@phdthesis{Fliesser2015,
  author    = {Fließer, Mirjam},
  title     = {Hypoxia and hypoxia-inducible factor 1α modulate the immune response of human dendritic cells against Aspergillus fumigatus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121392},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The mold Aspergillus fumigatus causes life-threatening infections in immunocompromised patients. Over the past decade new findings in research have improved our understanding of A. fumigatus-host interactions. One of them was the detection of localized areas of tissue hypoxia in the lungs of mice infected with A. fumigatus. The transcription factor hypoxia-inducible factor 1α (HIF 1α) is known as the central regulator of cellular responses to hypoxia. Under normoxia, this constitutively expressed protein is degraded by oxygen-dependent mechanisms in most mammalian cell types. Interaction with pathogens can induce HIF 1α stabilization under normoxic conditions in innate immune cells. Bacterial infection models revealed that hypoxic microenvironments and signaling via HIF 1α modulate functions of host immune cells. Moreover, it was recently described that in murine phagocytes, HIF 1α expression is essential to overcome an A. fumigatus infection. However, the influence of hypoxia and the role of HIF 1α signaling for anti-A. fumigatus immunity is still poorly understood, especially regarding dendritic cells (DCs), which are important regulators of anti-fungal immunity. In this study, the functional relevance of hypoxia and HIF 1α signaling in the response of human DCs against A. fumigatus has been investigated. Hypoxia attenuated the pro-inflammatory response of DCs against A. fumigatus during the initial infection as shown by genome-wide microarray expression analyses and cytokine quantification. The up-regulation of maturation-associated molecules on DCs stimulated with A. fumigatus under hypoxia was reduced; however, these DCs possessed an enhanced capacity to stimulate T cells. This study thereby revealed divergent influence of hypoxia on anti-A. fumigatus DC functions that included both, inhibiting and enhancing effects. HIF-1α was stabilized in DCs following stimulation with A. fumigatus under normoxic and hypoxic conditions. This stabilization was partially dependent on Dectin-1, the major receptor for A. fumigatus on human DCs. Using siRNA-based HIF 1α silencing combined with gene expression microarrays, a modulatory effect of HIF-1α on the anti-fungal immune response of human DCs was identified. Specifically, the transcriptomes of HIF-1α silenced DCs indicated that HIF-1α enhanced DC metabolism and cytokine release in response to A. fumigatus under normoxic and hypoxic conditions. This was confirmed by further down-stream analyses that included quantification of glycolytic activity and cytokine profiling of DCs. By that, this study demonstrated functional relevance of HIF 1α expression in DCs responding to A. fumigatus. The data give novel insight into the cellular functions of HIF 1α in human DCs that include regulation of the anti-fungal immune response under normoxia and hypoxia. The comprehensive transcriptome datasets in combination with the down-stream protein analyses from this study will promote further investigations to further characterize the complex interplay between hypoxia, activation of Dectin-1 and HIF-1α signaling in host responses against A. fumigatus.},
  subject      = {Immunologie},
  language  = {en}
}
@phdthesis{Fischer2015,
  author    = {Fischer, Robin},
  title     = {Generating useful tools for future studies in the center of the circadian clock - defined knockout mutants for PERIOD and TIMELESS},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119141},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {To unravel the role of single genes underlying certain biological processes, scientists often use amorphic or hypomorphic alleles. In the past, such mutants were often created by chance. Enormous approaches with many animals and massive screening effort for striking phenotypes were necessary to find a needle in the haystack. Therefore at the beginning chemical mutagens or radiation were used to induce mutations in the genome. Later P-element insertions and inaccurate jump-outs enabled the advantage of potential larger deletions or inversions. The mutations were characterized and subsequently kept in smaller populations in the laboratories. Thus additional mutations with unknown background effects could accumulate. The precision of the knockout through homologous recombination and the additional advantage of being able to generate many useful rescue constructs that can be easily reintegrated into the target locus made us trying an ends-out targeting procedure of the two core clock genes period and timeless in Drosophila melanogaster. Instead of the endogenous region, a small fragment of approximately 100 base pairs remains including an attP-site that can be used as integration site for in vitro created rescue constructs. After a successful ends-out targeting procedure, the locus will be restored with e.g. flies expressing the endogenous gene under the native promoter at the original locus coupled to a fluorescence tag or expressing luciferase. We also linked this project to other research interests of our work group, like the epigenetic related ADAR-editing project of the Timeless protein, a promising newly discovered feature of time point specific timeless mRNA modification after transcription with yet unexplored consequences. The editing position within the Timeless protein is likewise interesting and not only noticed for the first time. This will render new insights into the otherwise not-satisfying investigation and quest for functional important sequences of the Timeless protein, which anyway shows less homology to other yet characterized proteins. Last but not least, we bothered with the question of the role of Shaggy on the circadian clock. The impact of an overexpression or downregulation of Shaggy on the pace of the clock is obvious and often described. The influence of Shaggy on Period and Timeless was also shown, but for the latter it is still controversially discussed. Some are talking of a Cryptochrome stabilization effect and rhythmic animals in constant light due to Shaggy overexpression, others show a decrease of Cryptochrome levels under these conditions. Also the constant light rhythmicity of the flies, as it was published, could not be repeated so far. We were able to expose the conditions behind the Cryptochrome stabilization and discuss possibilities for the phenomenon of rhythmicity under constant light due to Shaggy overexpression.},
  subject      = {Biologische Uhr},
  language  = {en}
}
@phdthesis{RamosTirado2015,
  author    = {Ramos Tirado, Mario},
  title     = {Stammzellbasierte Behandlungsstrategien zur Stimmlippenaugmentation und laryngealen Defektrekonstruktion},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117528},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Der Kehlkopf ist ein stimmerzeugendes knorpelhaltiges Organ und spielt eine wichtige Rolle in der Atemfunktion und beim aspirationsfreien Schluckakt. Funktionsst{\"o}rungen des Kehlkopfs wie Stimmbandl{\"a}hmungen werden durch Sch{\"a}digungen des Kehlkopfnervs nach operativen Eingriffen und Halsverletzungen hervorgerufen. Des Weiteren f{\"u}hren durch Traumen, Teil- und komplette Resektionen verursachte Substanzdefekte des Kehlkopfs zu Funktionsverlusten. Die hierf{\"u}r notwendigen und komplexen Rekonstruktionen werden durch das schlechte Regenerationspotential von Knorpelgewebe eingeschr{\"a}nkt und k{\"o}nnen nur bedingt durch synthetische Ersatzmaterialen oder k{\"o}rpereigenes Ersatzgewebe bewerkstelligt werden. Ist es m{\"o}glich, mit Hilfe des Tissue Engineerings aus k{\"o}rpereigenen Stammzellen und biokompatiblen Tr{\"a}germaterialien implantierbares Knorpelersatzgewebe herzustellen, welches zur dauerhaften Wiederherstellung der Kehlkopffunktionen eingesetzt werden kann? Die zus{\"a}tzliche Markierung von Stammzellen mit superparamagnetischen Eisenoxidnanopartikeln (VSOP) als Zellmarker bietet die M{\"o}glichkeit der Detektion und der Verfolgung der Zellen mittels nicht-invasiver Nachweismethoden nach deren Implantation. Ist die Verwendung dieser Nanopartikel ohne negative Folgen f{\"u}r die Stammzellen m{\"o}glich und sind diese f{\"u}r den Einsatz in der Laryngologie geeignet? Fettgewebsstammzellen (ASC) wurden aus humanem Liposuktionsmaterial und Kaninchen-Nackenfett isoliert und expandiert. Die Zellen wurden in Hydrogelkombinationen aus Kollagen Typ-I, Agarose, Fibrin und Hyalurons{\"a}ure eingebettet und mit den chondrogenen Wachstumsfaktoren TGF-β3, BMP-6 und IGF-I {\"u}ber 14 Tage differenziert. Anschließend wurden diese Zell-Hydrogelkonstrukte bez{\"u}glich Morphologie, extrazellul{\"a}rer Matrixanreicherung und knorpelspezifischer Genexpression histologisch, immunhistochemisch und molekularbiologisch analysiert. In einem weiteren Schritt wurden die Integration der Zell-Hydrogelkonstrukte in natives Knorpelgewebe sowie die Defektdeckung in einem in vitro- und einem in vivo-Knorpeldefektmodell mit vor- und nicht-vordifferenzierten Zell-Hydrogelkonstrukten untersucht. Die Analyse m{\"o}glicher zyto- und genotoxischer Effekte von VSOP sowie des Einflusses der Markierung von ASC mit VSOP auf die Proliferation, Migration und das Multidifferenzierungspotential erfolgte nach der Markierung der Zellen mit unterschiedlichen VSOP-Konzentrationen. Außerdem wurden VSOP-markierte ASC in Kaninchenstimmlippen injiziert und die Nachweisbarkeit dieser Zellen im Injektionsareal histologisch und mittels Magnetresonanztomographie (MRT) untersucht. Nach 14-t{\"a}giger chondrogener Differenzierung wurde in den Zell-Hydrogelkonstrukten eine knorpel{\"a}hnliche Morphologie, die Anreicherung knorpelspezifischer Matrixproteine und die Expression chondrogener Markergene nachgewiesen. Die Kombination der chondrogenen Wachstumsfaktoren zeigte keinen verst{\"a}rkenden Einfluss auf die Chondrogenese von ASC. Hydrogele aus Kollagen Typ I und Hyalurons{\"a}ure wiesen die st{\"a}rkste extrazellul{\"a}re Matrixanreicherung auf. Bei den agarosefreien Hydrogelen war eine ausgepr{\"a}gte Gelschrumpfung auff{\"a}llig. In den beiden Knorpeldefektmodellen konnte weder eine Integration der Zell-Hydrogelkonstrukte in den Nativknorpel noch eine vollst{\"a}ndige Defektdeckung nachgewiesen werden. Nach der Markierung von ASC mit VSOP zeigte sich bei der h{\"o}chsten Konzentration von 1,5 mM eine genotoxische Wirkung. Zytotoxische Effekte sowie Einfl{\"u}sse der Markierung auf die Proliferation, Migration und das Multidifferenzierungspotential von ASC waren nicht nachweisbar. VSOP-markierte ASC konnten nach deren Injektion in Kaninchenstimmlippen im Injektionsareal nur vereinzelt mittels MRT und histologisch nachgewiesen werden. Es ist m{\"o}glich, mit Hilfe des Tissue Engineerings aus k{\"o}rpereigenen Stammzellen und biokompatiblen Tr{\"a}germaterialien implantierbares knorpel{\"a}hnliches Gewebe herzustellen. Dabei beg{\"u}nstigen agarosefreie Tr{\"a}germaterialien die chondrogene Differenzierung von ASC. Diese k{\"o}nnte durch die jeweilige Erh{\"o}hung der Zelldichte und Wachstumsfaktorkonzentrationen sowie die Verl{\"a}ngerung der Induktionszeit verst{\"a}rkt werden. Eine m{\"o}gliche klinische Anwendung dieser knorpel{\"a}hnlichen Gewebe in der Laryngologie ist jedoch durch deren Schrumpfung wie auch mangelnde Integration und Defektdeckung noch weit entfernt. Aufgrund ihrer genotoxischen Wirkung kann eine Verwendung von VSOP als Zellmarker auch unterhalb von 1,5 mM ohne negative Folgen f{\"u}r den Organismus nicht sicher ausgeschlossen werden. Der inhomogene Gewebekontrast im Kehlkopf, die schlechte Aufl{\"o}sung im MRT und die geringe Gr{\"o}ße von VSOP erschweren die Nachweisbarkeit und Verfolgung markierter Zellen mittels MRT. Daher sind andere nicht-invasive Nachweismethoden f{\"u}r die Verwendung von VSOP im Kehlkopf zu evaluieren. Der m{\"o}glichen Anwendung dieser knorpel{\"a}hnlichen Gewebe und VSOP in der rekonstruktiven Laryngologie muss eine erfolgreiche Optimierung und ausf{\"u}hrliche positive Validierung in klinischen Tests vorausgehen.},
  subject      = {Tissue Engineering},
  language  = {de}
}
@phdthesis{Graver2015,
  author    = {Graver, Shannon},
  title     = {Molecular and cellular cross talk between angiogenic, immune and DNA mismatch repair pathways},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108302},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {VEGF is a main driver of tumor angiogenesis, playing an important role not only in the formation of new blood vessels, but also acts as a factor for cell migration, proliferation, survival and apoptosis. Angiogenesis is a universal function shared by most solid tumors and its inhibition was thought to have the potential to work across a broad patient population. Clinical evidence has shown that inhibiting pathological angiogenesis only works in a subset of patients and the identification of those patients is an important step towards personalized cancer care. The first approved antiangiogenic therapy was bevacizumab (Avastin®), a monoclonal antibody targeting VEGF in solid tumors including CRC, BC, NSCLC, RCC and others. In addition to endothelial cells, VEGF receptors are present on a number of different cell types including tumor cells, monocytes and macrophages. The work presented in this thesis looked at the in vitro cellular changes in tumor cells and leukocytes in response to the inhibition of VEGF signaling with the use of bevacizumab. In the initial experiments, VEGF was induced by hypoxia in tumor cells to evaluate changes in survival, proliferation, migration and changes in gene or protein expression. There was a minimal direct response of VEGF inhibition in tumor cells that could be attributed to bevacizumab treatment, with minor variations in some of the cell lines screened but no uniform or specific response noted. MMR deficiency often results in microsatellite instability (MSI) in tumors, as opposed to microsatellite stable (MSS) tumors, and accounts for up to 15\% of colorectal carcinomas (CRCs). It has been suggested in clinical data that MMR deficient tumors responded better to bevacizumab regimens, therefore further research used isogenic paired CRC tumor cell lines (MMR deficient and proficient). Furthermore, a DNA damaging agent was added to the treatment regimen, the topoisomerase inhibitor SN-38 (the active metabolite of irinotecan). Inhibiting VEGF using bevacizumab significantly inhibited the ability of MMR deficient tumor cells to form anchor dependent colonies, however conversely, bevacizumab treatment before damaging cells with SN-38, showed a significant increase in colony numbers. Moreover, VEGF inhibition by bevacizumab pretreatment also significantly increased the mutation fraction in MMR deficient cells as measured by transiently transfecting a dinucleotide repeat construct, suggesting VEGF signaling may have an intrinsic role in MMR deficient cells. A number of pathways were analyzed in addition to changes in gene expression profiles resulting in the identification of JNK as a possible VEGF targeted pathway. JUN expression was also reduced in these conditions reinforcing this hypothesis, however the intricate molecular mechanisms remain to be elucidated. In order to remain focused on the clinical application of the findings, it was noted that some cytokines were differentially regulated by bevacizumab between MMR proficient and deficient cells. Treatment regimens employed in vitro attempted to mimic the clinical setting by inducing DNA damage, then allowing cells to recover with or without VEGF using bevacizumab treatment. Inflammatory cytokines, CCL7 and CCL8, were found to have higher expression in the MMR deficient cell line with bevacizumab after DNA damage, therefore the cross talk via tumor derived factors to myeloid cells was analyzed. Gene expression changes in monocytes induced by tumor conditioned media showed CCL18 to be a bevacizumab regulated gene by MMR deficient cells and less so in MMR proficient cells. CCL18 has been described as a prognostic marker in gastric, colorectal and ovarian cancers, however the significance is dependent on tumor type. CCL18 primarily exerts its function on the adaptive immune system to trigger a TH2 response in T cells, but is also described to increase non-specific phagocytosis. The results of this study did show an increase in the phagocytic activity of macrophages in the presence of bevacizumab that was significantly more apparent in MMR deficient cells. Furthermore, after DNA damage MMR deficient cells treated with bevacizumab released a cytokine mix that induced monocyte migration in a bevacizumab dependent manner, showing a functional response with the combination of MMR deficiency and bevacizumab. In summary, the work in this thesis has shown evidence of immune cell modulation that is specific to MMR deficient tumor cells that may translate into a marker for the administration of bevacizumab in a clinical setting. VEGF ist ein zentraler Regulator der Tumor-Angiogenese, und spielt eine wichtige Rolle nicht nur in der Bildung von neuen Blutgef{\"a}ßen, sondern ist auch f{\"u}r die Migration, Proliferation, das {\"U}berleben und Apoptose von Tumorzellen essentiell. Angiogenese ist eine der universellen Funktionen, welche das Wachstum der meisten soliden Tumoren charakterisiert. Eine der klassischen therapeutischen Ideen wurde auf der Basis entwickelt, dass die spezifische Hemmung der Angiogenese das Potenzial hat in einer breiten Patientenpopulation einen klinischen Effekt zu zeigen. Die klinische Erfahrung und Anwendung hat jedoch gezeigt, dass die Hemmung der pathologischen Angiogenese nur in einem Teil der Patienten einen therapeutischen Nutzen aufweist. Somit stellt die Identifikation derjenigen Patienten, welche von der anti-angiogenen Therapie profitieren, einen wichtiger Schritt zur personalisierten Krebsbehandlung dar. Die erste zugelassene antiangiogene Therapie war Bevacizumab (Avastin®), ein monoklonaler Antik{\"o}rper gegen VEGF, welcher unter anderem in soliden Tumoren wie CRC, BC, nicht-kleinzelligem Lungenkrebs (NSCLC) und dem Nierenzellkarzinom angewandt wird. VEGF-Rezeptoren befinden sich nicht nur auf Endothelzellen, sondern sind auch auf einer Anzahl von verschiedenen Zelltypen, einschließlich Tumorzellen, Monozyten und Makrophagen nachweisbar. Die in dieser Arbeit vorgestellten Ergebnisse befassen sich mit den zellul{\"a}ren Ver{\"a}nderungen an Tumorzellen und Leukozyten als Reaktion auf die Hemmung der VEGF-Signalkaskade durch Bevacizumab in-vitro. In den Initialen Experimenten wurde VEGF durch Hypoxie in Tumorzellen induziert und Ver{\"a}nderungen der {\"U}berlebensrate, der Proliferation, Migration als auch in der Gen- oder Protein-Expression gemessen. Es konnte eine minimale direkte Reaktion der VEGF-Hemmung auf Tumorzellen beobachtet werden, welche auf die Bevacizumab Behandlung zur{\"u}ckgef{\"u}hrt werden k{\"o}nnte. Es zeigten sich aber auch geringf{\"u}gige Abweichungen in einigen der verwendeten Zellinien, die keine einheitliche Interpretation erlauben oder auf eine uniformelle Reaktion hinweisen w{\"u}rden. Das ph{\"a}notypische Korrelat einer „Mismatch" Reparatur (MMR)-Defizienz ist die Mikrosatelliteninstabilit{\"a}t im Gegensatz zu mikrosatellitenstabilen Tumoren und findet sich bei bis zu 15\% der kolorektalen Karzinomen (CRC) wieder. Klinischen Daten deuten daraufhin, dass Bevacizumab besser in MMR-defizienten Tumoren wirkt. Daher wurden die weiteren Untersuchungen in gepaarten MMR stabilen und MMR instabilen CRC-Tumorzelllinien (MMR defizient und kompetent) durchgef{\"u}hrt. Weiterhin wurde ein DNA-sch{\"a}digendes Agens, SN-38, ein Topoisomerase-Inhibitor (der aktive Metabolit von Irinotecan) dem Behandlungsschema zugef{\"u}gt. Es zeigte sich, dass die Hemmung von VEGF mittels Bevacizumab die F{\"a}higkeit der MMR defizienten Tumorzellen Kolonien zu bilden signifikant inhibiert. Im Gegensatz dazu, hatte die Behandlung von Bevacizumab vor der Zugabe des DNA sch{\"a}digenden Agens zu einer vermehrten Kolonienzahl gef{\"u}hrt. Außerdem erh{\"o}hte die Vorbehandlung mit Bevacizumab deutlich die Mutationsrate in MMR-defizienten Zellen, was durch die transiente Transfektion eines Dinukleotid-Repeat-Konstrukts nachgewiesen werden konnte. Dies deutete darauf hin, dass VEGF eine intrinsische Rolle in der Signalkaskade des MMR-Systems haben k{\"o}nnte. Deshalb wurde eine Anzahl von Signalalkaskaden zus{\"a}tzlich zu Ver{\"a}nderungen von Genexpressionsprofilen untersucht und JNK als m{\"o}gliche Verbindungsstelle der beiden Signalkaskaden, VEGF und MMR, identifiziert. Diese Hypothese wurde zus{\"a}tzlich unterst{\"u}tzt durch die Tatsache, dass die JUN Expression unter diesen experimentellen Bedingungen reduziert war. Die Aufkl{\"a}rung der komplexen molekularen Mechanismen der potentiellen Interaktion bleibt zuk{\"u}nftigen Untersuchungen vorbehalten. In Hinblick auf die klinische Konsequenz der erhaltenen Ergebnisse war es auff{\"a}llig, dass einige Zytokine durch Bevacizumab in den MMR defizienten Zellen im Gegensatz zu den MMR kompetenten Zellen unterschiedlich reguliert wurden. Die in-vitro verwendeten Behandlungsschemata waren den klinisch zur Anwendung kommenden Protokollen nachempfunden. Zuerst wurde ein DNA-Schaden gesetzt, und den Zellen erm{\"o}glicht, sich mit oder ohne Bevacizumab zu erholen. Es konnte gezeigt werden, dass die inflammatorischen Zytokine CCL7 und CCL8 eine h{\"o}here Expression in der MMR-defiziente Zelllinie in Kombination mit Bevacizumab aufweisen. Daher wurde ein m{\"o}glicher Crosstalk zwischen von Tumorzellen sezernierten Faktoren und myeloischen Zellen weiter verfolgt. Ver{\"a}nderungen der Genexpression in Monozyten durch Tumorzell- konditionierte Medien zeigte CCL18 als ein Bevacizumab reguliertes Gen in MMR-defizienten Zellen, aber nicht in MMR kompetenten Zellen. CCL18 {\"u}bt seine Funktion prim{\"a}r im adaptiven Immunsystems aus um eine TH2-Antwort in T-Zellen auszul{\"o}sen Ausserdem wird eine Erh{\"o}hung der nicht-spezifische Phagozytose als weitere Funktion beschrieben. CCL18 wurde bereits als prognostischer Marker in Magen-, Dickdarm- und Eierstockkrebsarten beschrieben; die klinische Bedeutung ist jedoch abh{\"a}ngig von Tumortyp. Die Ergebnisse dieser Arbeit zeigen, dass eine Erh{\"o}hung der phagozytischen Aktivit{\"a}t von Makrophagen in Gegenwart von Bevacizumab wesentlich deutlicher in MMR-defizienten Zellen ausgepr{\"a}gt war. Weiterhin wurde gefunden, dass nach DNA-Sch{\"a}digung in Bevacizumab behandelten MMR-defizienten Zellen Zytokine freigesetzt werden, welche eine Monozytenmigration in einer Bevacizumab-abh{\"a}ngigen Weise induzieren. Dies weist auf eine funktionelle Interaktion von MMR-Defizienz und Bevacizumab hin. Zus{\"a}tzlich zeigen die Ergebnisse dieser Arbeit eine Immunzellmodulation, die spezifisch f{\"u}r Mismatch-Reparatur defiziente Tumorzellen ist und in der klinischen Praxis als Marker f{\"u}r die Verabreichung von Bevacizumab verwendet werden k{\"o}nnte.},
  subject      = {Vascular endothelial Growth Factor},
  language  = {en}
}
@phdthesis{Zhu2015,
  author    = {Zhu, Ana Cheng},
  title     = {Metagenomic analysis of genetic variation in human gut microbial species},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113890},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Microbial species (bacteria and archaea) in the gut are important for human health in various ways. Not only does the species composition vary considerably within the human population, but each individual also appears to have its own strains of a given species. While it is known from studies of bacterial pan-genomes, that genetic variation between strains can differ considerably, such as in Escherichia coli, the extent of genetic variation of strains for abundant gut species has not been surveyed in a natural habitat. This is mainly due to the fact that most of these species cannot be cultured in the laboratory. Genetic variation can range from microscale genomic rearrangements such as small nucleotide polymorphism (SNP) to macroscale large genomic rearrangements like structural variations. Metagenomics offers an alternative solution to study genetic variation in prokaryotes, as it involves DNA sequencing of the whole community directly from the environment. However, most metagenomic studies to date only focus on variation in gene abundance and hence are not able to characterize genetic variation (in terms of presence or absence of SNPs and genes) of gut microbial strains of individuals. The aim of my doctorate studies was therefore to study the extent of genetic variation in the genomic sequence of gut prokaryotic species and its phenotypic effects based on: (1) the impact of SNP variation in gut bacterial species, by focusing on genes under selective pressure and (2) the gene content variation (as a proxy for structural variation) and their effect on microbial species and the phenotypic traits of their human host. In the first part of my doctorate studies, I was involved in a project in which we created a catalogue of 10.3 million SNPs in gut prokaryotic species, based on metagenomes. I used this to perform the first SNP-based comparative study of prokaryotic species evolution in a natural habitat. Here, I found that strains of gut microbial species in different individuals evolve at more similar rates than the strains within an individual. In addition, I found that gene evolution can be uncoupled from the evolution of its originating species, and that this could be related to selective pressure such as diet, exemplified by galactokinase gene (galK). Despite the individuality (i.e. uniqueness of each individual within the studied metagenomic dataset) in the SNP profile of the gut microbiota that we found, for most cases it is not possible to link SNPs with phenotypic differences. For this reason I also used gene content as a proxy to study structural variation in metagenomes. In the second part of my doctorate studies, I developed a methodology to characterize the variability of gene content in gut bacterial species, using metagenomes. My approach is based on gene deletions, and was applied to abundant species (demonstrated using a set of 11 species). The method is sufficiently robust as it captures a similar range of gene content variability as has been detected in completely sequenced genomes. Using this procedure I found individuals differ by an average of 13\% in their gene content of gut bacterial strains within the same species. Interestingly no two individuals shared the same gene content across bacterial species. However, this variation corresponds to a lower limit, as it is only accounts for gene deletion and not insertions. This large variation in the gene content of gut strain was found to affect important functions, such as polysaccharide utilization loci (PULs) and capsular polysaccharide synthesis (CPS), which are related with digestion of dietary fibers. In summary, I have shown that metagenomics based approaches can be robust in characterizing genetic variation in gut bacterial species. I also illustrated, using examples both for SNPs and gene content (galK, PULs and CPS), that this genetic variation can be used to predict the phenotypic characteristics of the microbial species, as well as predicting the phenotype of their human host (for example, their capacity to digest different food components). Overall, the results of my thesis highlight the importance of characterizing the strains in the gut microbiome analogous to the emerging variability and importance of human genomics.},
  subject      = {Darmflora},
  language  = {en}
}
@phdthesis{Muenz2015,
  author    = {M{\"u}nz, Thomas Sebastian},
  title     = {Aspects of neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee Apis mellifera},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111611},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Division of labor represents a major advantage of social insect communities that accounts for their enormous ecological success. In colonies of the honeybee, Apis mellifera, division of labor comprises different tasks of fertile queens and drones (males) and, in general, sterile female workers. Division of labor also occurs among workers in form of an age-related polyethism. This helps them to deal with the great variety of tasks within the colony. After adult eclosion, workers spend around three weeks with various duties inside the hive such as tending the brood or cleaning and building cells. After this period workers switch to outdoor tasks and become foragers collecting nectar, pollen and water. With this behavioral transition, workers face tremendous changes in their sensory environment. In particular, visual sensory stimuli become important, but also the olfactory world changes. Foragers have to perform a completely new behavioral repertoire ranging from long distance navigation based on landmark orientation and polarized-skylight information to learning and memory tasks associated with finding profitable food sources. However, behavioral maturation is not a purely age-related internal program associated with a change, for example, in juvenile hormone titers. External factors such as primer pheromones like the brood pheromone or queen mandibular pheromone can modulate the timing of this transition. In this way colonies are able to flexibly adjust their work force distribution between indoor and outdoor tasks depending on the actual needs of the colony. Besides certain physiological changes, mainly affecting glandular tissue, the transition from indoor to outdoor tasks requires significant adaptations in sensory and higher-order integration centers of the brain. The mushroom bodies integrate olfactory, visual, gustatory and mechanosensory information. Furthermore, they play important roles in learning and memory processes. It is therefore not surprising that the mushroom bodies, in particular their main input region, the calyx, undergo volumetric neuronal plasticity. Similar to behavioral maturation, plastic changes of the mushroom bodies are associated with age, but are also to be affected by modulating factors such as task and experience. In my thesis, I analyzed in detail the neuronal processes underlying volumetric plasticity in the mushroom body. Immunohistochemical labeling of synaptic proteins combined with quantitative 3D confocal imaging revealed that the volume increase of the mushroom body calyx is largely caused by the growth of the Kenyon cell dendritic network. This outgrowth is accompanied by changes in the synaptic architecture of the mushroom body calyx, which is organized in a distinct pattern of synaptic complexes, so called microglomeruli. During the first week of natural adult maturation microglomeruli remain constant in total number. With subsequent behavioral transition from indoor duties to foraging, microglomeruli are pruned while the Kenyon cell dendritic network is still growing. As a result of these processes, the mushroom body calyx neuropil volume enlarges while the total number of microgloumeruli becomes reduced in foragers compared to indoor workers. In the visual subcompartments (calyx collar) this process is induced by visual sensory stimuli as the beginning of pruning correlates with the time window when workers start their first orientation flights. The high level of analysis of cellular and subcellular process underlying structural plasticity of the mushroom body calyx during natural maturation will serve as a framework for future investigations of behavioral plasticity in the honeybee. The transition to foraging is not purely age-dependent, but gets modulated, for example, by the presence of foragers. Ethyl oleate, a primer pheromone that is present only in foragers, was shown to delay the onset of foraging in nurse bees. Using artificial application of additional ethyl oleate in triple cohort colonies, I tested whether it directly affects adult neuronal plasticity in the visual input region of the mushroom body calyx. As the pheromonal treatment failed to induce a clear behavioral phenotype (delayed onset of foraging) it was not possible to show a direct link between the exposure to additional ethyl oleate and neuronal plasticity in mushroom body calyx. However, the general results on synaptic maturation confirmed my data of natural maturation processes in the mushroom body calyx. Given the result that dendritic plasticity is a major contributor to neuronal plasticity in the mushroom body calyx associated with division of labor, the question arose which proteins could be involved in mediating these effects. Calcium/calmodulin-dependent protein kinase II (CaMKII) especially in mammals, but also in insects (Drosophila, Cockroach), was shown to be involved in facilitating learning and memory processes like long-term synaptic potentiation. In addition to presynaptic effects, the protein was also revealed to directly interact with cytoskeleton elements in the postsynapse. It therefore is a likely candidate to mediate structural synaptic plasticity. As part of my thesis, the presence and distribution of CaMKII was analyzed, and the results showed that the protein is highly concentrated in a distinct subpopulation of the mushroom body intrinsic neurons, the noncompact Kenyon cells. The dendritic network of this population arborizes in two calyx subregions: one receiving mainly olfactory input - the lip - and the collar receiving visual input. This distribution pattern did not change with age or task. The high concentration of CaMKII in dendritic spines and its overlap with f-actin indicates that CaMKII could be a key player inducing structural neuronal plasticity associated with learning and memory formation and/or behavioral transitions related to division of labor. Interestingly CaMKII immunoreactivity was absent in the basal ring, another subregion of the mushroom body calyx formed almost exclusively by the inner compact Kenyon cells and known to receive combined visual and olfactory input. This indicates differences of this mushroom body subregion regarding the molecular mechanisms controlling plastic changes in corresponding Kenyon cells. How is timing of behavioral and neuronal plasticity regulated? The primer pheromone ethyl oleate was found in high concentrations on foragers and was shown to influence behavioral maturation by delaying the onset of foraging when artificially applied in elevated concentrations. But how is ethyl oleate transferred and how does it shift the work force distribution between indoor and outdoor tasks? Previous work showed that ethyl oleate concentrations are highest in the honeycrop of foragers and suggested that it is transferred and communicated inside the colony via trophallaxis. The results of this thesis however clearly show, that ethyl oleate was not present inside the honey crop or the regurgitate, but rather in the surrounding tissue of the honey crop. As additionally the second highest concentration of ethyl oleate was measured on the surface of the cuticle of forgers, trophallaxis was ruled out as a mode of transmission. Neurophysiological measurements at the level of the antennae (electroantennogram recordings) and the first olfactory neuropil (calcium imaging of activity in the antennal lobe) revealed that the primer pheromone ethyl oleate is received and processed as an olfactory stimulus. Appetitive olfactory conditioning using the proboscis extension response as a behavioral paradigm showed that ethyl oleate can be associated with a sugar reward. This indicates that workers are able to perceive, learn and memorize the presence of this pheromone. As ethyl oleate had to be presented by a heated stimulation device at close range, it can be concluded that this primer pheromone acts via close range/contact chemoreception through the olfactory system. This is also supported by previous behavioral observations. Taken together, the findings presented in this thesis revealed structural changes in the synaptic architecture of the mushroom body calyx associated with division of labor. For the primer pheromone ethyl oleate, which modulates the transition from nursing to foraging, the results clearly showed that it is received via the olfactory system and presumably acts via this pathway. However, manipulation experiments did not indicate a direct effect of ethyl oleate on synaptic plasticity. At the molecular level, CaMKII is a prime candidate to mediate structural synaptic plasticity in the mushroom body calyx. Future combined structural and functional experiments are needed to finally link the activity of primer pheromones like ethyl oleate to the molecular pathways mediating behavioral and synaptic plasticity associated with division of labor in Apis mellifera. The here identified underlying processes will serve as excellent models for a general understanding of fundamental mechanisms promoting behavioral plasticity.},
  subject      = {Biene},
  language  = {en}
}
@phdthesis{Stoll2015,
  author    = {Stoll, Georg},
  title     = {Identification of the mRNA-associated TOP3β- TDRD3-FMRP (TTF) -complex and its implication for neurological disorders},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111440},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The propagation of the genetic information into proteins is mediated by messenger- RNA (mRNA) intermediates. In eukaryotes mRNAs are synthesized by RNA- Polymerase II and subjected to translation after various processing steps. Earlier it was suspected that the regulation of gene expression occurs primarily on the level of transcription. In the meantime it became evident that the contribution of post- transcriptional events is at least equally important. Apart from non-coding RNAs and metabolites, this process is in particular controlled by RNA-binding proteins, which assemble on mRNAs in various combinations to establish the so-called "mRNP- code". In this thesis a so far unknown component of the mRNP-code was identified and characterized. It constitutes a hetero-trimeric complex composed of the Tudor domain-containing protein 3 (TDRD3), the fragile X mental retardation protein (FMRP) and the Topoisomerase III beta (TOP3β) and was termed TTF (TOP3β-TDRD3-FMRP) -complex according to its composition. The presented results also demonstrate that all components of the TTF-complex shuttle between the nucleus and the cytoplasm, but are predominantly located in the latter compartment under steady state conditions. Apart from that, an association of the TTF-complex with fully processed mRNAs, not yet engaged in productive translation, was detected. Hence, the TTF-complex is a component of „early" mRNPs. The defined recruitment of the TTF-complex to these mRNPs is not based on binding to distinct mRNA sequence-elements in cis, but rather on an interaction with the so-called exon junction complex (EJC), which is loaded onto the mRNA during the process of pre-mRNA splicing. In this context TDRD3 functions as an adapter, linking EJC, FMRP and TOP3β on the mRNP. Moreover, preliminary results suggest that epigenetic marks within gene promoter regions predetermine the transfer of the TTF-complex onto its target mRNAs. Besides, the observation that TOP3β is able to catalytically convert RNA-substrates disclosed potential activities of the TTF-complex in mRNA metabolism. In combination with the already known functions of FMRP, this finding primarily suggests that the TTF-complex controls the translation of bound mRNAs. In addition to its role in mRNA metabolism, the TTF-complex is interesting from a human genetics perspective as well. It was demonstrated in collaboration with researchers from Finland and the US that apart from FMRP, which was previously linked to neurocognitive diseases, also TOP3β is associated with neurodevelopmental disorders. Understanding the function of the TTF-complex in mRNA metabolism might hence provide important insight into the etiology of these diseases.},
  subject      = {Messenger-RNS},
  language  = {en}
}
@phdthesis{Kuhtz2015,
  author    = {Kuhtz, Juliane},
  title     = {Epimutationen humaner Keimzellen und Infertilit{\"a}t},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108248},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Infertilit{\"a}t stellt in unserer heutigen Gesellschaft ein zunehmendes Problem dar. Bei der Suche nach den der Infertilit{\"a}t zugrunde liegenden Ursachen ger{\"a}t immer mehr die Epigenetik in den Fokus. Epigenetische Prozesse sind nicht nur in die Embryo-nalentwicklung und Wachstumsprozesse des Kindes involviert, sondern auch in korrekte Funktionsweisen von Gameten. St{\"o}rungen k{\"o}nnen die Fertilit{\"a}t beeintr{\"a}ch-tigen. Eine besondere Rolle spielen gepr{\"a}gte Gene, die auf einem ihrer Allele, je nach parentaler Herkunft, ein Imprint in Form von DNA-Methylierung tragen. Fehl-regulationen solcher gepr{\"a}gter Gene k{\"o}nnen zu Imprinting-Erkrankungen f{\"u}hren. Seit Einf{\"u}hrung der In-vitro-Fertilisation (IVF) wurden verschiedene assistierte Re-produktionstechniken (ART) entwickelt, um infertilen Paaren zu helfen. Die Sicher-heit dieser Techniken ist nicht abschließend gekl{\"a}rt. Immer wieder wird von nach ART-Behandlung geh{\"a}uft auftretenden Imprinting-Erkrankungen berichtet. Diese Erkrankungen stehen jedoch eher in Zusammenhang mit der zugrunde liegenden Infertilit{\"a}t, als mit ART selbst. Dennoch ist es notwendig zu untersuchen inwieweit sich ART eventuell auf den Gesundheitszustand dieser Kinder auswirken k{\"o}nnte. In der hier vorgelegten Arbeit wurde der Zusammenhang von Epigenetik, Infertilit{\"a}t und ART von verschiedenen Standpunkten aus beleuchtet. In humanen Spermien wurde die DNA-Methylierung verschiedener gepr{\"a}gter Gene hinsichtlich Epimutationen untersucht. ICSI (intracytoplasmatic sperm injection) und IMSI (intracytoplasmic morphologically selected sperm injection) sind verschiedene Techniken zur Selektion von Spermien f{\"u}r eine ART-Behandlung. Hier wurde un-tersucht, ob IMSI epigenetisch bessere Spermien selektiert als die konventionelle ICSI-Methode. Außerdem ist bekannt, dass in Spermienk{\"o}pfen fertiler und infertiler M{\"a}nner Vakuolen vorkommen k{\"o}nnen, deren epigenetische Bedeutung jedoch un-bekannt ist. Ob diese Vakuolen in Zusammenhang mit Epimutationen stehen k{\"o}nn-ten, wurde ebenfalls {\"u}berpr{\"u}ft. Dazu wurde bisulfitkonvertierte DNA weniger Sper-mien (11 je Probe) mithilfe der Limiting Dilution (LD)-Technik und Pyrosequenzie-rung analysiert. Insgesamt standen 880 Spermien f{\"u}r diese Untersuchung zur Ver-f{\"u}gung. Es konnte kein Unterschied zwischen IMSI- und ICSI-selektierten Spermien gefunden werden. Vorhandene Vakuolen im Spermienkopf beeintr{\"a}chtigten nicht die DNA-Methylierung der Gene hGTL2, hLIT1 und hPEG3. Ein weiteres Projekt befasste sich mit der Frage, inwieweit die Technik der In-vitro-Maturation (IVM) DNA-Methylierung in humanen Oocyten beeinflussen k{\"o}nnte. Bisulfitkonvertierte DNA einzelner humaner Oocyten wurde mit LD und Pyrose-quenzierung analysiert. Verglichen wurden IVM und in vivo gereifte Oocyten. Hier-f{\"u}r standen 139 Oocyten zur Verf{\"u}gung, wovon 90 mittels IVM und 49 in vivo gereift waren. Untersucht wurden vier gepr{\"a}gte Gene (hGTL2, hLIT1, hPEG3 und hSNRPN) und drei nicht gepr{\"a}gte Gene (hDNMT3Lo, hNANOG und hOCT4). Es konnten keine IVM-bedingten Epimutationen gefunden werden. Im dritten Projekt wurde untersucht, ob sich die DNA-Methylierung normaler Sper-mien von Spermien aus Oligozoospermie-Asthenozoospermie-Teratozoospermie (OAT)-Syndrom-Patienten unterscheidet. Eine weitere Frage war, ob Epimutationen einen Einfluss auf den ART-Ausgang haben. Untersucht wurden 54 Spermienpro-ben von Paaren in ART-Behandlung. Zur Untersuchung der DNA-Methylierungsmuster der gepr{\"a}gten Gene hGTL2 und hPEG3 sowie der beiden nicht gepr{\"a}gten Pluripotenzgene hNANOG und hOCT4 wurde die Methode Deep Bisulfite Sequencing (DBS) verwendet. Dies ist eine Next Generation Sequencing (NGS)-Technik, angewandt an bisulfitkonvertierter DNA. Diese Technik erm{\"o}glicht es mehrere Proben sowie Gene gleichzeitig zu analysieren. Es zeigte sich, dass OAT-Spermien, die zu einer Lebendgeburt gef{\"u}hrt hatten, sich epigenetisch nicht von normalen Spermien unterschieden. Besonders viele Epimutationen konnten hingegen in OAT-Spermien gefunden werden, die zu keiner Schwangerschaft ge-f{\"u}hrt hatten. Zwischen Spermien die zu einer Lebendgeburt oder keiner Schwan-gerschaft gef{\"u}hrt hatten, zeigten sich Unterschiede in der H{\"a}ufigkeit von hGTL2-Epimutationen. {\"U}ber die H{\"a}ufigkeit von Epimutationen konnte eine pr{\"a}diktive Aus-sage zum ART-Ausgang getroffen werden. Zusammenfassend konnte in dieser Arbeit festgestellt werden, dass sich eine H{\"a}u-fung von Epimutationen darauf auswirkt, ob eine Schwangerschaft erreicht werden kann oder nicht. Diese Epimutationen liegen bereits im parentalen Genom vor. Sie werden nicht durch ART verursacht. Allerdings muss man Techniken finden, mit denen man Gameten mit m{\"o}glichst wenig Epimutationen selektiert, um eine {\"U}ber-tragung solcher auf das Kind zu verhindern.},
  subject      = {Epigenetik},
  language  = {de}
}