotic and cytokinetic target genes, which are important in cancer. Furthermore, the activation of mitotic genes and the induction of entry into mitosis by YAP were strongly dependent on MMB. By ChIP-seq and 4C-seq, the genome-wide binding of MMB upon YAP overexpression was analyzed and long-range chromatin interaction sites of selected MMB target gene promoters were identified. Strikingly, YAP strongly promoted chromatin-association of B-MYB through binding to distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. Together, the findings of this thesis provide a so far unknown molecular mechanism by which YAP and MMB cooperate to regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.}, subject = {Krebs }, language = {en} } @phdthesis{Seo2012, author = {Seo, Ean Jeong}, title = {Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72005}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Der probiotische Escherichia coli Stamm Nissle 1917 (EcN) ist eines der wenigen Probiotika, die als aktive Komponente eines Medikaments in mehreren L{\"a}ndern zugelassen sind. Am besten ist die Wirksamkeit des EcN f{\"u}r die Remissionserhaltung von an Colitis Ulcerosa leidenden Patienten dokumentiert. Diese F{\"a}higkeit ist vermutlich darauf zur{\"u}ckzuf{\"u}hren, dass EcN in der Lage ist die Produktion des humanen beta-Defensins 2 (HBD2) mittels seiner Flagelle zu Induzieren. In dieser Studie wurden rekombinante EcN St{\"a}mme konstruiert, die ein Defensin zu produzieren verm{\"o}gen. Zu diesem Zweck wurden Kodon-optimierte Defensingene in Expressionsplasmidvektoren kloniert, die entweder die Proform mit der Signalsequenz oder die reife Defensinform des humanen -Defensins 5 (HD5) oder des humanen -Defensins 2 (HBD2) unter der Kontrolle des T7-Promotors kodieren. Die Synthese dieser Defensine wurde mittels Western-Blot nach der Induktion der Expression und der Lyse der rekombinanten EcN St{\"a}mme demonstriert. Das rekombinante reife HBD2 mit einem N-terminalen His-Tag konnte mittels Ni-S{\"a}ulen-Chromatographie aufgereinigt werden. Das so gewonnene HBD2 zeigte antimikrobielle Aktivit{\"a}t gegen E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes. In einem zweiten Ansatz wurde der Teil des HBD2-Gens mit dem yebF-Gen fusioniert, der das reife HBD2 kodiert. Das resultierende Fusionsprotein YebFMHBD2 wurde von dem entsprechenden EcN Stamm nach Induktion der Expression sekretiert. Die Pr{\"a}senz von YebFMHBD2 im Medium war nicht das Ergebnis von Zellyse wie Western-Blots spezifisch f{\"u}r die -Galaktosidase und das Maltose-Bindeprotein mit dem Kultur{\"u}berstand zeigten. Dieser Kultur{\"u}berstand inhibierte das Wachstum von E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes nach Dialyse und Aufkonzentration sowohl in Agardiffusionsassays als auch in Fl{\"u}ssigcokultur. Damit konnte gezeigt werden, dass EcN ein f{\"u}r die Produktion von bestimmten humanen Defensinen geeignetes Probiotikum darstellt. EcN ist bei der Behandlung von Morbus Crohn Patienten nicht aktiv. Dies ist vermutlich in der genetisch bedingten Unf{\"a}higkeit zur ausreichenden Defensinproduktion solcher Individuen begr{\"u}ndet. Als ein erster Schritt in der Entwicklung von alternativen Ans{\"a}tzen zur Behandlung Morbus Crohn Patienten wurden in dieser Arbeit EcN St{\"a}mme konstruiert, die in der Lage sind HD5 oder HBD2 zu produzieren.}, subject = {Escherichia coli}, language = {en} } @phdthesis{Kirchmaier2010, author = {Kirchmaier, Bettina Carmen}, title = {Characterization of the Popeye domain containing gene family in zebrafish}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-49413}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {The Popeye domain containing (Popdc) gene family of membrane proteins is predominantly expressed in striated and smooth muscle tissues and has been shown to act as novel cAMP-binding proteins. In mice, loss of Popdc1 and Popdc2, respectively, affects sinus node function in the postnatal heart in an age and stress-dependent manner. In this thesis, I examined gene expression pattern and function of the Popdc gene family during zebrafish development with an emphasis on popdc2. Expression of the zebrafish popdc2 was exclusively present in cardiac and skeletal muscle during cardiac development, whereas popdc3 was expressed in striated muscle tissue and in distinct regions of the brain. In order to study the function of these genes, an antisense morpholino-based knockdown approach was used. Knockdown of popdc2 resulted in aberrant development of facial and tail musculature. In the heart, popdc2 morphants displayed irregular ventricular contractions with 2:1 and 3:1 ventricular pauses. Recordings of calcium transients using a transgenic indicator line Tg(cmlc2:gCaMP)s878 and selective plane illumination microscopy (SPIM) revealed the presence of an atrioventricular (AV) block in popdc2 morphants as well as a complete heart block. Interestingly, preliminary data revealed that popdc3 morphants developed a similar phenotype. In order to find a morphological correlate for the observed AV conduction defect, I studied the structure of the AV canal in popdc2 morphants using confocal analysis of hearts of the transgenic line Tg(cmlc2:eGFP-ras)s883, which outlines individual cardiac myocytes with the help of membrane-localized GFP. However, no evidence for morphological alterations was obtained. To ensure that the observed arrhythmia phenotype in the popdc2 morphant was based on a myocardial defect and not caused by defective valve development, live imaging was performed revealing properly formed valves. Thus, in agreement with the data obtained in knockout mice, popdc2 and popdc3 genes in zebrafish are involved in the regulation of cardiac electrical activity. However, both genes are not required for cardiac pacemaking, but they play essential roles in AV conduction. In order to elucidate the biological importance of cAMP-binding, wild type Popdc1 as well as mutants with a significant reduction in binding affinity for cAMP in vitro were overexpressed in zebrafish embryos. Expression of wild type Popdc1 led to a cardiac insufficiency phenotype characterized by pericardial edema and venous blood retention. Strikingly, the ability of the Popdc1 mutants to induce a cardiac phenotype correlated with the binding affinity for cAMP. These data suggest that cAMP-binding represents an important biological property of the Popdc protein family.}, subject = {Zebrab{\"a}rbling}, language = {en} } @phdthesis{FetivaMora2023, author = {Fetiva Mora, Maria Camila}, title = {Changes in chromatin accessibility by oncogenic YAP and its relevance for regulation of cell cycle gene expression and cell migration}, doi = {10.25972/OPUS-30291}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302910}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes. The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration. Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.}, subject = {Chromatin}, language = {en} } @phdthesis{Blenk2007, author = {Blenk, Steffen}, title = {Bioinformatical analysis of B-cell lymphomas}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-27421}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Background: The frequency of the most observed cancer, Non Hodgkin Lymphoma (NHL), is further rising. Diffuse large B-cell lymphoma (DLBCL) is the most common of the NHLs. There are two subgroups of DLBCL with different gene expression patterns: ABC ("Activated B-like DLBCL") and GCB ("Germinal Center B-like DLBCL"). Without therapy the patients often die within a few months, the ABC type exhibits the more aggressive behaviour. A further B-cell lymphoma is the Mantle cell lymphoma (MCL). It is rare and shows very poor prognosis. There is no cure yet. Methods: In this project these B-cell lymphomas were examined with methods from bioinformatics, to find new characteristics or undiscovered events on the molecular level. This would improve understanding and therapy of lymphomas. For this purpose we used survival, gene expression and comparative genomic hybridization (CGH) data. In some clinical studies, you get large data sets, from which one can reveal yet unknown trends. Results (MCL): The published proliferation signature correlates directly with survival. Exploratory analyses of gene expression and CGH data of MCL samples (n=71) revealed a valid grouping according to the median of the proliferation signature values. The second axis of correspondence analysis distinguishes between good and bad prognosis. Statistical testing (moderate t-test, Wilcoxon rank-sum test) showed differences in the cell cycle and delivered a network of kinases, which are responsible for the difference between good and bad prognosis. A set of seven genes (CENPE, CDC20, HPRT1, CDC2, BIRC5, ASPM, IGF2BP3) predicted, similarly well, survival patterns as proliferation signature with 20 genes. Furthermore, some bands could be associated with prognosis in the explorative analysis (chromosome 9: 9p24, 9p23, 9p22, 9p21, 9q33 and 9q34). Results (DLBCL): New normalization of gene expression data of DLBCL patients revealed better separation of risk groups by the 2002 published signature based predictor. We could achieve, similarly well, a separation with six genes. Exploratory analysis of gene expression data could confirm the subgroups ABC and GCB. We recognized a clear difference in early and late cell cycle stages of cell cycle genes, which can separate ABC and GCB. Classical lymphoma and best separating genes form a network, which can classify and explain the ABC and GCB groups. Together with gene sets which identify ABC and GCB we get a network, which can classify and explain the ABC and GCB groups (ASB13, BCL2, BCL6, BCL7A, CCND2, COL3A1, CTGF, FN1, FOXP1, IGHM, IRF4, LMO2, LRMP, MAPK10, MME, MYBL1, NEIL1 and SH3BP5; Altogether these findings are useful for diagnosis, prognosis and therapy (cytostatic drugs).}, subject = {Bioinformatik}, language = {en} } @phdthesis{Tyrsin2008, author = {Tyrsin, Dmitry}, title = {Autoregulation of NFATc1 gene}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-26544}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Die Familie der NFAT-Transkriptionsfaktoren (NFATc1-c4) ist im Zuge einer Immunreaktion endscheidend an der transkriptionellen Regulation der Genexpression beteiligt. Wurden NFAT-Faktoren zun{\"a}chst als T-zell-spezifische Aktivatoren von Zytokinpromotoren beschrieben, so hat sich inzwischen gezeigt, dass sie in einer Vielzahl von Geweben eine wichtige Rolle spielen. Als Beispiele seien die Herzklappenentwicklung, die Bildung von Blutgef{\"a}ssen, die Ausbildung neuronaler Axone oder die Osteoklastendifferenzierung genannt [10, 24]. In der hier vorliegenden Arbeit zeigen wir, dass die starke Expression der kurzen Isoform NFATc1/\&\#945;A in Effektor-T-Lymphozyten durch die induzierbare Aktivit{\"a}t des Promoters P1 kontrolliert wird. Die P1 Aktivierung f{\"u}hrt zum Splicing des Exon 1 zu 3 (\&\#945;-Isoformen) und endet meist durch Benutzung der Polyadenylierungsstelle pA1 hinter Exon 9 (A-Isoformen). Der zweite, schw{\"a}cherer Promoter P2 befindet sich vor dem zweiten Exon und ist f{\"u}r die konstitutive Synthese der \&\#946;-Isoformen verantwortlich. Der Transkriptionstart am zweiten Exon geht meist mit der Benutzung einer zweiten, hinter dem 11. Exon gelegenen Polyadenylierungsstelle pA2 einher, die durch alternatives Splicing zur Synthese der Isoformen B und C f{\"u}hrt. Insgesamt k{\"o}nnen so vom nfatc1-Lokus sechs verschiedene Isoformen (\&\#945;A, \&\#945;B, \&\#945;C, \&\#946;A, \&\#946;B und \&\#946;C) generiert werden. Die induzierbare Aktivit{\"a}t des P1-Promoters ist, im Gegensatz zum eher konstitutiv aktiven P2-Promoter, NFAT-abh{\"a}ngig und somit eine Form der Autoregulation. In ruhenden T-Lymphozyten sind einzig die Transkripte der NFATc1/\&\#946;-Isoformen nachweisbar. Nach einer T-Zell-Aktivierung nimmt ihre H{\"a}ufigkeit dann ab, w{\"a}hrend nun die \&\#945;-Isoformen dominant werden. In dieser Arbeit wird gezeigt, dass es nach Induktion prim{\"a}rer Effektor-T-Helfer-Zellen oder in T-Zell-Linien zu einer 15-20-fachen Akkumulation der NFATc1/\&\#945;A mRNA bzw. einer 2-5-fachen Zunahme der NFATc1/\&\#945;B und C mRNAs kommt. Zur maximalen Induktion des P1-Promotors bedarf es zum einen eines anhaltenden Anstiegs der intrazellul{\"a}ren Kalziumkonzentration, die zur Aktivierung der Phosphatase Calcineurin und damit zur Kernlokalisation der NFAT-Faktoren f{\"u}hrt. Zum anderen ist die Aktivierung der Proteinkinase C-Enzyme und der MAP-Kinasen notwendig, wie sie durch Phorbolester in der Zelle vermittelt wird. Dies l{\"a}sst darauf schließen, dass f{\"u}r eine optimale Aktivierung des P1-Promotors sowohl Signale des T-Zell-Rezeptors als auch Signale von Korezeptoren - wie von CD28 - notwendig sind. Da die Induktion von NFATc1/\&\#945;A in NFATc2/NFATc3 doppeldefizienten M{\"a}usen normal erfolgt, kann man schlussfolgern, dass NFATc1 in Form einer Autoregulation die Aktivit{\"a}t des P1-Promoters und damit die Synthese der \&\#945;-Isoformen kontrolliert. Die NFAT-vermittelte Aktivierung des P1-Promoters erfolgt {\"u}ber zwei tandemartig angeordnete NFAT-Bindungsstellen der Nukleotidsequenz TGGAAA, an die jeweils ein NFAT-Protein binden kann. Daneben enth{\"a}lt der Promoter konservierte Bindemotive f{\"u}r CREB-, AP-1, Sp-, NF-kB- und GATA-Faktoren, die wahrscheinlich an der komplexen Kontrolle dieses induzierbaren NFATc1-Promoters beteiligt sind. Zusammengefasst ergibt sich aus diesen Daten das folgende Modell. Die Transkription im nfatc1-Genlokus erfolgt in naiven und in ruhenden Effektor-T-Zellen konstitutiv und gesteuert durch den P2-Promotor. In Folge einer Aktivierung der Zelle verringert sich die Aktivit{\"a}t des P2-Promotors, w{\"a}hrend gleichzeitig der P1-Promotor induziert wird, der zusammen mit einer verst{\"a}rkten Nutzung der pA1-Polyadenylierungssequenz f{\"u}r die massive Zunahme der NFATc1/\&\#945;A-Isoform verantwortlich ist. Dies deutet auf eine besondere Bedeutung dieser kurzen Isoform in der Effektorphase der T-Zell-Aktivierung hin, insbesondere in Th1-Zellen, die NFATc1/\&\#945;A in hohen Konzentrationen produzieren.}, subject = {Autoregulation}, language = {en} } @phdthesis{Froehlich2012, author = {Fr{\"o}hlich, Kathrin}, title = {Assigning functions to Hfq-dependent small RNAs in the model pathogen Salmonella Typhimurium}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85488}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators' individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5' end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader.}, subject = {Small RNA}, language = {en} } @phdthesis{Vainshtein2010, author = {Vainshtein, Yevhen}, title = {Applying microarray-based techniques to study gene expression patterns: a bio-computational approach}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51967}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {The regulation and maintenance of iron homeostasis is critical to human health. As a constituent of hemoglobin, iron is essential for oxygen transport and significant iron deficiency leads to anemia. Eukaryotic cells require iron for survival and proliferation. Iron is part of hemoproteins, iron-sulfur (Fe-S) proteins, and other proteins with functional groups that require iron as a cofactor. At the cellular level, iron uptake, utilization, storage, and export are regulated at different molecular levels (transcriptional, mRNA stability, translational, and posttranslational). Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5'- or 3'- untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Methods In this project response to the iron treatment was examined under different conditions using bioinformatical methods. This would improve our understanding of an iron regulatory network. For these purposes we used microarray gene expression data. To identify novel IRE-containing mRNAs biochemical, biocomputational, and microarray-based experimental approaches were integrated. IRP/IRE messenger ribonucleoproteins were immunoselected and their mRNA composition was analysed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Analysis of IronChip microarray data requires specialized tool which can use all advantages of a customized microarray platform. Novel decision-tree based algorithm was implemented using Perl in IronChip Evaluation Package (ICEP). Results IRE-like motifs were identified from genomic nucleic acid databases by an algorithm combining primary nucleic acid sequence and RNA structural criteria. Depending on the choice of constraining criteria, such computational screens tend to generate a large number of false positives. To refine the search and reduce the number of false positive hits, additional constraints were introduced. The refined screen yielded 15 IRE-like motifs. A second approach made use of a reported list of 230 IRE-like sequences obtained from screening UTR databases. We selected 6 out of these 230 entries based on the ability of the lower IRE stem to form at least 6 out of 7 bp. Corresponding ESTs were spotted onto the human or mouse versions of the IronChip and the results were analysed using ICEP. Our data show that the immunoselection/microarray strategy is a feasible approach for screening bioinformatically predicted IRE genes and the detection of novel IRE-containing mRNAs. In addition, we identified a novel IRE-containing gene CDC14A (Sanchez M, et al. 2006). The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip, but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls (Vainshtein Y, et al., 2010).}, subject = {Microarray}, language = {en} } @phdthesis{Zimmermann2020, author = {Zimmermann, Henriette}, title = {Antigenic variation and stumpy development in \(Trypanosoma\) \(brucei\)}, doi = {10.25972/OPUS-14690}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146902}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {The eukaryotic parasite Trypanosoma brucei has evolved sophisticated strategies to persist within its mammalian host. Trypanosomes evade the hosts' immune system by antigenic variation of their surface coat, consisting of variant surface glycoproteins (VSGs). Out of a repertoire of thousands of VSG genes, only one is expressed at any given time from one of the 15 telomeric expression sites (ES). The VSG is stochastically exchanged either by a transcriptional switch of the active ES (in situ switch) or by a recombinational exchange of the VSG within the active ES. However, for infections to persist, the parasite burden has to be limited. The slender (sl) bloodstream form secretes the stumpy induction factor (SIF), which accumulates with rising parasitemia. SIF induces the irreversible developmental transition from the proliferative sl to the cell cycle-arrested but fly-infective stumpy (st) stage once a concentration threshold is reached. Thus, antigenic variation and st development ensure persistent infections and transmissibility. A previous study in monomorphic cells indicated that the attenuation of the active ES could be relevant for the development of trypanosomes. The present thesis investigated this hypothesis using the inducible overexpression of an ectopic VSG in pleomorphic trypanosomes, which possess full developmental competence. These studies revealed a surprising phenotypic plasticity: while the endogenous VSG was always down-regulated upon induction, the ESactivity determined whether the VSG overexpressors arrested in growth or kept proliferating. Full ES-attenuation induced the differentiation of bona fide st parasites independent of the cell density and thus represents the sole natural SIF-independent differentiation trigger to date. A milder decrease of the ES-activity did not induce phenotypic changes, but appeared to prime the parasites for SIF-induced differentiation. These results demonstrate that antigenic variation and development are linked and indicated that the ES and the VSG are independently regulated. Therefore, I investigated in the second part of my thesis how ES-attenuation and VSG-silencing can be mediated. Integration of reporters with a functional or defective VSG 3'UTR into different genomic loci showed that the maintenance of the active state of the ES depends on a conserved motif within the VSG 3'UTR. In situ switching was only triggered when the telomere-proximal motif was partially deleted, suggesting that it serves as a DNA-binding motif for a telomere-associated protein. The VSG levels seem to be additionally regulated in trans based on the VSG 3'UTR independent of the genomic context, which was reinforced by the regulation of a constitutively expressed reporter with VSG 3' UTR upon ectopic VSG overexpression.}, subject = {Trypanosoma brucei}, language = {en} }