@article{DegenHovestadtMitesseretal.2015, author = {Degen, Tobias and Hovestadt, Thomas and Mitesser, Oliver and H{\"o}lker, Franz}, title = {High female survival promotes evolution of protogyny and xexual conflict}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0118354}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143586}, pages = {e0118354}, year = {2015}, abstract = {Existing models explaining the evolution of sexual dimorphism in the timing of emergence (SDT) in Lepidoptera assume equal mortality rates for males and females. The limiting assumption of equal mortality rates has the consequence that these models are only able to explain the evolution of emergence of males before females, i.e. protandry-the more common temporal sequence of emergence in Lepidoptera. The models fail, however, in providing adaptive explanations for the evolution of protogyny, where females emerge before males, but protogyny is not rare in insects. The assumption of equal mortality rates seems too restrictive for many insects, such as butterflies. To investigate the influence of unequal mortality rates on the evolution of SDT, we present a generalised version of a previously published model where we relax this assumption. We find that longer life-expectancy of females compared to males can indeed favour the evolution of protogyny as a fitness enhancing strategy. Moreover, the encounter rate between females and males and the sex-ratio are two important factors that also influence the evolution of optimal SDT. If considered independently for females and males the predicted strategies can be shown to be evolutionarily stable (ESS). Under the assumption of equal mortality rates the difference between the females' and males' ESS remains typically very small. However, female and male ESS may be quite dissimilar if mortality rates are different. This creates the potential for an 'evolutionary conflict' between females and males. Bagworm moths (Lepidoptera: Psychidae) provide an exemplary case where life-history attributes are such that protogyny should indeed be the optimal emergence strategy from the males' and females' perspectives: (i) Female longevity is considerably larger than that of males, (ii) encounter rates between females and males are presumably low, and (iii) females mate only once. Protogyny is indeed the general mating strategy found in the bagworm family.}, language = {en} } @phdthesis{Carstensen2018, author = {Carstensen, Anne Carola}, title = {Identification of novel N-MYC interacting proteins reveals N-MYC interaction with TFIIIC}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143658}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated. To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif. Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.}, subject = {Biologie}, language = {en} } @article{SchwedeJonesEngstleretal.2011, author = {Schwede, Angela and Jones, Nicola and Engstler, Markus and Carrington, Mark}, title = {The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes}, series = {Molecular \& Biochemical Parasitology}, volume = {175}, journal = {Molecular \& Biochemical Parasitology}, number = {2}, doi = {10.1016/j.molbiopara.2010.11.004}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142746}, pages = {201-204}, year = {2011}, abstract = {In the mammalian host, the Trypanosoma brucei cell surface is covered with a densely packed protein coat of a single protein, the variant surface glycoprotein (VSG). The VSG is believed to shield invariant surface proteins from host antibodies but there is limited information on how far antibodies can penetrate into the VSG monolayer. Here, the VSG surface coat was probed to determine whether it acts as a barrier to binding of antibodies to the membrane proximal VSG C-terminal domain. The binding of C-terminal domain antibodies to VSG221 or VSG118 was compared with antibodies recognising the cognate whole VSGs. The C-terminal VSG domain was inaccessible to antibodies on live cells but not on fixed cells. This provides further evidence that the VSG coat acts as a barrier and protects the cell from antibodies that would otherwise bind to some of the other externally disposed proteins.}, language = {en} } @article{SchleuningFarwigPetersetal.2011, author = {Schleuning, Matthias and Farwig, Nina and Peters, Marcell K. and Bergsdorf, Thomas and Bleher, B{\"a}rbel and Brandl, Roland and Dalitz, Helmut and Fischer, Georg and Freund, Wolfram and Gikungu, Mary W. and Hagen, Melanie and Garcia, Francisco Hita and Kagezi, Godfrey H. and Kaib, Manfred and Kraemer, Manfred and Lung, Tobias and Naumann, Clas M. and Schaab, Gertrud and Templin, Mathias and Uster, Dana and W{\"a}gele, J. Wolfgang and B{\"o}hning-Gaese, Katrin}, title = {Forest Fragmentation and Selective Logging Have Inconsistent Effects on Multiple Animal-Mediated Ecosystem Processes in a Tropical Forest}, series = {PLoS ONE}, volume = {6}, journal = {PLoS ONE}, number = {11}, doi = {10.1371/journal.pone.0027785}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140093}, pages = {e27785}, year = {2011}, abstract = {Forest fragmentation and selective logging are two main drivers of global environmental change and modify biodiversity and environmental conditions in many tropical forests. The consequences of these changes for the functioning of tropical forest ecosystems have rarely been explored in a comprehensive approach. In a Kenyan rainforest, we studied six animal-mediated ecosystem processes and recorded species richness and community composition of all animal taxa involved in these processes. We used linear models and a formal meta-analysis to test whether forest fragmentation and selective logging affected ecosystem processes and biodiversity and used structural equation models to disentangle direct from biodiversity-related indirect effects of human disturbance on multiple ecosystem processes. Fragmentation increased decomposition and reduced antbird predation, while selective logging consistently increased pollination, seed dispersal and army-ant raiding. Fragmentation modified species richness or community composition of five taxa, whereas selective logging did not affect any component of biodiversity. Changes in the abundance of functionally important species were related to lower predation by antbirds and higher decomposition rates in small forest fragments. The positive effects of selective logging on bee pollination, bird seed dispersal and army-ant raiding were direct, i.e. not related to changes in biodiversity, and were probably due to behavioural changes of these highly mobile animal taxa. We conclude that animal-mediated ecosystem processes respond in distinct ways to different types of human disturbance in Kakamega Forest. Our findings suggest that forest fragmentation affects ecosystem processes indirectly by changes in biodiversity, whereas selective logging influences processes directly by modifying local environmental conditions and resource distributions. The positive to neutral effects of selective logging on ecosystem processes show that the functionality of tropical forests can be maintained in moderately disturbed forest fragments. Conservation concepts for tropical forests should thus include not only remaining pristine forests but also functionally viable forest remnants.}, language = {en} } @phdthesis{Gupta2018, author = {Gupta, Shishir Kumar}, title = {Re-annotation of Camponotus floridanus Genome and Characterization of Innate Immunity Transcriptome Responses to Bacterial Infections}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140168}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {The sequencing of several ant genomes within the last six years open new research avenues for understanding not only the genetic basis of social species but also the complex systems such as immune responses in general. Similar to other social insects, ants live in cooperative colonies, often in high densities and with genetically identical or closely related individuals. The contact behaviours and crowd living conditions allow the disease to spread rapidly through colonies. Nevertheless, ants can efficiently combat infections by using diverse and effective immune mechanisms. However, the components of the immune system of carpenter ant Camponotus floridanus and also the factors in bacteria that facilitate infection are not well understood. To form a better view of the immune repository and study the C. floridanus immune responses against the bacteria, experimental data from Illumina sequencing and mass-spectrometry (MS) data of haemolymph in normal and infectious conditions were analysed and integrated with the several bioinformatics approaches. Briefly, the tasks were accomplished in three levels. First, the C. floridanus genome was re-annotated for the improvement of the existing annotation using the computational methods and transcriptomics data. Using the homology based methods, the extensive survey of literature, and mRNA expression profiles, the immune repository of C. floridanus were established. Second, large-scale protein-protein interactions (PPIs) and signalling network of C. floridanus were reconstructed and analysed and further the infection induced functional modules in the networks were detected by mapping of the expression data over the networks. In addition, the interactions of the immune components with the bacteria were identified by reconstructing inter-species PPIs networks and the interactions were validated by literature. Third, the stage-specific MS data of larvae and worker ants were analysed and the differences in the immune response were reported. Concisely, all the three omics levels resulted to multiple findings, for instance, re-annotation and transcriptome profiling resulted in the overall improvement of structural and functional annotation and detection of alternative splicing events, network analysis revealed the differentially expressed topologically important proteins and the active functional modules, MS data analysis revealed the stage specific differences in C. floridanus immune responses against bacterial pathogens. Taken together, starting from re-annotation of C. floridanus genome, this thesis provides a transcriptome and proteome level characterization of ant C. floridanus, particularly focusing on the immune system responses to pathogenic bacteria from a biological and a bioinformatics point of view. This work can serve as a model for the integration of omics data focusing on the immuno-transcriptome of insects.}, subject = {Camponotus floridanus}, language = {en} } @article{SeherNickelMuelleretal.2011, author = {Seher, Axel and Nickel, Joachim and Mueller, Thomas D. and Kneitz, Susanne and Gebhardt, Susanne and Meyer ter Vehn, Tobias and Schlunck, Guenther and Sebald, Walter}, title = {Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro}, series = {Molecular Vision}, volume = {17}, journal = {Molecular Vision}, number = {08. Okt}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140189}, pages = {53-62}, year = {2011}, abstract = {Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix (TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix (TM) oligonucleotide array technology. Results were validated by real time RT-PCR. Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.}, language = {en} } @article{UppaluriNaglerStellamannsetal.2011, author = {Uppaluri, Sravanti and Nagler, Jan and Stellamanns, Eric and Heddergott, Niko and Herminghaus, Stephan and Pfohl, Thomas and Engstler, Markus}, title = {Impact of Microscopic Motility on the Swimming Behavior of Parasites: Straighter Trypanosomes are More Directional}, series = {PLoS Computational Biology}, volume = {7}, journal = {PLoS Computational Biology}, number = {6}, doi = {10.1371/journal.pcbi.1002058}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140814}, pages = {e1002058}, year = {2011}, abstract = {Microorganisms, particularly parasites, have developed sophisticated swimming mechanisms to cope with a varied range of environments. African Trypanosomes, causative agents of fatal illness in humans and animals, use an insect vector (the Tsetse fly) to infect mammals, involving many developmental changes in which cell motility is of prime importance. Our studies reveal that differences in cell body shape are correlated with a diverse range of cell behaviors contributing to the directional motion of the cell. Straighter cells swim more directionally while cells that exhibit little net displacement appear to be more bent. Initiation of cell division, beginning with the emergence of a second flagellum at the base, correlates to directional persistence. Cell trajectory and rapid body fluctuation correlation analysis uncovers two characteristic relaxation times: a short relaxation time due to strong body distortions in the range of 20 to 80 ms and a longer time associated with the persistence in average swimming direction in the order of 15 seconds. Different motility modes, possibly resulting from varying body stiffness, could be of consequence for host invasion during distinct infective stages.}, language = {en} } @phdthesis{Michel2020, author = {Michel, Konstanze}, title = {Die kardiale Bedeutung des Hormons C-Typ natriuretisches Peptid (CNP) und dessen Guanylylcyclase B (GC-B) Rezeptor}, doi = {10.25972/OPUS-20021}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200211}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {In der vorliegenden Dissertationsarbeit wurden die kardialen Effekte des C-Typ natriuretischen Peptids (CNP) an wildtypischen M{\"a}usen (Studie 1) und an einem neuen genetischen Mausmodell, mit einer Kardiomyozyten-spezifischen Deletion des Guanylyl-Cyclase B (GC-B) Rezeptors (Studie 2) untersucht. In Studie 1 wurden die Wirkungen von exogenem, synthetischem CNP auf eine durch Druckbelastung-induzierte Herzinsuffizienz in wildtypischen M{\"a}usen (C57Bl6 Hintergrund) untersucht. Daf{\"u}r wurde CNP parallel zu einer operativen transversen Aortenkonstriktion (TAC) {\"u}ber osmotische Minipumpen in einer Dosierung von 50 ng/kg/min {\"u}ber 14 Tage appliziert. Die 14 Tage TAC f{\"u}hrten zu einer ausgepr{\"a}gten Linksherzhypertrophie. Diese wurde durch exogenes CNP auf zellul{\"a}rer (verringerte Kardiomyozytenfl{\"a}chen) und molekularer (verringerte BNP mRNA Expression) Ebene signifikant gehemmt. Auch die durch TAC-induzierte linksventrikul{\"a}re Dilatation wurde durch exogenes CNP fast vollst{\"a}ndig verhindert. Diese kardialen protektiven Effekte von CNP traten ohne eine wesentliche Ver{\"a}nderung des arteriellen Blutdrucks auf. M{\"o}gliche mechanistische Ursachen f{\"u}r die sch{\"u}tzende Wirkung von CNP k{\"o}nnte die PKG-abh{\"a}ngige Phosphorylierung des sarkomerischen Proteins Titin sein. Eine gesteigerte Phosphorylierung von Titin an der elastischen N2B-Dom{\"a}ne verringert die Steifigkeit der Kardiomyozyten und verbessert somit deren Relaxationsf{\"a}higkeit (Hudson 2011). Die erh{\"o}hten linksventrikul{\"a}ren Volumina nach TAC (end-diastolische und end-systolische Volumina) wurden m{\"o}glicherweise durch eine erh{\"o}hte Steifigkeit der Kardiomyozyten provoziert. Dies k{\"o}nnte durch den akuten IL-6 mRNA Anstieg nach TAC beg{\"u}nstigt werden, da Kruger et al. einen Zusammenhang zwischen passiver Steifigkeit der Kardiomyozyten und IL-6-Expression postulierten (Kotter 2016, Kruger 2009). Diese Ver{\"a}nderungen wurden durch exogenes CNP verhindert. Es ist wahrscheinlich, dass die CNP-induzierte Phosphorylierung von Titin an Serin 4080 in die Relaxationsf{\"a}higkeit der Kardiomyozyten und somit die diastolische Funktion des linken Ventrikels verbesserte. Aufgrund dieser Beobachtungen wurde in Studie 2 untersucht, ob auch endogenes CNP als parakrines Hormon im Herzen eine TAC-induzierte Herzhypertrophie und die kontraktile Funktion von Kardiomyozyten bei einer hypertensiven Herzerkrankung beeinflussen kann. Daf{\"u}r wurde ein neues genetisches Mausmodell mit einer Kardiomyozyten-spezifischen Deletion des GC-B Rezeptors generiert (CM GC-B KO). Da vorangegangene Studien in unserer Arbeitsgruppe zeigten, dass die basale CNP-Expression im Herzen sehr gering ist, nach 3-t{\"a}giger TAC aber akut ansteigt und nach 14-t{\"a}giger TAC wieder abf{\"a}llt, haben wir CM GC-B KO M{\"a}use und deren Geschwister-Kontrolltiere an beiden Zeitpunkten nach TAC untersucht. Die TAC f{\"u}hrte Genotyp-unabh{\"a}ngig zu einem Anstieg der kardialen Nachlast nach 3 Tagen und weiter nach 14 Tagen. Diese Druckbelastung provozierte eine progressive, signifikante Linksherzhypertrophie. Allerdings reagierten die CM GC-B KO M{\"a}use im Vergleich zu den Kontrolltieren bereits nach 3-t{\"a}giger TAC mit einer ausgepr{\"a}gten Kardiomyozyten-Hypertrophie. Zudem beobachteten wir nach 3-t{\"a}giger TAC in den Knockout-M{\"a}usen eine Abnahme der Ejektionsfraktion und gleichzeitig eine signifikante Zunahme der beiden linksventrikul{\"a}ren Volumina (end-diastolische und end-systolische Volumen). Diese fr{\"u}he linksventrikul{\"a}re Dilatation wurde in den Kontrolltieren nicht beobachtet. Daraus schlussfolgerten wir, dass endogenes kardiales CNP, dessen Expression zu fr{\"u}hen Zeitpunkten nach Druckbelastung ansteigt, das Herz vor kontraktiler Dysfunktion und Dilatation sch{\"u}tzen kann. Um m{\"o}gliche Mechanismen f{\"u}r die protektive Wirkung von endogenem CNP zu erkl{\"a}ren, untersuchten wir die IL-6 mRNA Expression sowie die Titin-Phosphorylierung im Herzen. Der akute Anstieg der IL-6 mRNA Expression nach 3-t{\"a}giger TAC in den CM GC-B KO M{\"a}usen korreliert mit der verminderten Phosphorylierung von Titin an der PGK-spezifischen Phosphorylierungsstelle (Serin 4080). Somit k{\"o}nnte der CNP/GC-B/cGMP-Signalweg zu einer Inhibition pro-inflammatorischer Gene beitragen, da der akute IL-6 mRNA Anstieg in den Kontrollen nicht beobachtet wurde. Auch die gesteigerte NOX4 Expression 3 Tage nach TAC, k{\"o}nnte zu der fr{\"u}hen dilatativen Kardiomyopathie in den Knockout-M{\"a}usen beigetragen haben. Die verringerte STAT3 Aktivierung in den CM GC-B KO M{\"a}usen w{\"u}rde laut Literatur zu vermehrter Apoptose f{\"u}hren, indem pro-apoptotische Gene wie Bcl oder Bax vermehrt transkribiert werden. Auch die erh{\"o}hte Cxcl-1 mRNA Expression in den Knockout-M{\"a}usen deutet zusammen mit dem IL-6 Anstieg auf vermehrte Entz{\"u}ndungsreaktionen 3 Tage nach TAC hin. Zusammengenommen deuten die Ergebnisse dieser Dissertationsarbeit darauf hin, dass der CNP/GC-B/cGMP-Signalweg in fr{\"u}hen Stadien einer erh{\"o}hten kardialen Druckbelastung und der Entstehung einer dilatativen Kardiomyopathie entgegenwirken kann. Die Phosphorylierung des sarkomerischen Proteins Titin und die Hemmung der Expression pro-inflammatorischer Zytokine (speziell IL-6) k{\"o}nnten zu diesem protektiven Effekt beitragen.}, subject = {Herzinsuffizienz}, language = {de} } @phdthesis{Goetz2020, author = {G{\"o}tz, Ralph}, title = {Super-resolution microscopy of plasma membrane receptors and intracellular pathogens}, doi = {10.25972/OPUS-20716}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207165}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Humans tend to believe in what they can see with their own eyes. Hence, visualization methods like microscopy have always been extremely popular since their invention in the 17th century. With the advent of super-resolution microscopy, the diffraction limit of ~200 - 250 nm could be overcome to enable more detailed insights into biological samples. Especially the single molecule localization microscopy method dSTORM offers the possibility of quantitative bioimaging. Hereby, the repetitive photoswitching of organic dyes in the presence of thiols is exploited to enable a lateral resolution of 20 nm. Another, recently introduced super-resolution method is expansion microscopy (ExM) which physically expands the sample to increase the resolution by the expansion factor from four to even twenty. To enable this, the sample is embedded into a hydrogel, homogenized using an unspecific proteinase and expanded in distilled water. Within this thesis, both methods were used to shed light on plasma membrane receptor distributions and different bacterial and fungal pathogens. In the first part of this thesis dSTORM was used to elucidate the "Receptome", the entirety of all membrane receptors, of the cell line Jurkat T-cells and primary T-cells. Within this project we could successfully visualize and quantify the distribution of the plasma membrane receptors CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 and CD105 with receptor densities ranging from 0.8 cluster/µm² in case of CD20 and 81.4 cluster/µm² for the highly abundant CD45 in activated primary T-cells at the basal membrane. Hereby, we could also demonstrate a homogeneous distribution of most receptors, while only few were clustered. In the case of CD3-clusters were detected in Jurkat T-cells and in primary activated T-cells, but not in na{\"i}ve ones, demonstrating the activation of this receptor. This was followed by the application of dSTORM to three different clinical projects involving the receptors CD38, BCMA and CD20 which are immunotherapeutic targets by monoclonal antibodies and CAR T-cells. In the first two projects dSTORM was applied to determine the receptor upregulation upon exposure of various drugs to MM1.S cells or primary multiple myeloma patient cells. This increase in membrane receptor expression can subsequently enhance the efficacy of therapies directed against these receptors. Within the CD20-project, the superior sensitivity of dSTORM compared to flow cytometry could be demonstrated. Hereby, a substantially higher fraction of CD20-positive patient cells was detected by dSTORM than by flow cytometry. In addition, we could show that by dSTORM CD20-positive evaluated cells were eradicated by immunotherapeutic CAR T-cell treatment. These studies were followed by whole cell super-resolution imaging using both LLS-3D dSTORM and 10x ExM to exclude any artifacts caused by interactions with the glass surface. In 10x ExM signal amplification via biotinylated primary antibodies and streptavidin ATTO 643 was essential to detect even single antibodies directed against the heterodimer CD11a with standard confocal microscopes. Albeit probably not quantitative due to the process of gelation, digestion and expansion during the ExM protocol, even some putative dimers of the receptor CD2 could be visualized using 10x ExM-SIM, similar to dSTORM experiments. Within the second part of this thesis, expansion microscopy was established in bacterial and fungal pathogens. ExM enabled not only an isotropic fourfold expansion of Chlamydia trachomatis, but also allowed the discrimination between the two developmental forms by the chlamydial size after expansion into reticulate and elementary bodies. Hereafter, a new α-NH2-ω-N3-C6-ceramide was introduced enabling an efficient fixation and for the first time the use of lipids in both, 4x and 10x ExM, termed sphingolipid ExM. This compound was used to investigate the ceramide uptake and incorporation into the cell membrane of Chlamydia trachomatis and Simkania negevensis. For Chlamydia trachomatis the combined resolution power of 10x ExM and SIM even allowed the visualization of both bacterial membranes within a distance of ~30 nm. Finally, ExM was applied to the three different fungi Ustilago maydis, Fusarium oxysporum and Aspergillus fumigatus after enzymatic removal of the fungal cell wall. In case of Ustilago maydis sporidia this digestion could be applied to both, living cells resulting in protoplasts and to fixed cells, preserving the fungal morphology. This new protocol could be demonstrated for immunostainings and fluorescent proteins of the three different fungi.}, subject = {Mikroskopie}, language = {en} } @phdthesis{Krimmer2021, author = {Krimmer, Elena}, title = {Agri-environment schemes and ecosystem services: The influence of different sown flower field characteristics on pollination, natural pest control and crop yield}, doi = {10.25972/OPUS-20657}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206577}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Insects are responsible for the major part of the ecosystem services pollination and natural pest control. If insects decline, these ecosystem services can not longer be reliably delivered. Agricultural intensification and the subsequent loss and fragmentation of habitats has among others been identified to cause insect decline. Ecological intensification aims to promote alternative and sustainable management practices in agricultural farming, for example to decrease the use of external inputs such as pesticides. Agri-environment schemes make amends for farmers if they integrate ecologically beneficial measures into their farming regime and can therefore promote ecological intensification. There is a wide variety of agri-environment schemes, but the implementation of sown flower fields on crop fields is often included. Flower fields offer foraging resources as well as nesting sites for many different insect species and should be able to support insect populations as well as to increase ecosystem services to adjacent fields. However, the potential of flower fields to exhibit these effects is depending on many factors. Among others, the age and size of the flower field can influence if and how different insects profit from the measure. Additionally, the complexity of the surrounding landscape and therefore the existing biodiversity is influencing the potential of flower fields to increase ecosystem services locally. The goal of this study is to disentangle to which degree these factors influence the ecosystem services pollination and natural pest control and if these factors interact with each other. Furthermore, it will be examined if and how flower fields and ecosystem services influence crop yield. Additional factors examined in this study are distance decay and pesticide use. The abundance of beneficial insects can decrease strongly with increasing distance to suitable habitats. Pesticide use in turn could abrogate positive effects of flower fields on beneficial insects. To examine these different aspects and to be able to make recommendations for flower field implementation, field experiments were conducted on differently composed sown flower fields and adjacent oilseed rape fields. Flower fields differed in their age and continuity as well as in their size. Additionally, flower and oilseed rape fields were chosen in landscapes with different amounts of semi-natural habitat. Oilseed rape fields adjacent to calcareous grasslands and conventional crop fields served as controls. Pollinator observations and pollen beetle and parasitism surveys were conducted in the oilseed rape fields. Additionally, different yield parameters of the oilseed rape plants were recorded. Observations were conducted and samples taken in increasing distance to the flower fields to examine distance decay functions. Spray windows were established to inspect the influence of pesticides on ecosystem services and crop yields. Linear mixed models were used for statistical analysis. The results show, that newly established flower fields with high amounts of flower cover are very attractive for pollinators. If the flower fields reached a certain size (> 1.5ha), the pollinators tended to stay in these fields and did not distribute into the surroundings. High amounts of semi-natural habitat in the surrounding landscape increased the value of small flower fields as starting points for pollinators and their subsequent spillover into crop fields. Additionally, high amounts of semi-natural habitat decreased the decay of pollinators with increasing distance to the flower fields. Based on these results, it can be recommended to establish many small flower fields in landscapes with high amounts of semi-natural habitat and large flower fields in landscapes with low amounts of semi-natural habitat. However, it is mentionable that flower fields are no substitute for perennial semi-natural habitats. These still must be actively conserved to increase pollination to crop fields. Furthermore, the lowest amount of pollen beetle infestation was found on oilseed rape fields adjacent to continuous flower fields aged older than 6 years. Flower fields and calcareous grasslands in general increased pollen beetle parasitism in adjacent oilseed rape fields compared to conventional crop fields. The threshold for effective natural pest control could only be reached in the pesticide free areas in the oilseed rape fields adjacent to continuous flower fields and calcareous grasslands. Parasitism and superparasitism declined with increasing distance to the adjacent fields in pesticide treated areas of the oilseed rape fields. However, they remained on a similar level in spray windows without pesticides. Large flower fields increased parasitism and superparasitism more than small flower fields. Flower fields generally have the potential to increase pollen beetle parasitism rates, but pesticides can abrogate these positive effects of flower fields on natural pest control. Last but not least, effects of flower fields and ecosystem services on oilseed rape yield were examined. No positive effects of pollination on oilseed rape yield could be found. Old and continuous flower fields increased natural pest control in oilseed rape fields, which in turn increased seed set and total seed weight of oilseed rape plants. The pesticide treatment had negative effects on natural pest control, but positive effects on crop yield. Pollination and natural pest control decreased with increasing distance to the field edge, but fruit set slightly increased. The quality of the field in terms of soil and climatic conditions did not influence the yield parameters examined in this study. Yield formation in oilseed rape plants is a complex process with many factors involved, and it is difficult to disentangle indirect effects of flower fields on yield. However, perennial flower fields can promote ecological intensification by increasing crop yield via natural pest control. This study contributes to a better understanding of the effects of differently composed flower fields on pollination, natural pest control and oilseed rape yield.}, subject = {{\"O}kologie}, language = {en} } @phdthesis{Schuster2021, author = {Schuster, Sarah}, title = {Analysis of \(Trypanosoma\) \(brucei\) motility and the infection process in the tsetse fly vector}, doi = {10.25972/OPUS-19269}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192691}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {African trypanosomes are protist pathogens that are infective for a wide spectrum of mammalian hosts. Motility has been shown to be essential for their survival and represents an important virulence factor. Trypanosoma brucei is transmitted by the bite of the bloodsucking tsetse fly, the only vector for these parasites. The voyage through the fly is complex and requires several migration, proliferation and differentiation steps, which take place in a defined order and in specific fly tissues. The first part of this doctoral thesis deals with the establishment of the trypanosome tsetse system as a new model for microswimmer analysis. There is an increasing interdisciplinary interest in microbial motility, but a lack of accessible model systems. Therefore, this work introduces the first enclosed in vivo host parasite system that is suitable for analysis of diverse microswimmer types in specific microenvironments. Several methods were used and adapted to gain unprecedented insights into trypanosome motion, the fly´s interior architecture and the physical interaction between host and parasite. This work provides a detailed overview on trypanosome motile behavior as a function of development in diverse host surroundings. In additional, the potential use of artificial environments is shown. This can be used to partly abstract the complex fly architecture and analyze trypanosome motion in defined nature inspired geometries. In the second part of the thesis, the infection of the tsetse fly is under investigation. Two different trypanosome forms exist in the blood: proliferative slender cells and cell cycle arrested stumpy cells. Previous literature states that stumpy cells are pre adapted to survive inside the fly, whereas slender cells die shortly after ingestion. However, infection experiments in our laboratory showed that slender cells were also potentially infective. During this work, infections were set up so as to minimize the possibility of stumpy cells being ingested, corroborating the observation that slender cells are able to infect flies. Using live cell microscopy and fluorescent reporter cell lines, a comparative analysis of the early development following infection with either slender or stumpy cells was performed. The experiments showed, for the first time, the survival of slender trypanosomes and their direct differentiation to the procyclic midgut stage, contradicting the current view in the field of research. Therefore, we can shift perspectives in trypanosome biology by proposing a revised life cycle model of T. brucei, where both bloodstream stages are infective for the vector.}, subject = {Motilit{\"a}t}, language = {en} } @phdthesis{Waeldchen2020, author = {W{\"a}ldchen, Sina}, title = {Super-Resolution-Mikroskopie zur Visualisierung und Quantifizierung von Glutamatrezeptoren und ADHS-assoziierten Proteinen}, doi = {10.25972/OPUS-19283}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192834}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Die Entwicklung hochaufl{\"o}sender Fluoreszenzmikroskopiemethoden hat die Lichtmikroskopie revolutioniert. Einerseits erm{\"o}glicht die h{\"o}here erzielte r{\"a}umliche Aufl{\"o}sung die Abbildung von Strukturen, die deutlich unterhalb der beugungsbedingten Aufl{\"o}sungsgrenze liegen. Andererseits erh{\"a}lt man durch Einzelmolek{\"u}llokalisationsmikroskopiemethoden wie dSTORM (Direct Stochastic Optical Reconstruction Microscopy) Informationen, welche man f{\"u}r quantitative Analysen heranziehen kann. Aufgrund der sich dadurch bietenden neuen M{\"o}glichkeiten, hat sich die hochaufl{\"o}sende Fluoreszenzmikroskopie rasant entwickelt und kommt mittlerweile zur Untersuchung einer Vielzahl biologischer und medizinischer Fragestellungen zum Einsatz. Trotz dieses Erfolgs ist jedoch nicht zu verleugnen, dass auch diese neuen Methoden ihre Nachteile haben. Dazu z{\"a}hlt die Notwendigkeit relativ hoher Laserleistungen, welche Voraussetzung f{\"u}r hohe Aufl{\"o}sung ist und bei lebenden Proben zur Photosch{\"a}digung f{\"u}hren kann. Diese Arbeit widmet sich sowohl dem Thema der Photosch{\"a}digung durch Einzelmolek{\"u}llokalisationsmikroskopie, als auch der Anwendung von dSTORM und SIM (Structured Illumination Microscopy) zur Untersuchung neurobiologischer Fragestellungen auf Proteinebene. Zur Ermittlung der Photosch{\"a}digung wurden lebende Zellen unter typischen Bedingungen bestrahlt und anschließend f{\"u}r 20-24 h beobachtet. Als quantitatives Maß f{\"u}r den Grad der Photosch{\"a}digung wurde der Anteil sterbender Zellen bestimmt. Neben der zu erwartenden Intensit{\"a}ts- und Wellenl{\"a}ngenabh{\"a}ngigkeit, zeigte sich, dass die Schwere der Photosch{\"a}digung auch von vielen weiteren Faktoren abh{\"a}ngt und dass sich Einzelmolek{\"u}llokalisationsmikroskopie bei Ber{\"u}cksichtigung der gewonnenen Erkenntnisse durchaus mit Lebendzellexperimenten vereinbaren l{\"a}sst. Ein weiteres Projekt diente der Untersuchung der A- und B-Typ-Glutamatrezeptoren an der neuromuskul{\"a}ren Synapse von Drosophila melanogaster mittels dSTORM. Dabei konnte eine ver{\"a}nderte Anordnung beider Rezeptortypen infolge synaptischer Plastizit{\"a}t beobachtet, sowie eine absolute Quantifizierung des A-Typ-Rezeptors durchgef{\"u}hrt werden. Im Mittelpunkt eines dritten Projekts standen Cadherin-13 (CDH13) sowie der Glucosetransporter Typ 3 (GluT3), welche beide mit der Aufmerksamkeitsdefizit-Hyperaktivit{\"a}tsst{\"o}rung in Verbindung gebracht werden. CDH13 konnte mittels SIM in serotonergen Neuronen, sowie radi{\"a}ren Gliazellen der dorsalen Raphekerne des embryonalen Mausgehirns nachgewiesen werden. Die Rolle von GluT3 wurde in aus induzierten pluripotenten Stammzellen differenzierten Neuronen analysiert, welche verschiedene Kopienzahlvariation des f{\"u}r GluT3-codierenden SLC2A3-Gens aufwiesen. Die Proteine GluT3, Bassoon und Homer wurden mittels dSTORM relativ quantifiziert. W{\"a}hrend die Deletion des Gens zu einer erwartenden Verminderung von GluT3 auf Proteinebene f{\"u}hrte, hatte die Duplikation keinen Effekt auf die GluT3-Menge. F{\"u}r Bassoon und Homer zeigte sich weder durch die Deletion noch die Duplikation eine signifikante Ver{\"a}nderung.}, subject = {Mikroskopie}, language = {de} } @phdthesis{Neubert2019, author = {Neubert, Franziska}, title = {Markierung postsynaptischer Proteine f{\"u}r die hochaufl{\"o}sende Fluoreszenzmikroskopie}, doi = {10.25972/OPUS-19239}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192394}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Das menschliche Gehirn ist ein Organ, das aufgrund seiner Komplexit{\"a}t und zellul{\"a}ren Diversit{\"a}t noch am wenigsten verstanden ist. Eine der Ursachen daf{\"u}r sind zahlreiche Herausforderungen in diversen neurobiologischen Bild-gebungsverfahren. Erst seit der Erfindung der hochaufl{\"o}senden Fluoreszenz-mikroskopie ist es m{\"o}glich, Strukturen unterhalb der Beugungsgrenze zu visua-lisieren und somit eine maximale Aufl{\"o}sung von bis zu 20 nm zu erreichen. Zus{\"a}tzlich h{\"a}ngt die F{\"a}higkeit, biologische Strukturen aufzul{\"o}sen, von der Markierungs-gr{\"o}ße und -dichte ab. Derzeit ist die h{\"a}ufigste Methode zur Proteinf{\"a}rbung die indirekte Antik{\"o}rperf{\"a}rbung, bei der ein Fluorophor-markierter Sekund{\"a}rantik{\"o}rper an einen Epitop-spezifischen Prim{\"a}rantik{\"o}rper bindet. Dabei kann der Abstand von Zielstruktur und Fluorophor bis zu 30 nm betragen, was eine Aufl{\"o}sungs-verminderung zur Folge haben kann. Aufgrund dessen wurden in dieser Arbeit alternative Markierungsmethoden getestet, um postsynaptische Proteine sicht-bar zu machen. Zun{\"a}chst wurde der postsynaptische N-Methyl-D-Aspartat (NMDA)-Rezeptor mit Hilfe konventioneller indirekter Antik{\"o}rperf{\"a}rbung markiert. Hier war die NR1-Untereinheit des NMDA-Rezeptors von besonderem Interesse, da diese in der Autoimmunerkrankung Anti-NMDA-Rezeptor-Enzephalitis invol-viert ist. Patienten dieser seltenen Krankheit bilden Autoantik{\"o}rper gegen die NR1-Untereinheit, wodurch ein schneller reversibler Verlust der NMDA-Rezeptoren auf der Postsynapse induziert wird. Wichtige Informationen k{\"o}nnen nicht mehr ausreichend weitergegeben werden, was psychiatrische und neurologi-sche St{\"o}rungen zur Folge hat. In dieser Arbeit wurden sowohl kommerzielle NR1-Antik{\"o}rper, als auch rekombinante monoklonale NR1-Antik{\"o}rper von Patien-ten mit Anti-NMDA-Rezeptor-Enzephalitis getestet. In konfokalen und in hochaufgel{\"o}sten SIM- (engl. structured illumination microscopy) und dSTORM- (engl. direct stochastic optical reconstruction microscopy) Messun-gen konnten kommerzielle NR1-Antik{\"o}rper keine erfolgreichen F{\"a}rbungen erzielen. Dagegen erwiesen sich die rekombinanten monoklonalen NR1-Patientenantik{\"o}rper als sehr spezifisch, sowohl in prim{\"a}ren Neuronen als auch im Hippocampus von murinen Gehirnschnitten und lieferten gute Kolokalisati-onen mit dem postsynaptischen Markerprotein Homer. Um die optische Aufl{\"o}sung zu verbessern, wurde eine neue Markierungs-methode mit sog. „Super-Binde-Peptiden" (SBPs) getestet. SBPs sind modifi-zierte Peptide, die erh{\"o}hte Affinit{\"a}ten und Spezifit{\"a}ten aufweisen und mit ei-ner Gr{\"o}ße von ~ 2,5 nm wesentlich kleiner als Antik{\"o}rper sind. In dieser Arbeit best{\"a}tigte sich ein kleines hochspezifisches SPB, das an den Fluoreszenzfarb-stoff Tetra- methylrhodamin (TMR) gekoppelt ist, als effektiver Marker f{\"u}r das Ankerpro-tein Gephyrin. Gephyrin ist f{\"u}r die Lokalisation und Verankerung einiger post-synaptischer Rezeptoren zust{\"a}ndig, indem es sie mit dem Cytoskelett der Zelle verbindet. SIM-Messungen in prim{\"a}ren Neuronen zeigten eine bessere Clus-terrepr{\"a}sentation bei der F{\"a}rbung von Gephyrin mit SBPs, als mit Antik{\"o}rper-f{\"a}rbung. Zus{\"a}tzlich wurden Kolokalisationsanalysen von Gephyrin zusammen mit dem inhibito-rischen pr{\"a}synaptischen vesikul{\"a}ren GABA-Transporter VGAT durchgef{\"u}hrt. Eine weitere F{\"a}rbemethode stellte die bioorthogonale Click-F{\"a}rbung durch die Erweiterung des eukaryotischen genetischen Codes (engl. genetic code ex-pansion, GCE) dar. Dabei wurde eine unnat{\"u}rliche, nicht-kanonische Amino-s{\"a}ure (engl. non-canonical amino acid, ncAA) ins Zielprotein eingebaut und in Kombination mit der Click-Chemie ortsspezifisch mit organischen Tetrazin-Farbstoff-Konjugaten angef{\"a}rbt. Organische Fluorophore haben den Vorteil, dass sie mit einer Gr{\"o}ße von 0,5 - 2 nm sehr klein sind und damit die nat{\"u}rli-chen Funktionen der Proteine in der Zelle kaum beeinflussen. In dieser Arbeit wurde zum ersten Mal gezeigt, dass der tetramere postsynaptische NMDA-Rezeptor durch die Amber-Supres-sionsmethode bioorthogonal angef{\"a}rbt werden konnte. Aus sieben verschiede-nen Amber-Mutanten der NR1-Untereinheit stellte sich die Y392TAG-NR1-Mutante als diejenige mit der besten Proteinexpression, F{\"a}rbeeffizienz und rezeptorfunktionalit{\"a}t heraus. Dies konnte durch Fluoreszenzmikroskopie- und Whole-Cell Patch-Clamp-Experimenten gezeigt werden. Die bioorthogo-nale Click-F{\"a}rbung durch GCE eignete sich f{\"u}r die F{\"a}rbung des NMDA-Rezeptors in verschiedenen Zelllinien, mit unterschiedlichen Tetrazin-Farbstoff-Konjugaten und f{\"u}r Lebendzellexperimente. In dSTORM-Messungen erwies sich das Tetrazin-Cy5-Farbstoff-Konjugat als ideal aufgrund seiner Gr{\"o}-ße, Photostabilit{\"a}t, Helligkeit und seines geeigneten Blinkverhaltens, sodass eine homogene NMDA-Rezeptorverteilung auf der Zellmembran gezeigt wer-den konnte. NR1-Antik{\"o}rperf{\"a}rbungen wiesen dagegen starke Clusterbildun-gen auf. Die Ergebnisse konnten belegen, dass kleinere Farbstoffe eine deut-lich bessere Zug{\"a}nglichkeit zu ihrem Zielprotein haben und somit besser f{\"u}r die hochaufl{\"o}sende Fluoreszenzmikroskopie geeignet sind.}, subject = {hochaufl{\"o}sende Fluoreszenzmikroskopie}, language = {de} } @phdthesis{Grimm2019, author = {Grimm, Johannes}, title = {Autocrine and paracrine effects of BRAF inhibitor induced senescence in melanoma}, doi = {10.25972/OPUS-18116}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181161}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels. In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.}, subject = {Inhibitor}, language = {en} } @phdthesis{Heiby2021, author = {Heiby, Julia}, title = {Insight into molecular mechanisms of folding and self-association of spider silk protein domains}, doi = {10.25972/OPUS-19345}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193455}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Spider silk is a biomaterial of extraordinary toughness paired with elasticity. The assembly of silk proteins, so-called spidroins (from "spider" and "fibroin"), generates the silk threads we typically see in our garden or the corners of our houses. Although spider webs from different species vary considerably in geometry and size, many sections of spidroin sequences are conserved. Highly conserved regions, found in all spidroins, relate to the terminal domains of the protein, i.e., the N-terminal (NTD) and C-terminal domains (CTD). Both have an essential function in the silk fibre association and polymerisation. The NTD is a 14 kDa five-helix bundle, which self-associates via a pH-driven mechanism. This process is critical for starting the polymerisation of the fibre. However, detailed insights into how conserved this mechanism is in different species and the quantitative thermodynamic comparison between homologous NTDs was missing. For this reason, four homologous NTDs of the major ampullate gland (MaSp) from spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus were investigated. I analysed and quantified equilibrium thermodynamics, kinetics of folding, and self-association. Methods involved dynamic light scattering (MALS), stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. The results showed conserved, cooperative two-state folding on a sub-millisecond time scale. All homologous NTDs showed a similarly fast association in the order of 10^9 M^-1 s^-1, while the resulting equilibrium dissociation constants were in the low nanomolar range. Electrostatic forces were found to be of great importance for protein association. Monomeric protein stability increased with salt concentration while enhancing its folding speed. However, due to Debye-H{\"u}ckel effects, we found intermolecular electrostatics to be shielded, which reduced the NTDs association capacity significantly at high ionic strength. Altogether, the energetics and kinetics of the NTD dimerisation was conserved for all analysed homologs. Comparable to the NTD, the spider silks CTD is also a α-helix bundle, which covalently links two spidroins. The orientation of the domains predetermines the future fibre geometry. Here again, the detailed quantitative characterisation of the folding and dimerisation was missing. Therefore, the CTD from the E. australis was analysed in-depth. The protein folded via a three-state mechanism and was placed in the family of knotted proteins. By analysing the amino acid composition of the NTD of the MaSp1 of the Euprosthenops australis, we found an unusually high content of methionine residues (Met). To elucidate why this protein exhibits so many Met residues, I mutated all core Mets simultaneously to leucine (Leu). Results revealed a dramatically stabilised NTD, which now folded 50 times faster. After solving the tertiary structure of the mutant by NMR (nuclear magnetic resonance) spectroscopy, the structure of the monomeric mutant was found to be identical with the wild-type protein. However, when probing the dimerisation of the NTD, I could show that the association capacity was substantially impaired for the mutant. Our findings lead to the conclusion that Met provides the NTD with enhanced conformational dynamics and thus mobilises the protein, which results in tightly associated dimers. In additional experiments, I first re-introduced new Met residues into the Met-depleted protein at sequence positions containing native Leu. Hence, the mutated NTD protein was provided with the same number of Leu, which were previously removed by mutation. However, the protein did not regain wild-type characteristics. The functionality was not restored, but its stability was decreased as expected. To probe our hypothesis gained from the MaSp NTD, I transferred the experiment to another protein, namely the Hsp90 chaperone. Therefore, I incorporated methionine residues in the protein, which resulted in a slight improvement of its function. Finally, trial experiments were performed aiming at the synthesis of shortened spidroin constructs containing less repetitive middle-segments than the wild-type protein. The objective was to study the findings of the terminal domains in the context of an intact spidroin. The synthesis of these engineered spidroins was challenging. Nevertheless, preliminary results encourage the assumption that the characteristics observed in the isolated domains hold true in the context of a full-length spidroin.}, subject = {Spinnenseide}, language = {en} } @article{GrimmKleinKopeketal.2016, author = {Grimm, Jonathan B. and Klein, Teresa and Kopek, Benjamin G. and Shtengel, Gleb and Hess, Harald F. and Sauer, Markus and Lavis, Luke D.}, title = {Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy}, series = {Angewandte Chemie International Edition}, volume = {55}, journal = {Angewandte Chemie International Edition}, number = {5}, doi = {10.1002/anie.201509649}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191069}, pages = {1723-1727}, year = {2016}, abstract = {The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.}, language = {en} } @phdthesis{Hofrichter2020, author = {Hofrichter, Michaela Angelika Hedwig}, title = {Charakterisierung von angeborenen H{\"o}rst{\"o}rungen mit Hilfe von Hochdurchsatz-Sequenziermethoden}, doi = {10.25972/OPUS-18533}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185331}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Fast 500 Millionen Menschen weltweit sind von einer H{\"o}rst{\"o}rung betroffen. Es wird sogar angenommen, dass diese Anzahl laut der Weltgesundheitsorganisation (WHO) noch steigen und 2050 jeder zehnte Mensch eine H{\"o}rst{\"o}rung aufweisen wird. Mindestens in 50\% aller F{\"a}lle ist die H{\"o}rst{\"o}rung genetisch bedingt. Durch die j{\"u}ngsten Fortschritte der Sequenzierungstechnologien hat die genetische Analyse von H{\"o}rst{\"o}rungen an Bedeutung gewonnen, vor allem hinsichtlich Familienplanung, geeigneter Therapien und zuk{\"u}nftiger m{\"o}glichen Therapieans{\"a}tzen, um das H{\"o}rverm{\"o}gen wiederherzustellen. Die folgende Arbeit stellt 155 famili{\"a}re F{\"a}lle vor, die genetisch untersucht wurden. Diese F{\"a}lle konnten in zwei Kohorten unterteilt werden. Eine Kohorte (n = 74) umfasste Patienten mit kaukasischem Hintergrund, w{\"a}hrend die andere Kohorte (n = 81) Patienten beinhaltete, die aus dem Iran rekrutiert wurden. F{\"u}r die Untersuchung wurde zum einen eine Panel-Analyse mit dem TruSight One Panel (Illumina, San Diego, USA) und zum anderen eine Exom-Sequenzierung durchgef{\"u}hrt. Anschließend wurden die Daten mit Analyse-Programmen wie GensearchNGS (PhenoSystems, Wallonia, Belgien) ausgewertet. Insgesamt konnte f{\"u}r 55\% aller F{\"a}lle eine pathogene oder wahrscheinlich pathogene Variante durch Next Generation Sequencing diagnostiziert werden. Die meisten der gel{\"o}sten F{\"a}lle (ca. 73\%) stammten aus der iranischen Kohorte, was durch elterliche Blutsverwandtschaft und erh{\"o}hte Inzidenz von H{\"o}rst{\"o}rungen im Iran zu erkl{\"a}ren ist. 27\% der gel{\"o}sten F{\"a}lle geh{\"o}rten der zweiten Kohorte an. Mutationen in den Genen MYO15A, LHFPL5, TECTA und SLC26A4 konnten {\"u}berwiegend bei iranischen Patienten identifiziert werden. Varianten im Gen TECTA als auch im Gen SLC26A4 wurden ebenfalls in der kaukasischen Kohorte identifiziert. Beide Ethnien wiesen jeweils ein eigenes Mutationsspektrum auf. Jedoch wurden in beiden Gruppen {\"U}berschneidungen im klinischen Bild durch pathogene Varianten in einer Vielzahl von H{\"o}rst{\"o}rungsgenen, sowie unterschiedliche klinische Ph{\"a}notypen, deren Ursache pathogene Varianten im gleichen H{\"o}rst{\"o}rungsgen zugrunde liegen, und famili{\"a}re Locus-Heterogenit{\"a}t beobachtet.. In dieser Arbeit konnte eine De Novo Mutation im CEACAM16-Gen (DFNA4B) best{\"a}tigt und der Effekt von einer wiederholt betroffenen Aminos{\"a}ure im S1PR2-Gen (DFNB68) beschrieben werden. Dar{\"u}ber hinaus wurden mehrere Patienten mit X-chromosomalem H{\"o}rverlust aufgrund von Defekten im POU3F4-Gen (DFNX2) und Deletionen im SMPX-Gen (DFNX4) diagnostiziert. Zus{\"a}tzlich konnte mit Hilfe einer Exom-basierten Copy Number Variation-Analyse eine Deletion im OTOA-Gen (DFNB22) gefunden werden, welche sich bis in die Tandempseudogenregion erstreckte. Diese Untersuchung zeigt die enormen M{\"o}glichkeiten zur Detektion von Mutationen bei heterogenen Erkrankungen durch Anwendung von Next Generation Sequencing. Weiterhin konnte eine intragenische Deletion im Gen COL9A1 identifiziert werden, die im Zusammenhang mit einer scheinbar isolierten H{\"o}rst{\"o}rung steht und durch den komplexen Umlagerungsmechanismus FoSTeS/MMBIR (Fork Stalling und Template Switching/Microhomology-mediated Break-induced Replication) entstand, der so bei H{\"o}rst{\"o}rungen noch nicht beschrieben wurde. Auf der Suche nach Genen, die bisher noch nicht mit H{\"o}rst{\"o}rungen assoziiert werden konnten, wurden acht Familien in eine Kandidatengenuntersuchung miteinbezogen und eine Exom-weite Analyse durchgef{\"u}hrt. Bei f{\"u}nf Familien konnte noch keine urs{\"a}chliche Variante identifiziert werden. Jedoch wurde bei drei Familien mit einer autosomal dominanten Schwerh{\"o}rigkeit eine genetische Ursache identifiziert und TECTB, ATP11A und THBS2 konnten als Kandidatengene ermittelt werden. Diese Arbeit zeigt, wie wichtig es ist, die kausale Variante bei H{\"o}rst{\"o}rungspatienten zu detektieren. Eine genetische Diagnostik erm{\"o}glicht eine endg{\"u}ltige Diagnose eines Syndroms, ist f{\"u}r die Klassifizierung der H{\"o}rst{\"o}rung notwendig und tr{\"a}gt zu einer zuk{\"u}nftigen Therapie der Patienten bei.}, subject = {H{\"o}rst{\"o}rungen}, language = {de} } @article{HassounaOttWuestefeldetal.2016, author = {Hassouna, I. and Ott, C. and W{\"u}stefeld, L. and Offen, N. and Neher, R. A. and Mitkovski, M. and Winkler, D. and Sperling, S. and Fries, L. and Goebbels, S. and Vreja, I. C. and Hagemeyer, N. and Dittrich, M. and Rossetti, M. F. and Kr{\"o}hnert, K. and Hannke, K. and Boretius, S. and Zeug, A. and H{\"o}schen, C. and Dandekar, T. and Dere, E. and Neher, E. and Rizzoli, S. O. and Nave, K.-A. and Sir{\´e}n, A.-L. and Ehrenreich, H.}, title = {Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus}, series = {Molecular Psychiatry}, volume = {21}, journal = {Molecular Psychiatry}, number = {12}, doi = {10.1038/mp.2015.212}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186669}, pages = {1752-1767}, year = {2016}, abstract = {Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of similar to 20\%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a \(^{15}\)N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated \(^{15}\)N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.}, language = {en} } @article{SchneiderDittrichBoecketal.2016, author = {Schneider, Eberhard and Dittrich, Marcus and B{\"o}ck, Julia and Nanda, Indrajit and M{\"u}ller, Tobias and Seidmann, Larissa and Tralau, Tim and Galetzka, Danuta and El Hajj, Nady and Haaf, Thomas}, title = {CpG sites with continuously increasing or decreasing methylation from early to late human fetal brain development}, series = {Gene}, volume = {592}, journal = {Gene}, number = {1}, doi = {10.1016/j.gene.2016.07.058}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186936}, pages = {110-118}, year = {2016}, abstract = {Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny.}, language = {en} } @article{MarkertBritzProppertetal.2016, author = {Markert, Sebastian Matthias and Britz, Sebastian and Proppert, Sven and Lang, Marietta and Witvliet, Daniel and Mulcahy, Ben and Sauer, Markus and Zhen, Mei and Bessereau, Jean-Louis and Stigloher, Christian}, title = {Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome}, series = {Neurophotonics}, volume = {3}, journal = {Neurophotonics}, number = {4}, doi = {10.1117/1.NPh.3.4.041802}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187292}, pages = {041802}, year = {2016}, abstract = {Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.}, language = {en} } @phdthesis{Markert2021, author = {Markert, Sebastian Matthias}, title = {Enriching the understanding of synaptic architecture from single synapses to networks with advanced imaging techniques}, doi = {10.25972/OPUS-18993}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189935}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Because of its complexity and intricacy, studying the nervous system is often challenging. Fortunately, the small nematode roundworm Caenorhabditis elegans is well established as a model system for basic neurobiological research. The C. elegans model is also the only organism with a supposedly complete connectome, an organism-wide map of synaptic connectivity resolved by electron microscopy, which provides some understanding of how the nervous system works as a whole. However, the number of available data-sets is small and the connectome contains errors and gaps. One example of this concerns electrical synapses. Electrical synapses are formed by gap junctions and difficult to map due to their often ambiguous morphology in electron micrographs, leading to misclassification or omission. On the other hand, chemical synapses are more easily mapped, but many aspects of their mode of operation remain elusive and their role in the C. elegans connectome is oversimplified. A comprehensive understanding of signal transduction of neurons between each other and other cells will be indispensable for a comprehensive understanding of the nervous system. In this thesis, I approach these challenges with a combination of advanced light and electron microscopy techniques. First, this thesis describes a strategy to increase synaptic specificity in connectomics. Specifically, I classify gap junctions with a high degree of confidence. To achieve this, I utilized array tomography (AT). In this thesis, AT is adapted for high-pressure freezing to optimize for structure preservation and for super-resolution light microscopy; in this manner, I aim to bridge the gap between light and electron microscopy resolutions. I call this adaptation super-resolution array tomography (srAT). The srAT approach made it possible to clearly identify and map gap junctions with high precision and accuracy. The results from this study showcased the feasibility of incorporating electrical synapses into connectomes in a systematic manner, and subsequent studies have used srAT for other models and questions. As mentioned above, the C. elegans connectomic model suffers from a shortage of datasets. For most larval stages, including the special dauer larval stage, connectome data is completely missing up to now. To obtain the first partial connectome data-set of the C. elegans dauer larva, we used focused ion-beam scanning electron microscopy (FIB-SEM). This technique offers an excellent axial resolution and is useful for acquiring large volumes for connectomics. Together with our collaborators, I acquired several data-sets which enable the analysis of dauer stage-specific "re-wiring" of the nervous system and thus offer valuable insights into connectome plasticity/variability. While chemical synapses are easy to map relative to electrical synapses, signal transduction via chemical transmitters requires a large number of different proteins and molecular processes acting in conjunction in a highly constricted space. Because of the small spatial scale of the synapse, investigating protein function requires very high resolution, which electron tomography provides. I analyzed electron tomograms of a worm-line with a mutant synaptic protein, the serine/threonine kinase SAD-1, and found remarkable alterations in several architectural features. My results confirm and re-contextualize previous findings and provide new insight into the functions of this protein at the chemical synapse. Finally, I investigated the effectiveness of our methods on "malfunctioning," synapses, using an amyotrophic lateral sclerosis (ALS) model. In the putative synaptopathy ALS, the mechanisms of motor neuron death are mostly unknown. However, mutations in the gene FUS (Fused in Sarcoma) are one known cause of the disease. The expression of the mutated human FUS in C. elegans was recently shown to produce an ALS-like phenotype in the worms, rendering C. elegans an attractive disease model for ALS. Together with our collaboration partners, I applied both srAT and electron tomography methods to "ALS worms" and found effects on vesicle docking. These findings help to explain electrophysiological recordings that revealed a decrease in frequency of mini excitatory synaptic currents, but not amplitudes, in ALS worms compared to controls. In addition, synaptic endosomes appeared larger and contained electron-dense filaments in our tomograms. These results substantiate the idea that mutated FUS impairs vesicle docking and also offer new insights into further molecular mechanisms of disease development in FUS-dependent ALS. Furthermore, we demonstrated the broader applicability of our methods by successfully using them on cultured mouse motor neurons. Overall, using the C. elegans model and a combination of light and electron microscopy methods, this thesis helps to elucidate the structure and function of neuronal synapses, towards the aim of obtaining a comprehensive model of the nervous system.}, subject = {Caenorhabditis elegans}, language = {en} } @article{KilincEhrigPessianetal.2016, author = {Kilinc, Mehmet Okyay and Ehrig, Klaas and Pessian, Maysam and Minev, Boris R. and Szalay, Aladar A.}, title = {Colonization of xenograft tumors by oncolytic vaccinia virus (VACV) results in enhanced tumor killing due to the involvement of myeloid cells}, series = {Journal of Translational Medicine}, volume = {14}, journal = {Journal of Translational Medicine}, number = {340}, doi = {10.1186/s12967-016-1096-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168914}, year = {2016}, abstract = {Background The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment. Methods Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS\(^{+}\) myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student's t test was used for comparison between groups in all of the experiments. Results Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b\(^{+}\) ly6G\(^{+}\) myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS\(^{+}\) MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7-10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments. Conclusions We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.}, language = {en} } @article{VogtmannHuaZelleretal.2016, author = {Vogtmann, Emily and Hua, Xing and Zeller, Georg and Sunagawa, Shinichi and Voigt, Anita Y. and Hercog, Rajna and Goedert, James J. and Shi, Jianxin and Bork, Peer and Sinha, Rashmi}, title = {Colorectal Cancer and the Human Gut Microbiome: Reproducibility with Whole-Genome Shotgun Sequencing}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {5}, doi = {10.1371/journal.pone.0155362}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-166904}, pages = {e0155362}, year = {2016}, abstract = {Accumulating evidence indicates that the gut microbiota affects colorectal cancer development, but previous studies have varied in population, technical methods, and associations with cancer. Understanding these variations is needed for comparisons and for potential pooling across studies. Therefore, we performed whole-genome shotgun sequencing on fecal samples from 52 pre-treatment colorectal cancer cases and 52 matched controls from Washington, DC. We compared findings from a previously published 16S rRNA study to the metagenomics-derived taxonomy within the same population. In addition, metagenome-predicted genes, modules, and pathways in the Washington, DC cases and controls were compared to cases and controls recruited in France whose specimens were processed using the same platform. Associations between the presence of fecal Fusobacteria, Fusobacterium, and Porphyromonas with colorectal cancer detected by 16S rRNA were reproduced by metagenomics, whereas higher relative abundance of Clostridia in cancer cases based on 16S rRNA was merely borderline based on metagenomics. This demonstrated that within the same sample set, most, but not all taxonomic associations were seen with both methods. Considering significant cancer associations with the relative abundance of genes, modules, and pathways in a recently published French metagenomics dataset, statistically significant associations in the Washington, DC population were detected for four out of 10 genes, three out of nine modules, and seven out of 17 pathways. In total, colorectal cancer status in the Washington, DC study was associated with 39\% of the metagenome-predicted genes, modules, and pathways identified in the French study. More within and between population comparisons are needed to identify sources of variation and disease associations that can be reproduced despite these variations. Future studies should have larger sample sizes or pool data across studies to have sufficient power to detect associations that are reproducible and significant after correction for multiple testing.}, language = {en} } @article{Hoelldobler2016, author = {H{\"o}lldobler, Bert}, title = {Queen Specific Exocrine Glands in Legionary Ants and Their Possible Function in Sexual Selection}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0151604}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167057}, pages = {e0151604}, year = {2016}, abstract = {The colonies of army ants and some other legionary ant species have single, permanently wingless queens with massive post petioles and large gasters. Such highly modified queens are called dichthadiigynes. This paper presents the unusually rich exocrine gland endowment of dichthadiigynes, which is not found in queens of other ant species. It has been suggested these kinds of glands produce secretions that attract and maintain worker retinues around queens, especially during migration. However, large worker retinues also occur in non-legionary species whose queens do not have such an exuberance of exocrine glands. We argue and present evidence in support of our previously proposed hypothesis that the enormous outfit of exocrine glands found in dichthadiigynes is due to sexual selection mediated by workers as the main selecting agents}, language = {en} } @article{JoschinskiBeerHelfrichFoersteretal.2016, author = {Joschinski, Jens and Beer, Katharina and Helfrich-F{\"o}rster, Charlotte and Krauss, Jochen}, title = {Pea Aphids (Hemiptera: Aphididae) Have Diurnal Rhythms When Raised Independently of a Host Plant}, series = {Journal of Insect Science}, volume = {16}, journal = {Journal of Insect Science}, number = {1}, doi = {10.1093/jisesa/iew013}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168783}, pages = {31}, year = {2016}, abstract = {Seasonal timing is assumed to involve the circadian clock, an endogenous mechanism to track time and measure day length. Some debate persists, however, and aphids were among the first organisms for which circadian clock involvement was questioned. Inferences about links to phenology are problematic, as the clock itself is little investigated in aphids. For instance, it is unknown whether aphids possess diurnal rhythms at all. Possibly, the close interaction with host plants prevents independent measurements of rhythmicity. We reared the pea aphid Acyrthosiphon pisum (Harris) on an artificial diet, and recorded survival, moulting, and honeydew excretion. Despite their plant-dependent life style, aphids were independently rhythmic under light-dark conditions. This first demonstration of diurnal aphid rhythms shows that aphids do not simply track the host plant's rhythmicity.}, language = {en} } @phdthesis{Blaettner2016, author = {Bl{\"a}ttner, Sebastian}, title = {The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146662}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis. In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70\% of the CA infections. The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur. S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient. In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae. The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria.}, subject = {Staphylococcus aureus}, language = {en} } @article{HornKellerHildebrandtetal.2016, author = {Horn, Hannes and Keller, Alexander and Hildebrandt, Ulrich and K{\"a}mpfer, Peter and Riederer, Markus and Hentschel, Ute}, title = {Draft genome of the \(Arabidopsis\) \(thaliana\) phyllosphere bacterium, \(Williamsia\) sp. ARP1}, series = {Standards in Genomic Sciences}, volume = {11}, journal = {Standards in Genomic Sciences}, number = {8}, doi = {10.1186/s40793-015-0122-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146008}, year = {2016}, abstract = {The Gram-positive actinomycete \(Williamsia\) sp. ARP1 was originally isolated from the \(Arabidopsis\) \(thaliana\) phyllosphere. Here we describe the general physiological features of this microorganism together with the draft genome sequence and annotation. The 4,745,080 bp long genome contains 4434 protein-coding genes and 70 RNA genes. To our knowledge, this is only the second reported genome from the genus \(Williamsia\) and the first sequenced strain from the phyllosphere. The presented genomic information is interpreted in the context of an adaptation to the phyllosphere habitat.}, language = {en} } @phdthesis{Mekala2019, author = {Mekala, SubbaRao}, title = {Generation of cardiomyocytes from vessel wall-resident stem cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146046}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Myocardial infarction (MI) is a major cause of health problems and is among the leading deadly ending diseases. Accordingly, regenerating functional myocardial tissue and/or cardiac repair by stem cells is one of the most desired aims worldwide. Indeed, the human heart serves as an ideal target for regenerative intervention, because the capacity of the adult myocardium to restore itself after injury or infarct is limited. Thus, identifying new sources of tissue resident adult stem or progenitor cells with cardiovascular potential would help to establish more sophisticated therapies in order to either prevent cardiac failure or to achieve a functional repair. Ongoing research worldwide in this field is focusing on a) induced pluripotent stem (iPS) cells, b) embryonic stem (ES) cells and c) adult stem cells (e. g. mesenchymal stem cells) as well as cardiac fibroblasts or myofibroblasts. However, thus far, these efforts did not result in therapeutic strategies that were transferable into the clinical management of MI and heart failure. Hence, identifying endogenous and more cardiac-related sources of stem cells capable of differentiating into mature cardiomyocytes would open promising new therapeutic opportunities. The working hypothesis of this thesis is that the vascular wall serves as a niche for cardiogenic stem cells. In recent years, various groups have identified different types of progenitors or mesenchymal stem cell-like cells in the adventitia and sub-endothelial zone of the adult vessel wall, the so called vessel wall-resident stem cells (VW-SCs). Considering the fact that heart muscle tissue contains blood vessels in very high density, the physiological relevance of VW-SCs for the myocardium can as yet only be assumed. The aim of the present work is to study whether a subset of VW-SCs might have the capacity to differentiate into cardiomyocyte-like cells. This assumption was challenged using adult mouse aorta-derived cells cultivated in different media and treated with selected factors. The presented results reveal the generation of spontaneously beating cardiomyocyte-like cells using specific media conditions without any genetic manipulation. The cells reproducibly started beating at culture days 8-10. Further analyses revealed that in contrast to several publications reporting the Sca-1+ cells as cardiac progenitors the Sca-1- fraction of aortic wall-derived VW-SCs reproducibly delivered beating cells in culture. Similar to mature cardiomyocytes the beating cells developed sarcomeric structures indicated by the typical cross striated staining pattern upon immunofluorescence analysis detecting α-sarcomeric actinin (α-SRA) and electron microscopic analysis. These analyses also showed the formation of sarcoplasmic reticulum which serves as calcium store. Correspondingly, the aortic wall-derived beating cardiomyocyte-like cells (Ao-bCMs) exhibited calcium oscillations. This differentiation seems to be dependent on an inflammatory microenvironment since depletion of VW-SC-derived macrophages by treatment with clodronate liposomes in vitro stopped the generation of Ao bCMs. These locally generated F4/80+ macrophages exhibit high levels of VEGF (vascular endothelial growth factor). To a great majority, VW-SCs were found to be positive for VEGFR-2 and blocking this receptor also stopped the generation VW-SC-derived beating cells in vitro. Furthermore, the treatment of aortic wall-derived cells with the ß-receptor agonist isoproterenol or the antagonist propranolol resulted in a significant increase or decrease of beating frequency. Finally, fluorescently labeled aortic wall-derived cells were implanted into the developing chick embryo heart field where they became positive for α-SRA two days after implantation. The current data strongly suggest that VW-SCs resident in the vascular adventitia deliver both progenitors for an inflammatory microenvironment and beating cells. The present study identifies that the Sca-1- rather than Sca-1+ fraction of mouse aortic wall-derived cells harbors VW-SCs differentiating into cardiomyocyte-like cells and reveals an essential role of VW-SCs-derived inflammatory macrophages and VEGF-signaling in this process. Furthermore, this study demonstrates the cardiogenic capacity of aortic VW-SCs in vivo using a chimeric chick embryonic model.}, subject = {Herzmuskelzelle}, language = {en} } @phdthesis{Pischimarov2016, author = {Pischimarov, Jordan Ivanov}, title = {Bioinformatische Methoden zur Identifizierung und Klassifizierung somatischer Mutationen in h{\"a}matologischen Erkrankungen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147773}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Die Sequenzierungstechnologien entwickeln sich stetig weiter, dies erm{\"o}glicht eine zuvor nicht erreichte Ausbeute an experimentellen Daten und auch an Neuentwicklungen von zuvor nicht realisierbaren Experimenten. Zugleich werden spezifische Datenbanken, Algorithmen und Softwareprogramme entwickelt, um die neu entstandenen Daten zu analysieren. W{\"a}hrend der Untersuchung bioinformatischer Methoden f{\"u}r die Identifizierung und Klassifizierung somatischer Mutationen in h{\"a}matologischen Erkrankungen, zeigte sich eine hohe Vielfalt an alternativen Softwaretools die f{\"u}r die jeweiligen Analyseschritte genutzt werden k{\"o}nnen. Derzeit existiert noch kein Standard zur effizienten Analyse von Mutationen aus Next-Generation-Sequencing (NGS)-Daten. Die unterschiedlichen Methoden und Pipelines generieren Kandidaten, die zum gr{\"o}ßten Anteil in allen Ans{\"a}tzen identifiziert werden k{\"o}nnen, jedoch werden Software spezifische Kandidaten nicht einheitlich detektiert. Um eine einheitliche und effiziente Analyse von NGS-Daten durchzuf{\"u}hren war im Rahmen dieser Arbeit die Entwicklung einer benutzerfreundlichen und einheitlichen Pipeline vorgesehen. Hierf{\"u}r wurden zun{\"a}chst die essentiellen Analysen wie die Identifizierung der Basen, die Alignierung und die Identifizierung der Mutationen untersucht. Des Weiteren wurden unter Ber{\"u}cksichtigung von Effizienz und Performance diverse verf{\"u}gbare Softwaretools getestet, ausgewertet und sowohl m{\"o}gliche Verbesserungen als auch Erleichterungen der bisherigen Analysen vorgestellt und diskutiert. Durch Mitwirken in Konsortien wie der klinischen Forschergruppe 216 (KFO 216) und International Cancer Genome Consortium (ICGC) oder auch bei Haus-internen Projekten wurden Datens{\"a}tze zu den Entit{\"a}ten Multiples Myelom (MM), Burkitt Lymphom (BL) und Follikul{\"a}res Lymphom (FL) erstellt und analysiert. Die Selektion geeigneter Softwaretools und die Generierung der Pipeline basieren auf komparativen Analysen dieser Daten, sowie auf geteilte Ergebnisse und Erfahrungen in der Literatur und auch in Foren. Durch die gezielte Entwicklung von Skripten konnten biologische und klinische Fragestellungen bearbeitet werden. Hierzu z{\"a}hlten eine einheitliche Annotation der Gennamen, sowie die Erstellung von Genmutations-Heatmaps mit nicht Variant-Calling-File (VCF)-Syntax konformen Dateien. Des Weiteren konnten nicht abgedeckte Regionen des Genoms in den NGS-Daten identifiziert und analysiert werden. Neue Projekte zur detaillierten Untersuchung der Verteilung von wiederkehrender Mutationen und Funktionsassays zu einzelnen Mutationskandidaten konnten basierend auf den Ergebnissen initiiert werden. Durch eigens erstellte Python-Skripte konnte somit die Funktionalit{\"a}t der Pipeline erweitert werden und zu wichtigen Erkenntnissen bei der biologischen Interpretation der Sequenzierungsdaten f{\"u}hren, wie beispielsweise zu der Detektion von drei neuen molekularen Subgruppen im MM. Die Erweiterungen, der in dieser Arbeit entwickelten Pipeline verbesserte somit die Effizienz der Analyse und die Vergleichbarkeit unserer Daten. Des Weiteren konnte durch die Erstellung eines eigenen Skripts die Analyse von unbeachteten Regionen in den NGS-Daten erfolgen.}, subject = {Pipeline-Rechner}, language = {de} } @article{SommerlandtSpaetheRoessleretal.2016, author = {Sommerlandt, Frank M. J. and Spaethe, Johannes and R{\"o}ssler, Wolfgang and Dyer, Adrian G.}, title = {Does Fine Color Discrimination Learning in Free-Flying Honeybees Change Mushroom-Body Calyx Neuroarchitecture?}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0164386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147932}, pages = {e0164386}, year = {2016}, abstract = {Honeybees learn color information of rewarding flowers and recall these memories in future decisions. For fine color discrimination, bees require differential conditioning with a concurrent presentation of target and distractor stimuli to form a long-term memory. Here we investigated whether the long-term storage of color information shapes the neural network of microglomeruli in the mushroom body calyces and if this depends on the type of conditioning. Free-flying honeybees were individually trained to a pair of perceptually similar colors in either absolute conditioning towards one of the colors or in differential conditioning with both colors. Subsequently, bees of either conditioning groups were tested in non-rewarded discrimination tests with the two colors. Only bees trained with differential conditioning preferred the previously learned color, whereas bees of the absolute conditioning group, and a stimuli-na{\"i}ve group, chose randomly among color stimuli. All bees were then kept individually for three days in the dark to allow for complete long-term memory formation. Whole-mount immunostaining was subsequently used to quantify variation of microglomeruli number and density in the mushroom-body lip and collar. We found no significant differences among groups in neuropil volumes and total microglomeruli numbers, but learning performance was negatively correlated with microglomeruli density in the absolute conditioning group. Based on these findings we aim to promote future research approaches combining behaviorally relevant color learning tests in honeybees under free-flight conditions with neuroimaging analysis; we also discuss possible limitations of this approach.q}, language = {en} } @article{AnkenbrandWeberBeckeretal.2016, author = {Ankenbrand, Markus J. and Weber, Lorenz and Becker, Dirk and F{\"o}rster, Frank and Bemm, Felix}, title = {TBro: visualization and management of de novo transcriptomes}, series = {Database}, volume = {2016}, journal = {Database}, doi = {10.1093/database/baw146}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147954}, pages = {baw146}, year = {2016}, abstract = {RNA sequencing (RNA-seq) has become a powerful tool to understand molecular mechanisms and/or developmental programs. It provides a fast, reliable and cost-effective method to access sets of expressed elements in a qualitative and quantitative manner. Especially for non-model organisms and in absence of a reference genome, RNA-seq data is used to reconstruct and quantify transcriptomes at the same time. Even SNPs, InDels, and alternative splicing events are predicted directly from the data without having a reference genome at hand. A key challenge, especially for non-computational personnal, is the management of the resulting datasets, consisting of different data types and formats. Here, we present TBro, a flexible de novo transcriptome browser, tackling this challenge. TBro aggregates sequences, their annotation, expression levels as well as differential testing results. It provides an easy-to-use interface to mine the aggregated data and generate publication-ready visualizations. Additionally, it supports users with an intuitive cart system, that helps collecting and analysing biological meaningful sets of transcripts. TBro's modular architecture allows easy extension of its functionalities in the future. Especially, the integration of new data types such as proteomic quantifications or array-based gene expression data is straightforward. Thus, TBro is a fully featured yet flexible transcriptome browser that supports approaching complex biological questions and enhances collaboration of numerous researchers.}, language = {en} } @article{OthmanNaseemAwadetal.2016, author = {Othman, Eman M. and Naseem, Muhammed and Awad, Eman and Dandekar, Thomas and Stopper, Helga}, title = {The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0168386}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147983}, pages = {e0168386}, year = {2016}, abstract = {Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.}, language = {en} } @article{KunzWolfSchulzeetal.2016, author = {Kunz, Meik and Wolf, Beat and Schulze, Harald and Atlan, David and Walles, Thorsten and Walles, Heike and Dandekar, Thomas}, title = {Non-Coding RNAs in Lung Cancer: Contribution of Bioinformatics Analysis to the Development of Non-Invasive Diagnostic Tools}, series = {Genes}, volume = {8}, journal = {Genes}, number = {1}, doi = {10.3390/genes8010008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147990}, pages = {8}, year = {2016}, abstract = {Lung cancer is currently the leading cause of cancer related mortality due to late diagnosis and limited treatment intervention. Non-coding RNAs are not translated into proteins and have emerged as fundamental regulators of gene expression. Recent studies reported that microRNAs and long non-coding RNAs are involved in lung cancer development and progression. Moreover, they appear as new promising non-invasive biomarkers for early lung cancer diagnosis. Here, we highlight their potential as biomarker in lung cancer and present how bioinformatics can contribute to the development of non-invasive diagnostic tools. For this, we discuss several bioinformatics algorithms and software tools for a comprehensive understanding and functional characterization of microRNAs and long non-coding RNAs.}, language = {en} } @article{Kramer2016, author = {Kramer, Susanne}, title = {Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution}, series = {Nucleic Acids Research}, journal = {Nucleic Acids Research}, doi = {10.1093/nar/gkw1245}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148002}, pages = {gkw1245}, year = {2016}, abstract = {The detection of mRNAs undergoing transcription or decay is challenging, because both processes are fast. However, the relative proportion of an mRNA in synthesis or decay increases with mRNA size and decreases with mRNA half-life. Based on this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5΄ and 3΄ ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH probes, respectively. In the resulting fluorescence images, yellow spots represent intact mRNAs; red spots are mRNAs in transcription or 3΄-5΄ decay, and green spots are mRNAs in 5΄-3΄ degradation. Most red spots were nuclear and insensitive to transcriptional inhibition and thus likely transcription intermediates. Most green spots were cytoplasmic, confirming that the majority of cytoplasmic decay in trypanosomes is 5΄-3΄. The system showed the expected changes at inhibition of transcription or translation and RNAi depletion of the trypanosome homologue to the 5΄-3΄ exoribonuclease Xrn1. The method allows to monitor changes in mRNA metabolism both on cellular and on population/tissue wide levels, but also to study the subcellular localization of mRNA transcription and decay pathways. I show that the system is applicable to mammalian cells.}, language = {en} } @article{MildnerRoces2016, author = {Mildner, Stephanie and Roces, Flavio}, title = {Plasticity of Daily Behavioral Rhythms in Foragers and Nurses of the Ant Camponotus rufipes: Influence of Social Context and Feeding Times}, series = {PLoS One}, volume = {12}, journal = {PLoS One}, number = {1}, doi = {10.1371/journal.pone.0169244}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148010}, pages = {e0169244}, year = {2016}, abstract = {Daily activities within an ant colony need precise temporal organization, and an endogenous clock appears to be essential for such timing processes. A clock drives locomotor rhythms in isolated workers in a number of ant species, but its involvement in activities displayed in the social context is unknown. We compared locomotor rhythms in isolated individuals and behavioral rhythms in the social context of workers of the ant Camponotus rufipes. Both forager and nurse workers exhibited circadian rhythms in locomotor activity under constant conditions, indicating the involvement of an endogenous clock. Activity was mostly nocturnal and synchronized with the 12:12h light-dark-cycle. To evaluate whether rhythmicity was maintained in the social context and could be synchronized with non-photic zeitgebers such as feeding times, daily behavioral activities of single workers inside and outside the nest were quantified continuously over 24 hours in 1656 hours of video recordings. Food availability was limited to a short time window either at day or at night, thus mimicking natural conditions of temporally restricted food access. Most foragers showed circadian foraging behavior synchronized with food availability, either at day or nighttime. When isolated thereafter in single locomotor activity monitors, foragers mainly displayed arrhythmicity. Here, high mortality suggested potential stressful effects of the former restriction of food availability. In contrast, nurse workers showed high overall activity levels in the social context and performed their tasks all around the clock with no circadian pattern, likely to meet the needs of the brood. In isolation, the same individuals exhibited in turn strong rhythmic activity and nocturnality. Thus, endogenous activity rhythms were inhibited in the social context, and timing of daily behaviors was flexibly adapted to cope with task demands. As a similar socially-mediated plasticity in circadian rhythms was already shown in honey bees, the temporal organization in C. rufipes and honey bees appear to share similar basic features.}, language = {en} } @article{AdolfiHerpinRegensburgeretal.2016, author = {Adolfi, Mateus C. and Herpin, Amaury and Regensburger, Martina and Sacquegno, Jacopo and Waxman, Joshua S. and Schartl, Manfred}, title = {Retinoic acid and meiosis induction in adult versus embryonic gonads of medaka}, series = {Scientific Reports}, volume = {6}, journal = {Scientific Reports}, doi = {10.1038/srep34281}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147843}, pages = {34281}, year = {2016}, abstract = {In vertebrates, one of the first recognizable sex differences in embryos is the onset of meiosis, known to be regulated by retinoic acid (RA) in mammals. We investigated in medaka a possible meiotic function of RA during the embryonic sex determination (SD) period and in mature gonads. We found RA mediated transcriptional activation in germ cells of both sexes much earlier than the SD stage, however, no such activity during the critical stages of SD. In adults, expression of the RA metabolizing enzymes indicates sexually dimorphic RA levels. In testis, RA acts directly in Sertoli, Leydig and pre-meiotic germ cells. In ovaries, RA transcriptional activity is highest in meiotic oocytes. Our results show that RA plays an important role in meiosis induction and gametogenesis in adult medaka but contrary to common expectations, not for initiating the first meiosis in female germ cells at the SD stage.}, language = {en} } @article{SerenGrimmFitzetal.2016, author = {Seren, {\"U}mit and Grimm, Dominik and Fitz, Joffrey and Weigel, Detlef and Nordborg, Magnus and Borgwardt, Karsten and Korte, Arthur}, title = {AraPheno: a public database for Arabidopsis thaliana phenotypes}, series = {Nucleic Acids Research}, volume = {45}, journal = {Nucleic Acids Research}, number = {D1}, doi = {10.1093/nar/gkw986}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147909}, pages = {D1054-D1059}, year = {2016}, abstract = {Natural genetic variation makes it possible to discover evolutionary changes that have been maintained in a population because they are advantageous. To understand genotype-phenotype relationships and to investigate trait architecture, the existence of both high-resolution genotypic and phenotypic data is necessary. Arabidopsis thaliana is a prime model for these purposes. This herb naturally occurs across much of the Eurasian continent and North America. Thus, it is exposed to a wide range of environmental factors and has been subject to natural selection under distinct conditions. Full genome sequencing data for more than 1000 different natural inbred lines are available, and this has encouraged the distributed generation of many types of phenotypic data. To leverage these data for meta analyses, AraPheno (https://arapheno.1001genomes.org) provide a central repository of population-scale phenotypes for A. thaliana inbred lines. AraPheno includes various features to easily access, download and visualize the phenotypic data. This will facilitate a comparative analysis of the many different types of phenotypic data, which is the base to further enhance our understanding of the genotype-phenotype map.}, language = {en} } @article{BargulJungMcOdimbaetal.2016, author = {Bargul, Joel L. and Jung, Jamin and McOdimba, Francis A. and Omogo, Collins O. and Adung'a, Vincent O. and Kr{\"u}ger, Timothy and Masiga, Daniel K. and Engstler, Markus}, title = {Species-Specific Adaptations of Trypanosome Morphology and Motility to the Mammalian Host}, series = {PLoS Pathogens}, volume = {12}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1005448}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146513}, pages = {e1005448}, year = {2016}, abstract = {African trypanosomes thrive in the bloodstream and tissue spaces of a wide range of mammalian hosts. Infections of cattle cause an enormous socio-economic burden in sub-Saharan Africa. A hallmark of the trypanosome lifestyle is the flagellate's incessant motion. This work details the cell motility behavior of the four livestock-parasites Trypanosoma vivax, T. brucei, T. evansi and T. congolense. The trypanosomes feature distinct swimming patterns, speeds and flagellar wave frequencies, although the basic mechanism of flagellar propulsion is conserved, as is shown by extended single flagellar beat analyses. Three-dimensional analyses of the trypanosomes expose a high degree of dynamic pleomorphism, typified by the 'cellular waveform'. This is a product of the flagellar oscillation, the chirality of the flagellum attachment and the stiffness of the trypanosome cell body. The waveforms are characteristic for each trypanosome species and are influenced by changes of the microenvironment, such as differences in viscosity and the presence of confining obstacles. The distinct cellular waveforms may be reflective of the actual anatomical niches the parasites populate within their mammalian host. T. vivax displays waveforms optimally aligned to the topology of the bloodstream, while the two subspecies T. brucei and T. evansi feature distinct cellular waveforms, both additionally adapted to motion in more confined environments such as tissue spaces. T. congolense reveals a small and stiff waveform, which makes these parasites weak swimmers and destined for cell adherence in low flow areas of the circulation. Thus, our experiments show that the differential dissemination and annidation of trypanosomes in their mammalian hosts may depend on the distinct swimming capabilities of the parasites.}, language = {en} } @article{RosenbaumSchickWollbornetal.2016, author = {Rosenbaum, Corinna and Schick, Martin Alexander and Wollborn, Jakob and Heider, Andreas and Scholz, Claus-J{\"u}rgen and Cecil, Alexander and Niesler, Beate and Hirrlinger, Johannes and Walles, Heike and Metzger, Marco}, title = {Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {3}, doi = {10.1371/journal.pone.0151335}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146544}, pages = {e0151335}, year = {2016}, abstract = {Background Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. Methods and Results In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. Conclusion and Significance Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.}, language = {en} } @phdthesis{Auer2021, author = {Auer, Daniela}, title = {Impact of the chlamydial deubiquitinase ChlaDUB1 on host cell defense}, doi = {10.25972/OPUS-17846}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178462}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The human pathogen Chlamydia trachomatis is the main cause of sexually transmitted infections worldwide. The obligate intracellular bacteria are the causative agent of several diseases that reach from conjunctivitis causing trachoma and blindness as well as salpingitis and urethritis which can lead to infertility if left untreated. In order to gain genetically engineered Chlamydia that inducible knock down specific gene expression, the CRISPRi system was established in C. trachomatis. In a proof of principle experiment it was shown that C. trachomatis pCRISPRi:gCdu1III target ChlaDUB1 expression and reduce the protein amount up to 50 \%. Knock-down of the DUB did not influence protein levels of anti-apoptotic Mcl-1 and did not make cells susceptible for apoptosis. However, reduced dCas9 protein size, bacterial growth impairment and off target effects interfering with the GFP signal, form obstacles in CRISPRi system in Chlamydia. For routinely use of the CRISPRi method in C. trachomatis further investigation is needed. Since the bacterial life cycle includes two morphological and functional distinct forms, it is essential for chlamydial spread to complete the development cycle and form infectious progeny. Therefore, Chlamydia has evolved strategies to evade the host immune system in order to stay undetected throughout the developmental cycle. The bacteria prevent host cell apoptosis via stabilization of anti-apoptotic proteins like Mcl-1, Survivin and HIF-1α and activate pro-survival pathways, inhibiting invasion of immune cells to the site of infection. The host cell itself can destroy intruders via cell specific defense systems that involve autophagy and recruitment of professional immune cells. In this thesis the role of the chlamydial deubiuqitinase ChlaDUB1 upon immune evasion was elucidated. With the mutant strain Ctr Tn-cdu1 that encodes for a truncated DUB due to transposon insertion, it was possible to identify ChlaDUB1 as a potent opponent of the autophagic system. Mutant inclusions were targeted by K48 and K63 chain ubiquitination. Subsequently the inclusion was recognized by autophagic receptors like p62, NBR1 and NDP52 that was reversed again by complementation with the active DUB. Xenophagy was promoted so far as LC3 positive phagosomes formed around the inclusion of Ctr Tn-cdu1, which did not fuse with the lysosome. The detected growth defect in human primary cells of Chlamydia missing the active DUB was not traced back to autophagy, but was due to impaired development and replication. It was possible to identify Ankib1, the E3 ligase, that ubiquitinates the chlamydial inclusion in a siRNA based screen. The activating enzyme Ube1 and the conjugating enzyme Ube2L3 are also essential in this process. Chlamydia have a reduced genome and depend on lipids and nutrients that are translocated from the host cell to the inclusion to proliferate. Recruitment of fragmented Golgi stacks to the inclusion surface was prevented when ChlaDUB1 was inactive, probably causing diminished bacterial growth. Additionally, the modification of the inclusion by Ankib1 and subsequent decoration by autophagic markers was not only present in human but also murine cells. Comparison of other Chlamydia strains and species revealed Ankib1 to be located at the proximity of the inclusion in C. trachomatis strains only but not in C. muridarum or C. pneumoniae, indicating that Ankib1 is specifically the E3 ligase of C. trachomatis. Moreover, the role of ChlaDUB1 in infected tissue was of interest, since ChlaDUB1 protein was also found in early EB stage and so might get in contact with invading immune cells after cell lysis. While bacteria spread and infect new host cells, Chlamydia can also infect immune cells. Infection of human neutrophils with Ctr Tn-cdu1 shows less bacterial survival and affirms the importance of the DUB for bacterial fitness in these cells.}, subject = {Chlamydia}, language = {en} } @article{ChenReiherHermannLuibletal.2016, author = {Chen, Jiangtian and Reiher, Wencke and Hermann-Luibl, Christiane and Sellami, Azza and Cognigni, Paola and Kondo, Shu and Helfrich-F{\"o}rster, Charlotte and Veenstra, Jan A. and Wegener, Christian}, title = {Allatostatin A Signalling in Drosophila Regulates Feeding and Sleep and Is Modulated by PDF}, series = {PLoS Genetics}, volume = {12}, journal = {PLoS Genetics}, number = {9}, doi = {10.1371/journal.pgen.1006346}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178170}, year = {2016}, abstract = {Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.}, language = {en} } @article{DjuzenovaFiedlerKatzeretal.2016, author = {Djuzenova, Cholpon S. and Fiedler, Vanessa and Katzer, Astrid and Michel, Konstanze and Deckert, Stefanie and Zimmermann, Heiko and Sukhorukov, Vladimir L. and Flentje, Michael}, title = {Dual PI3K-and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule}, series = {Oncotarget}, volume = {7}, journal = {Oncotarget}, number = {25}, doi = {10.18632/oncotarget.9501}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177770}, pages = {38191-38209}, year = {2016}, abstract = {Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.}, language = {en} } @phdthesis{CastilloCajas2020, author = {Castillo Cajas, Ruth}, title = {Evolution and diversity of cuticular hydrocarbon profiles of cuckoo wasps}, doi = {10.25972/OPUS-17341}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173418}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2020}, abstract = {Cuticular hydrocarbons (CHC) abound on the surface of arthropods. In spite of their simple structure (molecules of carbon and hydrogen atoms), they provide pivotal functions in insects: their hydrophobic properties confer the insects a means to regulate water balance and avoid desiccation, whereas their diversity has enhanced their use as signals and cues in a wide range of communication and recognition processes. Although the study of CHC in insects over the past two decades has provided great insight into the wide range of functions they play, there is still a gap in understanding how they diversify and evolve. In this thesis, I have used members of the family Chrysididae to explore patterns of diversification of CHC. Most of the species of cuckoo wasps in this study are specialized parasitoids or kleptoparasites of mainly solitary hymenopteran hosts. Other hosts of the family include butterflies or stick insects. Cuckoo wasps are a particular interesting model to study the evolution of cuticular hydrocarbons because of their chemical adaptations that allow them to remain unrecognized by their hosts. Chemical insignificance (the reduction of the total amount of CHC on the cuticle) and chemical mimicry (the de novo production of CHC profiles resembling those of their female host) have been described in some representatives of the family and unpublished evidence suggests chemical deception is widespread in Chrysididae (Chapter 2). Nonetheless, to trace the evolution of any trait of interest, a reliable phylogenetic reconstruction of the family is required. Therefore, the first study of this thesis constitutes the largest and to-date most reliable phylogenetic reconstruction of the family Chrysididae, which includes representatives of 186 species of cuckoo wasps. While the results of this phylogenetic reconstruction are consistent with previous ideas on the relationships of subfamilies and tribes, it shows the existence of several non-monophyletic genera (Chapter 3). CHC are involved in intraspecific recognition, often acting as contact sex pheromones. Nevertheless, it is not yet understood to what extent CHC profiles differ between the two sexes and whether some compound classes are more prevalent in one or the other sex. So far, no comparison of CHC profiles of males and females has been done for more than a dozen of related species. In Chapter 4, I describe and compare CHC profiles of females and males of 58 species of cuckoo wasps in order to evaluate whether and to what extent CHC profiles of these species differ between the sexes. I demonstrated that CHC profiles of cuckoo wasps are frequently (more than 90\% of the species analyzed) and strongly dimorphic (both sexes of a given species tend to produce very different CHC compounds). Methyl-branched compounds tend to be more prevalent in males (especially dimethyl-branched compounds) and unsaturated compounds prevail in females. Moreover, a sex-specific pattern in the distribution of the double bond position of alkenes was evident: internal double bond positions (> 11) occur predominantly in males, whereas alkenes with the doubl{\´e} bond at position 9 were more abundant and frequent in females (Chapter4). In Chapter5, I investigated how CHC profiles of cuckoo wasps differ across species. Are CHC profiles of cuckoo wasps species-specific, enabling their use as cues for species recognition? How do CHC profiles resemble phylogenetic relatedness? In Chapter 5, I try to answer these questions by comparing CHC profiles of 59 species of cuckoo wasps. CHC profiles of cuckoo wasps are shown to be species (and sex-) specific. I show that CHC profiles are useful as a complementary tool to help delimiting taxonomically difficult sibling species. Moreover, the evaluation of CHC profiles of five commonly occurring species within a genus, showed little or no geographical variation. However, CHC profiles of closely related species may differ strongly among each other, not being useful to track the evolutionary history of species (Chapter 5). Sexual selection is generally credited for generating striking sexual dimorphism by causing changes in male traits. Most often, sexual selection has a stronger effect on males, who compete for access to and may be selected by females, thus male traits may rapidly evolve. Nevertheless, in cuckoo wasps, it appears that it is the female sex the one evolving faster changes, with females of very closely related species showing extremely divergent profiles. One plausible reason for this disparity is that natural selection acting on female's CHC profiles may be stronger than sexual selection on males (Chapter 6). Since females of cuckoo wasps are most probably engaged in an evolutionary arms race with their female hosts, CHC profiles of female cuckoo wasps are likely rapidly evolving, thus explaining part of the strong observed sexual dimorphism of CHC (Chapter 6). In fact, Chapter 7 shows evidence of a possible ongoing evolutionary arms race between five cuckoo wasps of the genus Hedychrum and their hosts. Hedychrum species parasitize either Coleoptera-hunting or Hymenoptera-hunting digger wasps. Since the coleopteran prey of the former digger wasps is naturally better protected against fungus infestation, these wasps do not embalm their prey with alkene-enriched secretions as do the Hymenoptera-hunting digger wasps. Thus, Coleoptera-hunting digger wasps can apparently diversify their profiles to escape chemical mimicry. Interestingly, only female cuckoo wasps of these hosts have started producing the same compound classes and even the same CHC compounds as those of their hosts. Male cuckoo wasps, however retain an alkene-enriched CHC profile that reflects the molecular phylogeny of the genus (Chapter 7). Whereas, a larger number of parasite-host comparisons may be needed to further conclude that an arms race between cuckoo wasps and their hosts is capable of generating sexual dimorphism of cuckoo wasps, this thesis constitutes the first effort towards this, providing a starting point for further studies. Finally, I provide some methodological tools that may help in speeding up the sometimes cumbersome process of analyzing and identifying CHC profiles. One of the most time-demanding steps in the processing of CHC data is the alignment of CHC chromatograms. This process is often done manually, because alignment programs are mostly designed for metabolomics or are just recently being developed. I analyzed CHC profiles using a combined approach with two freely available programs. I used AMDIS (Automated Mass Spectral Deconvolution and Identification System, http://chemdata.nist.gov/mass-spc/amdis/) to deconvolute and automatically identify all CHC of interest present in a chromatogram. I then developed a series of R scripts to correct for potential, unavoidable errors while processing CHC chromatograms with AMDIS. Chapter 8 explains this procedure. In the next chapter, I developed a program that helps in the identification of one commonly occurring class of hydrocarbons. The limited number of linear alkanes (only one per carbon atom) and their characteristic diagnostic ion allows a rapid and unambigous identification of these substances. In opposition, unsaturated and methyl-branched compounds are more difficult to identify, as a result of the much larger diversity of existing compounds. To identify unsaturated compounds a derivatization is necessary to determine the position of the double bond. Methyl-branched alkanes, however can be identified from the original chromatogram if their diagnostic ions are known. Nonetheless, polymethyl-branched alkanes (e.g., compounds with two or more methyl groups along the chain) are often difficult to identify, because they may appear in mixes (e.g., 3,7 diMeC27 and 3,9 diMeC27), and tables containing the diagnostic ions are not easily available. Therefore, I developed a program that creates a table with all possiblemethyl-branched compounds containing up to 4 methyl groups, and that provides their diagnostic ions and a calculated retention index. This may allow a much faster identification of the methyl-branched compound a researcher is dealing with, without having to lose time in the tedious calculations by hand. The program is able to correctly identify, or at least, greatly reduce the number of possible options for the identification of an unknown methyl-branched compound. Thus, using this tool, most methyl-branched compounds can be readily identified (Chapter 9). This thesis ends with a general discussion (Chapter 10). Overall, this work provides a comprehensive overview of the diversity of cuticular hydrocarbons of cuckoo wasps. The analyses presented here shed light on the emergence and evolution of interspecific diversity and intraspecific sexual dimorphism of CHC profiles. In addition, two technical methods have been developed that could greatly facilitate the CHC analysis of insects.}, language = {en} } @phdthesis{Kuehl2022, author = {K{\"u}hl, Julia}, title = {FAAP100, der FA/BRCA-Signalweg f{\"u}r genomische Stabilit{\"a}t und das DNA-Reparatur-Netzwerk}, doi = {10.25972/OPUS-17166}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171669}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Die Fanconi-An{\"a}mie (FA) ist eine seltene, heterogene Erbkrankheit. Sie weist ein sehr variables klinisches Erscheinungsbild auf, das sich aus angeborenen Fehlbildungen, h{\"a}matologischen Funktionsst{\"o}rungen, einem erh{\"o}hten Risiko f{\"u}r Tumorentwicklung und endokrinen Pathologien zusammensetzt. Die Erkrankung z{\"a}hlt zu den genomischen Instabilit{\"a}tssyndromen, welche durch eine fehlerhafte DNA-Schadensreparatur gekennzeichnet sind. Bei der FA zeigt sich dies vor allem in einer charakteristischen Hypersensitivit{\"a}t gegen{\"u}ber DNA-quervernetzenden Substanzen (z. B. Mitomycin C, Cisplatin). Der zellul{\"a}re FA-Ph{\"a}notyp zeichnet sich durch eine erh{\"o}hte Chromosomenbr{\"u}chigkeit und einen Zellzyklusarrest in der G2-Phase aus. Diese Charakteristika sind bereits spontan vorhanden und werden durch Induktion mit DNA-quervernetzenden Substanzen verst{\"a}rkt. Der Gendefekt ist dabei in einem der 22 bekannten FA-Gene (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U, -V, -W) oder in noch unbekannten FA-Genen zu finden. Die FA-Gendefekte werden mit Ausnahme von FANCR (dominant-negative de novo Mutationen) und FANCB (X-chromosomal) autosomal rezessiv vererbt. Die FA-Genprodukte bilden zusammen mit weiteren Proteinen den FA/BRCA-Signalweg. Das Schl{\"u}sselereignis dieses Signalwegs stellt die Monoubiquitinierung von FANCD2 und FANCI (ID2-Komplex) dar. Ausgehend davon l{\"a}sst sich zwischen upstream- und downstream-gelegenen FA-Proteinen unterscheiden. Letztere sind direkt an der DNA-Schadensreparatur beteiligt. Zu den upstream-gelegenen Proteinen z{\"a}hlt der FA-Kernkomplex, der sich aus bekannten FA-Proteinen und aus FA-assoziierten-Proteinen (FAAPs) zusammensetzt und f{\"u}r die Monoubiquitinierung des ID2-Komplexes verantwortlich ist. F{\"u}r FAAPs wurden bisher keine pathogenen humanen Mutationen beschrieben. Zu diesen Proteinen geh{\"o}rt auch FAAP100, das mit FANCB und FANCL innerhalb des FA-Kernkomplexes den Subkomplex LBP100 bildet. Durch die vorliegende Arbeit wurde eine n{\"a}here Charakterisierung dieses Proteins erreicht. In einer Amnion-Zelllinie konnte eine homozygote Missense-Mutation identifiziert werden. Der Fetus zeigte einen typischen FA-Ph{\"a}notyp und auch seine Zellen wiesen charakteristische FA-Merkmale auf. Der zellul{\"a}re Ph{\"a}notyp ließ sich durch FAAP100WT komplementieren, sodass die Pathogenit{\"a}t der Mutation bewiesen war. Unterst{\"u}tzend dazu wurden mithilfe des CRISPR/Cas9-Systems weitere FAAP100-defiziente Zelllinien generiert. Diese zeigten ebenfalls einen typischen FA-Ph{\"a}notyp, welcher sich durch FAAP100WT komplementieren ließ. Die in vitro-Modelle dienten als Grundlage daf{\"u}r, die Funktion des FA-Kernkomplexes im Allgemeinen und die des Subkomplexes LBP100 im Besonderen besser zu verstehen. Dabei kann nur durch intaktes FAAP100 das LBP100-Modul gebildet und dieses an die DNA-Schadensstelle transportiert werden. Dort leistet FAAP100 einen essentiellen Beitrag f{\"u}r den FANCD2-Monoubiquitinierungsprozess und somit f{\"u}r die Aktivierung der FA-abh{\"a}ngigen DNA-Schadensreparatur. Um die Funktion von FAAP100 auch in vivo zu untersuchen, wurde ein Faap100-/--Mausmodell generiert, das einen mit anderen FA-Mausmodellen vergleichbaren, relativ schweren FA-Ph{\"a}notyp aufwies. Aufgrund der Ergebnisse l{\"a}sst sich FAAP100 als neues FA-Gen klassifizieren. Zudem wurde die Rolle des Subkomplexes LBP100 innerhalb des FA-Kernkomplexes weiter aufgekl{\"a}rt. Beides tr{\"a}gt zu einem besseren Verst{\"a}ndnis des FA/BRCA-Signalweges bei. Ein weiterer Teil der vorliegenden Arbeit besch{\"a}ftigt sich mit der Charakterisierung von FAAP100138, einer bisher nicht validierten Isoform von FAAP100. Durch dieses Protein konnte der zellul{\"a}re FA-Ph{\"a}notyp von FAAP100-defizienten Zelllinien nicht komplementiert werden, jedoch wurden Hinweise auf einen dominant-negativen Effekt von FAAP100138 auf den FA/BRCA-Signalweg gefunden. Dies k{\"o}nnte zu der Erkl{\"a}rung beitragen, warum und wie der Signalweg, beispielsweise in bestimmtem Gewebearten, herunterreguliert wird. Zudem w{\"a}re eine Verwendung in der Krebstherapie denkbar.}, subject = {Fanconi-An{\"a}mie}, language = {de} } @phdthesis{Njovu2019, author = {Njovu, Henry Kenneth}, title = {Patterns and drivers of herbivore diversity and invertebrate herbivory along elevational and land use gradients at Mt. Kilimanjaro, Tanzania}, doi = {10.25972/OPUS-17254}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172544}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {This thesis elucidates patterns and drivers of invertebrate herbivory, herbivore diversity, and community-level biomass along elevational and land use gradients at Mt. Kilimanjaro, Tanzania. Chapter I provides background information on the response and predictor variables, study system, and the study design. First, I give an overview of the elevational patterns of species diversity/richness and herbivory published in the literature. The overview illuminates existing debates on elevational patterns of species diversity/richness and herbivory. In connection to these patterns, I also introduce several hypotheses and mechanisms put forward to explain macroecological patterns of species richness. Furthermore, I explain the main variables used to test hypotheses. Finally, I describe the study system and the study design used. Chapter II explores the patterns of invertebrate herbivory and their underlying drivers along extensive elevational and land use gradients on the southern slopes of Mt. Kilimanjaro. I recorded standing leaf herbivory from leaf chewers, leaf miners and gall-inducing insects on 55 study sites located in natural and anthropogenic habitats distributed from 866 to 3060 meters above sea level (m asl) on Mt. Kilimanjaro. Standing leaf herbivory was related to climatic variables [mean annual temperature - (MAT) and mean annual precipitation - (MAP)], net primary productivity (NPP) and plant functional traits (leaf traits) [specific leaf area (SLA), carbon to nitrogen ratio (CN), and nitrogen to phosphorous ratio (NP)]. Results revealed an unimodal pattern of total leaf herbivory along the elevation gradient in natural habitats. Findings also revealed differences in the levels and patterns of herbivory among feeding guilds and between anthropogenic and natural habitats. Changes in NP and CN ratios which were closely linked to NPP were the strongest predictors of leaf herbivory. Our study uncovers the role of leaf nutrient stoichiometry and its linkages to climate in explaining the variation in leaf herbivory along climatic gradients. Chapter III presents patterns and unravels direct and indirect effects of resource (food) abundance (NPP), resource (food) diversity [Functional Dispersion (FDis)], resource quality (SLA, NP, and CN rations), and climate variables (MAT and MAP) on species diversity of phytophagous beetles. Data were collected from 65 study sites located in natural and anthropogenic habitats distributed from 866 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Sweep net and beating methods were used to collect a total of 3,186 phytophagous beetles representing 21 families and 304 morphospecies. Two groups, weevils (Curculionidae) and leaf beetles (Chrysomelidae) were the largest and most diverse families represented with 898 and 1566 individuals, respectively. Results revealed complex (bimodal) and dissimilar patterns of Chao1-estimated species richness (hereafter referred to as species diversity) along elevation and land use gradients. Results from path analysis showed that temperature and climate-mediated changes in NPP had a significant positive direct and indirect effect on species diversity of phytophagous beetles, respectively. The results also revealed that the effect of NPP (via beetles abundance and diversity of food resources) on species diversity is stronger than that of temperature. Since we found that factors affecting species diversity were intimately linked to climate, I concluded that predicted climatic changes over the coming decades will likely alter the species diversity patterns which we observe today. Chapter IV presents patterns and unravels the direct and indirect effects of climate, NPP and anthropogenic disturbances on species richness and community-level biomass of wild large mammals which represent endothermic organisms and the most important group of vertebrate herbivores. Data were collected from 66 study sites located in natural and anthropogenic habitats distributed from 870 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Mammals were collected using camera traps and used path analysis to disentangle the direct and indirect effects of climatic variables, NPP, land use, land area, levels of habitat protection and occurrence of domesticated mammals on the patterns of richness and community-level biomass of wild mammals, respectively. Results showed unimodal patterns for species richness and community-level biomass of wild mammals along elevation gradients and that the patterns differed depending on the type of feeding guild. Findings from path analysis showed that net primary productivity and levels of habitat protection had a strong direct effect on species richness and community-level biomass of wild mammals whereas temperature had an insignificant direct effect. Findings show the importance of climate-mediated food resources in determining patterns of species richness of large mammals. While temperature is among key predictors of species richness in several ectotherms, its direct influence in determining species richness of wild mammals was insignificant. Findings show the sensitivity of wild mammals to anthropogenic influences and underscore the importance of protected areas in conserving biodiversity. In conclusion, despite a multitude of data sets on species diversity and ecosystem functions along broad climatic gradients, there is little mechanistic understanding of the underlying causes. Findings obtained in the three studies illustrate their contribution to the scientific debates on the mechanisms underlying patterns of herbivory and diversity along elevation gradients. Results present strong evidence that plant functional traits play a key role in determining invertebrate herbivory and species diversity along elevation gradients and that, their strong interdependence with climate and anthropogenic activities will shape these patterns in future. Additionally, findings from path analysis demonstrated that herbivore diversity, community-level biomass, and herbivory are strongly influenced by climate (either directly or indirectly). Therefore, the predicted climatic changes are expected to dictate ecological patterns, biotic interactions, and energy and nutrient fluxes in terrestrial ecosystems in the coming decades with stronger impacts probably occurring in natural ecosystems. Furthermore, findings demonstrated the significance of land use effects in shaping ecological patterns. As anthropogenic pressure is advancing towards more pristine higher elevations, I advocate conservation measures which are responsive to and incorporate human dimensions to curb the situation. Although our findings emanate from observational studies which have to take several confounding factors into account, we have managed to demonstrate global change responses in real ecosystems and fully established organisms with a wide range of interactions which are unlikely to be captured in artificial experiments. Nonetheless, I recommend additional experimental studies addressing the effect of top-down control by natural enemies on herbivore diversity and invertebrate herbivory in order to deepen our understanding of the mechanisms driving macroecological patterns along elevation gradients.  }, subject = {Species richness}, language = {en} } @article{LukešGlatzovaKvičalovaetal.2017, author = {Lukeš, Tom{\´a}š and Glatzov{\´a}, Daniela and Kv{\´i}čalov{\´a}, Zuzana and Levet, Florian and Benda, Aleš and Letschert, Sebastian and Sauer, Markus and Brdička, Tom{\´a}š and Lasser, Theo and Cebecauer, Marek}, title = {Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, doi = {10.1038/s41467-017-01857-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172993}, year = {2017}, abstract = {Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.}, language = {en} } @article{EndresKneitzOrthetal.2016, author = {Endres, Marcel and Kneitz, Susanne and Orth, Martin F. and Perera, Ruwan K. and Zernecke, Alma and Butt, Elke}, title = {Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1)}, series = {Oncotarget}, volume = {7}, journal = {Oncotarget}, number = {39}, doi = {10.18632/oncotarget.11720}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176920}, pages = {64244-64259}, year = {2016}, abstract = {The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines. By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines. In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown. Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression. The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies.}, language = {en} } @phdthesis{Burgert2018, author = {Burgert, Anne}, title = {Untersuchung von Sphingolipiden und anderen Membrankonjugaten mittels hochaufl{\"o}sender Fluoreszenzmikroskopie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145725}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Methoden der Fluoreszenz-Lokalisationsmikroskopie (engl. single-molecule localization microscopy, SMLM) erm{\"o}glichen es Molek{\"u}le zu quantifizieren und deren Verteilung zu analysieren. Im Rahmen dieser Arbeit wurden verschiedene Membranmolek{\"u}le auf unterschiedlichen eukaryotischen Zellen, aber auch auf Prokaryoten mit dSTORM (engl. direct stochastic optical reconstruction microscopy) oder PALM (engl.: photoactivated localization microscopy) aufgenommen und quantifiziert. Bevor jedoch diese hochaufl{\"o}sende fluoreszenzbasierte Technik f{\"u}r biologische Fragestellungen angewendet werden konnten, mussten zun{\"a}chst potentielle Artefakt-ausl{\"o}sende Quellen identifiziert und Strategien gefunden werden, um diese zu eliminieren. Eine m{\"o}gliche Artefakt-Quelle ist eine zu niedrige Photonenzahl, die von Fluorophoren emittiert wird. Werden zu wenige Photonen detektiert, kann die Lokalisation eines Fluorophors weniger pr{\"a}zise bestimmt werden. Dies kann zu einer falschen Abbildung von Strukturen f{\"u}hren oder zu falschen R{\"u}ckschl{\"u}ssen {\"u}ber die Verteilung von Molek{\"u}len. Eine M{\"o}glichkeit die Anzahl der emittierten Photonen zu erh{\"o}hen, ist chemische Additive als Triplettl{\"o}scher einzusetzen. Sie bewirken, dass die Fluorophore wieder in den Grundzustand relaxieren und somit wieder angeregt werden k{\"o}nnen. Es wurden verschiedene Additive, die in der Literatur als Triplettl{\"o}scher beschrieben sind, getestet. Dazu wurden zun{\"a}chst ihre Auswirkungen auf den Triplettzustand verschiedener Fluorophore (Alexa Fluor (Al) 488, 532 und 647 und Atto655) mit Hilfe von Fluoreszenzkorrelationsspektroskopie (FCS) untersucht. Cyclooctatetraen (COT) bewirkte dabei eine Abnahme der Triplettausbeute von Al488, Al532 und Al647 um ~ 40-60\%, bei Atto655 ver{\"a}nderte sie sich nicht. Obwohl die Ergebnisse der FCS-Messungen darauf hindeuten, dass COT in einer erh{\"o}hten Anzahl an emittierten Photonen resultiert, konnte dies bei dSTORM-Messungen nicht best{\"a}tigt werden. Hier hatte COT nur einen gr{\"o}ßeren positiven Effekt auf das Fluorophor Al647 (Zunahme um ~ 60\%). Eine Erkl{\"a}rung f{\"u}r diese Widerspr{\"u}chlichkeit zu den Ergebnissen aus den FCS-Messungen, k{\"o}nnte das Vorhandensein des Schaltpuffers bei dSTORM-Messungen sein. Dieser bewirkt den {\"U}bergang der Fluorophore in den Aus-Zustand bzw. entzieht dem Puffer Sauerstoff. Bei der Zugabe von 5 mM Kaliumiodid (KI) nahm die Triplettamplitude bei FCS-Messungen nur bei Al488 ab (um ~ 80\%). Eine geringe Steigerung (um ~ 10\%) der Intensit{\"a}t von Al488 mit KI konnte bei dSTORM-Messungen mit niedrigen Konzentrationen (~ 0,5 mM) erzielt werden. Bei einer Konzentration von 5 mM sank die Intensit{\"a}t jedoch wieder um 40\%. Deuteriumoxid (D2O) soll, anders als die Triplettl{\"o}scher, eine Verbesserung der Photonenausbeute dadurch bewirken, dass strahlungslose Relaxationsprozesse minimiert werden. Mit dSTORM-Messungen konnte gezeigt werden, dass Atto655 und Al647 in D2O zwar pro An-Zustand mehr Photonen emittieren als in Schaltpuffer ohne D2O, da die Fluorophore hier jedoch schneller bleichen, letztendlich die gleiche Anzahl an Photonen detektiert werden. Um die Anzahl an emittierten Photonen zu erh{\"o}hen, eignet sich also nur COT bei dSTORM-Messungen mit AL647 und KI in sehr geringen Konzentrationen bei Al488. D2O kann eingesetzt werden, wenn eine Probe schnell vermessen werden muss, wie zum Beispiel bei Lebendzellmessungen. Nicht nur eine zu niedrige Photonenzahl, auch eine zu geringe Photoschaltrate kann Artefakte bei dSTORM-Messungen erzeugen. Dies wurde anhand von verschiedenen biologischen Strukturen, die mit unterschiedlichen Anregungsintensit{\"a}ten aufgenommen wurden, deutlich gemacht. Besonders die Aufnahmen von Plasmamembranen sind anf{\"a}llig f{\"u}r die Generierung von Artefakten. Sie weisen viele inhomogene und lokal dichte Regionen auf. Wenn nun mehr als ein Emitter pro µm² gleichzeitig an ist, erzeugt das Auswertungsprogramm große artifizielle Cluster. Die hier durchgef{\"u}hrten Messungen machen deutlich, wie wichtig es ist, dSTORM-Bilder immer auf m{\"o}gliche Artefakte hin zu untersuchen, besonders wenn Molek{\"u}le quantifiziert werden sollen. Daf{\"u}r m{\"u}ssen die unbearbeiteten Rohdaten sorgf{\"a}ltig gesichtet werden und notfalls die Messungen mit einer h{\"o}heren Laserleistung wiederholt werden. Da dSTORM mittlerweile immer mehr zur Quantifizierung eingesetzt wird und Clusteranalysen durchgef{\"u}hrt werden, w{\"a}re es sinnvoll bei Ver{\"o}ffentlichungen die Rohdaten von entscheidenden Aufnahmen der {\"O}ffentlichkeit zur Verf{\"u}gung zu stellen. Die F{\"a}rbemethode ist ein weiterer Punkt, durch den Artefakte bei der Abbildung von Molek{\"u}len mittels SMLM entstehen k{\"o}nnen. H{\"a}ufig werden Antik{\"o}rper zum Markieren verwendet. Dabei sollte darauf geachtet werden, dass m{\"o}glichst kleine Antik{\"o}rper oder Antik{\"o}rperfragmente verwendet werden, besonders wenn Clusteranalysen durchgef{\"u}hrt werden sollen. Anderenfalls leidet die Aufl{\"o}sung darunter, bzw. erh{\"o}ht sich die Gefahr der Kreuzvernetzung von Molek{\"u}len. Im zweiten Teil der vorliegenden Arbeit, wurden Plasmamembran-Ceramide untersucht. Ceramide geh{\"o}ren zu den Sphingolipiden und regulieren diverse zellul{\"a}re Prozesse. Verschiedene Stimuli bewirken eine Aktivierung von Sphingomyelinasen (SMasen), die Ceramide in der Plasmamembran synthetisieren. Steigt die Konzentration von Ceramiden in der Plasmamembran an, kondensieren diese zu Ceramid-reichen Plattformen (CRPs). Bisher ist noch wenig {\"u}ber die Verteilung der Ceramide und die Gr{\"o}ße der CRPs bekannt. Sie wurden hier {\"u}ber IgG-Antik{\"o}rper in der Plasmamembran von Jurkat-, U2OS-, HBME- und prim{\"a}ren T-Zellen angef{\"a}rbt und erstmals mit dSTORM hochaufgel{\"o}st, um sie dann zu quantifizieren. Unabh{\"a}ngig von der Zelllinie befanden sich 50\% aller Ceramidmolek{\"u}le in ~ 75 nm großen CRPs. Im Mittel bestanden die CRPs aus ~ 20 Ceramiden. Mit Hilfe einer Titrationsreihe konnte ausgeschlossen werden, dass diese Cluster nur durch die Antik{\"o}rper-F{\"a}rbung artifiziell erzeugt wurden. Bei Inkubation der Zellen mit Bacillus cereus Sphingomyelinase (bSMase) stieg die Gesamtkonzentration der Ceramide in der Plasmamembran an, ebenso wie die Ceramidanzahl innerhalb der CRPs, außerdem die Anzahl und Gr{\"o}ße der CRPs. Dies k{\"o}nnte zu einer Ver{\"a}nderung der L{\"o}slichkeit von Membrankomponenten f{\"u}hren, was wiederum eine Akkumulation bestimmter Rezeptoren oder eine Kompartimentierung bestimmter Proteine erleichtern k{\"o}nnte. Die Anh{\"a}ufung der Ceramide in den CRPs k{\"o}nnte ebenfalls die lokale Interaktion mit anderen Membranmolek{\"u}len erleichtern und dadurch m{\"o}glicherweise die Reaktivit{\"a}t von Rezeptoren ver{\"a}ndern. Mittels Azid-modifizierten Ceramidanaloga und kupferfreier Click-Chemie wurden Plasmamembran-Ceramide auch in lebenden Jurkat-Zellen mit Hilfe konfokaler Laser-Raster-Mikroskopie (CLSM, engl. confocal laser scanning microscopy) und Strukturierter Beleuchtungsmikroskopie (SIM, engl. structured illumination microscopy) untersucht. Dabei konnte gezeigt werden, dass die Fetts{\"a}ure-Kettenl{\"a}nge und die Position des Azids bei den Ceramidanaloga eine entscheidende Rolle spielt, wie hoch das detektierte Signal in der Plasmamembran letztendlich ist. Die Versuche machen auch deutlich, dass die klickbaren Ceramidanaloga lebendzellkompatibel sind, sodass sie eine hervorragende M{\"o}glichkeit darstellen, zellul{\"a}re Reaktionen zu verfolgen. Es wurden hier nicht nur Ceramide in eukaryotischen Zellen analysiert, sondern auch in Bakterien. Neisseria meningitidis (N. meningitidis) sind gramnegative Bakterien, die im Menschen eine Sepsis oder eine Meningitis ausl{\"o}sen k{\"o}nnen. Es wurde mittels immunhistochemischen F{\"a}rbungen mit dem anti-Ceramid IgG-Antik{\"o}rper, aber auch mit den klickbaren Ceramidanaloga, ein Signal in der Membran erhalten, was mit dSTORM hochaufgel{\"o}st wurde. In anderen Bakterien wurden ebenfalls schon Sphingolipide nachgewiesen. Studien zu Ceramiden in N. meningitidis wurden bisher jedoch noch nicht ver{\"o}ffentlicht. Im Rahmen dieser Arbeit konnten erstmals Ergebnisse erhalten werden, die darauf hinweisen, dass N. meningitidis ebenfalls Ceramide besitzen k{\"o}nnten. In einem dritten Projekt wurde die Interaktion zwischen NK-Zellen und Aspergillus fumigatus untersucht. Der Schimmelpilz kann eine Invasive Aspergillose in immunsupprimierten Menschen ausl{\"o}sen, was zum Tod f{\"u}hren kann. Verschiedene Studien konnten schon zeigen, dass NK-Zellen eine wichtige Rolle bei der Bek{\"a}mpfung des Pilzes spielen. Der genaue Mechanismus ist jedoch noch unbekannt. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass der NK-Zell-Marker CD56 entscheidend f{\"u}r die Pilzerkennung ist. Mit immunhistochemischen F{\"a}rbungen und LSM-, aber auch dSTORM-Messungen, konnte gezeigt werden, dass die normalerweise homogen verteilten CD56-Rezeptoren auf der Plasmamembran von NK-Zellen aktiv an die Interaktionsstelle zu A. fumigatus transportiert werden. Mit der Zeit akkumulieren hier immer mehr CD56-Proteine, w{\"a}hrend das Signal in der restlichen Membran immer weiter abnimmt. Es konnte erstmals CD56 als wichtiger Erkennungsrezeptor f{\"u}r A. fumigatus identifiziert werden. In dem letzten bearbeiteten Projekt, wurde die Bindung von Anti-N-Methyl-D-Aspartat (NMDA)-Rezeptor Enzephalitis Autoantik{\"o}rper an Neuronen untersucht. Bei einer Anti-NMDA-Rezeptor Enzephalitis bilden die Patienten Autoantik{\"o}rper gegen die NR1-Untereinheit ihrer eigenen postsynaptischen NMDA-Rezeptoren. Da die Krankheit oft sehr sp{\"a}t erkannt wird und die Behandlungsm{\"o}glichkeiten noch sehr eingeschr{\"a}nkt sind, f{\"u}hrt sie noch oft zum Tod. Sie wurde erst vor wenigen Jahren beschrieben, sodass der genaue Mechanismus noch unbekannt ist. Im Rahmen dieser Arbeit, konnten erste F{\"a}rbungen mit aufgereinigten Antik{\"o}rper aus Anti-NMDA-Rezeptor Enzephalitis Patienten an NMDA-Rezeptor-transfizierte HEK-Zellen und hippocampalen Maus-Neuronen durchgef{\"u}hrt und mit dSTORM hochaufgel{\"o}st werden. Mit den Messungen der HEK-Zellen konnte best{\"a}tigt werden, dass die Autoantik{\"o}rper an die NR1-Untereinheit der Rezeptoren binden. Es konnten erstmals auch die Bindung der Antik{\"o}rper an Neuronen hochaufgel{\"o}st werden. Dabei wurde sichtbar, dass die Antik{\"o}rper zum einen dicht gepackt in den Synapsen vorliegen, aber auch d{\"u}nner verteilt in den extrasynaptischen Regionen. Basierend auf der Ripley's H-Funktion konnten in den Synapsen große Cluster von ~ 90 nm Durchmesser und im Mittel ~ 500 Lokalisationen und extrasynaptisch kleinere Cluster mit einem durchschnittlichen Durchmesser von ~ 70 nm und ~ 100 Lokalisationen ausgemacht werden. Diese ersten Ergebnisse legen den Grundstein f{\"u}r weitere Messungen, mit denen der Mechanismus der Krankheit untersucht werden kann.}, subject = {Ceramide}, language = {de} } @phdthesis{Bucher2018, author = {Bucher, Hannes}, title = {Pre-clinical modeling of viral- and bacterial-induced exacerbations of chronic obstructive pulmonary disease}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144368}, school = {Universit{\"a}t W{\"u}rzburg}, pages = {XIII, 105}, year = {2018}, abstract = {Chronic Obstructive Pulmonary Disease (COPD) exacerbations are a considerable reason for increased morbidity and mortality in patients. Infections with influenza virus (H1N1), respiratory syncytial virus (RSV) or nontypeable Haemophilus influenzae (NTHi) are important triggers of exacerbations. To date, no treatments are available which can stop the progression of COPD. Novel approaches are urgently needed. Pre-clinical models of the disease are crucial for the development of novel therapeutic options. In order to establish pre-clinical models which mimic aspects of human COPD exacerbations, mice were exposed to cigarette smoke (CS) and additionally infected with H1N1, RSV and/or NTHi. Clinically relevant treatments such as the corticosteroids Fluticasone propionate and Dexamethasone, the phosphodiesterase-4 (PDE-4) inhibitor Roflumilast and the long-acting muscarinic receptor antagonist Tiotropium were tested in the established models. Furthermore, a novel treatment approach using antibodies (Abs) directed against IL-1α, IL-1β or IL-1R1 was examined in the established CS/H1N1 model. Levels of IFN-γ, IL-1β, IL-2, IL-6, KC, TNF-α, RANTES, IL-17, MCP-1, MIP 1α and MIP-1β were measured in lung homogenate. Numbers of total cells, neutrophils and macrophages were assessed in bronchoalveolar lavage (BAL) fluid. Hematoxylin- and eosin- (H\&E-) stained lung slices were analyzed to detect pathological changes. Quantitative polymerase-chain-reaction (qPCR) was used to investigate gene expression of ICAM-1 and MUC5 A/C. The viral/bacterial load was investigated in lung homogenate or BAL fluid. In addition to the in vivo studies, the effects of the above mentioned treatments were investigated in vitro in H1N1, RSV or NTHi-infected (primary) human bronchial epithelial cells using submerged or air-liquid-interface (ALI) cell culture systems. Four pre-clinical models (CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi) were established depicting clinically relevant aspects of COPD exacerbations such as increased inflammatory cells and cytokines in the airways and impaired lung function. In the CS/H1N1 model, Tiotropium improved lung function and was superior in reducing inflammation in comparison to Fluticasone or Roflumilast. Moreover, Fluticasone increased the loss of body-weight, levels of IL-6, KC and TNF-α and worsened lung function. In CS/RSV-exposed mice Tiotropium but not Fluticasone or Roflumilast treatment reduced neutrophil numbers and IL-6 and TNF α levels in the lung. The viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after Fluticasone and Dexamethasone treatment. The results from these studies demonstrate that Tiotropium has anti-inflammatory effects on CS/virus-induced inflammation and might help to explain the observed reduction of exacerbation rates in Tiotropium-treated COPD patients. Furthermore, the findings from this work indicate that treatment with Fluticasone or Dexamethasone might not be beneficial to reduce inflammation in the airways of COPD patients and supports clinical studies that link treatment with corticosteroids to an increased risk for pneumonia. Testing of anti-IL-1α, anti-IL-1β or anti-IL-1R1 Abs in the CS/H1N1 model suggests that, in line with clinical data, antagonization of IL-1β is not sufficient to reduce pulmonary inflammation and indicates a predominant role of IL-1α in CS/virus-induced airway inflammation. In line with the in vivo findings, anti-IL-1α but not anti-IL-1β Abs reduced levels of TNF-α and IL-6 in H1N1-infected primary human bronchial epithelial ALI cell culture. Blocking the IL-1R1 provided significant inhibitory effects on inflammatory cells in vivo but was inferior compared to inhibiting both its soluble ligands IL-1α and IL-1β. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1β were significantly reduced and ICAM-1 mRNA expression was attenuated. These results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce inflammation and exacerbations in COPD patients. Moreover, combined targeting of both IL-1α/IL-1β might be more efficient compared to inhibition of the IL-1R1. As in the CS/virus models, corticosteroid treatment failed to reduce inflammatory cells in the CS/NTHi and CS/H1N1/NTHi models, increased the loss of body-weight and the bacterial load. Furthermore, Roflumilast administration had no significant effects on cell counts or cytokines. However, it improved compliance in the CS/NTHi model. Treatment with Azithromycin reduced the bacterial load in the CS/NTHi model and reduced numbers of total cells, neutrophils, macrophages and levels of KC and TNF-α in the CS/H1N1/NTHi model. In conclusion, the established CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi models depict clinically relevant aspects of human COPD exacerbations in mice and provide the opportunity to investigate underlying disease mechanisms and to test novel therapies.}, subject = {Obstruktive Ventilationsst{\"o}rung}, language = {en} } @phdthesis{Letschert2019, author = {Letschert, Sebastian}, title = {Quantitative Analysis of Membrane Components using Super-Resolution Microscopy}, doi = {10.25972/OPUS-16213}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162139}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and -lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60-1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.}, subject = {Fluoreszenzmikroskopie}, language = {en} } @phdthesis{Griffoni2019, author = {Griffoni, Chiara}, title = {Towards advanced immunocompetent skin wound models for in vitro drug evaluation}, doi = {10.25972/OPUS-19212}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192125}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models.}, subject = {Haut}, language = {en} } @phdthesis{daCruzGueerisoli2021, author = {da Cruz G{\"u}erisoli, Irene Maria}, title = {Investigating the murine meiotic telomere complex TERB1-TERB2-MAJIN: spatial organization and evolutionary history}, doi = {10.25972/OPUS-21056}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210562}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {Einess der faszinierenden Merkmale der meiotischen Prophase I sind die hochkonservierten kr{\"a}ftigen Bewegungen homologer Chromosomen. Diese Bewegungen sind entscheidend f{\"u}r den Erfolg von Schl{\"u}sselereignissen wie die Ausrichtung, Paarung und Rekombination der homologen Chromosomen. Mehrere bisher untersuchte Organismen, darunter S{\"a}ugetiere, W{\"u}rmer, Hefen und Pflanzen, erreichen diese Bewegungen, indem sie die Chromosomenenden an spezialisierten Stellen in der Kernh{\"u}lle verankern. Diese Verankerung erfordert Telomer-Adapterproteine, die bisher in der Spalthefe und der Maus identifiziert wurden. Die meiosespezifischen Telomer-Adapterproteine der Maus, TERB1, TERB2 und MAJIN, sind an der Verankerung des ubiquit{\"a}ren Telomer-Shelterin-protein an den LINC-Komplex beteiligt, mit einem analogen Mechanismus, wie er die Spalthefe beschrieben wird. Obgleich die meiose-spezifischen TelomerAdapterproteine eine wesentliche Rolle spielen, ist der genaue Mechanismus der Verankerung der Telomere an die Kernh{\"u}lle sowie ihre evolution{\"a}re Geschichte bisher noch wenig verstanden. Das Hauptziel dieser Arbeit ist daher die Untersuchung der Organisation des meiosespezifischen TelomerAdapterkomplexes TERB1-TERB2-MAJIN der Maus und dessen Evolutionsgeschichte. Im ersten Teil dieser Arbeit wurde die Organisation des TERB1-TERB2-MAJIN Komplexes mittels hochaufl{\"o}sender Mikroskopie (SIM), an Mausspermatozyten untersucht, sowie die Lokalisation in Bezug auf TRF1 des Telomer-ShelterinKomplexes und die telomerische DNA analysiert. In den Stadien Zygot{\"a}n und Pachyt{\"a}n zeigten die Fluoreszenzsignale eine starke {\"U}berlappung der Verteilung der meiotischen Telomer-Komplex-Proteine, wobei die Organisation von TERB2 an den Chromosomenenden heterogener war als die von TERB1 und MAJIN. Außerdem konnte die TRF1-Lokalisation an den Enden der Lateralelemente (LEs) mit einer griffartigen Anordnung um die TERB1- und MAJIN-Signale im Zygot{\"a}n- und Pachyt{\"a}n-Stadium gezeigt werden. Interessanterweise erwies sich die telomerische DNA als lateral verteilt und teilweise {\"u}berlappend mit der zentralen Verteilung der meiotischen Telomer-Komplex-Proteine an den Enden der LEs. Die Kombination dieser Ergebnisse erlaubte die Beschreibung eines alternativen Modells der Verankerung der Telomer an die Kernh{\"u}lle w{\"a}hrend der meiotischen Prophase I. Der zweite Teil dieser Arbeit analysiert die Evolutionsgeschichte der Mausproteine von TERB1, TERB2 und MAJIN. Die fehlende {\"U}bereinstimmung zwischen den Meiose-spezifische Telomer-Adapteproteinen der Maus und der Spalthefe hat die Frage nach dem evolutionsbedingten Ursprung dieses spezifischen Komplexes aufgeworfen. Um vermeintliche Orthologen der Mausproteinevon TERB1, TERB2 und MAJIN {\"u}ber Metazoen hinweg zu identifizieren, wurden computergest{\"u}tzte Verfahren und phylogenetische Analysen durchgef{\"u}hrt. Dar{\"u}ber hinaus wurden Expressionsstudien implementiert, um ihre potenzielle Funktion w{\"a}hrend der Meiose zu testen. Die Analysen haben ergeben, dass der Meiose-spezifische Telomer-Komplex der Maus sehr alt ist, da er bereits in den Eumetazoen entstand, was auf einen einzigen Ursprung hindeutet. Das Fehlen jeglicher Homologen des meiosespezifischen Telomerkomplexes in Nematoden und die einigen wenigen in Arthropoden nachgewiesenen Kandidaten, deuten darauf hin, dass die Telomer-Adapterproteine in diesen Abstammungslinien verloren/ersetzt oder stark diversifiziert worden sind. Bemerkenswerterweise zeigten Proteindom{\"a}nen von TERB1, TERB2 und MAJIN, die an der Bildung des Komplexes sowie an der Interaktion mit dem Telomer-Shelterin-Protein und den LINC-Komplexen beteiligt sind, eine hohe Sequenz{\"a}hnlichkeit {\"u}ber alle Kladen hinweg. Abschließend lieferte die Genexpression im Nesseltier Hydra vulgaris den Beweis, dass der TERB1-TERB2-MAJIN-Komplex selektiv in der Keimbahn exprimiert wird, was auf die Konservierung meiotischer Funktionen {\"u}ber die gesamte Metazoen-Evolution hinweg hindeutet. Zusammenfassend bietet diese Arbeit bedeutende neue Erkenntnisse hinsichtlich des Meiose-spezifischen Telomer-Adapterkomplex, seines Mechanismus zur Verankerung der Telomer an die Kernh{\"u}lle und die Entschl{\"u}sselung seines Ursprungs in den Metazoen.}, language = {en} } @phdthesis{Jahn2012, author = {Jahn, Daniel}, title = {Die Bedeutung von verk{\"u}rzten Spleißvarianten des Lamin A-Gens f{\"u}r die Meiose und f{\"u}r die Pathogenese von Laminopathien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74123}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Lamina ist ein dichtes Netzwerk aus Intermedi{\"a}r-Filamenten, den Laminen, an der nucleoplasmatischen Seite der inneren Kernmembran. Hier interagieren Lamine sowohl mit Transmembran-Proteinen der Kernh{\"u}lle als auch mit dem Chromatin. Diese Wechselwirkungen mit Interaktionspartnern verschiedener zellul{\"a}rer Kompartimente macht die Lamina, neben einer Ger{\"u}ststruktur mit wichtigen mechanische Aufgaben, auch zu einer zentralen Schnittstelle von Signalwegen, die eine intrazellul{\"a}re Kommunikation zwischen Nucleus und Cytoplasma erm{\"o}glichen. Die Lamina ist somit ein entscheidender Regulator der funktionellen Organisation des Chromatins und der differentiellen Genexpression. Das Expressionsmuster der Lamine w{\"a}hrend der Spermatogenese von S{\"a}ugern unterscheidet erheblich von der Lamin-Expression somatischer Zellen und weist einige Besonderheiten auf. Dies schließt unter anderem die spezifische Expression der verk{\"u}rzten A-Typ Lamin-Spleißvariante C2 w{\"a}hrend der meiotischen Phase der Spermatogenese ein. Diese und andere Beobachtungen deuteten bereits l{\"a}nger darauf hin, dass der speziellen Zusammensetzung der Lamina und vor allem dem meiosespezifischen Lamin C2 w{\"a}hrend der Gametogenese im m{\"a}nnlichen Organismus eine entscheidende Rolle zukommen k{\"o}nnte. Neuere Studien im Mausmodell bekr{\"a}ftigen diese Hypothese und leisten dar{\"u}ber hinaus einen entscheidenden Betrag dazu, die Funktion der Lamina w{\"a}hrend der Meiose auf molekularer Ebene pr{\"a}zise zu definieren. Im deutlichen Gegensatz zu den weitreichenden Kenntnissen zur Situation in M{\"a}nnchen lagen zu Beginn der vorliegenden Arbeit keine Daten {\"u}ber die Zusammensetzung der Lamina in weiblichen Keimzellen vor. Konsequenterweise existierten auch keine funktionellen Untersuchungen zur Relevanz der Lamina f{\"u}r die Oogenese. In der vorliegenden Arbeit wurden diese reproduktionsbiologisch hoch interessanten Fragestellungen detailliert untersucht. Dabei zeigte sich unter anderem, dass Lamin C2 auch in weiblichen Keimzellen spezifisch w{\"a}hrend der Meiose exprimiert wird. Durch Studien an einer Lamin C2-defizienten Mauslinie wurde die Funktion von Lamin C2 in der Meiose in Weibchen genau untersucht. Dabei wurde eine erhebliche Beeintr{\"a}chtigung der strukturellen Paarung der homologen Chromosomen und der homologen Rekombination in Lamin C2-defizienten Weibchen festgestellt. Da die genannten Prozesse Schl{\"u}sselereignisse f{\"u}r die korrekte Segregation der Homologen in sp{\"a}teren Stadien der Meiose sind, deuten die erzielten Ergebnisse auf eine erhebliche qualitative Beeintr{\"a}chtigung der reifen Gameten in Lamin C2-defizienten Weibchen hin. Ein weiterer zentraler Aspekt der Arbeit war die Analyse der molekularen Eigenschaften des meiosespezifischen Lamin C2 in vitro. Diese Experimente definieren wichtige Unterschiede hinsichtlich seiner Polymerisationseigenschaften im Vergleich zu Laminen somatischer Zellen und tragen, zusammen mit anderen Studien, dadurch erheblich dazu bei, die Funktion von Lamin C2 in der Meiose im mechanistischen Sinne besser zu verstehen. Zudem deckt die vorliegende Arbeit erstmals einen funktionellen Zusammenhang zwischen der Lamina-Zusammensetzung und der Qualit{\"a}t der Keimzellen weiblicher S{\"a}uger auf und erm{\"o}glicht dadurch zuk{\"u}nftige Studien zur Rolle der Lamine in der Oogenese, die m{\"o}glicherweise auch f{\"u}r die menschliche Fertilit{\"a}t sehr interessant sein k{\"o}nnte. Der zweite Teil der Dissertation besch{\"a}ftigt sich mit der Beschreibung einer trunkierten A-Typ Lamin-Spleißvariante in einer Mauslinie, die bislang als A-Typ Lamin-defizient angesehen wurde (Lmna-/-). Die durchgef{\"u}hrten Untersuchungen besitzen vor allem dadurch hohe Relevanz, dass die untersuchte Lmna-/- Mauslinie seit Jahren als das wichtigste Modell zur funktionellen Untersuchung der A-Typ Lamine gilt und bereits in einer Vielzahl von Publikationen eingesetzt wurde. In den hierzu durchgef{\"u}hrten Versuchen konnte das in der Lmna-/- Mauslinie persistierende A-Typ Lamin mittels diverser methodischer Ans{\"a}tze als C-terminale Deletionsmutante definiert werden, der die Exons 8-11 der insgesamt 12 Exons des Lmna-Gens fehlen. Daher wurde diese Lamin A-Mutante als Lamin AΔ8-11 bezeichnet. Die Konsequenzen der C-terminalen Deletion f{\"u}r die physiologischen Eigenschaften des Lamin Adelta8-11 sowie die Auswirkungen seiner Expression in der Lmna-/- Mauslinie auf aktuelle Modellvorstellungen zur Funktion der A-Typ Lamine und zur Entstehung Lamin-assoziierter, humaner Erkrankungen (Laminopathien) werden in der Arbeit ausf{\"u}hrlich diskutiert.}, subject = {Meiose}, language = {de} } @phdthesis{Sbiera2012, author = {Sbiera, Silviu}, title = {Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74183}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Primary contact with human polyomaviruses is followed by lifelong asymptomatic persistence of viral DNA. Under severe immunosuppression JCV activation may lead to unrestricted virus growth in the CNS followed by development of progressive multifocal leukoencephalopathy (PML). Besides the kidney and the brain, target cells of persistent infection were also found in the hematopoietic system. This included the presence of JCV genomes in peripheral blood cells (PBCs). In the attempt to understand the role of PBCs for the JCV infection in humans, we asked for the type of cells affected as well as for virus interaction with PBCs. Analysis of separated subpopulations by highly sensitive and specific polymerase chain reaction and Southern blot hybridization revealed the presence of JCV DNA mostly in circulating granulocytes. These cells have important functions in innate immunity and are professional phagocytes. This suggested that PCR amplified DNA might be the result of an extranuclear association of the virus due to membrane attachment or phagocytosis rather than JCV infection with presence of viral DNA in the nucleus. In the attempt to answer this question JCV DNA was subcellularly localized in the blood of 22 healthy donors by JCV specific fluorescence in situ hybridization (FISH). Granulocytes and peripheral blood mononuclear cells (PBMCs) were separated by Percoll gradient centrifugation. Intracellular JCV DNA was hybridized with Digoxigenin-labeled JCV specific DNA probes covering half of the viral genome. As the sensitivity of the anti-digoxigenin antibody system was lower than the PCR detection level, a chemical amplification step was included consisting of peroxidase labeled secondary antibody precipitating biotinylated tyramide followed by detection with streptavidin-Texas-Red and fluorescence microscopy. Comparison of the number of cells affected in healthy individuals with 15 HIV-1 infected patients with and without PML revealed that the rate of affected PBMCs was comparable in both groups (2.5±0.4 and 14.5±0.9 per 1000). In contrast, the rate of JCV positive granulocytes in the immunosuppressed group was 92.6±1.7\% compared to 4±1.4\% in healthy donors thus confirming that granulocytes are the major group of circulating cells affected by JCV and that HIV-1 associated immune impairment has an important effect on the virus-cell association. Localization revealed that JCV DNA was predominantly located within the cytoplasm, although hybridizing signals occasionally covered the nuclear compartment. The fluorescent glow of chemical amplification combined with classical fluorescence microscopy did not allow an unequivocal localization of viral DNA. However, confocal microscopy of 24 sections through single cells combined with FISH without chemical amplification confirmed cytoplasmic localization of JCV DNA in a large number of cells. Additionally, it clearly demonstrated that JCV DNA was also located in the nucleus and nuclear localization directly correlated with the number of cells affected. Calculation of the virus load in subcellular compartments revealed that up to 50\% of the JCV genomes were located in the nucleus thus pointing to viral infection at least in the granulocytes of HIV-1 infected patients. This may contribute to the distribution of the virus from sites of peripheral infection to the CNS and may promote the development of active PML in the severely immune impaired patients.}, subject = {Polyomaviren}, language = {en} } @article{AlbrechtSharmaReinhardtetal.2010, author = {Albrecht, Marco and Sharma, Cynthia M. and Reinhardt, Richard and Vogel, Joerg and Rudel, Thomas}, title = {Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68389}, year = {2010}, abstract = {Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNAseq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semiquantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.}, subject = {Biologie}, language = {en} } @article{KoetschanFoersterKelleretal.2010, author = {Koetschan, Christian and Foerster, Frank and Keller, Alexander and Schleicher, Tina and Ruderisch, Benjamin and Schwarz, Roland and Mueller, Tobias and Wolf, Matthias and Schultz, Joerg}, title = {The ITS2 Database III-sequences and structures for phylogeny}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68390}, year = {2010}, abstract = {The internal transcribed spacer 2 (ITS2) is a widely used phylogenetic marker. In the past, it has mainly been used for species level classifications. Nowadays, a wider applicability becomes apparent. Here, the conserved structure of the RNA molecule plays a vital role. We have developed the ITS2 Database (http://its2.bioapps .biozentrum.uni-wuerzburg.de) which holds information about sequence, structure and taxonomic classification of all ITS2 in GenBank. In the new version, we use Hidden Markov models (HMMs) for the identification and delineation of the ITS2 resulting in a major redesign of the annotation pipeline. This allowed the identification of more than 160 000 correct full ength and more than 50 000 partial structures. In the web interface, these can now be searched with a modified BLAST considering both sequence and structure, enabling rapid taxon sampling. Novel sequences can be annotated using the HMM based approach and modelled according to multiple template structures. Sequences can be searched for known and newly identified motifs. Together, the database and the web server build an exhaustive resource for ITS2 based phylogenetic analyses.}, subject = {Biologie}, language = {en} } @phdthesis{Pleiser2012, author = {Pleiser, Sandra}, title = {Mouse genetic analyses of Spir functions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73634}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Das Aktin-Zytoskelett ist f{\"u}r viele zellul{\"a}re Funktionen unerl{\"a}sslich, dazu geh{\"o}ren der strukturelle Aufbau von Zellen, die Zellwanderung und Vesikeltransportprozesse. Die funktionelle Vielfalt der Aktinstrukturen spiegelt sich in einer Vielzahl verschiedener molekularer Mechanismen wieder, welche die Polymerisierung von Aktinfilamenten regulieren. Die sponante Aktinpolymerisierung wird jedoch verhindert aufgrund der Instabilit{\"a}t von kleinen Aktin Oligomeren und durch Aktin Monomer bindende Proteine, welche die Bildung solcher Oligomere unterbinden. Aktinnukleationsfaktoren helfen diese kinetische Barriere der Filamentbildung zu {\"u}berwinden und sind wesentlich f{\"u}r die Erzeugung von neuen Aktinfilamenten an bestimmten subzellul{\"a}ren Kompartimenten. Spir Proteine sind die ersten beschriebenen Mitglieder der neuen Klasse von WH2 Dom{\"a}nen Aktinnukleationsfaktoren. Sie leiten die Polymerisierung von Aktin ein, indem sie Aktinmonomere an die vier WH2 Dom{\"a}nen im Zentrum des Proteins binden. Trotz ihrer Eigenschaft Aktinpolymerisation in vitro selber zu nukleieren, bilden Spir Proteine einen regulatorischen Komplex mit anderen Aktinnukleatoren der formin Untergruppe von forminen. Spir hat eine Funktion bei der Regulierung von vesikul{\"a}r erzeugten filament{\"o}sen Aktinstrukturen, Vesikeltransportprozessen und der Bildung der Teilungsfurche w{\"a}hrend der asymmetrischen meiotischen Zellteilung. Das S{\"a}ugetiergenom kodiert zwei spir Gene, spir-1 und spir-2. Die entsprechenden Proteine haben einen identischen strukturellen Aufbau und sind zu einem großen Teil homolog zueinander. Um die Spir Funktion im sich entwickelnden und adulten Nervensystem zu untersuchen, wurde die bisher unbekannte Expression des Maus spir-2 Gens analysiert. Real-time PCR Analysen haben ergeben, dass spir-2 in adulten M{\"a}usen in Oozyten, dem Gehirn, im Gastrointestinaltrakt, den Hoden und der Niere exprimiert wird. In situ Hybridisierungen wurden durchgef{\"u}hrt um die zellul{\"a}re Natur der spir Expression nachzuweisen. W{\"a}hrend der Embryogenese haben in situ Hybridisierungen gezeigt, dass spir-2 im sich entwickelnden Nervensytem und Darmtrakt exprimiert wird. In adulten Mausgeweben, wurde die h{\"o}chste Expression von spir-2 in Epithelzellen des Verdauungstraktes, in neuronalen Zellen des Nervensystems und in Spermatocyten gefunden. Im Gegensatz zur eher begrenzten Expression des Maus spir-1 Gens, welches {\"u}berwiegend im Nervensystem, den Oozyten und Hoden zu finden ist, zeigen die hier aufgef{\"u}hrten Daten ein breiteres Expressionsmuster des spir-2 Gens und unterst{\"u}tzen damit eine allgemeinere zellbiologische Funktion der neuen Aktinnukleatoren. Um die Funktion des Spir Proteins im sich entwickelnden und adulten Nervensystem zu untersuchen, wurden Spir-1 defiziente M{\"a}use mit Hilfe der gene trap Methode generiert. Spir-1 defiziente M{\"a}use sind lebensf{\"a}hig und eignen sich daher perfekt um die Neurobiologie des Spir-1 Aktinnukleators zu untersuchen. Die Analyse von prim{\"a}ren kortikalen Neuronen von Spir-1 defizienten M{\"a}usen zeigte eine Reduktion dendritischer Verzweigungen und ist die erste Beschreibung einer neuronalen Funktion von Spir-1. Desweiteren wurde eine transgene Mauslinie (thy1-GFP-M) eingesetzt, die das gr{\"u}ne Fluoreszenzprotein (GFP) unter der Kontrolle von Neuronen-spezifischen Elementen des thy1 Promoters exprimiert. GFP ist dabei nur in einer Teilmenge von Neuronen exprimiert, f{\"a}rbt diese Neuronen jedoch in ihrer Gesamtheit an. Spir-1 defiziente M{\"a}use, die das GFP Transgen exprimieren wurden generiert und analysiert. Es wurde herausgefunden, dass Spir-1 defiziente M{\"a}use eine reduzierte Anzahl an dendritischen Dornen im entorhinalen Kortex im Vergleich zu Wildtyp- Geschwistertieren aufweisen. Zusammengefasst gibt diese Studie neue Erkenntnisse {\"u}ber die zellbiologische Funktion von Spir und liefert Einsichten wie das neuronale Netzwerk sturkturiert wird.}, subject = {Actin-bindende Proteine}, language = {en} } @article{GoebMeyerNatusBenaventeetal.2011, author = {G{\"o}b, Eva and Meyer-Natus, Elisabeth and Benavente, Ricardo and Alsheimer, Manfred}, title = {Expression of individual mammalian Sun1 isoforms depends on the cell type}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68750}, year = {2011}, abstract = {Mammalian Sun1 belongs to an evolutionarily conserved family of inner nuclear membrane proteins, which are known as SUN domain proteins. SUN domain proteins interact with KASH domain partners to form bridging complexes, so-called LINC complexes, that physically connect the nuclear interior to the cytoskeleton. LINC complexes are critical for nuclear integrity and play fundamental roles in nuclear positioning, shaping and movement. The mammalian genome codes for at least five different SUN domain proteins used for the formation of a number of different LINC complexes. Recently, we reported on the identification of everal Sun1 isoforms, which tremendously enlarges the alternatives to form functional LINC complexes. We now confirmed that Sun1 actually exists in at least seven distinct splice variants. Besides that, we observed that expression of individual Sun1 isoforms remarkably depends on the cell type, suggesting a cell type-specific adaption of Sun1 dependent LINC complexes to specific cellular and physiological requirements.}, subject = {Biologie}, language = {en} } @phdthesis{Bartl2012, author = {Bartl, Jasmin}, title = {Impairment of insulin signaling pathway in Alzheimer's disease}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74197}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {The neurodegenerative disorder Alzheimer's disease (AD) is the cause of approximately 60\% of the world's 35 million patients suffering from dementia. Current research focuses here are on association with other diseases such as diabetes type 2 (T2DM), possible genetic markers, specific signal transduction pathways within the brain and potential protein modification, because the pathogenesis and etiology of AD are still not fully understood. Specifically association of T2DM with AD came to the focus with the so-called "Rotterdam study" in 1999, indicating that T2DM doubles the risk of developing AD. In the meantime, it is known that the prevalence rate in patients with T2DM is 30\%. Drugs commonly used in the treatment of T2DM such as peroxisome proliferator-activated receptors gamma (PPARγ) agonists show improvement of the cognitive abilities in patients with early stage of dementia, with potential therapeutically relevance. Therefore it is important not only to investigate a link between these diseases, but also to investigate the insulin signaling pathway in the brain of AD patients. In order to investigate this complex issue in more details and demonstrate additional links between T2DM and AD, the present study used several basic biological methods to clarify the question: "Is impaired insulin signaling pathway within the brain crucial for the development of AD?" from several points of view. The methods used in this work have been i) an analysis of single nucleotide (SNP) polymorphism of the insulin-degrading enzyme gene (IDE) in relation to risk of AD and / or of T2DM, ii) post-mortem histochemical studies of brain tissue of patients with only AD, with AD combined with T2DM and with only T2DM compared with an age-matched control group, and iii.) investigations of neurochemical pathways and gene/protein expression changes of a human cell culture as a consequences of amyloid β (Aβ) treatment. After analysis of the IDE SNP polymorphism in the selected VITA (Vienna Trans Danube Aging) cohort disease-specific effects were discovered. The upstream polymorphism (IDE2) was found to influence AD risk in a protective manner, while the downstream polymorphism (IDE7) modified the T2DM risk. Based on the SNP results, the presented study delineate the model that IDE promoter and 3‟ untranslated region/downstream variation can have different effects on IDE expression, maybe a relevant endophenotype with disorder-specific effects on AD and T2DM susceptibility. Furthermore, the human post-mortem studies could show that both AD as well as T2DM patients had a significantly lower density of the insulin receptor (IR) in the hippocampus, whereas a significantly increased density of inactive phosphorylated PPARγ has been found and this persisted even in patients with both diseases. Summarizing the histological study, it was possible to reveal common histological features of AD and T2DM, but no direct connection between the two diseases. Although AD is nowadays not only characterized by amyloid-containing plaque deposits and by the hyperphosphorylation of tau protein, the excessive Aβ42 presence in the brains of AD patients is still playing a key role. Up to date it is still not entirely clear which physical form of Aβ42 is responsible for the development of AD. The present work investigated, what impact has the state of aggregation of Aβ42 on genes and proteins of the insulin signaling pathway and the amyloid cascade. It could be shown that the oligomeric variant enhanced specifically the gene and protein expression of glycogen synthase kinase (GSK) 3β and also the enzyme activity was significantly increased, but has in turn strongly inhibited the IR gene and protein expression. Additionally, the effect of Aβ42 on monoamine oxidase B (MAO-B) was examined. An effect of both aggregated forms of Aβ42 had on enzyme activity was discovered. However, the fibrillar variants led to significantly increased activity of MAO-B while the oligomeric variants inhibited the enzyme activity. Several previous studies have demonstrated the involvement of increased MAO-B activity in AD, but the present work provides for the first time a direct link between the states of aggregation of Aβ42 to enzyme activity. Finally the results of the presented thesis can be summarized to following conclusion: Although AD and T2DM sharing some degrees of common features, still there is a lack of direct association, and therefore the diseases must be considered more independent rather than linked. But the impaired cerebral insulin signaling pathway seems to be another manifested hallmark of AD.}, subject = {Alzheimer-Krankheit}, language = {en} } @article{NiewaldaVoellerEschbachetal.2011, author = {Niewalda, Thomas and V{\"o}ller, Thomas and Eschbach, Claire and Ehmer, Julia and Chou, Wen-Chuang and Timme, Marc and Fiala, Andr{\´e} and Gerber, Bertram}, title = {A Combined Perceptual, Physico-Chemical, and ImagingApproach to 'Odour-Distances' Suggests a CategorizingFunction of the Drosophila Antennal Lobe}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74769}, year = {2011}, abstract = {How do physico-chemical stimulus features, perception, and physiology relate? Given the multi-layered and parallel architecture of brains, the question specifically is where physiological activity patterns correspond to stimulus features and/ or perception. Perceived distances between six odour pairs are defined behaviourally from four independent odour recognition tasks. We find that, in register with the physico-chemical distances of these odours, perceived distances for 3-octanol and n-amylacetate are consistently smallest in all four tasks, while the other five odour pairs are about equally distinct. Optical imaging in the antennal lobe, using a calcium sensor transgenically expressed in only first-order sensory or only second-order olfactory projection neurons, reveals that 3-octanol and n-amylacetate are distinctly represented in sensory neurons, but appear merged in projection neurons. These results may suggest that within-antennal lobe processing funnels sensory signals into behaviourally meaningful categories, in register with the physico-chemical relatedness of the odours.}, subject = {Drosophila Antennal Lobe}, language = {en} } @phdthesis{Zeeshan2012, author = {Zeeshan, Ahmed}, title = {Bioinformatics Software for Metabolic and Health Care Data Management}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73926}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Computer Science approaches (software, database, management systems) are powerful tools to boost research. Here they are applied to metabolic modelling in infections as well as health care management. Starting from a comparative analysis this thesis shows own steps and examples towards improvement in metabolic modelling software and health data management. In section 2, new experimental data on metabolites and enzymes induce high interest in metabolic modelling including metabolic flux calculations. Data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific strengths is now possible by an advantageous combination of specific software. How can available software for metabolic modelling be improved from a computational point of view? A number of available and well established software solutions are first discussed individually. This includes information on software origin, capabilities, development and used methodology. Performance information is obtained for the compared software using provided example data sets. A feature based comparison shows limitations and advantages of the compared software for specific tasks in metabolic modeling. Often found limitations include third party software dependence, no comprehensive database management and no standard format for data input and output. Graphical visualization can be improved for complex data visualization and at the web based graphical interface. Other areas for development are platform independency, product line architecture, data standardization, open source movement and new methodologies. The comparison shows clearly space for further software application development including steps towards an optimal user friendly graphical user interface, platform independence, database management system and third party independence especially in the case of desktop applications. The found limitations are not limited to the software compared and are of course also actively tackled in some of the most recent developments. Other improvements should aim at generality and standard data input formats, improved visualization of not only the input data set but also analyzed results. We hope, with the implementation of these suggestions, metabolic software applications will become more professional, cheap, reliable and attractive for the user. Nevertheless, keeping these inherent limitations in mind, we are confident that the tools compared can be recommended for metabolic modeling for instance to model metabolic fluxes in bacteria or metabolic data analysis and studies in infection biology. ...}, subject = {Stoffwechsel}, language = {en} } @phdthesis{Seida2012, author = {Seida, Ahmed Adel}, title = {The Immunomodulatory Role of Endogenous Glucocorticoids in Ovarian Cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73901}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Ovarian cancer currently causes ~6,000 deaths per year in Germany alone. Since only palliative treatment is available for ovarian carcinomas that have developed resistance against platinum-based chemotherapy and paclitaxel, there is a pressing medical need for the development of new therapeutic approaches. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. The research of our group thus aims at overcoming tumour immune escape by counteracting immunosuppressive mechanisms in the tumour microenvironment. In this context, we found that tumour-infiltrating myeloid-derived suppressor cells (MDSC) or tumour associated macrophages (TAM) which are abundant in ovarian cancer express high levels of the enzyme 11β-hydroxysteroid dehydrogenase1 (11-HSD1). This oxido-reductase enzyme is essential for the conversion of biologically inactive cortisone into active cortisol. In line with this observation, high endogenous cortisol levels could be detected in serum, ascitic fluid and tumour exudates from ovarian cancer patients. Considering that cortisol exerts strong anti-inflammatory and immunosuppressive effects on immune cells, it appears likely that high endogenous cortisol levels contribute to immune escape in ovarian cancer. We thus hypothesised that local activation of endogenous glucocorticoids could suppress beneficial immune responses in the tumour microenvironment and thereby prevent a successful immunotherapy. To investigate the in vivo relevance of this postulated immune escape mechanism, irradiated PTENloxP/loxP loxP-Stop-loxP-krasG12D mice were reconstituted with hematopoietic stem cells from either glucocorticoid receptor (GR) expressing mice (GRloxP/loxP) or from mice with a T cell-specific glucocorticoid receptor knock-out (lck-Cre GRloxP/loxP) mice. In the host mice, the combination of a conditional PTEN knock-out with a latent oncogenic kras leads to tumour development when a Cre-encoding adenovirus is injected into the ovarian bursa. Using this model, mice that had been reconstituted with GC-insensitive T cells showed better intratumoural T cell infiltration than control mice that had received functionally unaltered GRloxP/loxP cells via adoptive transfer. However, tumour-infiltrating T cells mostly assumed a Foxp3+ (regulatory) phenotype and survival was even shortened in mice with cortisol-insensitive T cells. Thus, endogenous cortisol seems to inhibit immune cell infiltration in ovarian cancer, but productive anti-tumour immune responses might still be prevented by further factors from the tumour microenvironment. Thus, our data did not provide a sufficiently strong rationale to further pursue the antagonisation of glucocorticoid signalling in ovarian cancer patients, Moreover, glucocorticoids are frequently administered to cancer patients to reduce inflammation and swelling and to prevent chemotherapy-related toxic side effects like nausea or hypersensitivity reactions associated with paclitaxel therapy. Thus, we decided to address the question whether specific signalling pathways in innate immune cells, preferentially in NK cells, could still be activated even in the presence of GC. A careful investigation of the various activating NK cell receptors (i.e. NKp30, NKp44, NKp46), DNAM-1 and NKG2D) was thus performed which revealed that NKp30, NKp44 and NKG2D are all down-regulated by cortisol whereas NKp46 is actually induced by cortisol. Interestingly, NKp46 is the only known receptor that is strictly confined to NK cells. Its activation via crosslinking leads to cytokine release and activation of cytotoxic activity. Stimulation of NK cells via NKp46 may contribute to immune-mediated tumour destruction by triggering the lysis of tumour cells and by altering the cytokine pattern in the tumour microenvironment, thereby generating more favourable conditions for the recruitment of antigen-specific immune cells. Accordingly, our observation that even cortisol-treated NK cells can still be activated via NKp46 and CD2 might become valuable for the design of immunotherapies that can still be applied in the presence of endogenous or therapeutically administered glucocorticoids.}, subject = {Cortison}, language = {en} } @phdthesis{Schneider2011, author = {Schneider, Christof}, title = {Detecting the influence of different potential stress factors on the behavior of the honeybee Apis mellifera using Radiofrequency Identification (RFID)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71344}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {This study was conducted to determine the influence of different stress factors on the honeybee Apis mellifera. The investigation was motivated by previous experiments that suggested the existence of an unspecific defense mechanism causing a generalized change of flight behavior after the onset of different diseases. This mechanism is thought to impede the ability of flight bees to return to their respective colonies thereby removing the disease from the colony over time. During the last years, the existence of such a "suicidal behavior" was supported by further studies. Thus, an unnoticed, potentially highly effective defense mechanism of social insects was revealed whose spectrum of activity and physiological basics require further investigation. Suggesting that the reaction by the bees is unspecific to different diseases as well as to other potential stress factors, this study was designed to investigate the influence of pathogens, insecticides, and different brood rearing temperatures on different parameters like lifespan, foraging activity, and foraging trip duration of worker bees.}, subject = {Biene}, language = {en} } @article{WangorschButtMarketal.2011, author = {Wangorsch, Gaby and Butt, Elke and Mark, Regina and Hubertus, Katharina and Geiger, J{\"o}rg and Dandekar, Thomas and Dittrich, Marcus}, title = {Time-resolved in silico modeling of fine-tuned cAMP signaling in platelets: feedback loops, titrated phosphorylations and pharmacological modulation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69145}, year = {2011}, abstract = {Background: Hemostasis is a critical and active function of the blood mediated by platelets. Therefore, the prevention of pathological platelet aggregation is of great importance as well as of pharmaceutical and medical interest. Endogenous platelet inhibition is predominantly based on cyclic nucleotides (cAMP, cGMP) elevation and subsequent cyclic nucleotide-dependent protein kinase (PKA, PKG) activation. In turn, platelet phosphodiesterases (PDEs) and protein phosphatases counterbalance their activity. This main inhibitory pathway in human platelets is crucial for countervailing unwanted platelet activation. Consequently, the regulators of cyclic nucleotide signaling are of particular interest to pharmacology and therapeutics of atherothrombosis. Modeling of pharmacodynamics allows understanding this intricate signaling and supports the precise description of these pivotal targets for pharmacological modulation. Results: We modeled dynamically concentration-dependent responses of pathway effectors (inhibitors, activators, drug combinations) to cyclic nucleotide signaling as well as to downstream signaling events and verified resulting model predictions by experimental data. Experiments with various cAMP affecting compounds including antiplatelet drugs and their combinations revealed a high fidelity, fine-tuned cAMP signaling in platelets without crosstalk to the cGMP pathway. The model and the data provide evidence for two independent feedback loops: PKA, which is activated by elevated cAMP levels in the platelet, subsequently inhibits adenylyl cyclase (AC) but as well activates PDE3. By multi-experiment fitting, we established a comprehensive dynamic model with one predictive, optimized and validated set of parameters. Different pharmacological conditions (inhibition, activation, drug combinations, permanent and transient perturbations) are successfully tested and simulated, including statistical validation and sensitivity analysis. Downstream cyclic nucleotide signaling events target different phosphorylation sites for cAMP- and cGMP-dependent protein kinases (PKA, PKG) in the vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation as well as cAMP levels resulting from different drug strengths and combined stimulants were quantitatively modeled. These predictions were again experimentally validated. High sensitivity of the signaling pathway at low concentrations is involved in a fine-tuned balance as well as stable activation of this inhibitory cyclic nucleotide pathway. Conclusions: On the basis of experimental data, literature mining and database screening we established a dynamic in silico model of cyclic nucleotide signaling and probed its signaling sensitivity. Thoroughly validated, it successfully predicts drug combination effects on platelet function, including synergism, antagonism and regulatory loops.}, subject = {Vasodilatator-stimuliertes Phosphoprotein}, language = {en} } @phdthesis{Krueger2012, author = {Kr{\"u}ger, Beate}, title = {Integration und Kombination bioinformatischer Methoden in Biotechnologie, synthetischer Biologie und Pharmaindustrie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70702}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Bioinformatik ist eine interdisziplin{\"a}re Wissenschaft, welche Probleme aus allen Lebenswissenschaften mit Hilfe computergest{\"u}tzter Methoden bearbeitet. Ihr Ziel ist es, die Verarbeitung und Interpretation großer Datenmengen zu erm{\"o}glichen. Zudem unterst{\"u}tzt sie den Designprozess von Experimenten in der Synthetischen Biologie. Die synthetische Biologie besch{\"a}ftigt sich mit der Generierung neuer Komponenten und deren Eigenschaften, welche durch die Behandlung und Manipulation lebender Organismen oder Teilen daraus entstehen. Ein besonders interessantes Themengebiet hierbei sind Zweikomponenten-Systeme (Two-Component System, TCS). TCS sind wichtige Signalkaskaden in Bakterien, welche in der Lage sind Informationen aus der Umgebung in eine Zelle zu {\"u}bertragen und darauf zu reagieren. Die vorliegende Dissertation besch{\"a}ftigt sich mit der Beurteilung, Nutzung und Weiterentwicklung von bioinformatischen Methoden zur Untersuchung von Proteininteraktionen und biologischen Systemen. Der wissenschaftliche Beitrag der vorliegenden Arbeit kann in drei Aspekte unterteilt werden: - Untersuchung und Beurteilung von bioinformatischen Methoden und Weiterf{\"u}hrung der Ergebnisse aus der vorhergehenden Diplomarbeit zum Thema Protein-Protein-Interaktionsvorhersagen. - Analyse genereller evolution{\"a}rer Modifikationsm{\"o}glichkeiten von TCS sowie deren Design und spezifische Unterschiede. - Abstraktion bzw. Transfer der gewonnenen Erkenntnisse auf technische und biologische Zusammenh{\"a}nge. Mit dem Ziel das Design neuer Experimente in der synthetischen Biologie zu vereinfachen und die Vergleichbarkeit von technischen und biologischen Prozessen sowie zwischen Organismen zu erm{\"o}glichen. Das Ergebnis der durchgef{\"u}hrten Studie zeigte, dass Zweikomponenten-Systeme in ihrem Aufbau sehr konserviert sind. Nichtsdestotrotz konnten viele spezifische Eigenschaften und drei generelle Modifikationsm{\"o}glichkeiten entdeckt werden. Die Untersuchungen erm{\"o}glichten die Identifikation neuer Promotorstellen, erlaubten aber auch die Beschreibung der Beschaffenheit unterschiedlicher Signalbindestellen. Zudem konnten bisher fehlende Komponenten aus TCS entdeckt werden, ebenso wie neue divergierte TCS-Dom{\"a}nen im Organismus Mycoplasma. Eine Kombination aus technischen Ans{\"a}tzen und synthetischer Biologie vereinfachte die gezielte Manipulation von TCS oder anderen modularen Systemen. Die Etablierung der vorgestellten zweistufigen Modul-Klassifikation erm{\"o}glichte eine effizientere Analyse modular aufgebauter Prozesse und erlaubte somit das molekulare Design synthetischer, biologischer Anwendungen. Zur einfachen Nutzung dieses Ansatzes wurde eine frei zug{\"a}ngliche Software GoSynthetic entwickelt. Konkrete Beispiele demonstrierten die praktische Anwendbarkeit dieser Analysesoftware. Die vorgestellte Klassifikation der synthetisch-biologischen und technischen Einheiten soll die Planung zuk{\"u}nftiger Designexperimente vereinfachen und neue Wege f{\"u}r sinnverwandte Bereiche aufzeigen. Es ist nicht die Hauptaufgabe der Bioinformatik, Experimente zu ersetzen, sondern resultierende große Datenmengen sinnvoll und effizient auszuwerten. Daraus sollen neue Ideen f{\"u}r weitere Analysen und alternative Anwendungen gewonnen werden, um fehlerhafte oder falsche Ans{\"a}tze fr{\"u}hzeitig zu erkennen. Die Bioinformatik bietet moderne, technische Verfahren, um vertraute, aber oft m{\"u}hsame experimentelle Wege durch neue, vielversprechende Ans{\"a}tze zur Datenstrukturierung und Auswertung großer Datenmengen zu erg{\"a}nzen. Neue Sichtweisen werden durch die Erleichterung des Testprozederes gef{\"o}rdert. Die resultierende Zeitersparnis f{\"u}hrt zudem zu einer Kostenreduktion.}, subject = {Biotechnologie}, language = {de} } @phdthesis{Goehler2012, author = {G{\"o}hler, Antonia}, title = {Untersuchung Karbohydrat-bindender Proteine mit hoher zeitlicher und r{\"a}umlicher Aufl{\"o}sung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76665}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Das menschliche Genom verschl{\"u}sselt 30000 bis 40000 Proteine, von denen ein Großteil kovalent gebundene Karbohydrat-Gruppen an Asparagin-, Serin-, Threonin- oder Hydroxylysin-Resten tr{\"a}gt. Diese sogenannten Glykoproteine sind allgegenw{\"a}rtige Bestandteile der extrazellul{\"a}ren Matrix von Zelloberfl{\"a}chen. Sie steuern Zell-Zell- und Zell-Matrix-Kommunikationen, k{\"o}nnen bei der roteinfaltung helfen bzw. die Proteinstabilit{\"a}t erh{\"o}hen oder Immunantworten regulieren. Die Ausl{\"o}sung von biologischen Prozesse erfordert aber {\"U}bersetzer der zuckerbasierten Informationen. Solche Effektoren sind die Lektine, unter ihnen auch die Galektine. Galektine binden spezifisch β-Galaktosen, weisen strukturelle {\"U}bereinstimmungen in der Aminos{\"a}uresequenz ihrer Zuckererkennungsdom{\"a}nen (CRDs) auf und zeigen ein „jelly-roll"-Faltungsmuster, bestehend aus einem β-Sandwich mit zwei antiparallelen Faltbl{\"a}ttern. Strukturell werden die CRDs in drei verschiedenen, topologischen Formen pr{\"a}sentiert. Proto-Typen existieren als nicht-kovalent verkn{\"u}pfte Dimere der CRDs, Chimera-Typen besitzen neben der CRD eine Nicht-Lektin-Dom{\"a}ne und bei den Tandem-Repeat-Typen sind zwei verschiedene CRDs {\"u}ber ein kurzes Linker-Peptid kovalent verbunden. Galektine werden sowohl in normalem wie auch pathogenem Gewebe exprimiert und das zunehmende Wissen {\"u}ber die Beteiligung an verschiedenen Krankheiten und Tumorwachstum liefert die Motivation, strukturelle Aspekte und die Vernetzung von Lektinen detailliert, insbesondere im Hinblick auf ihre intrafamili{\"a}ren Unterschiede, zu untersuchen. Durch die Kombination verschiedener Spektroskopie-Techniken mit hoher zeitlicher und r{\"a}umlicher Aufl{\"o}sung, basierend auf der Verwendung von Fluorophoren (intrinsisch und extrinsisch), werden in dieser Arbeit die Eigenschaften von Galektinen n{\"a}her untersucht. Mit Fluoreszenz-Korrelations-Spektroskopie (FCS) und Anisotropie-Messungen wird gezeigt, dass eine Liganden-Bindung bei Proto-Typ-Galektinen mit einer Verringerung des hydrodynamischen Radius einhergeht. Bei Tandem-Repeat- und Chimera-Typen bleibt der Radius konstant. Daf{\"u}r skaliert die Diffusionskonstante von Tandem-Repeat-Typen anormal mit der molaren Masse. Die Anisotropie-Messungen werden parallel zu den FCS-Messungen durchgef{\"u}hrt, um einen Einfluss des Fluoreszenzmarkers auszuschließen. Mit Hilfe dieser Technik wird außerdem gezeigt, dass unterschiedliche Dissoziationskonstanten und Kinetiken f{\"u}r den Bindungsprozess innerhalb der Proto-Typ-Gruppe m{\"o}glichweise auf unterschiedliche Konformationsdynamiken zur{\"u}ckgehen. Der Vergleich von hGal-1 und cG-1B verdeutlicht, dass strukturelle {\"A}hnlichkeiten zwar ein identisches Bindungsverhalten hervorrufen k{\"o}nnen, der Oxidationsprozess der Proteine aber unterschiedlich ablaufen kann. Beide Methoden k{\"o}nnen so als sehr sensitive Techniken zur Untersuchung von Strukturmerkmalen bei Galektinen etabliert werden, wobei die {\"U}bertragbarkeit auf andere Glykoproteine gew{\"a}hrleistet ist. Weiterhin gilt Quervernetzung als eine der wichtigsten Eigenschaften von Galektinen, da durch die Vernetzung von Glykoproteinen auf der Zelloberfl{\"a}che Signalwege aktiviert und Immunantworten reguliert werden. Um die r{\"a}umliche organisation und Quervernetzung von hGal-1 auf den Oberfl{\"a}chen von Neuroblastomzellen nachzuweisen, eignet sich das hochaufl{\"o}sende Mikroskopieverfahren dSTORM sehr gut. Durch Verwendung des photoschaltbaren Fluorophors Alexa647 als spezifischem Marker f{\"u}r hGal-1, einem Standard-Weitfeld-Aufbau und verschiedenen Analyseverfahren, kann eine Clusterformation von hGal-1 auf der Zelloberfl{\"a}che best{\"a}tigt werden. hGal-1 bildet Cluster mit einem mittleren Durchmesser von 81±7 nm aus. Der Durchmesser ist unabh{\"a}ngig von der Konzentration, w{\"a}hrend die Anzahl der Cluster davon abh{\"a}ngt. F{\"u}r die Clusterausbildung ist ein Startpunkt, also eine minimale Dichte der Galektin-Molek{\"u}le, notwendig. Durch Blockierung der CRDs mit Laktose wird die Clusterbildung unterdr{\"u}ckt und die Spezifit{\"a}t der CRDs gegen{\"u}ber β-Galaktosen erneut herausgestellt. Anders als dimeres hGal-1 binden Monomere deutlich schlechter an die Membranrezeptoren. Es werden keine Cluster ausgebildet, eine Quervernetzung von Membranrezeptoren ist nicht m{\"o}glich. Außerdem kann es durch die Monomere zu einer vollst{\"a}ndigen Markierung und damit Abkugellung der Zellen kommen. M{\"o}glicherweise wird der Zelltod induziert. Hochaufl{\"o}sende Mikroskopieverfahren sind durch den Markierungsprozess limitiert. Die bioorthogonale Click-Chemie er{\"o}ffnet jedoch neue M{\"o}glichkeiten zur Markierung und Visualisierung von Biomolek{\"u}len, ohne die Notwenigkeit genetischer Manipulationen. Es werden modifizierte Zuckermolek{\"u}le in die Zellmembranen eingebaut, {\"u}ber eine 1,3-polare Cycloaddition mit einem Alkin markiert und ihre Verteilung mit Hilfe von dSTORM untersucht. Es wird nachgewiesen, dass die Zuckermolek{\"u}le in Clustern auftreten und Click-Chemie trotz dem Katalysator Kupfer an lebenden Zellen durchf{\"u}hrbar ist. Die Bewegung der Gesamtcluster wird mittels Mean Square Displacement aufgeschl{\"u}sselt und eine Diffusionskonstante f{\"u}r Cluster im Bereich von 40 - 250 nm bestimmt. Zusammenfassend stellt die Kombination verschiedener Spektroskopie-Techniken ein gutes Werkzeug zur Untersuchung von Karbohydrat-bindendenden Proteinen mit hoher r{\"a}umlicher und zeitlicher Aufl{\"o}sung dar und erm{\"o}glicht einen neuen Einblick in die Biologie der Galektine.}, subject = {Fluoreszenz}, language = {de} } @phdthesis{Knaf2012, author = {Knaf, Tobias}, title = {Spezifische Bindung von Aluminium und Eisen an den kationenselektiven Kanal MppA von Microthrix parvicella}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77011}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Schwermetallsalze wie beispielsweise Aluminium- oder Eisensalze werden in der Abwasserbehandlung zur Pr{\"a}vention und Bek{\"a}mpfung von Bl{\"a}hschlamm, Schwimmschlamm und Schaumbildung verwendet. Dadurch kann eine Verbesserung der Schlammabsetzeigenschaften im Nachkl{\"a}rbecken erreicht werden. {\"U}berm{\"a}ßiges Wachstum des grampositiven Bakteriums Microthrix parvicella gilt dabei als Hauptursache von Schlammabsetzproblemen und kann ebenfalls durch die Dosierung von schwermetallhaltigen Flockungs- und F{\"a}llungsmitteln vermieden werden. Da diese Verbindungen in Wasser gel{\"o}st sind, m{\"u}ssen sie die Außenmembran bestimmter Bakterien passieren. Nur der Einbau von wassergef{\"u}llten Kan{\"a}len erlaubt den gel{\"o}sten Salzen das Passieren der durch hydrophobe Fetts{\"a}uren aufgebauten zus{\"a}tzlichen Permeabilit{\"a}tsbarriere. In dieser Arbeit wurden wassergef{\"u}llten Kan{\"a}le von Microthrix parvicella isoliert, aufgereinigt und mit Hilfe der Black-Lipid-Bilayer-Technik charakterisiert. Erg{\"a}nzend wurde der Einfluss und der Durchlass der Flockungs- und F{\"a}llungsmittel in Titrationsexperimenten untersucht. Dabei konnte ein wassergef{\"u}llter Kanal, der die Bezeichnung MppA erhielt, gefunden werden, welcher eine Leitf{\"a}higkeit von 600 pS in 1 M Kaliumchlorid und eine Bindestelle f{\"u}r mehrwertige Kationen wie Eisen oder Aluminium zeigte. Die Bindung dieser mehrwertigen Kationen f{\"u}hrte zu einer {\"A}nderung der Ionenselektivit{\"a}t. Ohne Bindung mehrwertiger Kationen zeigte der Kanal eine leichte Kationenselektivit{\"a}t. Nach der Bindung wechselte die Ionenselektivit{\"a}t zu einer Anionenselektivit{\"a}t, was auf eine spezifische Ladungsverteilung im Kanal hinweist. Der Kanal MppA zeigte gleichwertige Bindekonstanten f{\"u}r Aluminium und Eisen. Beide Metalle werden als F{\"a}llungs- und Flockungsmittel in Kl{\"a}ranlagen zum Verhindern von Schwimm- und Bl{\"a}hschlamm verwendet. Fr{\"u}here Arbeiten offenbarten bereits, dass haupts{\"a}chlich der Aluminiumanteil entscheidend f{\"u}r die Wirkung dieser Mittel ist. Diese Beobachtungen in Verbindung mit den Ergebnissen dieser Arbeit f{\"u}hrten zu der Annahme, dass Eisen und Aluminium eine kompetitive Bindung an der Bindestelle im Kanalinneren zeigen k{\"o}nnten. So k{\"o}nnte in manchen F{\"a}llen Aluminium anstelle des sonst als Spurenelement ben{\"o}tigten Eisens durch den Kanal transportiert werden und in Enzym-Substrat-Komplexen eingebaut werden. Dadurch k{\"o}nnten toxische Effekte auftreten, die letztlich ein Absterben des Organismus zur Folge h{\"a}tten. F{\"u}r die Bindung der Metallsalze konnte zus{\"a}tzlich eine pH-Abh{\"a}ngigkeit beobachtet werden. Nur eine Zugabe von Metalll{\"o}sungen mit einem pH-Wert kleiner 6 f{\"u}hrte zu einer Bindung im Kanal. Die Zugabe von Metalll{\"o}sungen mit einem pH-Wert gr{\"o}ßer 6 zeigte keinen Effekt auf die Leitf{\"a}higkeit des Kanals. Diese Ergebnisse best{\"a}tigen die auf Kl{\"a}ranlagen und in vorherigen Arbeiten get{\"a}tigte Beobachtung, dass der pH-Wert f{\"u}r die Wirksamkeit der Verbindungen entscheidend ist. In dieser Arbeit konnte jedoch erstmals gezeigt werden, dass der pH-Wert direkt die Bindung der Metallsalze beeinflusst.}, subject = {Aluminium}, language = {de} } @phdthesis{CardosoeCastro2012, author = {Cardoso e Castro, In{\^e}s Sofia}, title = {Epigenetic switch induced by MYC in Non-Small-Cell Lung Cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76713}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Non-Small-Cell Lung Cancer (NSCLC) is the most frequent human lung cancer and a major cause of death due to its high rate of metastasis1. These facts emphasize the urgent need for the investigation of new targets for anti-metastatic therapy. Up to now a number of genes and gene products have been identified that positively or negatively affect the probability of established human tumor cell lines to metastasize2. Previously, together with the group of Professor Ulf Rapp, we have described the first conditional mouse model for metastasis of NSCLC and identified a gene, c-MYC, that is able to orchestrate all steps of this process. We could identify potential markers for detection of metastasis and highlighted GATA4, which is exclusively expressed during lung development, as a target for future therapeutic intervention2. However, the mechanism underlying this metastatic conversion remained to be identified, and was therefore the focus of the present work. Here, GATA4 is identified as a MYC target in the development of metastasis and epigenetic alterations at the GATA4 promoter level are shown after MYC expression in NSCLC in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. This work identifies a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in NSCLC, suggesting novel potential targets for the development of anti-metastatic therapy.}, subject = {Nicht-kleinzelliges Bronchialkarzinom}, language = {en} } @phdthesis{Hillebrand2013, author = {Hillebrand, Frank}, title = {Der Einfluss des PI3-Kinase Signalwegs auf die Regulation des alternativen HIV-1 pr{\"a}-mRNA Spleißens}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76914}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {In der vorliegenden Arbeit wurden ausgehend von HIV-1 basierten Minigenkonstrukten und der proviralen NL4-3 DNA die Einfl{\"u}sse der PI3K Signalwegmodulation auf das alternative Spleißen der HIV-1 pr{\"a}-mRNA sowie auf die Virus Replikation untersucht. Mittels RT-PCR Analysen konnte gezeigt werden, dass die PI3K Inhibition im Falle der HIV-1 basierten Minigenkonstrukte in einer erh{\"o}hten Abundanz ungespleißter bzw. intronhaltiger mRNAs resultierte, w{\"a}hrend im Kontext des Virus die Induktion alternativer Tat Transkriptvarianten nachgewiesen werden konnte. Als Folge der Inhibition des PI3K Signalwegs kam es zu einem vermehrten Einschluss der HIV-1 Leader Exone2/2b und 3. Da der Einschluss dieser Exone durch die hnRNP A/B- und F/H-abh{\"a}ngigen Silencer Elemente ESSV und GI2-1 negativ reguliert wird, wurde vermutet, dass die PI3K Inhibition mit der Funktionalit{\"a}t dieser spleißregulatorischen Aktivit{\"a}t interferiert. Unterst{\"u}tzt wurde diese Hypothese durch Replikationsexperimente mit ESSV und GI2-1 Mutanten in Gegenwart und Abwesenheit des PI3K-Inhibitors. Zus{\"a}tzlich wurde auch der Einfluss des Inhibitors unter {\"U}berexpressionsbedingungen von hnRNP H auf das alternative HIV-1 Spleißen analysiert. In dieser Arbeit konnte ebenfalls gezeigt werden, dass die PI3K Inhibition ein ver{\"a}ndertes hnRNP H Spleißmuster bedingt sowie die SR-Protein Phosphorylierung und Expression beeinflusst. Des Weiteren war es im Verlauf der vorliegenden Arbeit m{\"o}glich, eine Interferenz der PI3K Modulation mit der Virus Replikation nachzuweisen. Die {\"U}berexpression der aktivierten Akt-Kinase lies hier nur eine sehr geringe Virus Produktion zu w{\"a}hrend die PI3K Inhibition diese auf ca. die H{\"a}lfte reduzierte. Weiterf{\"u}hrende Experimente zeigten, dass die {\"U}berexpression der aktivierten Akt-Kinase den nuklearen Export Rev-abh{\"a}ngiger HIV-1 mRNAs zu blockieren scheint. Dar{\"u}ber hinaus beeinflusste die PI3K Inhibition neben dem alternativen HIV-1 Spleißen auch die virale Transkription sowie die zellul{\"a}re Translation. Zusammen k{\"o}nnten diese Effekte die reduzierte virale Replikation erkl{\"a}ren. Der PI3K Signalweg spielt somit eine zentrale Rolle bei dem alternativen HIV-1 Spleißen und der viralen Replikation und bietet so die M{\"o}glichkeit der Entwicklung neuer Ans{\"a}tze einer antiviralen Therapie.  }, subject = {RNS-Spleißen}, language = {de} } @article{HsiehLinsenmair2012, author = {Hsieh, Yu-Lung and Linsenmair, Karl Eduard}, title = {Seasonal dynamics of arboreal spider diversity in a temperate forest}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75158}, year = {2012}, abstract = {Measuring and estimating biodiversity patterns is a fundamental task of the scientist working to support conservation and informmanagement decisions.Most biodiversity studies in temperate regions were often carried out over a very short period of time (e.g., a single season) and it is often—at least tacitly—assumed that these short-termfindings are representative of long-termgeneral patterns.However, should the studied biodiversity pattern in fact contain significant temporal dynamics, perhaps leading to contradictory conclusions. Here, we studied the seasonal diversity dynamics of arboreal spider communities dwelling in 216 European beeches (Fagus sylvatica L.) to assess the spider community composition in the following seasons: two cold seasons (I:November 2005-January 2006; II: February-April) and two warm seasons (III: May-July; IV: August-October). We show that the usually measured diversity of the warmseason community (IV: 58 estimated species) alone did not deliver a reliable image of the overall diversity present in these trees, and therefore, we recommend it should not be used for sampling protocols aimed at providing a full picture of a forest's biodiversity in the temperate zones. In particular, when the additional samplings of other seasons (I, II, III) were included, the estimated species richness nearly doubled (108). Community I possessed the lowest diversity and evenness due to the harsh winter conditions: this community was comprised of one dominant species together with several species low in abundance. Similarity was lowest (38.6\%) between seasonal communities I and III, indicating a significant species turnover due to recolonization, so that community III had the highest diversity. Finally, using nonparametric estimators, we found that further sampling in late winter (February-April) is most needed to complete our inventory. Our study clearly demonstrates that seasonal dynamics of communities should be taken into account when studying biodiversity patterns of spiders, and probably forest arthropods in general.}, subject = {Biologie}, language = {en} } @article{BeisserGrohmeKopkaetal.2012, author = {Beisser, Daniela and Grohme, Markus A. and Kopka, Joachim and Frohme, Marcus and Schill, Ralph O. and Hengherr, Steffen and Dandekar, Thomas and Klau, Gunnar W. and Dittrich, Marcus and M{\"u}ller, Tobias}, title = {Integrated pathway modules using time-course metabolic profiles and EST data from Milnesium tardigradum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75241}, year = {2012}, abstract = {Background: Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress. Results: In this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurablemetabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration. Conclusions: The functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.}, subject = {Milnesium tardigradum}, language = {en} } @article{OndruschKreft2011, author = {Ondrusch, Nicolai and Kreft, J{\"u}rgen}, title = {Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75451}, year = {2011}, abstract = {Background: In a number of gram-positive bacteria, including Listeria, the general stress response is regulated by the alternative sigma factor B (SigB). Common stressors which lead to the activation of SigB and the SigB-dependent regulon are high osmolarity, acid and several more. Recently is has been shown that also blue and red light activates SigB in Bacillus subtilis. Methodology/Principal Findings: By qRT-PCR we analyzed the transcriptional response of the pathogen L. monocytogenes to blue and red light in wild type bacteria and in isogenic deletion mutants for the putative blue-light receptor Lmo0799 and the stress sigma factor SigB. It was found that both blue (455 nm) and red (625 nm) light induced the transcription of sigB and SigB-dependent genes, this induction was completely abolished in the SigB mutant. The blue-light effect was largely dependent on Lmo0799, proving that this protein is a genuine blue-light receptor. The deletion of lmo0799 enhanced the red-light effect, the underlying mechanism as well as that of SigB activation by red light remains unknown. Blue light led to an increased transcription of the internalin A/B genes and of bacterial invasiveness for Caco-2 enterocytes. Exposure to blue light also strongly inhibited swimming motility of the bacteria in a Lmo0799- and SigB-dependent manner, red light had no effect there. Conclusions/Significance: Our data established that visible, in particular blue light is an important environmental signal with an impact on gene expression and physiology of the non-phototrophic bacterium L. monocytogenes. In natural environments these effects will result in sometimes random but potentially also cyclic fluctuations of gene activity, depending on the light conditions prevailing in the respective habitat.}, subject = {Listeria monocytogenes}, language = {en} } @phdthesis{Chaianunporn2012, author = {Chaianunporn, Thotsapol}, title = {Evolution of dispersal and specialization in systems of interacting species}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76779}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {A metacommunity approach will be a useful framework to assess and predict changes in biodiversity in spatially structured landscapes and changing environments. However, the relationship between two core elements of metacommunity dynamics, dispersal and species interaction are not well understood. Most theoretical studies on dispersal evolution assume that target species are in isolation and do not interact with other species although the species interactions and community structure should have strong interdependence with dispersal. On the one hand, a species interaction can change the cost and benefit structure of dispersing in relation to non-dispersing individuals. On the other hand, with dispersal, an individual can follow respectively avoid species partners. Moreover, it is also important to explore the interdependence between dispersal and species interaction with spatial and temporal heterogeneity of environment because it would allow us to gain more understanding about responses of community to disturbances such as habitat destruction or global climate change, and this aspect is up to now not well-studied. In this thesis, I focus on the interactive and evolutionary feedback effects between dispersal and various types of interspecific interactions in different environmental settings. More specifically, I contrast dispersal evolution in scenarios with different types of interactions (chapter 2), explore the concurrent evolution of dispersal and habitat niche width (specialization) in spatial heterogeneous landscape (chapter 3) and consider (potential) multidimensional evolutionary responses under climate change (chapter 4). Moreover, I investigate consequences of different dispersal probability and group tolerance on group formation respectively group composition and the coexistence of 'marker types' (chapter 5). For all studies, I utilize individual-based models of single or multiple species within spatially explicit (grid-based) landscapes. In chapter 5, I also use an analytical model in addition to an individual-based model to predict phenomenon in group recognition and group formation. ...}, subject = {Tiergesellschaft}, language = {en} } @article{WagnerFischerThomaetal.2011, author = {Wagner, Toni U. and Fischer, Andreas and Thoma, Eva C. and Schartl, Manfred}, title = {CrossQuery : A Web Tool for Easy Associative Querying of Transcriptome Data}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76088}, year = {2011}, abstract = {Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deepsequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.}, subject = {CrossQuery}, language = {en} } @article{VandenHoveJakobSchrautetal.2011, author = {Van den Hove, Daniel and Jakob, Sissi Brigitte and Schraut, Karla-Gerlinde and Kenis, Gunter and Schmitt, Angelika Gertrud and Kneitz, Susanne and Scholz, Claus-J{\"u}rgen and Wiescholleck, Valentina and Ortega, Gabriela and Prickaerts, Jos and Steinbusch, Harry and Lesch, Klaus-Peter}, title = {Differential Effects of Prenatal Stress in 5-Htt Deficient Mice: Towards Molecular Mechanisms of Gene x Environment Interactions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75795}, year = {2011}, abstract = {Prenatal stress (PS) has been shown to influence the development of the fetal brain and to increase the risk for the development of psychiatric disorders in later life. Furthermore, the variation of human serotonin transporter (5-HTT, SLC6A4) gene was suggested to exert a modulating effect on the association between early life stress and the risk for depression. In the present study, we used a 5-Htt6PS paradigm to investigate whether the effects of PS are dependent on the 5-Htt genotype. For this purpose, the effects of PS on cognition, anxiety- and depression-related behavior were examined using a maternal restraint stress paradigm of PS in C57BL6 wild-type (WT) and heterozygous 5-Htt deficient (5-Htt +/2) mice. Additionally, in female offspring, a genome-wide hippocampal gene expression profiling was performed using the Affymetrix GeneChipH Mouse Genome 430 2.0 Array. 5-Htt +/2 offspring showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. In contrast, exposure of 5-Htt +/2 mice to PS was associated with increased depressive-like behavior, an effect that tended to be more pronounced in female offspring. Further, 5-Htt genotype, PS and their interaction differentially affected the expression of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signaling were regulated by both the 5-Htt +/2 genotype and PS exposure, whereas cytokine and Wnt signaling were affected in a 5-Htt genotype6PS manner, indicating a gene6environment interaction at the molecular level. In conclusion, our data suggest that although the 5-Htt +/2 genotype shows clear adaptive capacity, 5-Htt +/2 mice -particularly females- at the same time appear to be more vulnerable to developmental stress exposure when compared to WT offspring. Moreover, hippocampal gene expression profiles suggest that distinct molecular mechanisms mediate the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction.}, subject = {Medizin}, language = {en} } @article{KronauerPetersSchoningetal.2011, author = {Kronauer, Daniel J. C. and Peters, Marcell K. and Schoning, Caspar and Boomsma, Jacobus J.}, title = {Hybridization in East African swarm-raiding army ants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68798}, year = {2011}, abstract = {Background: Hybridization can have complex effects on evolutionary dynamics in ants because of the combination of haplodiploid sex-determination and eusociality. While hybrid non-reproductive workers have been found in a range of species, examples of gene-flow via hybrid queens and males are rare. We studied hybridization in East African army ants (Dorylus subgenus Anomma) using morphology, mitochondrial DNA sequences, and nuclear microsatellites. Results: While the mitochondrial phylogeny had a strong geographic signal, different species were not recovered as monophyletic. At our main study site at Kakamega Forest, a mitochondrial haplotype was shared between a "Dorylus molestus-like" and a "Dorylus wilverthi-like" form. This pattern is best explained by introgression following hybridization between D. molestus and D. wilverthi. Microsatellite data from workers showed that the two morphological forms correspond to two distinct genetic clusters, with a significant proportion of individuals being classified as hybrids. Conclusions: We conclude that hybridization and gene-flow between the two army ant species D. molestus and D. wilverthi has occurred, and that mating between the two forms continues to regularly produce hybrid workers. Hybridization is particularly surprising in army ants because workers have control over which males are allowed to mate with a young virgin queen inside the colony.}, subject = {Zoologie}, language = {en} } @article{WegertBausenweinKneitzetal.2011, author = {Wegert, Jenny and Bausenwein, Sabrina and Kneitz, Susanne and Roth, Sabine and Graf, Norbert and Geissinger, Eva and Gessler, Manfred}, title = {Retinoic acid pathway activity in Wilms tumors and characterization of biological responses in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69137}, year = {2011}, abstract = {Background: Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90\% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear. Results: The association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/ intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however. Conclusions: Altered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.}, subject = {Krebs}, language = {en} } @article{EndesfelderMalkuschFlottmannetal.2011, author = {Endesfelder, Ulrike and Malkusch, Sebastian and Flottmann, Benjamin and Mondry, Justine and Liguzinski, Piotr and Verveer, Peter J. and Heilemann, Mike}, title = {Chemically Induced Photoswitching of Fluorescent Probes - A General Concept for Super-Resolution Microscopy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74896}, year = {2011}, abstract = {We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.}, subject = {Super-Resolution Microscopy}, language = {en} } @phdthesis{Heidbreder2012, author = {Heidbreder, Meike}, title = {Association and Activation of TNF-Receptor I Investigated with Single-Molecule Tracking and Super-Resolution Microscopy in Live Cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73191}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cellular responses to outer stimuli are the basis for all biological processes. Signal integration is achieved by protein cascades, recognizing and processing molecules from the environment. Factors released by pathogens or inflammation usually induce an inflammatory response, a signal often transduced by Tumour Necrosis Factor alpha (TNF). TNFα receptors TNF-R1 and TNF-R2 can in turn lead to apoptosis or proliferation via NF-B. These processes are closely regulated by membrane compartimentalization, protein interactions and trafficking. Fluorescence microscopy offers a reliable and non-invasive method to probe these cellular events. However, some processes on a native membrane are not resolvable, as they are well below the diffraction limit of microscopy. The recent development of super-resolution fluorescence microscopy methods enables the observation of these cellular players well below this limit: by localizing, tracking and counting molecules with high spatial and temporal resolution, these new fluorescence microscopy methods offer a previously unknown insight into protein interactions at the near-molecular level. Direct stochastic optical reconstruction microscopy (dSTORM) utilizes the reversible, stochastic blinking events of small commercially available fluorescent dyes, while photoactivated localization microscopy (PALM) utilizes phototransformation of genetically encoded fluorescent proteins. By photoactivating only a small fraction of the present fluorophores in each observation interval, single emitters can be localized with high precision and a super-resolved image can be reconstructed. Quantum Dot Triexciton imaging (QDTI) utilizes the three-photon absorption (triexcitonic) properties of quantum dots (QD) and to achieve a twofold resolution increase using conventional confocal microscopes. In this thesis, experimental approaches were implemented to achieve super-resolution microscopy in fixed and live-cells to study the spatial and temporal dynamics of TNF and other cellular signaling events. We introduce QDTI to study the three-dimensional cellular distribution of biological targets, offering an easy method to achieve resolution enhancement in combination with optical sectioning, allowing the preliminary quantification of labeled proteins. As QDs are electron dense, QDTI can be used for correlative fluorescence and transmission electron microscopy, proving the versatility of QD probes. Utilizing the phototransformation properties of fluorescent proteins, single-receptor tracking on live cells was achieved, applying the concept of single particle tracking PALM (sptPALM) to track the dynamics of a TNF-R1-tdEos chimera on the membrane. Lateral receptor dynamics can be tracked with high precision and the influences of ligand addition or lipid disruption on TNF-R1 mobility was observed. The results reveal complex receptor dynamics, implying internalization processes in response to TNFα stimulation and a role for membrane domains with reduced fluidity, so-called lipid raft domains, in TNF-R1 compartimentalization prior or post ligand induction. Comparisons with previously published FCS data show a good accordance, but stressing the increased data depth available in sptPALM experiments. Additionally, the active transport of NF-κB-tdEos fusions was observed in live neurons under chemical stimulation and/or inhibition. Contrary to phototransformable proteins that need no special buffers to exhibit photoconversion or photoactivation, dSTORM has previously been unsuitable for in vivo applications, as organic dyes relied on introducing the probes via immunostaining in concert with a reductive, oxygen-free medium for proper photoswitching behaviour. ATTO655 had been previously shown to be suitable for live-cell applications, as its switching behavior can be catalyzed by the reductive environment of the cytoplasm. By introducing the cell-permeant organic dye via a chemical tag system, a high specificity and low background was achieved. Here, the labeled histone H2B complex and thus single nucleosome movements in a live cell can be observed over long time periods and with ~20 nm resolution. Implementing these new approaches for imaging biological processes with high temporal and spatial resolution provides new insights into the dynamics and spatial heterogeneities of proteins, further elucidating their function in the organism and revealing properties that are usually only detectable in vitro.  }, subject = {Fluoreszenzmikrosopie}, language = {en} } @phdthesis{Englberger2012, author = {Englberger, Eva}, title = {Gene regulation in hearts of Hey-mutant mouse embryos and monitoring of sub-cellular Hey1 distribution}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73395}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Hey-mutant mouse hearts at embryonic day E14.5 were shown to react to the knock out of Hey2 with several up-regualted genes. This up-regulation is due to the lack of Hey2 and cannot be explained by the structural changes in heart morphology as shown using control animals. Part of the gene regulation was further validated using in situ hybridization. Hey1 was located to the nucleus in immunofluorescence experiments. However, experiments on protein level showed also amount of Hey1 within the cytoplasm. The nuclear localization of Hey1 was unchanged during all cell cycle phases as well as when CaMKII was co-expressed or other cellular pathways were inhibited or stimulated. Hey1 does not seem to interact with the nuclear transport proteins importin-alpha and -beta, therefore it still needs to be elucidated how Hey1 is transported into the nucleus.}, subject = {Maus}, language = {en} } @phdthesis{Schlegelmilch2012, author = {Schlegelmilch, Katrin}, title = {Molecular function of WISP1/CCN4 in the musculoskeletal system with special reference to apoptosis and cell survival}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73430}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.}, subject = {Knorpelzelle}, language = {en} } @phdthesis{Mihlan2012, author = {Mihlan, Sabrina [geb. Jasper]}, title = {Identifikation von Zonula Occludens 2 (ZO-2) als neuen LASP-1 Interaktionspartner und Aufkl{\"a}rung der LASP-1/ZO-2 Kern-Zytosol Translokation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73442}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {LASP-1 (LIM und SH3 Dom{\"a}nen Protein) ist ein in Zellen ubiquit{\"a}r vorkommendes Protein, welches in verschiedenen Tumorgeweben eine pathophysiologische {\"U}berexpression aufweist. Das Protein besitzt eine LIM Dom{\"a}ne, zwei Aktinbindungsregionen sowie eine SH3 Dom{\"a}ne und bindet einerseits an dynamischen Aktinstrukturen wie den fokalen Kontakten, Lamellopodien und Membranforts{\"a}tzen, kann andererseits aber auch in den Zellkern translokalisieren. F{\"u}r Aktinstrukturen wirkt LASP-1 als Ger{\"u}stprotein und ist wichtig f{\"u}r die Migration und Proliferation der Zellen. Die Funktion von LASP-1 im Zellkern ist noch nicht bekannt, da aber in Tumorzellen eine erh{\"o}hte nukleare Akkumulation von LASP-1 beobachtet werden konnte, deren Intensit{\"a}t mit der Tumorgr{\"o}ße sowie dem Langzeit{\"u}berleben der Patientinnen korreliert, ist LASP-1, zus{\"a}tzlich zu seiner Funktion als Strukturprotein, vermutlich auch ein Transkriptionsfaktor oder ein transkriptioneller Kofaktor. Eine Herunterregulation von LASP-1 in verschiedenen Tumorentit{\"a}ten f{\"u}hrt zur Inhibition der Proliferation und Migration. In dieser Arbeit konnte der bisher unbekannte Zellkernimport und -export von LASP-1 aufgekl{\"a}rt werden. Maßgeblich daran beteiligt ist ein durch Pulldown Experimente neu identifizierter LASP-1 Bindungspartner: das Zonula Occludens 2 Protein (ZO-2). Mittels Immunpr{\"a}zipitationen und Immunfluoreszenzen wurde diese Interaktion best{\"a}tigt. Nach Phosphorylierung von LASP-1 an Ser-146 durch Aktivierung der cAMP-abh{\"a}ngigen Proteinkinase (PKA) kommt es zu einer partiellen Abl{\"o}sung des LASP-1/ZO-2 Komplexes aus den fokalen Kontakten hin zu einer vermehrten Kernlokalisation beider Proteine. Dies l{\"a}sst sich durch Kern/Zytosol Trennungen belegen. Dabei ist die Bindung von LASP-1 an ZO-2 essentiell f{\"u}r die Translokation in den Zellkern, da bei einem ZO-2 Knockdown auch nach PKA Aktivierung LASP-1 zytosolisch lokalisiert bleibt. Wie Mutationsanalysen zeigen, findet die Interaktion zwischen der C-terminalen SH3 Dom{\"a}ne im LASP-1 und der Prolin-reichen SH3-Bindungssequenz im Bereich der Aminos{\"a}uren 1103-1121 am C-Terminus im ZO-2 statt. Die Translokation des Komplexes in den Kern erfolgt dabei {\"u}ber das Kernlokalisationssignal im ZO-2, da die LASP-1 Sequenz selbst keine nukleare Importsequenz aufweist. Im Zellkern konnte die direkte Interaktion von LASP-1 und ZO-2 mittels Duolink® Proximity Ligation Assay sichtbar gemacht werden. Der Export der Proteine erfolgt {\"u}ber das Protein CRM1. Eine Inhibition der Kernexportmaschinerie mit Leptomycin B erh{\"o}ht die Konzentration beider Proteine im Zellkern. Das nukleare Exportsignal (NES) im LASP-1 konnte durch Punktmutationen N-terminal der Leucin-reichen Aminos{\"a}uresequenz 70-77 zugeordnet werden (NLRLKQQS). Im letzten Schritt dieses Zyklus erfolgt die Relokalisation von LASP-1 zur{\"u}ck an die Zellmembranstrukturen. Der neu gefundene Signalweg dient wahrscheinlich zur Weiterleitung von externen Stimuli in den Kern und zur Genregulation - mit LASP-1 als Transkriptionsfaktor oder transkriptionellen Kofaktor.}, subject = {Tumorzelle}, language = {de} } @phdthesis{Kistenpfennig2012, author = {Kistenpfennig, Christa}, title = {Rhodopsin 7 and Cryptochrome - circadian photoreception in Drosophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72209}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Many organisms evolved an endogenous clock to adapt to the daily environmental changes caused by the earth's rotation. Light is the primary time cue ("Zeitgeber") for entrainment of circadian clocks to the external 24-h day. In Drosophila, several visual pigments are known to mediate synchronization to light: The blue-light photopigment Cryptochrome (CRY) and six well-described rhodopsins (Rh1-Rh6). CRY is present in the majority of clock neurons as well as in the compound eyes, whereas the location of rhodopsins is restricted to the photoreceptive organs - the compound eyes, the ocelli and the HB-eyelets. CRY is thought to represent the key photoreceptor of Drosophila's circadian clock. Nevertheless, mutant flies lacking CRY (cry01) are able to synchronize their locomotor activity rhythms to light-dark (LD) cycles, but need significantly longer than wild-type flies. In this behavior, cry01 mutants strongly resemble mammalian species that do not possess any internal photoreceptors and perceive light information exclusively through their photoreceptive organs (eyes). Thus, a mammalian-like phase-shifting behavior would be expected in cry01 flies. We investigated this issue by monitoring a phase response curve (PRC) of cry01 and wild-type flies to 1-h light pulses of 1000 lux irradiance. Indeed, cry01 mutants produced a mammalian-similar so called type 1 PRC of comparatively low amplitude (< 25\% of wild-type) with phase delays to light pulses during the early subjective night and phase advances to light pulses during the late subjective night (~1 h each). Despite the predominant role of CRY, the visual system contributes to the light sensitivity of the fly's circadian clock, mainly around dawn and dusk. Furthermore, this phase shifting allows for the slow re-entrainment which we observed in cry01 mutants to 8-h phase delays of the LD 12 h:12 h cycle. However, cry01 also showed surprising differences in their shifting ability: First of all, their PRC was characterized by a second dead zone in the middle of the subjective night (ZT17-ZT19) in addition to the usual unresponsiveness during the subjective day. Second, in contrast to wild-type flies, cry01 mutants did not increase their shift of activity rhythms neither in response to longer stimuli nor to light pulses of higher irradiance. In contrast, both 6-h light pulses of 1000 lux and 1-h light pulses of 10,000 lux light intensity during the early subjective night even resulted in phase advances instead of the expected delays. Thus, CRY seems to be not only responsible for the high light sensitivity of the wild-type circadian clock, but is apparently also involved in integrating and processing light information. Rhodopsin 7 (Rh7) is a yet uncharacterized protein, but became a good photoreceptor candidate due to sequence similarities to the six known Drosophila Rhs. The second part of this thesis investigated the expression pattern of Rh7 and its possible functions, especially in circadian photoreception. Furthermore, we were interested in a potential interaction with CRY and thus, tested cry01 and rh70 cry01 mutants as well. Rh1 is the main visual pigment of the Drosophila compound eye and expressed in six out of eight photoreceptors cells (R1-R6) in each of the ~800 ommatidia. Motion vision depends exclusively on Rh1 function but, moreover, Rh1 plays an important structural role and assures proper photoreceptor cell development and maintenance. In order to investigate its possible photoreceptive function, we expressed Rh7 in place of Rh1. Rh7 was indeed able to overtake the role of Rh1 in both aspects: It prevented retinal degeneration and mediated the optomotor response (OR), a motion vision-dependent behavior. At the transcriptional level, rh7 is expressed at approximately equal amounts in adult fly brains and retinas. Due to a reduced specificity of anti-Rh7 antibodies, we could not verify this result at the protein level. However, analysis of rh7 null mutants (rh70) suggested different Rh7 functions in vivo. Previous experiments strongly indicated an increased sensitivity of the compound eyes in the absence of Rh7 and suggested impaired light adaptation. We aimed to test this hypothesis at the levels of circadian photoreception. Locomotor activity rhythms are a reliable output of the circadian clock. Rh70 mutant flies generally displayed a wild-type similar bimodal activity pattern comprising morning (M) and evening (E) activity bouts. Activity monitoring supported the proposed "shielding" function, since rh70 mutants behaved like wild-type flies experiencing high irradiances. Under all investigated conditions, their activity peaks lay further apart resulting in a prolonged midday break. The behavior of cry01 mutants was mainly characterized by an unexpectedly high flexibility in the timing of M and E activity bouts which allowed tracking of lights-on and lights-off even under extreme photoperiods. Activity profiles of the corresponding rh70 cry01 double mutants reflected neither synergistic nor antagonistic effects of Rh7 and CRY and were dominated by a broad E activity peak. In the future, the different circadian phenotypes will be further investigated on the molecular level by analysis of clock protein cycling in the underlying pacemaker neurons. The work of this thesis confirmed that Rh7 is indeed able to work as a photoreceptor and to initiate the classical phototransduction cascade. On the other hand, it provided further evidence at the levels of circadian photoreception that Rh7 might serve as a shielding pigment for Rh1 in vivo, thereby mediating proper light adaptation.}, language = {en} } @phdthesis{Thoma2011, author = {Thoma, Eva Christina}, title = {Directed differentiation of pluripotent stem cells induced by single genes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54706}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Pluripotency describes the ability of stem cells to form every cell type of the body.. Pluripotent stem cells are e.g. embryonic stem cells (ESCs), but also the so called induced pluripotent stem cells (IPS cells), that are generated by reprogramming differentiated somatic cells into a pluripotent state. Furthermore, it has been shown that spermatogonia (SG) derived from adult testes of mouse or human are pluripotent. Because of their ability to differentiate into every somatic cell type, pluripotent stem cells have a unique status in research and regenerative medicine. For the latter, they offer a valuable opportunity to replace destroyed tissues or organs. For basic research, stem cells represent a useful system to study differentiation or developmental processes that are difficult to access in the physiological situation e.g. during embryogenesis. Both applications, however, require methods that allow efficient and directed differentiation of stem cells into defined specialized cell types. This study first aims to investigate the differentiation potential of SG derived from the teleost fish medaka (Oryzias latipes). My results demonstrate that medaka SG are able to form different somatic cell types, namely adipocytes, melanocytes, osteoblasts, and neurons. This indicates that medake SG have retained a broad differentiation potential suggesting that pluripotency is not restricted to mouse and human SG but might be conserved among vertebrates. Next, I wanted to establish a differentiation method that is solely based on ectopic expression of genes known to be essential for the formation of certain somatic cell types - so called master regulators (MRs). My findings show that ectopic expression of the melanocyte-specific transcription factor mitf-m that has previously been shown to induce differentiation of medaka ESCs into pigment cells resulted in the formation of the same cell type in medaka SG. This approach could be used to generate other somatic cell types. Thus, ectopic expression of the MRs cbfa1 and mash1 in MF-SG was sufficient to induce differentiation into osteoblasts and neurons, respectively. Interestingly, these differentiation processes included the activation of genes that are expressed earlier during embryogenesis than the differentiation-inducing MR. Furthermore, my findings show that the approach of MR-induced differentiation can be transferred to mammalian stem cell systems. Ectopic expression of the neural transcription factor ngn2 was sufficient to induce efficient and rapid differentiation of neurons in mouse ESCs. This differentiation process also included the induction of genes that in vivo are activated at earlier stages that ngn2. By generating a transgenic cell line allowing induction of ectopic ngn2 expression, it was possible to obtain a relatively pure culture of functional neurons. Ngn2-induced differentiation did not require any additional signals and occurred even under pluripotency promoting conditions. Moreover, ectopic expression of ngn2 did also induce the formation of cells with neuronal morphology in IPS cells indicating that MR-induced differentiation is operative in different stem cell types. Furthermore, protein transduction of Ngn2 into mouse ESCs also resulted in a neuronal differentiation process up to the appearance of neural precursor cells. Last, my results show that MR-induced differentiation can also be used to generate other cell types than neurons from mouse ESCs. Myoblasts and macrophage-like cells were generated by ectopic expression of the MRs myoD and cebpa, respectively. Using transgenic cell lines enabling induction of MR expression it was possible to obtain mixed cultures with two different differentiation processes occurring in parallel. Altogether this study shows that ectopic expression of single genes is sufficient to induce directed differentiation of stem cells into defined cell types. The feasibility of this approach was demonstrated for different MRs and consequently different somatic cell types. Furthermore, MR induced differentiation was operative in different stem cell types from fish and mouse. Thus, one can conclude that certain genes are able to define cell fates in in vitro stem cell systems and that this cell fate defining potential appears to be a conserved feature in vertebrates. These findings therefore provide new insights in the role of MRs in cell commitment and differentiation processes. Furthermore, this study presents a new method to induce directed differentiation of stem cells that offers several advantages regarding efficiency, rapidness, and reproducibility. MR-induced differentiation therefore represents a promising tool for both stem cell research and regenerative medicine.}, subject = {Stammzelle}, language = {en} } @phdthesis{Philippi2011, author = {Philippi, Nicole}, title = {Modellierung von Signalwegen in verschiedenen biologischen Systemen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-57690}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Die Apoptose der Leberzellen ist abh{\"a}ngig von externen Signalen wie beispielsweise Komponenten der Extrazellul{\"a}ren Matrix sowie anderen Zell-Zell-Kontakten, welche von einer Vielfalt und Vielzahl an Knoten verarbeitet werden. Einige von ihnen wurden im Rahmen dieser Arbeit auf ihre Systemeffekte hin unter- sucht. Trotz verschiedener {\"a}ußerer Einfl{\"u}sse und nat{\"u}rlicher Selektion ist das System daraufhin optimiert, eine kleine Anzahl verschiedener und klar voneinander unterscheidbarer Systemzust{\"a}nde anzunehmen. Die verschiedenartigen Einfl{\"u}sse und Crosstalk-Mechanismen dienen der Optimierung der vorhandenen Systemzust{\"a}nde. Das in dieser Arbeit vorgestellte Modell zeigt zwei apoptotische sowie zwei nicht-apoptotische stabile Systemzust{\"a}nde, wobei der Grad der Aktivierung eines Knotens bis zu dem Moment stark variieren kann, in welchem der absolute Systemzustand selbst ver{\"a}ndert wird (Philippi et al., BMC Systems Biology,2009) [1]. Dieses Modell stellt zwar eine Vereinfachung des gesamten zellul{\"a}ren Netzwerkes und seiner verschiedenen Zust{\"a}nde dar, ist aber trotz allem in der Lage, unabh{\"a}ngig von detaillierten kinetischen Daten und Parametern der einzelnen Knoten zu agieren. Gleichwohl erlaubt das Modell mit guter qualitativer {\"U}bereinstimmung die Apoptose als Folge einer Stimulation mit FasL zu modellieren. Weiterhin umfasst das Modell sowohl Crosstalk-M{\"o}glichkeiten des Collagen-Integrin-Signalwegs, ebenso ber{\"u}cksichtigt es die Auswirkungen der genetischen Deletion von Bid sowie die Konsequenzen einer viralen Infektion. In einem zweiten Teil werden andere Anwendungsm{\"o}glichkeiten dargestellt. Hormonale Signale in Pflanzen, Virusinfektionen und intrazellul{\"a}re Kommunikation werden semi-quantitativ modelliert. Auch hier zeigte sich eine gute Ubereinstimmung der Modelle mit den experimentellen Daten.}, subject = {Systembiologie}, language = {de} } @phdthesis{Hancock2012, author = {Hancock, Christine [geb. Herbst]}, title = {Influence of land use on Plantago lanceolata L. and its higher trophic levels at different spatial scales and in three geographic regions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73877}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Heutzutage pr{\"a}gen landwirtschaftlich genutzte Fl{\"a}chen einen großen Teil der deutschen Landschaft. Die Umwandlung von nat{\"u}rlichen Lebensr{\"a}umen zu bewirtschaftetem Gr{\"u}nland beeinflusst grundlegend die Vielfalt von Pflanzen und Tieren. Zwar erh{\"o}ht die intensive Nutzung dieser Fl{\"a}chen die Produktivit{\"a}t der Pflanzen oder die Biomasse als Viehfutter auf den Wiesen. Wie diese Einfl{\"u}sse auf die Artenvielfalt, {\"O}kosysteme und trophische Interaktionen, im Laufe der Jahre wirken ist jedoch immer noch nicht vollst{\"a}ndig verstanden. Um die Funktionen der Biodiversit{\"a}t in einer landwirtschaftlich genutzten Fl{\"a}che zu verstehen konzentrierte sich meine Arbeit auf den Einfluss der Landnutzung (D{\"u}ngung, Beweidung und Mahd) auf ein Herbivor-Parasitoid-System von Plantago lanceolata. Der Spitzwegerich ist ein generalistisches Kraut mit kosmopolitischem Vorkommen. Er kann in einem sehr breiten Spektrum von Bodenverh{\"a}ltnissen (sowohl in nassen und auch in trockenen Lebensr{\"a}umen) vorkommen und ist daher ein ideales Modellsystem zur Untersuchung tritrophischer Systeme in einem Landnutzungs-intensit{\"a}tsgradienten. Die R{\"u}sselk{\"a}fer Mecinus labilis und M. pascuorum ern{\"a}hren sich von P. lanceolata und legen dort ihre Eier ab. Mesopolobus incultus ist ein generalistisch lebender Parasitoid, der verschiedenen Insektenordnungen parasitiert. Die einzigen Wirte auf P. lanceolata sind jedoch die beiden erw{\"a}hnten R{\"u}sselk{\"a}ferarten. Das Ziel meiner Studie war es, den Einfluss der Landnutzung auf ein tritrophisches System und seiner umgebenden Vegetation (Struktur, Dichte und Artenreichtum) auf unterschiedlichen r{\"a}umlichen Skalen wie Subplot, Plot und Landschaftebene in drei verschiedenen Regionen (Nord-, Mittel- und S{\"u}ddeutschland) zu untersuchen. Ich untersuchte den Einfluss der Nutzungsintensit{\"a}t nicht nur korrelativ, sondern auch experimentell. Zus{\"a}tzlich zielte ich darauf ab, aufzuzeigen wie die Vegetationszusammensetzung die Metabolite der Wirtspflanze ver{\"a}ndert und ob diese Ver{\"a}nderungen Auswirkungen auf h{\"o}here trophische Ebenen im Feld haben.}, subject = {Landnutzung}, language = {en} } @article{VogelLoeschbergerSaueretal.2011, author = {Vogel, Benjamin and L{\"o}schberger, Anna and Sauer, Markus and Hock, Robert}, title = {Cross-linking of DNA through HMGA1 suggests a DNA scaffold}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68865}, year = {2011}, abstract = {Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.}, subject = {DNA}, language = {en} } @article{NeupertSebaldSchwabetal.1969, author = {Neupert, W. and Sebald, Walter and Schwab, A. J. and Pfaller, A. and B{\"u}cher, T.}, title = {Puromycin sensitivity of ribosomal label after incorporation of \(^{14}\)C-labelled amino acids into isolated mitochondria from Neurospora crassa}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62899}, year = {1969}, abstract = {Radioactive amino acids were incorporated into isolated mitochondria from Neurospora crassa. Then the mitochondrial ribosomes were isolated and submitted to density gradient centrifugation. A preferential labelling of polysomes was observed. However, when the mitochondrial suspension was treated with puromycin after amino acid incorporation, no radioactivity could be detected in either the monosomes or the polysomes. The conclusion is drawn that isolated mitochondria under these conditions do not incorporate significant amounts of amino acids into proteins of their ribosomes.}, subject = {Biochemie}, language = {en} } @article{SebaldSchwabBuecher1969, author = {Sebald, Walter and Schwab, A. J. and B{\"u}cher, T.}, title = {Cycloheximide resistant amino acid incorporation into mitochondrial protein from Neurospora crassa in vivo}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62900}, year = {1969}, abstract = {No abstract available}, subject = {Biochemie}, language = {en} } @article{SebaldHofstoetterHackeretal.1969, author = {Sebald, Walter and Hofst{\"o}tter, T. and Hacker, D. and B{\"u}cher, T.}, title = {Incorporation of amino acids into mitochondrial protein of the flight muscle of Locusta migratoria in vitro and in vivo in the presence of cycloheximide}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62919}, year = {1969}, abstract = {No abstract available}, subject = {Biochemie}, language = {en} } @article{SebaldBuecherOlbrichetal.1968, author = {Sebald, Walter and B{\"u}cher, T. and Olbrich, B. and Kaudewitz, F.}, title = {Electrophoretic pattern of and amino acid incorporation in vitro into the insoluble mitochondrial protein of neurospora crassa wild type and mi-1 mutant}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-62926}, year = {1968}, abstract = {No abstract available}, subject = {Biochemie}, language = {en} } @phdthesis{Oberlaender2012, author = {Oberl{\"a}nder, Uwe}, title = {Untersuchung der immunstimulatorischen Effekte von Neuromelanin (NM) auf dendritische Zellen und deren Bedeutung in der Pathogenese von Morbus Parkinson}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73684}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Hintergrund: Das Absterben Neuromelanin (NM)-haltiger Zellen in der substantia nigra (SN), und die daraus resultierende Erniedrigung des Dopaminspiegels im striatum, ist ein pathologisches Hauptmerkmal der Parkinsonschen Krankheit. Ein neuerlicher Nachweis von Anti-Melanin-Antik{\"o}rpern gibt Anlass zur Vermutung, dass NM ein Autoantigen sein k{\"o}nnte. In dieser Arbeit wurde gezeigt, dass NM tats{\"a}chlich von dendritischen Zellen (DZ), die in vivo hauptverantwortlich f{\"u}r die Ausl{\"o}sung von T- und B-Zellantworten sind, erkannt wird. Die Erkennung von NM durch DZ ist eine unabdingbare Voraussetzung f{\"u}r die Einleitung einer adaptiven Immunantwort. Methoden: Murine dendritische Zellen (mDZ) wurden aus Knochenmarkszellen generiert und mit NM aus humaner SN oder synthetischem Dopaminmelanin (DAM) behandelt, nachdem beide Melanine endotoxinfrei getestet wurden. Die Phagozytose von NM wurde mittels konfokaler Mikroskopie dokumentiert. Die Expression von MHC II und CD86 wurde mittels Durchflusszytometrie (FACS) analysiert. Zytokinkonzentrationen von TNF- und dem Interleukin IL-6 wurden mit ELISA-Assays bestimmt. Abschließend wurde die Funktion der durch NM aktivierten DZ mit einer allogenen mixed lymphocyte reaction (MLR) {\"u}berpr{\"u}ft. Ergebnisse: NM wurde von den mDZ effektiv phagozytiert, woraufhin die mDZ einen reifen Phenotyp (CD86high/MHC IIhigh) zeigten. Zus{\"a}tzlich sekretierten durch NM aktivierte mDZ die Zytokine IL-6 and TNF-. Schließlich ließen die mDZ T-Zellen in einer MLR proliferieren, und beweisen so ihre Funktionalit{\"a}t und die F{\"a}higkeit eine prim{\"a}re T-Zellantwort auszul{\"o}sen. Im Gegenteil dazu konnte DAM, dem die Protein- und Lipidkomponenten von NM fehlen und nur das Melaninr{\"u}ckrat mit NM gemeinsam hat, nur einen kleinen Effekt bei den mDZ hervorrufen. Diskussion: NM wird von DZ in vitro erkannt und bewirkt deren Reifung. Sollte der Vorgang auch in vivo stattfinden, besteht die M{\"o}glichkeit, dass SN-Antigene dem adaptiven Immunsystem pr{\"a}sentiert werden, was in einzelnen F{\"a}llen zur Einleitung einer adaptiven Immunantwort f{\"u}hren k{\"o}nnte. NM k{\"o}nnte also der Ausl{\"o}ser f{\"u}r einen autoimmunen Pathomechanismus in der parkinsonschen Krankheit sein.}, subject = {Parkinson-Krankheit}, language = {de} } @article{MeierjohannHufnagelWendeetal.2010, author = {Meierjohann, Svenja and Hufnagel, Anita and Wende, Elisabeth and Kleinschmidt, Markus A. and Wolf, Katarina and Friedl, Peter and Gaubatz, Stefan and Schartl, Manfred}, title = {MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68335}, year = {2010}, abstract = {Background: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTKdriven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.}, subject = {Medizin}, language = {en} } @article{Zeeshan2010, author = {Zeeshan, Ahmed}, title = {Towards Performance Measurement and Metrics Based Analysis of PLA Applications}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68188}, year = {2010}, abstract = {This article is about a measurement analysis based approach to help software practitioners in managing the additional level complexities and variabilities in software product line applications. The architecture of the proposed approach i.e. ZAC is designed and implemented to perform preprocessesed source code analysis, calculate traditional and product line metrics and visualize results in two and three dimensional diagrams. Experiments using real time data sets are performed which concluded with the results that the ZAC can be very helpful for the software practitioners in understanding the overall structure and complexity of product line applications. Moreover the obtained results prove strong positive correlation between calculated traditional and product line measures.}, subject = {Programmierbare logische Anordnung}, language = {en} } @article{BollazziRoces2010, author = {Bollazzi, Martin and Roces, Flavio}, title = {The thermoregulatory function of thatched nests in the South American grass-cutting ant, Acromyrmex heyeri}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68225}, year = {2010}, abstract = {The construction of mound-shaped nests by ants is considered as a behavioral adaptation to low environmental temperatures, i.e., colonies achieve higher and more stables temperatures than those of the environment. Besides the well-known nests of boreal Formica wood-ants, several species of South American leaf-cutting ants of the genus Acromyrmex construct thatched nests. Acromyrmex workers import plant fragments as building material, and arrange them so as to form a thatch covering a central chamber, where the fungus garden is located. Thus, the degree of thermoregulation attained by the fungus garden inside the thatched nest largely depends on how the thatch affects the thermal relations between the fungus and the environment. This work was aimed at studying the thermoregulatory function of the thatched nests built by the grass-cutting ant Acromyrmex heyeri Forel (Hymenoptera: Formicidae: Myrmicinae). Nest and environmental temperatures were measured as a function of solar radiation on the long-term. The thermal diffusivity of the nest thatch was measured and compared to that of the surrounding soil, in order to assess the influence of the building material on the nest's thermoregulatory ability. The results showed that the average core temperature of thatched nests was higher than that of the environment, but remained below values harmful for the fungus. This thermoregulation was brought about by the low thermal diffusivity of the nest thatch built by workers with plant fragments, instead of the readily-available soil particles that have a higher thermal diffusivity. The thatch prevented diurnal nest overheating by the incoming solar radiation, and avoided losses of the accumulated daily heat into the cold air during the night. The adaptive value of thatching behavior in Acromyrmex leaf-cutting ants occurring in the southernmost distribution range is discussed.}, subject = {Acromyrmex heyeri}, language = {en} } @article{SchwarzHameisterGessleretal.1994, author = {Schwarz, Klaus and Hameister, Horst and Gessler, Manfred and Grzeschik, Karl-Heinz and Hansen-Hagge, Thomas E. and Bartram, Claus R.}, title = {Confirmation of the localization of the human recombination activating gene 1 (RAG1) to chromosome 11p13}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59136}, year = {1994}, abstract = {The human recombination activating gene 1 (RAGl) has previously been mapped to chromosomes 14q and 11 p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to llp13.}, subject = {Biochemie}, language = {en} } @article{GesslerKonigMooreetal.1993, author = {Gessler, Manfred and Konig, Anja and Moore, Jay and Qualman, Steven and Arden, Karen and Cavenee, Webster and Bruns, Gail}, title = {Homozygous inactivation of WTI in a Wilms' tumor associated with the WAGR syndrome}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59146}, year = {1993}, abstract = {Wilms' tumor is a childhood nephroblastoma that is postulated to arise through the inactivation of a tumor suppressor gene by a two-hit mechanism. A candidate II p 13 Wilms' tumor gene, WTI, has been cloned and shown to encode a zinc finger protein. Patients with the WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) have a high risk of developing Wilms' tumor and they carry constitutional deletions of one chromosome II allele encompassing the WTI gene. Analysis of the remaining WTI allele in a Wilms' tumor from a WAGR patient revealed the deletion of a single nucleotide in exon 7. This mutation likely played a key role in tumor formation, as it prevents translation of the DNA-binding zinc finger domain that is essential for the function of the WTI polypeptide as a transcriptional regulator.}, subject = {Biochemie}, language = {en} } @article{HenryHooversBarichardetal.1993, author = {Henry, Isabelle and Hoovers, Jan and Barichard, Fernande and Berth{\´e}as, Marie-Francoise and Puech, Anne and Prieur, Fabienne and Gessler, Manfred and Bruns, Gail and Mannens, Marcel and Junien, Claudine}, title = {Pericentric intrachromosomal insertion responsible for recurrence of del(11)(p13p14) in a family}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59157}, year = {1993}, abstract = {The combined use of qualitative and quantitative analysis of I I p I 3 polymorphic markers tagether with chromosomal in situ suppression hybridization (CISS) with biotin labeled probes mapping to I I p allowed us to characterize a complex rearrangement segregating in a family. We detected a pericentric intrachromosomal insertion responsible (or recurrence of del( I I )(p 13p 14) in the family: an insertion of band I I p 13-p 14 carrying the genes for predisposition to Wilms' tumor, WT I, and for aniridia, AN2, into the long arm of chromosome I I in II q 13-q 1<4. Asymptomatic balanced carriers were observed over three generations. Classical cytogenetics had failed to detect this anomaly in the balanced carriers, who were first considered to be somatic mosaics for del( II )(p 13). Two of these women gave birth to children carrying a deleted chromosome II. most likely resulting from the loss of the I I p 13 band inserted in I I q. Although in both cases the deletion encompassed exactly the same maternally inherited markers, there was a wide Variation in clinical expression. One child, with the karyotype 46,XY,del(ll)(pllpl4), presented the full-blown WAGR syndrome with anlridia, mental retardation, Wilms' tumor, and pseudohermaphroditism, but also had proteinuria and glomerular sclerosis reminiscent of Drash syndrome. In contrast, the other one, a girl with the karyotype 46,XX,del( I I )(p I 3), only had aniridia. Although a specific set of mutational sites has been observed in Drash patients, these findings suggest that the loss of one copy of the WTI gene can result in similar genital and kidney abnormalities.}, subject = {Biochemie}, language = {en} } @phdthesis{Schneider2011, author = {Schneider, Matthias}, title = {Characterisation of Metalloprotease-mediated EGFR Signal Transactivation after GPCR Stimulation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65105}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {In the context of metalloprotease-mediated transactivation of the epidermal growth factor receptor, different monoclonal antibodies against ADAM17 / TACE were characterized for their ability to block the sheddase. Activity of some of them was observed at doses between 2µg/mL and 10µg/mL. Kinetic analyses showed their activity starting at around 30 minutes. In cellular assays performed with the antibodies, especially upon treatment of cells with sphingosine-1-phosphate a reduction in proliferation was observed with some candidates. Moreover this study provides potential new roles for ß-Arrestins. Their involvement in the triple membrane-passing signal pathway of EGFR transactivation was shown. Furthermore, in overexpressing cellular model systems, an interaction between ADAM17 and ß-Arrestin1 could be observed. Detailed analysis discovered that phosphorylation of ß-Arrestin1 is crucial for this interaction. Additionally, the novel mechanism of UV-induced EGFR transactivation was extended to squamous cell carcinoma. The mechanism happens in a dose dependent manner and requires a metalloprotease to shed the proligand Amphiregulin. The involvement of both ADAM9 and ADAM17, being the metalloproteases responsible for this cleavage, was shown for SCC9 cells.}, subject = {Epidermaler Wachstumsfaktor-Rezeptor}, language = {en} } @phdthesis{Heisig2011, author = {Heisig, Julia}, title = {Identifizierung neuer Zielgene der Hey bHLH Transkriptionsfaktoren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65053}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Der Notch Signalweg spielt w{\"a}hrend der Embryonalentwicklung eine zentrale Rolle in der Spezifizierung des Zellschicksales, der Proliferation und der Kommunikation benachbarter Zellen. Die Hey bHLH Transkriptionsfaktoren sind Zielgene des Notch-Signalweges und besitzen wichtige Funktionen in der kardiovaskul{\"a}ren Entwicklung. Hey2 Knockout (KO) M{\"a}use und Hey1/HeyL Doppelknockout-M{\"a}use (DKO) sind gekennzeichnet durch eine fehlerhafte Ausbildung der Herzscheidewand und der Herzklappen und durch eine unzureichende Differenzierung w{\"a}hrend der Blutgef{\"a}ßentwicklung. Ziel dieser Arbeit war es, neue Zielgene der Hey Proteine zu finden, um ihre Funktion in der Organentwicklung und die Auspr{\"a}gung der Hey KO Maus-Ph{\"a}notypen besser verstehen zu k{\"o}nnen. Dazu wurde als Methode eine Kombination aus Microarray-Analyse und Chromatinimmunpr{\"a}zipitation (ChIP) gew{\"a}hlt, um gleichzeitig einen {\"U}berblick {\"u}ber die regulierten Zielgene und der direkt gebundenen Promotoren zu gewinnen. Als Zellkulturmodell wurden HEK293-Zellen genutzt, die doxyzyklin-induzierbar Flag-markiertes Hey1, bzw. Hey2 Protein {\"u}berexprimieren. Eine Microarray-Analyse nach {\"U}berexpression von Hey1, bzw. Hey2 ergab insgesamt ca. 100 bis zu 5-fach herunterregulierte Zielgene und nur f{\"u}r Hey2 15 Gene, die st{\"a}rker als 2-fach hochreguliert waren. Eine ChIP mit αFlag-Antik{\"o}rper zeigte eine direkte DNA-Bindung von Hey1, bzw. Hey2, im proximalen Promotorbereich von 4 herunterregulierten Zielgenen (HEY1, BMP2, KLF10 und FOXC1). Ist jedoch die DNA-bindende basische Dom{\"a}ne des Hey1-Proteins deletiert, bzw. durch Aminos{\"a}ureaustausche (3 Arginine zu 3 Lysine) vermutlich nicht mehr DNA-bindend, kann eine Herunterregulation der Zielgene nach {\"U}berexpression der Hey1-Mutanten nicht mehr festgestellt werden. Ebenso kann eine Bindung der Hey1-Mutanten an die ausgew{\"a}hlten Promotoren von HEY1, BMP2, KLF10 oder FOXC1 mit ChIP nicht mehr nachgewiesen werden. Dies deutet darauf hin, dass die basische Dom{\"a}ne essentiell f{\"u}r die DNA-Bindung und f{\"u}r die Funktion der Hey Proteine ist. Mit ChIP-PET und anschließender Hochdurchsatz-Sequenzierung wurde ein genomweiter Screen der Hey1- und der Hey2-Bindungsstellen in HEK293-Zellen durchgef{\"u}hrt. F{\"u}r Hey1 wurden 1453 Zielgene, f{\"u}r Hey2 4288 Zielgene bestimmt, wobei 1147 Gene gemeinsame Zielgene von Hey1 und Hey2 waren. Obwohl die Bindungsstellen in 5'- und 3'-Richtung von kodierenden Sequenzen und auch in Exons und Introns lokalisiert waren, waren 55 \%, bzw. 49 \% aller Bindungsstellen f{\"u}r Hey1, bzw. Hey2 im proximalen Promotorbereich von -0,5 kb und im ersten Exon lokalisiert. Eine in silico Analyse des Bindemotivs deutete auf eine repetitive GC-haltige Sequenz hin, die vermutlich in CpG Inseln lokalisiert ist. Diese Ergebnisse weisen auf eine direkte Regulation der Transkriptionsmaschinerie durch die Hey Proteine hin. Ein Vergleich der Zielgene aus den Microarray-Analysen mit den ChIP-PET Daten zeigte einen hohen Anteil an herunterregulierten Genen mit Bindestellen, die direkt von Hey gebunden waren. W{\"a}hrend 60 \% der herunterregulierten Hey2 Zielgene in der ChIP-PET Analyse eine direkte DNA-Bindung zeigen, weisen nur 20 \% der hochregulierten Gene Bindestellen f{\"u}r Hey2 auf. Dies spricht f{\"u}r eine {\"u}berwiegende Repressorfunktion der Hey Proteine. Um zu {\"u}berpr{\"u}fen, inwieweit die Hey Proteine zelltypspezifisch verschiedene Zielgene regulieren, wurden embryonale Stammzellen (ES-Zellen) generiert, die ebenfalls doxyzyklin-induzierbar Hey1, bzw. Hey2 {\"u}berexprimieren. Diese ES-Zellen konnten effektiv zu Kardiomyozyten differenziert werden, so dass auch in diesen Zellen eine Hey {\"U}berexpression induziert und somit eine Genexpressionsanalyse durchgef{\"u}hrt werden konnte. Microarray Analysen der ES-Zellen und Kardiomyozyten ergaben mehr hoch- als herunterregulierte Gene im Vergleich zu HEK293-Zellen. Die {\"U}berlappung an gemeinsam regulierten Zielgenen in HEK293, ES-Zellen und Kardiomyozyten war sehr gering. Nur zwei Hey2-Zielgene wurden gleichzeitig in HEK293 und ES-Zellen st{\"a}rker als 2-fach reguliert (Hes1, Zic2). Diese geringe {\"U}berlappung deutet auf ein enges zelltypspezifische Regulationspotential hin. Eine Genontologie-Analyse aller Zielgene zeigte Interaktionen der Hey Proteine mit verschiedenen Signalwegen (z.B. TGFβ-, Id- oder Wnt-Signalweg), die alle unersetzlich in fr{\"u}hen Entwicklungsprozessen sind. Diese Ergebnisse deuten darauf hin, dass die Hey Proteine zelltypspezifisch die Expression von Genen aus verschiedenen Signalwegen beeinflussen und modulieren k{\"o}nnen. Weiterhin er{\"o}ffnen diese Daten neue M{\"o}glichkeiten f{\"u}r zuk{\"u}nftige Forschung, um die Rolle der Hey Proteine in der fr{\"u}hen Organentwicklung genauer ergr{\"u}nden.}, subject = {Gen notch}, language = {de} }