@phdthesis{Gulve2019, author = {Gulve, Nitish}, title = {Subversion of Host Genome Integrity by Human Herpesvirus 6 and \(Chlamydia\) \(trachomatis\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162026}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Ovarian cancer is one of the most common gynecological malignancies in the world. The prevalence of a microbial signature in ovarian cancer has been reported by several studies till date. In these microorganisms, Human herpesvirus 6 (HHV-6) and Chlamydia trachomatis (C.tr) are especially important as they have significantly high prevalence rate. Moreover, these pathogens are directly involved in causing DNA damage and thereby disrupting the integrity of host genome which is the underlying cause of any cancer. This study focuses on how the two pathogens, HHV-6 and C. trachomatis can affect the genome integrity in their individual capacities and thereby may drive ovarian epithelial cells towards transformation. HHV-6 has unique tendency to integrate its genome into the host genome at subtelomeric regions and achieve a state of latency. This latent virus may get reactivated during the course of life by stress, drugs such as steroids, during transplantation, pregnancy etc. The study presented here began with an interesting observation wherein the direct repeat (DR) sequences flanking the ends of double stranded viral genome were found in unusually high numbers in human blood samples as opposed to normal ratio of two DR copies per viral genome. This study was corroborated with in vitro data where cell lines were generated to mimic the HHV-6 status in human samples. The same observation of unusually high DR copies was found in these cell lines as well. Interestingly, fluorescence in situ hybridization (FISH) and inverse polymerase chain reaction followed by southern blotting showed that DR sequences were found to be integrated in nontelomeric regions as opposed to the usual sub-telomeric integration sites in both human samples and in cell lines. Sanger sequencing confirmed the non-telomeric integration of viral DR sequences in the host genome. Several studies have shown that C. trachomatis causes DNA damage and inhibits the signaling cascade of DNA damage response. However, the effect of C. trachomatis infection on process of DNA repair itself was not addressed. In this study, the effect of C. trachomatis infection on host base excision repair (BER) has been addressed. Base excision repair is a pathway which is responsible for replacing the oxidized bases with new undamaged ones. Interestingly, it was found that C. trachomatis infection downregulated polymerase β expression and attenuated polymerase β- mediated BER in vitro. The mechanism of the polymerase β downregulation was found to be associated with the changes in the host microRNAs and downregulation of tumor suppressor, p53. MicroRNA-499 which has a binding site in the polymerase β 3'UTR was shown to be upregulated during C. trachomatis infection. Inhibition of miR-499 using synthetic miR-499 inhibitor indeed improved the repair efficiency during C. trachomatis infection in the in vitro repair assay. Moreover, p53 transcriptionally regulates polymerase β and stabilizing p53 during C. trachomatis infection enhanced the repair efficiency. Previous studies have shown that C. trachomatis can reactivate latent HHV-6. Therefore, genomic instability due to insertions of unstable 'transposon-like' HHV-6 DR followed by compromised BER during C. trachomatis infection cumulatively support the hypothesis of pathogenic infections as a probable cause of ovarian cancer}, subject = {Chlamydia trachomatis}, language = {en} } @phdthesis{Blocka2019, author = {Blocka, Joanna}, title = {Molecular mechanisms underlying Woodhouse-Sakati syndrome: characterization of DCAF17 with specific, polyclonal antibodies}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174766}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Woodhouse-Sakati syndrome (WSS) is a rare multisystemic, autosomal recessive disease. The underlying cause of WSS are mutations of C2orf37 gene, which result in a truncated protein. Little is known about the function of C2orf37 (DDB1-CUL4A-associated factor 17, DCAF17) apart from it being part of the DDB1-CUL4-ROC1 E3 ubiquitin ligase complex, specifically binding directly to DDB1 and serving as a substrate recruiter for E3. There are two major isoforms of DCAF17: beta (65 kDa, 520 amino acids) and alpha (27 kDa, 240 amino acids), which is a C-terminal part of beta. The intracellular localization of the WSS protein is thought to be primarily the nucleolus. A murine ortholog protein was found to be expressed in all tissues with a relatively higher expression in the brain, liver, and skin.The aim of this work was to investigate DCAF17 in HeLa cells in more detail, in particular the redistribution of both WSS isoforms on the subcellular and -nuclear level as well as their chemical features. For these experiments, I developed, through recombinant expression and affinity purification, a specific polyclonal antibody against a WSS-epitope 493-520. Furthermore, three other specific polyclonal antibodies were obtained through affinity purification with help of commercially produced high-affinity epitope peptides.By means of these antibodies, I determined- through immunofluorescence and subcellular protein fractionation- that, apart from the redistribution of the WSS protein within the non-soluble = chromatin-bound nuclear fraction, a significant amount of both WSS isoforms is present in the soluble nuclear fraction. Indeed, treatment of purified nuclear envelopes with an increasing concentration of NaCl as well as urea confirmed a non-covalent binding of the WSS protein to the nuclear envelope with the detachment ofbeta-WSS at a lower NaCl concentration than alpha-WSS. In regard to the chromatin-bound WSS protein, I performed hydrolysis of nuclear and nucleolar extract with DNase and RNase. The results indicate that the WSS protein is bound to DNA but not RNA, with alpha-WSS being possibly located more abundantly in the nucleolus, whereas beta-WSS within other subnuclear departments. Furthermore, in all the above-mentioned experiments, a presence of an 80-kDa protein, which specifically reacted with the polyclonal high-affinity antibodies and showed similar redistribution and chemical features as alpha- and beta-WSS, was observed. In order to investigate whether this protein is a posttranslationally modified WSS isoform, I performed deglycosylation and dephosphorylation of nuclear extract, which showed no disappearance or change in abundance of the 80-kDa band on Western blot. While other ways of poststranslational modification cannot be excluded as the cause of occurrence of the 80-kDa protein, an existence of a third, yet undescribed, major isoform is also conceivable. Summarizing, this work contributed to a deeper characterization of the WSS protein, which can help future investigators in developing new experimental ideas to better understand the pathology of WSS.}, subject = {Humangenetik}, language = {en} } @phdthesis{Bertho2016, author = {Bertho, Sylvain}, title = {Biochemical and molecular characterization of an original master sex determining gene in Salmonids}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139130}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Sexual development is a fundamental and versatile process that shapes animal morphology, physiology and behavior. The underlying developmental process is composed of the sex determination and the sex differentiation. Sex determination mechanisms are extremely labile among taxa. The initial triggers of the sex determination process are often genetics called sex determining genes. These genes are expressed in the bipotential gonad and tilt the balance to a developmental program allowing the differentiation of either a testis or an ovary. Fish represent a large and fascinating vertebrate group to study both sex determination and sex differentiation mechanisms. To date, among the known sex determining genes, three gene families namely sox, dmrt and TGF-β factors govern this developmental program. As exception to this rule, sdY "sexually dimorphic on the Y" does not belong to one of these families as it comes from the duplication / evolution of an ancestor gene related to immunity, i.e., the interferon related factor 9, irf9. sdY is the master sex determining gene in salmonids, a group of fishes that include species such as rainbow trout and Atlantic salmon. The present study was aimed to firstly characterize the features of SdY protein. Results indicate that SdY is predominantly localized in the cytoplasm tested in various fish and mammalian cell lines and confirmed by different methods. Predictive in silico analysis revealed that SdY is composed of a β-sandwich core surrounded by three α-helices as well specific characteristics conferring a putative protein-protein interaction site. Secondly, the study was aimed to understand how SdY could trigger testicular differentiation. SdY is a truncated divergent version of Irf9 that has a conserved protein-protein domain but lost the DNA interaction domain of its ancestor gene. It was then hypothesized that SdY could initiate testicular differentiation by protein-protein interactions. To evaluate this we first conducted a yeast-two-hybrid screen that revealed a high proportion of transcription factors including fox proteins. Using various biochemical and cellular methods we confirm an interaction between SdY and Foxl2, a major transcription factor involved in ovarian differentiation and identity maintenance. Interestingly, the interaction of SdY with Foxl2 leads to nuclear translocation of SdY from the cytoplasm. Furthermore, this SdY translocation mechanism was found to be specific to fish Foxl2 and to a lesser extend Foxl3 and not other Fox proteins or mammalian FoxL2. In addition, we found that this interaction allows the stabilization of SdY and prevents its degradation. Finally, to better decipher SdY action we used as a model a mutated version of SdY that was identified in XY females of Chinook salmon natural population. Results show that this mutation induces a local conformation defect obviously leading to a misfolded protein and a quick degradation. Moreover, the mutated version compromised the interaction with Foxl2 defining a minimal threshold to induce testicular differentiation. Altogether results from my thesis propose that SdY would trigger testicular differentiation in salmonids by preventing Foxl2 to promote ovarian differentiation. Further research should be now carried out on how this interaction of SdY and Foxl2 acts in-vivo.}, subject = {Lachsartige }, language = {en} } @phdthesis{Chouhan2017, author = {Chouhan, Nitin Singh}, title = {Time-odor learning in \(Drosophila\) \(melanogaster\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145675}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2017}, abstract = {Endogenous clocks help animals to anticipate the daily environmental changes. These internal clocks rely on environmental cues, called Zeitgeber, for synchronization. The molecular clock consists of transcription-translation feedback loops and is located in about 150 neurons (Helfrich-F{\"o}rster and Homberg, 1993; Helfrich-F{\"o}rster, 2005). The core clock has the proteins Clock (CLK) and Cycle (CYC) that together act as a transcription activator for period (per) and timeless (tim) which then, via PER and TIM block their own transcription by inhibiting CLK/CYC activity (Darlington et al., 1998; Hardin, 2005; Dubruille and Emery, 2008). Light signals trigger the degradation of TIM through a blue-light sensing protein Cryptochrome (CRY) and thus, allows CLK/CYC to resume per and tim transcription (Emery et al., 1998; Stanewsky et al., 1998). Therefore, light acts as an important Zeitgeber for the clock entrainment. The mammalian clock consists of similarly intertwined feedback loops. Endogenous clocks facilitate appropriate alterations in a variety of behaviors according to the time of day. Also, these clocks can provide the phase information to the memory centers of the brain to form the time of day related associations (TOD). TOD memories promote appropriate usage of resources and concurrently better the survival success of an animal. For instance, animals can form time-place associations related to the availability of a biologically significant stimulus like food or mate. Such memories will help the animal to obtain resources at different locations at the appropriate time of day. The significance of these memories is supported by the fact that many organisms including bees, ants, rats and mice demonstrate time-place learning (Biebach et al. 1991; Mistlberger et al. 1997; Van der Zee et al. 2008; Wenger et al. 1991). Previous studies have shown that TOD related memories rely on an internal clock, but the identity of the clock and the underlying mechanism remain less well understood. The present study demonstrates that flies can also form TOD associated odor memories and further seeks to identify the appropriate mechanism. Hungry flies were trained in the morning to associate odor A with the sucrose reward and subsequently were exposed to odor B without reward. The same flies were exposed in the afternoon to odor B with and odor A without reward. Two cycles of the 65 reversal training on two subsequent days resulted in the significant retrieval of specific odor memories in the morning and afternoon tests. Therefore, flies were able to modulate their odor preference according to the time of day. In contrast, flies trained in a non-reversal manner were unable to form TOD related memories. The study also demonstrates that flies are only able to form time-odor memories when the two reciprocal training cycles occur at a minimum 6 h interval. This work also highlights the role of the internal state of flies in establishing timeodor memories. Prolonged starvation motivates flies to appropriate their search for the food. It increases the cost associated with a wrong choice in the T-maze test as it precludes the food discovery. Accordingly, an extended starvation promotes the TOD related changes in the odor preference in flies already with a single cycle of reversal training. Intriguingly, prolonged starvation is required for the time-odor memory acquisition but is dispensable during the memory retrieval. Endogenous oscillators promote time-odor associations in flies. Flies in constant darkness have functional rhythms and can form time-odor memories. In contrast, flies kept in constant light become arrhythmic and demonstrated no change in their odor preference through the day. Also, clock mutant flies per01 and clkAR, show compromised performance compared to CS flies when trained in the time-odor conditioning assay. These results suggest that flies need a per and clk dependent oscillator for establishing TOD related memories. Also, the clock governed rhythms are necessary for the timeodor memory acquisition but not for the retrieval. Pigment-Dispersing Factor (PDF) neuropeptide is a clock output factor (Park and Hall, 1998; Park et al., 2000; Helfrich-F{\"o}rster, 2009). pdf01 mutant flies are unable to form significant time-odor memories. PDF is released by 8 neurons per hemisphere in the fly brain. This cluster includes the small (s-LNvs) and large (l-LNvs) ventral lateral neurons. Restoring PDF in these 16 neurons in the pdf01 mutant background rescues the time-odor learning defect. The PDF neuropeptide activates a seven transmembrane G-protein coupled receptor (PDFR) which is broadly expressed in the fly brain (Hyun et al., 2005). The present study shows that the expression of PDFR in about 10 dorsal neurons (DN1p) is sufficient for robust time-odor associations in flies. 66 In conclusion, flies use distinct endogenous oscillators to acquire and retrieve time-odor memories. The first oscillator is light dependent and likely signals through the PDF neuropeptide to promote the usage of the time as an associative cue during appetitive conditioning. In contrast, the second clock is light independent and specifically signals the time information for the memory retrieval. The identity of this clock and the underlying mechanism are open to investigation.}, subject = {Taufliege}, language = {en} } @phdthesis{Fackler2014, author = {Fackler, Marc}, title = {Biochemical characterization of GAS2L3, a target gene of the DREAM complex}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-103394}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {GAS2L3 was identified recently as a target gene of the DREAM complex (Reichert et al., 2010; Wolter et al., 2012). It was shown that GAS2L3 is expressed in a cell cycle specific manner and that depletion of the protein leads to defects in cytokinesis and genomic instability (Wolter et al., 2012). Major aim of this thesis was, to further characterize the biochemical properties and physiological function of GAS2L3. By in vitro co-sedimentation and bundling assays, GAS2L3 was identified as a cytoskeleton associated protein which bundles, binds and crosslinks F-actin and MTs. GST pulldown assays and co-immunoprecipitation experiments revealed that GAS2L3 interacts in vitro and in vivo with the chromosomal passenger complex (CPC), a very important regulator of mitosis and cytokinesis, and that the interaction is mediated by the GAR domain of GAS2L3 and the C-terminal part of Borealin and the N-terminal part of Survivin. Kinase assays showed that GAS2L3 is not a substrate of the CPC but is strongly phosphorylated by CDK1 in vitro. Depletion of GAS2L3 by shRNA influenced protein stability and activity of the CPC. However pharmacological studies showed that the decreased CPC activity is not responsible for the observed cytokinesis defects upon GAS2L3 depletion. Immunofluorescence experiments revealed that GAS2L3 is localized to the constriction zone by the CPC in a GAR dependent manner and that the GAR domain is important for proper protein function. New interacting proteins of GAS2L3 were identified by stable isotope labelling by amino acids in cell culture (SILAC) in combination with tandem affinity purification and subsequent mass spectrometrical analysis. Co-immunoprecipitation experiments further confirmed the obtained mass spectrometrical data. To address the physiological function of GAS2L3 in vivo, a conditional and a non-conditional knockout mouse strain was established. The non-conditional mouse strain showed a highly increased mortality rate before weaning age probably due to heart failure. The physiological function of GAS2L3 in vivo as well as the exact reason for the observed heart phenotype is not known at the moment.}, subject = {Zellzyklus}, language = {en} } @phdthesis{Gerold2011, author = {Gerold, Kay}, title = {CTLA4 and CLEC16A in Type 1 Diabetes - Looking behind the association}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-66617}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Type 1 diabetes is an autoimmune disease that leads to the destruction of insulin-producing pancreatic beta cells and consequently to hyperglycemia. In the last 60 years, the prevalence of type 1 diabetes has been increasing constantly and is predicted to continue rising. About 80\% of the disease risk is attributable to the genetic variation. Thanks to genome wide association studies the number of known disease-associated polymorphisms climbed from five to 53 in the last 10 years. As these studies reveal possible candidate genes but not underlying mechanisms we strove to take the next step and explore the association of two genes suggested by these studies with type 1 diabetes. As a method of choice we decided to use lentiviral RNAi in non obese diabetic (NOD) mice, a widely-used model for type 1 diabetes, introducing a shRNA directed against the target message into the genome of this mouse strain via a lentivirus. This allowed us to study the partial loss-of-function of the target gene within the context of diabetes, directly seeing its effect on autoimmune mechanisms. In this thesis we examined two different genes in this manner, Ctla4 and Clec16a. A type 1 diabetes associated polymorphism in the CTLA4 gene had been found to alter the splicing ratio of its variants soluble CTLA-4 (sCTLA-4) and full length CTLA-4, the associated allele producing less sCTLA-4 than the protective allele. We mimicked this effect by specifically targeting the sCtla4 mRNA via lentiviral RNAi in the NOD model. As a result we could confirm the reduction of sCTLA-4 to accelerate type 1 diabetes development. Furthermore we could show a function of sCTLA-4 in regulatory T cells, more specifically at least partly in their ability to modulate costimulation by antigen presenting cells. The second candidate gene, Clec16a was targeted with the shRNA in a way that was designed to knock down most splice variants. As the gene function and the effect of the associated SUMMARY 10 polymorphism was unknown, we reasoned this method to be feasible to investigate its role in type 1 diabetes. The knockdown of Clec16a in NOD mice resulted in an almost complete protection from diabetes development that could be attributed to T cells dysfunction. However, as expression patterns and a study of the Drospophila orthologue suggested a possible role of CLEC16A in antigen presentation we also examined antigen presenting cells in the thymus and periphery. Although we did not detect any effect of the knockdown on peripheral antigen presenting cells, thymic epithelial cells were clearly affected by the loss of CLEC16A, rendering them more activated and shifting the ratio of cortical to medullary epithelial cells in favor of cortical cells. We therefore suggest a role of CLEC16A in the selection of T cells, that needs, however, to be further investigated. In this thesis we provided a feasible and fast method to study function of genes and even of single splice variants within the NOD mouse model. We demonstrate its usefulness on two candidate genes associated with type 1 diabetes by confirming and unraveling the cause of their connection to the disease.}, subject = {Diabetes mellitus}, language = {en} } @phdthesis{Cook2012, author = {Cook, Mandy}, title = {The neurodegenerative Drosophila melanogaster AMPK mutant loechrig}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72027}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {In dieser Doktorarbeit wird die Drosophila Mutante loechrig (loe), die progressive Degeneration des Nervensystems aufweist, weiter beschrieben. In der loe Mutante fehlt eine neuronale Isoform der γ- Untereinheit der Proteinkinase AMPK (AMP-activated protein kinase). Die heterotrimere AMPK (auch als SNF4Aγ bekannt) kontrolliert das Energieniveau der Zelle, was st{\"a}ndiges Beobachten des ATP/AMP- Verh{\"a}ltnis erfordert. AMPK wird durch niedrige Energiekonzentrationen und Beeintr{\"a}chtigungen im Metabolismus, wie zum Beispiel Sauerstoffmangel, aktiviert und reguliert mehrere wichtige Signaltransduktionswege, die den Zellmetabolismus kontrollieren. Jedoch ist die Rolle von AMPK im neuronalen {\"U}berleben noch unklar. Eines der Proteine, dass von AMPK reguliert wird, ist HMGR (hydroxymethylglutaryl-CoA- reductase), ein Schl{\"u}sselenzym in der Cholesterin- und Isoprenoidsynthese. Es wurde gezeigt, dass wenn die Konzentration von HMGR manipuliert wird, auch der Schweregrad des neurodegenerativen Ph{\"a}notyps in loe beeinflusst wird. Obwohl die regulatorische Rolle von AMPK auf HMGR in Drosophila konserviert ist, k{\"o}nnen Insekten Cholesterin nicht de novo synthetisieren. Dennoch ist der Syntheseweg von Isoprenoiden zwischen Vertebraten und Insekten evolution{\"a}r konserviert. Isoprenylierung von Proteinen, wie zum Beispiel von kleinen G-Proteinen, stellt den Proteinen einen hydophobischen Anker bereit, mit denen sie sich an die Zellmembran binden k{\"o}nnen, was in anschließender Aktivierung resultieren kann. In dieser Doktorarbeit wird gezeigt, dass die loe Mutation die Prenylierung von Rho1 und den LIM-Kinasesignalweg beeinflusst, was eine wichtige Rolle im Umsatz von Aktin und axonalem Auswachsen spielt. Die Ergebnisse weisen darauf hin, dass die Mutation in LOE, Hyperaktivit{\"a}t des Isoprenoidsynthesewegs verursacht, was zur erh{\"o}hten Farnesylierung von Rho1 und einer dementsprechend h{\"o}heren Konzentration von Phospho- Cofilin f{\"u}hrt. Eine Mutation in Rho1 verbessert den neurodegenerativen Ph{\"a}notyp und die Lebenserwartung von loe. Der Anstieg vom inaktiven Cofilin in loe f{\"u}hrt zu einer Zunahme von filament{\"o}sen Aktin. Aktin ist am Auswachen von Neuronen beteiligt und Experimente in denen loe Neurone analysiert wurden, gaben wertvolle Einblicke in eine m{\"o}gliche Rolle die AMPK, und dementsprechend Aktin, im Neuronenwachstum spielt. Des Weiteren wurde demonstriert, dass Neurone, die von der loe Mutante stamen, einen verlangsamten axonalen Transport aufweisen, was darauf hinweist dass Ver{\"a}nderungen, die durch den Einfluss von loe auf den Rho1 Signalweg im Zytoskelettnetzwerk hervorgerufen wurden, zur St{\"o}rung des axonalen Transports und anschließenden neuronalen Tod f{\"u}hren. Es zeigte außerdem, dass Aktin nicht nur am neuronalen Auswachsen beteiligt ist, sondern auch wichtig f{\"u}r die Aufrechterhaltung von Neuronen ist. Das bedeutet, dass {\"A}nderungen der Aktindynamik zur progressiven Degeneration von Neuronen f{\"u}hren kann. Zusammenfassend unterstreichen diese Ergebnisse die wichtige Bedeutung von AMPK in den Funktionen und im {\"U}berleben von Neuronen und er{\"o}ffnen einen neuartigen funktionellen Mechanismus in dem {\"A}nderungen in AMPK neuronale Degeneration hervorrufen kann.}, subject = {Taufliege}, language = {en} } @phdthesis{AlcantarinoMenescal2012, author = {Alcantarino Menescal, Luciana}, title = {In vivo characterization of genetic factors involved in Xmrk driven melanoma formation in Medaka (Oryzias latipes): a closer look at braf, Stat5 and c-myc}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70762}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100\% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15\% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.}, subject = {Japank{\"a}rpfling}, language = {en} } @phdthesis{Weissflog2011, author = {Weißflog, Lena}, title = {Molecular Genetics of Emotional Dysregulation in Attention-Deficit/Hyperactivity Disorder}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69345}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Attention-deficit/hyperactivity disorder (ADHD) is a genetically complex childhood onset neurodevelopmental disorder which is highly persistent into adulthood. Several chromo-somal regions associated with this disorder were identified previously in genome-wide linkage scans, association (GWA) and copy number variation (CNV) studies. In this work the results of case-control and family-based association studies using a can-didate gene approach are presented. For this purpose, possible candidate genes for ADHD have been finemapped using mass array-based SNP genotyping. The genes KCNIP4, CDH13 and DIRAS2 have been found to be associated with ADHD and, in addition, with cluster B and cluster C personality disorders (PD) which are known to be related to ADHD. Most of the associations found in this work would not withstand correction for multiple testing. However, a replication in several independent populations has been achieved and in conjunction with previous evidence from linkage, GWA and CNV studies, it is assumed that there are true associations between those genes and ADHD. Further investigation of DIRAS2 by quantitative real-time PCR (qPCR) revealed expression in the hippocampus, cerebral cortex and cerebellum of the human brain and a significant increase in Diras2 expression in the mouse brain during early development. In situ hybrid-izations on murine brain slices confirmed the results gained by qPCR in the human brain. Moreover, Diras2 is expressed in the basolateral amygdala, structures of the olfactory system and several other brain regions which have been implicated in the psychopatholo-gy of ADHD. In conclusion, the results of this work provide further support to the existence of a strong genetic component in the pathophysiology of ADHD and related disorders. KCNIP4, CDH13 and DIRAS2 are promising candidates and need to be further examined to get more knowledge about the neurobiological basis of this common disease. This knowledge is essential for understanding the molecular mechanisms underlying the emergence of this disorder and for the development of new treatment strategies.}, subject = {Aufmerksamkeits-Defizit-Syndrom}, language = {en} } @phdthesis{Niewalda2010, author = {Niewalda, Thomas}, title = {Neurogenetic analyses of pain-relief learning in the fruit fly}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65035}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {All animals learn in order to cope with challenges imposed on them by their environment. This is true also for both larval and adult fruit flies as exemplified in pavlovian conditioning. The focus of this Thesis is on various aspects of the fruit flies learning ability. My main project deals with two types of learning which we call punishment-learning and pain-relief learning. Punishment learning happens when fruit flies are exposed to an odour which is followed by electric shock. After such training, flies have learned that that odour signals pain and consequently will avoid it in the future. If the sequence of the two stimuli is reversed such that odour follows shock, flies learn the odour as a signal for relief and will later on approach it. I first report a series of experiments investigating qualitative and parametric features of relief-learning; I find that (i) relief learning does result from true associative conditioning, (ii) it requires a relatively high number of training trials, (iii) context-shock training is ineffective for subsequent shock-odour learning. A further question is whether punishment-learning and pain-relief learning share genetic determinants. In terms of genetics, I test a synapsin mutant strain, which lacks all Synapsin protein, in punishment and relief-learning. Punishment learning is significantly reduced, and relief-learning is abolished. Pan-neuronal RNAi-mediated knock-down of Synapsin results in mutant-like phenotypes, confirming the attribution of the phenotype to lack of Synapsin. Also, a rescue of Synapsin in the mushroom body of syn97 mutants restores both punishment- and relief-learning fully, suggesting the sufficiency of Synapsin in the mushroom body for both these kinds of learning. I also elucidate the relationship between perception and physiology in adult fruit flies. I use odour-shock conditioning experiments to identify degrees of similarity between odours; I find that those similarity measures are consistent across generalization and discrimination tasks of diverse difficulty. Then, as collaborator of T. V{\"o}ller and A. Fiala, I investigate how such behavioural similarity/dissimilarity is reflected at the physiological level. I combine the behaviour data with calcium imaging data obtained by measuring the activity patterns of those odours in either the sensory neurons or the projection neurons at the antennal lobe. Our interpretation of the results is that the odours perceptual similarity is organized by antennal lobe interneurons. In another project I investigate the effect of gustatory stimuli on reflexive behaviour as well as their role as reinforcer in larval learning. Drosophila larvae greatly alter their behaviour in presence of sodium chloride. Increasing salt concentration modulates choice behaviour from weakly appetitive to strongly aversive. A similar concentration-behaviour function is also found for feeding: larval feeding is slightly enhanced in presence of low salt concentrations, and strongly decreased in the presence of high salt concentrations. Regarding learning, relatively weak salt concentrations function as appetitive reinforcer, whereas high salt concentrations function as aversive reinforcer. Interestingly, the behaviour-concentration curves are shifted towards higher concentrations from reflexive behaviour (choice behaviour, feeding) as compared to associative learning. This dissociation may reflect a different sensitivity in the respective sensory-motor circuitry.}, subject = {Taufliege}, language = {en} } @phdthesis{Schulz2010, author = {Schulz, Sandra}, title = {The Contribution of Common and Rare Variants to the Complex Genetics of Psychiatric Disorders}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-50677}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Attention deficit/hyperactivity disorder (ADHD), one of the most frequent childhood-onset, chronic and lifelong neurodevelopmental diseases, affects 5 - 10\% of school - aged children and adolescents, and 4\% of adults. The classified basic symptoms are - according to the diagnostic system DSM-VI - inattentiveness, impulsivity and hyperactivity. Also daily life of patients is impaired by learning problems, relationship crises, conflicts with authority and unemployment, but also comorbidities like sleep - and eating problems, mood - and anxiety disorders, depression and substance abuse disorders are frequently observed. Although several twin and family studies have suggested heritability of ADHD, the likely involvement of multiple genes and environmental factors has hampered the elucidation of its etiology and pathogenesis. Due to the successful medication of ADHD with dopaminergic drugs like methylphenidate, up to now, the search for candidate genes has mainly focused on the dopaminergic and - because of strong interactions - the serotonergic system, including the already analyzed candidate genes DAT1, DRD4 and 5, DBH or 5-HTTLPR. Recently, DNA copy number changes have been implicated in the development of a number of neurodevelopmental diseases and the analysis of chromosomal gains and losses by Array Comparative Genomic Hybridization (Array CGH) has turned out a successful strategy to identify disease associated genes. Here we present the first systematic screen for chromosomal imbalances in ADHD using sub-megabase resolution Array CGH. To detect micro-deletions and -duplications which may play a role in the pathogenesis of ADHD, we carried out a genome-wide screen for copy number variations (CNVs) in a cohort of 99 children and adolescents with severe ADHD. Using high-resolution aCGH, a total of 17 potentially syndrome-associated CNVs were identified. The aberrations comprise four deletions and 13 duplications with approximate sizes ranging from 110 kb to 3 Mb. Two CNVs occurred de novo and nine were inherited from a parent with ADHD, whereas five are transmitted by an unaffected parent. Candidates include genes expressing acetylcholine-metabolising butyrylcholinesterase (BCHE), contained in a de novo chromosome 3q26.1 deletion, and a brain-specific pleckstrin homology domain-containing protein (PLEKHB1), with an established function in primary sensory neurons, in two siblings carrying a 11q13.4 duplication inherited from their affected mother. Other genes potentially influencing ADHD-related psychopathology and involved in aberrations inherited from affected parents are the genes for the mitochondrial NADH dehydrogenase 1 alpha subcomplex assembly factor 2 (NDUFAF2), the brain-specific phosphodiesterase 4D isoform 6 (PDE4D6), and the neuronal glucose transporter 3 (SLC2A3). The gene encoding neuropeptide Y (NPY) was included in a ~3 Mb duplication on chromosome 7p15.2-15.3, and investigation of additional family members showed a nominally significant association of this 7p15 duplication with increased NPY plasma concentrations (empirical FBAT, p = 0.023). Lower activation of the left ventral striatum and left posterior insula during anticipation of large rewards or losses elicited by fMRI links gene dose-dependent increases in NPY to reward and emotion processing in duplication carriers. Additionally, further candidate genes were examined via Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). This method enables the analysis of SNPs directly from human genomic DNA without the need for initial target amplification by PCR. All these findings implicate CNVs of behavior-related genes in the pathogenesis of ADHD and are consistent with the notion that both frequent and rare variants influence the development of this common multifactorial syndrome. The second part of this work concentrates on MLC1, a gene associated with Megalencephalic leukoencephalopathy with subcortical cysts, located on chromosome 22q13.33. To get more insight in the disease itself, a targeting vector for a conditional knockout mouse was constructed using homologous recombination. Furthermore, MLC1 has been suggested as a risk gene for schizophrenia, especially the periodic catatonia subtype. An initially identified missense mutation was found to be extremely rare in other patient cohorts; however, a recent report again argued for an association of two intronic MLC1 SNPs with schizophrenia and bipolar disorder. A case-control study of these polymorphisms as well as SNPs in the transcriptional control region of MLC1 was conducted in 212 chronic schizophrenic patients, 56 of which suffered from periodic catatonia, 106 bipolar patients, and 284 controls. Both intronic and promoter polymorphisms were specifically and significantly associated with periodic catatonia but not schizophrenia or bipolar disorder in general. A haplotype constructed from all polymorphisms was also associated with periodic catatonia. The MLC1 variation is associated with periodic catatonia; whether it constitutes a susceptibility or a modifier gene has to be determined.}, subject = {Aufmerksamkeits-Defizit-Syndrom}, language = {en} } @phdthesis{Zdziarski2008, author = {Zdziarski, Jaroslaw Maciej}, title = {Bacterial Genome Plasticity and its Role for Adaptation and Evolution of Asymptomatic Bacteriuria (ABU) Escherichia coli Strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-32879}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Asymptomatic bacteriuria (ABU) represents the long term bacterial colonization of the urinary tract, frequently caused by Escherichia coli (E. coli), without typical symptoms of a urinary tract infection (UTI). To investigate characteristics of ABU E. coli isolates in more detail, the geno- and phenotypes of eleven ABU isolates have been compared. Moreover, consecutive in vivo re-isolates of the model ABU strain 83972 were characterized with regard to transcriptomic, proteomic and genomic alterations upon long term in vivo persistence in the human bladder. Finally, the effect of the human host on bacterial adaptation/evolution was assessed by comparison of in vitro and in vivo-propagated strain 83972. ABU isolates represent a heterologous group of organisms. The comparative analysis of different ABU isolates elucidated the remarkable genetic and phenotypic flexibility of E. coli isolates. These isolates could be allocated to all four major E. coli phylogenetic lineages as well as to different clonal groups. Accordingly, they differed markedly in genome content, i.e., the genome size as well as the presence of typical UPEC virulence-associated genes. Multi locus sequence typing suggested that certain ABU strains evolved from UPEC variants that are able to cause symptomatic UTI by genome reduction. Consequently, the high E. coli genome plasticity does not allow a generalized view on geno- and phenotypes of individual isolates within a clone. Reductive evolution by point mutations, DNA rearrangements and deletions resulted in inactivation of genes coding for several UPEC virulence factors, thus supporting the idea that a reduced bacterial activation of host mucosal inflammation promotes the ABU lifestyle of these E. coli isolates. Gene regulation and genetic diversity are strategies which enable bacteria to live and survive under continuously changing environmental conditions. To study adaptational changes upon long term growth in the bladder, consecutive re-isolates of model ABU strain 83972 derived from a human colonisation study and from an in vitro long term cultivation experiment were analysed with regard to transcriptional changes and genome rearrangements. In this context, it could be demonstrated that E. coli, when exposed to different host backgrounds, is able to adapt its metabolic networks resulting in an individual bacterial colonisation strategy. Transcriptome and proteome analyses demonstrated distinct metabolic strategies of nutrients acquisition and energy production of tested in vivo re-isolates of strain 83972 that enabled them to colonise their host. Utilisation of D-serine, deoxy- and ribonucleosides, pentose and glucuronate interconversions were main up-regulated pathways providing in vivo re-isolates with extra energy for efficient growth in the urinary bladder. Moreover, this study explored bacterial response networks to host defence mechanisms: The class III alcohol dehydrogenase AdhC, already proven to be involved in nitric oxide detoxification in pathogens like Haemophilus influenzae, was shown for the first time to be employed in defending E. coli against the host response during asymptomatic bacteriuria. Consecutive in vivo and in vitro re-isolates of strain 83972 were also analysed regarding their genome structure. Several changes in the genome structure of consecutive re-isolates derived from the human colonisation study implied the importance of bacterial interactions with the host during bacterial microevolution. In contrast, the genome structure of re-isolates from the in vitro long term cultivation experiment, where strain 83972 has been propagated without host contact, was not affected. This suggests that exposure to the immune response promotes genome plasticity thus being a driving force for the development of the ABU lifestyle and evolution within the urinary tract.}, subject = {Escherichia coli}, language = {en} } @phdthesis{Mao2006, author = {Mao, Zhengrong}, title = {Clonality analysis in B-Cell Chronic Lymphocytic Leukemia (B-CLL) associated with Richter's syndrome}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-20596}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Die chronische lymphatische Leuk{\"a}mie vom B-Zell-Typ (B-CLL) macht ca. 90\% der chronischen lymphatischen Leuk{\"a}mien aus. Der klinische Verlauf der B-CLL ist heterogen, und bei 3-5\% der Patienten kommt es im Verlauf der Erkrankung zu einer Transformation in ein aggressives Lymphom, meist in ein diffuses großzelliges B-Zell-Lymphom (DLBCL) oder in ein Hodgkin-Lymphom (HL). Eine solche Transformation wird als Richter-Syndrom bezeichnet und ist mit einer ung{\"u}nstigen klinischen Prognose assoziiert. In B-Zell-Lymphomen (B-NHL) erm{\"o}glicht der Mutationsstatus der variablen Anteile der Immunglobulinschwerkettengene (IgVH) eine Aussage {\"u}ber das Entwicklungsstadium der B-Zelle, in dem sich die Transformation zu einem B-Zell-Lymphom ereignet hat. In der B-CLL stellt der Mutationsstatus auch einen bedeutenden prognostischen Faktor dar, wobei die Erkrankung bei Patienten mit unmutierten IgVH Genen meist einen ung{\"u}nstigen klinischen Verlauf zeigt. Die wenigen bisher durchgef{\"u}hrten molekularen Analysen an F{\"a}llen von Richter-Syndromen deuten darauf hin, dass ein Transformationsereignis sowohl bei B-CLL-Patienten mit mutierten als auch mit unmutierten IgVH Genen vorkommen kann, und dass die Tumorzellen des DLBCL oder HL sowohl klonal identisch mit dem B-CLL-Klon sein k{\"o}nnen als auch unabh{\"a}ngig als sekund{\"a}res Lymphom entstehen k{\"o}nnen. Um die klonale Beziehung zwischen DLBCL- bzw. Hodgkin/Reed-Sternberg (HRS)-Zellen und den Zellen der vorbestehenden B-CLL zu analysieren, den Mutationsstatus des IgVH Gens sowie m{\"o}gliche prognostische Faktoren in B-CLL-F{\"a}llen mit Richter-Transformation zu identifizieren, wurde eine gr{\"o}ßere Fallserie mit Hilfe eines PCR-basierten GeneScan Ansatzes mit anschließender Sequenzierung der IgVH-Gene untersucht. In F{\"a}llen mit HRS bzw. HRS-{\"a}hnlichen Zellen wurden die CD30-positiven Tumorzellen mittels Laser-Capture Mikrodissektion (LCM) isoliert. Weiterhin erfolgte eine morphologische und immunhistochemische Analyse der F{\"a}lle. Insgesamt wurden 48 Patientenproben untersucht, darunter 40 F{\"a}lle mit Richter-Syndrom sowie weitere 8 B-CLL-F{\"a}lle mit CD30-positiven HRS-{\"a}hnlichen Zellen. Unter den 40 Proben mit Richter-Syndrom zeigten 34 B-CLL-F{\"a}lle eine Transformation in ein DLBCL, in 6 F{\"a}llen erfolgte eine Transformation in ein HL. In 23 F{\"a}llen mit B-CLL und DLBCL wurde eine Sequenzierung der IgVH Gene durchgef{\"u}hrt. In 18 F{\"a}llen waren B-CLL und DLBCL klonal identisch, in 5 F{\"a}llen war das DLBCL als klonal unabh{\"a}ngige Neoplasie entstanden. Unter den klonal verwandten Paaren zeigten 11 von 15 Paaren unmutierte IgVH-Gene sowohl in der B-CLL als auch im DLBCL, wohingegen 5 von 6 F{\"a}lle mit Transformation einer B-CLL in ein HL mutierte IgVH Gene trugen. HRS-Zellen in 2 F{\"a}llen und HRS-{\"a}hnliche Zellen in einem Fall waren klonal verschieden vom B-CLL-Zellklon. Die VH-Gene VH3-23, VH3-74, VH1-2 und VH3-9 waren {\"u}berproportional h{\"a}ufig in B-CLL F{\"a}llen mit einer sp{\"a}teren Transformation in ein DLBCL vertreten, wohingegen VH4-34 und VH3-48 in mehr als der H{\"a}lfte der F{\"a}lle mit Transformation zu einem HL nachgewiesen werden konnten. Der immunhistochemische Nachweis von ZAP70 zeigte eine signifikante Assoziation mit unmutierten IgVH-Genen in B-CLL mit nachfolgender Richter-Transformation. Bei 24 Patienten konnte der klinische Verlauf eruiert werden. Die mediane {\"U}berlebenszeit von Patienten mit B-CLL mit stattgehabter Transformation in ein DLBCL oder ein HL betrug 7 beziehungsweise 21 Monate. Es fanden sich weder signifikante Unterschiede der {\"U}berlebenszeit zwischen klonal verwandten und nicht verwandten F{\"a}llen, noch zwischen IgVH-mutierten und -unmutierten F{\"a}llen. Zusammenfassend kann festgestellt werden, dass bei der Richter-Transformation einer B-CLL in ein DLBCL dieses einerseits aus dem pr{\"a}existenten B-CLL-Klon entstehen kann, andererseits aber auch als unabh{\"a}ngige, klonal nicht verwandte Neoplasie auftreten kann. In der Mehrzahl der F{\"a}lle (78\% in dieser Serie) sind B-CLL und DLBCL jedoch klonal identisch und nur gelegentlich entsteht ein DLBCL ohne klonale Beziehung zur B-CLL als unabh{\"a}ngige, sekund{\"a}re Neoplasie. Eine klonale Transformation in ein DLBCL tritt vorwiegend bei B-CLL-Patienten mit unmutierten IgVH-Genen auf, wohingegen die Mehrzahl der B-CLL-Patienten mit einer Transformation in ein HL oder CD30-positive HRS-{\"a}hnliche Zellen mutierte IgVH-Gene aufweist. Dieser Befund l{\"a}sst auf unterschiedliche Transformationswege der beiden Subtypen des Richter-Syndroms schließen. Weiterhin existieren vermutlich wesentliche Unterschiede in der Pathogenese zwischen DLBCL-F{\"a}llen, die sich aus einer vorbestehenden B-CLL entwickelt haben, und de novo DLBCL-F{\"a}llen, da de novo DLBCL zumeist durch mutierte IgVH-Gene charakterisiert sind.}, subject = {B-Zell-Leuk{\"a}mie}, language = {en} } @phdthesis{Prusko2006, author = {Prusko, Carsten Dietmar}, title = {Evolutionary Diversification of Protein Functions : From Translation in Prokaryotes to Innate Immunity in Invertebrates}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18517}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {With the progress in sequencing of the honey bee genome new data become available which allows the search and identification of genes coding for homologous proteins found in other organism. Two genes coding for c-type lysozymes were identified in the genome of A. mellifera through an online-based BLAST search. Expression of both intron-less genes seems not to be under the regulatory control of either of the two pathways involved in humoral insect immunity, i.e. Toll and Imd, since no NF-\&\#954;B transcription factor binding sites are found upstream of the genes. The encoded Lys-1 and Lys-2 are 157 and 143 amino acid long, respectively, and share a sequence similarity of 90\%. Further in silico analysis revealed a signal peptidase cleavage site at the N-terminus of each amino acid sequence, strongly suggesting a secretion of the enzymes into the surrounding environment of the producing cells. Sequence alignments of both amino acid sequences with other c-type lysozymes identified the highly conserved active site glutamic acid (Glu32) as well as eight highly conserved cysteine residues. However, an important aspartic acid (Asp50) in the active site that helps to stabilize a substrate intermediate during catalysis is replaced by a serine residue in the lysozymes of A. mellifera. The replacement of the active site aspartic acid in the honey bee lysozymes suggests a different catalytic mechanism and/or a different substrate-specificity in respect to other c-type lysozymes. Furthermore, 3D-models of Lys-1 and Lys-2 were generated based on the sequence similarity of A. mellifera lysozymes with other c-type lysozymes. The published 3D structure of the lysozyme from the silkmoth Bombyx mori, which shares the highest sequence similarity of all available structures with A. mellifera lysozymes, was used as template for the construction of the 3D-models. The models of Lys-1 and Lys-2 suggest that both enzymes resemble, in large part, the structure of B. mori lysozyme. In order to identify the set of AMPs in the hemolymph of A. mellifera, hemolymph of immunized bees was analyzed. Applying SDS-polyacrylamide gel electrophoresis and mass spectrometry on hemolymph from immunized bees, three out of the four peptides were identified, i.e. abaecin, defensin 1 and hymenoptaecin. Furthermore, Lys-2 was identified in the hemolymph by mass spectrometry, conclusively demonstrating the presence of a lysozyme in the hemolymph of A. mellifera for the first time. However, the protein levels of Lys-2 were not affected by bacterial injection, suggesting that the gene expression of the putative antibacterial protein is not under the regulatory control of the Imd and/or Toll pathway. Besides the abovementioned antimicrobial peptides, the 76 kDa large transferrin was also identified. Transferrin is an iron-binding protein that has been implicated in innate immunity in the honey bee. Furthermore, the effect of pathogenic dose, the timeline of peptide induction and the age-related accumulation of the aforementioned AMPs were studied. The intensity of expression of the antimicrobial peptides, abaecin, defensin 1, and hymenoptaecin as well as transferrin increased proportionally with the amount of bacteria injected into the hemocoel. No such effect was observed for the protein levels of Lys-2. Furthermore, up-regulation of the three antibacterial peptides and transferrin was observed within the first 24 h following infection with E. coli (gram-). Infection with the gram+ bacterium Micrococcus flavus resulted in high and moderate protein levels for transferrin and abaecin, respectively, whereas hardly any accumulation of hymenoptaecin was observed, indicating that the gene expression of abaecin and transferrin is somehow positively correlated, and would suggest a shared regulatory pathway that differs from that of hymenoptaecin. Although bacterial infections didn't seem to stimulate the production of Lys-2, different concentrations in the hemolymph were observed in bees of different ages, suggesting a correlation between the expression of Lys-2 and the age-related division of labor of adult worker honey bees, also known as age polyethism. The results further allow a proposed causal connection between the age-dependent accumulation of Lys-2 and the hemolymph titer of the gonotrophic hormone juvenile hormone, which is the "behavioral pacemaker" in adult honey bees.}, subject = {Biene}, language = {en} } @phdthesis{Cruz2006, author = {Cruz, Alexandre Bettencourt da}, title = {Molecular and functional characterization of the swiss-cheese and olk mutants in Drosophila melanogaster : two approaches to killing neurons}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-17734}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {In this thesis two genes involved in causing neurodegenerative phenotypes in Drosophila are described. olk (omb-like), a futsch allele, is a micotubule associated protein (MAP) which is homologous to MAP1B and sws (swiss cheese) a serine esterase of yet unknown function within the nervous system. The lack of either one of these genes causes progressive neurodegeneration in two different ways. The sws mutant is characterized by general degeneration of the adult nervous system, glial hyperwrapping and neuronal apoptosis. Deletion of NTE (neuropathy target esterase), the SWS homolog in vertebrates, has been shown to cause a similar pattern of progressive neural degeneration in mice. NTE reacts with organophosphates causing axonal degeneration in humans. Inhibition of vertebrate NTE is insufficient to induce paralyzing axonal degeneration, a reaction called "aging reaction" is necessary for the disease to set in. It is hypothesized that a second "non-esterase" function of NTE is responsible for this phenomenon. The biological function of SWS within the nervous system is still unknown. To characterize the function of this protein several transgenic fly lines expressing different mutated forms of SWS were established. The controlled expression of altered SWS protein with the GAL4/UAS system allowed the analysis of isolated parts of the protein that were altered in the respective constructs. The characterization of a possible non-esterase function was of particular interest in these experiments. One previously described aberrant SWS construct lacking the first 80 amino acids (SWS\&\#916;1-80) showed a deleterious, dominant effect when overexpressed and was used as a model for organophosphate (OP) intoxication. This construct retains part of its detrimental effect even without catalytically active serine esterase function. This strongly suggests that there is another characteristic to SWS that is not defined solely by its serine esterase activity. Experiments analyzing the lipid contents of sws mutant, wildtype (wt) and SWS overexpressing flies gave valuable insights into a possible biological function of SWS. Phosphatidylcholine, a major component of cell membranes, accumulates in sws mutants whereas it is depleted in SWS overexpressing flies. This suggests that SWS is involved in phosphatidylcholine regulation. The produced \&\#945;-SWS antibody made it possible to study the intracellular localization of SWS. Images of double stainings with ER (endoplasmic reticulum) markers show that SWS is in great part localized to the ER. This is consistent with findings of SWS/ NTE localization in yeast and mouse cells. The olk mutant also shows progressive neurodegeneration but it is more localized to the olfactory system and mushroom bodies. Regarding specific cell types it seemed that specifically the projection neurons (PNs) are affected. A behavioral phenotype consisting of poor olfactory memory compared to wt is also observed even before histologically visible neurodegeneration sets in. Considering that the projection neurons connect the antennal lobes to the mushroom bodies, widely regarded as the "learning center", this impairment was expected. Three mutants where identified (olk1-3) by complementation analysis with the previously known futschN94 allele and sequencing of the coding sequence of olk1 revealed a nonsense mutation early in the protein. Consistent with the predicted function of Futsch as a microtubule associated protein (MAP), abnormalities are most likely due to a defective microtubule network and defects in axonal transport. In histological sections a modified cytoskeletal network is observed and western blots confirm a difference in the amount of tubulin present in the olk1 mutant versus the wt. The elaboration of neuronal axons and dendrites is dependent on a functional cytoskeleton. Observation of transport processes in primary neural cultures derived from olk1 mutant flies also showed a reduction of mitochondrial transport. Interaction with the fragile X mental retardation gene (dfmr1) was observed with the olk mutant. A dfmr1/ olk1 double mutant shows an ameliorated phenotype compared to the olk1 single mutant. tau, another MAP gene, was also shown to be able to partially rescue the olk1 mutant.}, subject = {Taufliege}, language = {en} } @phdthesis{Motsch2005, author = {Motsch, Isabell}, title = {Lamin A and lamin C are differentially dysfunctional in autosomal dominant Emery-Dreifuss muscular dystrophy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15360}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterised by early contractures of the elbows, Achilles tendons and spine, slowly progressive muscle wasting and cardiomyopathy associated with cardiac conduction defect. The autosomal dominant form is caused by mutations in the LMNA gene which gives rise to lamin A and lamin C proteins by alternative splicing. These A-type lamins, together with B-type lamins, form the nuclear lamina, a network of intermediate filament proteins underlining the nuclear envelope. In order to ascertain the role lamin A and C separately contribute to the molecular phenotype, we analysed ten LMNA mutations and one single nucleotide polymorphism (SNP) in transfection studies in COS7 fibroblasts and, partially, in C2C12 myoblasts. The EGFP or DsRed2 tagged lamins were exogenously expressed either individually or both A-types together and examined by light and electron microscopy. The protein mobility of lamin A mutants was determined by FRAP analysis. Additionally, a co-immunoprecipitation binding assay of in vitro synthesised A-type lamins and emerin was performed.Eight of the LMNA mutations (R50S, R133P, E358K, E358K+C