@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{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{Buechsenschuetz2005,
  author    = {B{\"u}chsensch{\"u}tz, Kai},
  title     = {Struktur und r{\"a}umlich-zeitliches Expressionsverhalten von Kaliumkan{\"a}len bei Zea mays},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-17522},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {Bei Zea mays wurden neue Kaliumkan{\"a}le der Shaker-Familie isoliert, charakterisiert und zusammen mit bereits bekannten Vertretern dieser Familie hinsichtlich m{\"o}glicher Aufgaben und Interaktionen untersucht.},
  subject      = {Mais},
  language  = {de}
}
@phdthesis{Huth2012,
  author    = {Huth, Antje [geb. Koppelkamm]},
  title     = {Studien zum mRNA profiling an humanem postmortalem Gewebe},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74308},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Die vorliegende Arbeit besch{\"a}ftigt sich mit der Frage, inwieweit quantitative Genexpressionsstudien an postmortalen humanen Proben mit erh{\"o}htem Postmortalintervall und somit m{\"o}glicherweise verminderter RNA-Integrit{\"a}t realisierbar sind und in Zukunft als zus{\"a}tzliches diagnostisches Werkzeug zur Determinierung der Todesursache im forensischen Kontext herangezogen werden k{\"o}nnen. Daf{\"u}r wurden in mehreren Teilstudien Faktoren untersucht, die die Verl{\"a}sslichkeit quantitativer Genexpressionsdaten beeinflussen k{\"o}nnen. Es konnte zun{\"a}chst f{\"u}r postmortales Skelettmuskelgewebe festgestellt werden, dass Verstorbene mit erh{\"o}htem BMI statistisch signifikant niedrigere RIN-Werte aufweisen als normalgewichtige Personen. Zudem wurde eine Korrelation zwischen dem Gewebetyp und der Integrit{\"a}t der daraus extrahierten RNA gefunden. Unter Anwendung der in dieser Arbeit gew{\"a}hlten Extraktionsmethode scheint postmortales Skelettmuskelgewebe f{\"u}r Genexpressionsstudien an Autopsiematerial besonders geeignet. Dagegen wurde im vorliegenden Probengut kein Zusammenhang zwischen verminderter RNA-Integrit{\"a}t und Parametern wie Geschlecht, PMI, Sterbealter, Dauer der Agonie und Todesursache gefunden. In einer weiteren Teilstudie wurde anhand von postmortalem Herzmuskel-, Skelettmuskel- und Gehirngewebe aus einer Auswahl von zehn funktionell verschiedenen endogenen Kontrollgenen HMBS, UBC, SDHA und TBP als die Gene mit der gr{\"o}ßten postmortalen Transkriptstabilit{\"a}t identifiztiert. Zudem konnte gezeigt werden, dass die Verwendung von vier stabilen endogenen Kontrollen am untersuchten Probenmaterial eine verl{\"a}ssliche Datennormalisierung erlaubt. Die validierten Kontrollgene k{\"o}nnen auch zuk{\"u}nftig f{\"u}r quantitative Genexpressionsstudien eingesetzt werden, solange die untersuchte Probenzusammensetzung der hier vorgestellten {\"a}hnelt. F{\"u}r die Todesursache sowie f{\"u}r den Body Mass Index des Probenspenders wurde in der vorliegenden Arbeit ein statistisch signifikanter Einfluss auf das Expressionslevel instabiler Gene festgestellt. Diese Parameter sind daher geeignet, die gefundenen Instabilit{\"a}ten m{\"o}glicher Kontrollgene zu erkl{\"a}ren. Die Ergebnisse der vorliegenden Arbeit lassen des Weiteren darauf schließen, dass das Erstellen von Degradierungslinien aus kommerziell erh{\"a}ltlicher RNA f{\"u}r jedes verwendete qPCR-Assay ein wichtiges Instrument der Qualit{\"a}tspr{\"u}fung ist. Mit der Degradierungslinie kann die Detektionsgrenze des verwendeten qPCR-Assays validiert werden kann. Nur so ist die Festlegung des Bereichs m{\"o}glich, in dem ein ver{\"a}ndertes Expressionslevel tats{\"a}chlich mit dem Einfluss eines spezifischen Parameters in Verbindung gebracht werden kann und klar von einer nur scheinbaren Genexpressions{\"a}nderung unterscheidbar ist, die durch eine Degradierung der Probe vorget{\"a}uscht wird. Zudem scheint es praktikabel, in zuk{\"u}nftigen Studien nur Proben mit {\"a}hnlichen Integrit{\"a}ten miteinander zu vergleichen. Pathologische Prozesse im menschlichen K{\"o}rper, auch solche, die die Funktion von Organen sowie die Morphologie betreffen, sind hoch komplex und k{\"o}nnen interindividuell variieren, weshalb die Genexpression im forensischen Kontext erg{\"a}nzende Hinweise auf die Diagnose liefern k{\"o}nnte. Als erstes anwendungsbezogenes Beispiel wurde in der vorliegenden Arbeit der Einfluss von Hypoxie auf das Transkriptlevel von HIF-1α, VEGF und SLC2A1 untersucht. Bei Normalisierung der Daten gegen vier stabile Kontrollgene ergaben sich anhand der untersuchten Gewebeproben Hinweise auf eine todesursachenbezogene Hochregulierung der drei Zielgene. Die Studie unterstrich die besondere Bedeutung der gew{\"a}hlten Normalisierungsstrategie. Wurden die Daten nur gegen GAPDH als einzelnes, nicht validiertes Kontrollgen normalisiert, deuteten die Ergebnisse eine {\"u}berraschende Herunterregulierung der Zielgene an. Als m{\"o}gliche Ursache f{\"u}r diese scheinbare Diskrepanz kommt die im weiteren Verlauf dieser Studie nachgewiesene Instabilit{\"a}t infolge einer Co-Regulation von GAPDH unter hypoxischen Bedingungen in Betracht. Mit dem Fernziel postmortale Genexpressionsstudien als zus{\"a}tzliches Instrument in der forensischen Todesursachenbestimmung einzusetzen, schafft die vorliegende Arbeit durch die Einf{\"u}hrung von validierten Kontrollgenen sowie durch die Analyse weiterer Einfluss nehmender Faktoren eine Basis f{\"u}r die verl{\"a}ssliche Durchf{\"u}hrung k{\"u}nftiger Genexpressionsstudien an humanem Autopsiegewebe.},
  subject      = {Genexpression},
  language  = {de}
}
@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{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{LangjahrverhHeld2018,
  author    = {Langjahr [verh. Held], Melissa},
  title     = {Systemische Expression von Zytokinen bei schmerzhaften und schmerzlosen Polyneuropathien},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154445},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Die Pathophysiologie der PNP wie auch die Entstehung der oft assoziierten neuropathischen Schmerzen ist unklar. Gleichzeitig gibt es bislang keine geeigneten Biomarker, die die oft komplizierte Differentialdiagnose vereinfachen k{\"o}nnen. Einige Tiermodelle und klinische Studien lieferten bereits Hinweise auf die entscheidende Rolle pro- und anti-inflammatorischer Zytokine in diesen Prozessen. Ziel unserer Studie war es, die systemische Genexpression pro- und anti-inflammatorischer Zytokine in einer großen Kohorte von Patienten mit PNP verschiedener {\"A}tiologie zu charakterisieren. Insgesamt konnten 111 PNP-Patienten und 38 gesunde Kontrollpersonen prospektiv rekrutiert werden. Nach Isolation von PBMC aus Blutproben von 97 Patienten wurde die Genexpression der pro-inflammatorischen Zytokine TNF, IL1, IL2, IL6, IL8 und der anti-inflammatorischen Zytokine IL4 und IL10 mittels qRT-PCR bestimmt. Bei 47 Patienten und 12 Kontrollen wurde zudem die IL6-, IL-8- und TNF-Zytokinproduktion von PBMC in vitro nach Stimulation durch LPS mittels ELISA untersucht. Hauptbefund war ein pro-inflammatorisches Zytokinprofil der PNP-Patienten mit h{\"o}herer Genexpression von IL1, IL2, IL8 und TNF im Vergleich zu den gesunden Kontrollen. Im Falle der entz{\"u}ndlichen Neuropathien konnte zudem eine niedrigere Genexpression von IL10 im Vergleich zu Gesunden nachgewiesen werden. Sowohl schmerzhafte als auch schmerzlose Verlaufsformen wiesen ein pro-inflammatorisches Zytokingenexpressionsprofil im Vergleich zu Gesunden auf, das bei schmerzhaften PNP deutlich mehr beteiligte pro-inflammatorische Zytokine umfasste; relevante Unterschiede zwischen den PNP-Patienten mit und ohne Schmerz sowie der diagnostischen Subgruppen fanden sich nicht. Eine niedrigere Stimulationsschwelle der PBMC lag bei PNP-Patienten im Vergleich zu Gesunden nicht vor. Insgesamt erscheint die Rolle einzelner Zytokine als systemische Biomarker f{\"u}r die Differenzierung verschiedener PNP-Formen bzw. bez{\"u}glich neuropathischen Schmerzes aufgrund einer niedrigen Spezifit{\"a}t deutlich eingeschr{\"a}nkt. Dennoch sprechen unsere Ergebnisse f{\"u}r eine m{\"o}gliche Rolle eines pro-inflammatorischen Milieus bei der Entstehung bzw. des Verlaufes verschiedener entz{\"u}ndlicher und nicht-entz{\"u}ndlicher Neuropathien und neuropathischen Schmerzes.},
  subject      = {Polyneuropathie},
  language  = {de}
}
@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{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{Cam2006,
  author    = {Cam, Hakan},
  title     = {The role of p53 family members in myogenic differentiation and rhabdomyosarcoma development},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-20240},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {Krebserkrankungen zeichnen sich h{\"a}ufig durch St{\"o}rungen zellul{\"a}rer Differenzierungsprozesse aus. So weisen Rhabdomyosarkome, die aus Muskelvorl{\"a}uferzellen hervorgehen, Differenzierungsdefekte auf, die zur unkontrollierten Proliferation der Tumorzellen f{\"u}hren. Bislang ist ungekl{\"a}rt, ob die Differenzierungsdefekte auf der verst{\"a}rkten Expression von Inhibitoren, der defekten Funktion von Aktivatoren oder einer Kombination von beidem beruht. In dieser Arbeit wird gezeigt, dass im Unterschied zu normalen Muskelzellen RMS-Zellen verst{\"a}rkt DeltaNp73, einen Pan-Inhibitor der p53-Tumorsuppressorfamilie, exprimieren. Die experimentelle {\"U}berexpression von DeltaNp73 in normalen Myoblasten blockierte die Muskeldifferenzierung und f{\"o}rderte in Kombination mit klassischen RMS-Onkogenen wie IGF2 oder PAX3/FKHR die maligne Transformation. Umgekehrt f{\"u}hrte die Hemmung von DeltaNp73 durch RNAi zur Reduktion der Tumorigenit{\"a}t von RMS-Tumorzellen. Da DeltaNp73 als dominant-negativer Inhibitor der p53-Familie wirkt, lies die Hemmung von Differenzierungsprozessen durch DeltaNp73 vermuten, dass die p53-Familienmitglieder (p53, p63, und p73) an der Regulation der Muskeldifferenzierung beteiligt sind. Tats{\"a}chlich konnte in dieser Arbeit gezeigt werden, dass die drei p53-Familienmitglieder bei der Induktion sp{\"a}ter Differenzierungsstadien kooperieren, indem sie die Aktivit{\"a}t des Retinoblastoma-Proteins RB regulieren. Die Funktion von RB ist bekanntermassen sowohl f{\"u}r den permanenten Zellzyklusarrest als auch f{\"u}r die Aktivierung Muskel-spezifischer Gene notwendig. W{\"a}hrend p53 die Proteinspiegel von RB reguliert, kontrollieren p63 und p73 den Aktivierungsgrad von RB, indem sie dessen Phoshphorylierungszustand {\"u}ber den Zyklin-abh{\"a}ngigen Kinaseinhibitor p57KIP2 modifizieren. Eine Hemmung dieser Funktionen blockiert das Differenzierungsprogramm und f{\"o}rdert die Tumorentstehung. Die Aktivierung zellul{\"a}rer Differenzierungsprozesse stellt somit einen entscheidenden Bestandteil der Tumorsuppressoraktivit{\"a}t der p53-Familie dar und liefert eine Erkl{\"a}rung f{\"u}r die H{\"a}ufigkeit von Mutationen im p53-Signalweg bei Rhabdomyosarkom-Patienten.},
  subject      = {Rhabdomyosarkom},
  language  = {de}
}