@phdthesis{Weisert2024,
  author    = {Weisert, Nadine},
  title     = {Characterization of telomere-associated proteins in \(Trypanosoma\) \(brucei\)},
  doi       = {10.25972/OPUS-35273},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-352732},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The unicellular pathogen Trypanosoma brucei is the causative agent of African trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF. In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.},
  subject      = {Telomer <Molekulargenetik>},
  language  = {en}
}
@phdthesis{BakariSoale2024,
  author    = {Bakari Soale, Majeed},
  title     = {Regulation of the Variant Surface Glycoprotein (VSG) Expression and Characterisation of the Nucleolar DExD/H box Protein Hel66 in \(Trypanosoma\) \(brucei\)},
  doi       = {10.25972/OPUS-25809},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-258090},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The variant surface glycoprotein (VSG) of African trypanosomes plays an essential role in protecting the parasites from host immune factors. These trypanosomes undergo antigenic variation resulting in the expression of a single VSG isoform out of a repertoire of around 2000 genes. The molecular mechanism central to the expression and regulation of the VSG is however not fully understood. Gene expression in trypanosomes is unusual due to the absence of typical RNA polymerase II promoters and the polycistronic transcription of genes. The regulation of gene expression is therefore mainly post-transcriptional. Regulatory sequences, mostly present in the 3´ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 100 \% conserved 16mer motif within the 3´ UTR has been shown to modulate the stability of VSG transcripts and hence their expression. As a stability-associated sequence element, the absence of nucleotide substitutions in the motif is however unusual. It was therefore hypothesised that the motif is involved in other essential roles/processes besides stability of the VSG transcripts. In this study, it was demonstrated that the 100 \% conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. It was further shown that the intact motif in the active VSG 3´ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Crosstalk between the VSG and procyclin genes during differentiation to the insect vector stage is also unaffected in cells with a mutated 16mer motif. Ectopic overexpression of a second VSG however requires the intact motif to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The 16mer motif therefore plays a dual role in VSG in situ switching and stability of VSG transcripts. The additional role of the 16mer in the essential process of antigenic variation appears to be the driving force for the 100 \% conservation of this RNA motif. A screen aimed at identifying candidate RNA-binding proteins interacting with the 16mer motif, led to the identification of a DExD/H box protein, Hel66. Although the protein did not appear to have a direct link to the 16mer regulation of VSG expression, the DExD/H family of proteins are important players in the process of ribosome biogenesis. This process is relatively understudied in trypanosomes and so this candidate was singled out for detailed characterisation, given that the 16mer story had reached a natural end point. Ribosome biogenesis is a major cellular process in eukaryotes involving ribosomal RNA, ribosomal proteins and several non-ribosomal trans-acting protein factors. The DExD/H box proteins are the most important trans-acting protein factors involved in the biosynthesis of ribosomes. Several DExD/H box proteins have been directly implicated in this process in yeast. In trypanosomes, very few of this family of proteins have been characterised and therefore little is known about the specific roles they play in RNA metabolism. Here, it was shown that Hel66 is involved in rRNA processing during ribosome biogenesis. Hel66 localises to the nucleolus and depleting the protein led to a severe growth defect. Loss of the protein also resulted in a reduced rate of global translation and accumulation of rRNA processing intermediates of both the small and large ribosomal subunits. Hel66 is therefore an essential nucleolar DExD/H protein involved in rRNA processing during ribosome biogenesis. As very few protein factors involved in the processing of rRNAs have been described in trypanosomes, this finding represents an important platform for future investigation of this topic.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{Koenig2024,
  author    = {K{\"o}nig, Sebastian Thomas},
  title     = {Temperature-driven assembly processes of Orthoptera communities: Lessons on diversity, species traits, feeding interactions, and associated faecal microorganisms from elevational gradients in Southern Germany (Berchtesgaden Alps)},
  doi       = {10.25972/OPUS-35460},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-354608},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Chapter I: Introduction Temperature is a major driver of biodiversity and abundance patterns on our planet, which becomes particularly relevant facing the entanglement of an imminent biodiversity and climate crisis. Climate shapes the composition of species assemblages either directly via abiotic filtering mechanisms or indirectly through alterations in biotic interactions. Insects - integral elements of Earth's ecosystems - are affected by climatic variation such as warming, yet responses vary among species. While species' traits, antagonistic biotic interactions, and even species' microbial mutualists may determine temperature-dependent assembly processes, the lion's share of these complex relationships remains poorly understood due to methodological constraints. Mountains, recognized as hotspots of diversity and threatened by rapidly changing climatic conditions, can serve as natural experimental settings to study the response of insect assemblages and their trophic interactions to temperature variation, instrumentalizing the high regional heterogeneity of micro- and macroclimate. With this thesis, we aim to enhance our mechanistic understanding of temperature-driven assembly processes within insect communities, exemplified by Orthoptera, that are significant herbivores in temperate mountain grassland ecosystems. Therefore, we combined field surveys of Orthoptera assemblages on grassland sites with molecular tools for foodweb reconstruction, primarily leveraging the elevational gradients offered by the complex topography within the Berchtesgaden Alpine region (Bavaria, Germany) as surrogate for temperature variation (space-for-time substitution approach). In this framework, we studied the effects of temperature variation on (1) species richness, abundance, community composition, and interspecific as well as intraspecific trait patterns, (2) ecological feeding specialisation, and (3) previously neglected links to microbial associates found in the faeces. Chapter II: Temperature-driven assembly processes Climate varies at multiple scales. Since microclimate is often overlooked, we assessed effects of local temperature deviations on species and trait compositions of insect communities along macroclimatic temperature gradients in Chapter II. Therefore, we employed joint species distribution modelling to explore how traits drive variation in the climatic niches of Orthoptera species at grassland sites characterized by contrasting micro- and macroclimatic conditions. Our findings revealed two key insights: (1) additive effects of micro- and macroclimate on the diversity, but (2) interactive effects on the abundance of several species, resulting in turnover and indicating that species possess narrower climatic niches than their elevational distributions might imply. This chapter suggests positive effects of warming on Orthoptera, but also highlights that the interplay of macro- and microclimate plays a pivotal role in structuring insect communities. Thus, it underscores the importance of considering both elements when predicting the responses of species to climate change. Additionally, this chapter revealed inter- and intraspecific effects of traits on the niches and distribution of species. Chapter III: Dietary specialisation along climatic gradients A crucial trait linked to the position of climatic niches is dietary specialisation. According to the 'altitudinal niche-breadth hypothesis', species of high-elevation habitats should be less specialized compared to their low-elevation counterparts. However, empirical evidence on shifts in specialization is scarce for generalist insect herbivores and existing studies often fail to control for the phylogeny and abundance of interaction partners. In Chapter III, we used a combination of field observations and amplicon sequencing to reconstruct dietary relationships between Orthoptera and plants along an extensive temperature gradient. We did not find close but flexible links between individual grasshopper and plant taxa in space. While interaction network specialisation increased with temperature, the corrected dietary specialisation pattern peaked at intermediate elevations on assemblage level. These nuanced findings demonstrate that (1) resource availability, (2) phylogenetic relationships, and (3) climate can affect empirical foodwebs intra- and interspecifically and, hence, the dietary specialisation of herbivorous insects. In this context, we discuss that the underlying mechanisms involved in shaping the specialisation of herbivore assemblages may switch along temperature clines. Chapter IV: Links between faecal microbe communities, feeding habits, and climate Since gut microbes affect the fitness and digestion of insects, studying their diversity could provide novel insights into specialisation patterns. However, their association with insect hosts that differ in feeding habits and specialisation has never been investigated along elevational climatic gradients. In Chapter IV, we utilized the dietary information gathered in Chapter III to characterize links between insects with distinct feeding behaviour and the microbial communities present in their faeces, using amplicon sequencing. Both, feeding and climate affected the bacterial communities. However, the large overlap of microbes at site level suggests that common bacteria are acquired from the shared feeding environment, such as the plants consumed by the insects. These findings emphasize the influence of a broader environmental context on the composition of insect gut microbial communities. Chapter V: Discussion \& Conclusions Cumulatively, the sections of this dissertation provide support for the hypothesis that climatic conditions play a role in shaping plant-herbivore systems. The detected variation of taxonomic and functional compositions contributes to our understanding of assembly processes and resulting diversity patterns within Orthoptera communities, shedding light on the mechanisms that structure their trophic interactions in diverse climates. The combined results presented suggest that a warmer climate could foster an increase of Orthoptera species richness in Central European semi-natural grasslands, also because the weak links observed between insect herbivores and plants are unlikely to limit decoupled range shifts. However, the restructuring of Orthoptera communities in response to warmer temperatures depends on species' traits such as moisture preferences or phenology. Notably, we were able to demonstrate a crucial role of microclimate for many species, partly unravelling narrower climatic niches than their elevational ranges suggest. We found evidence that not only Orthoptera community composition, specialisation, and traits varied along elevational gradients, but even microbial communities in the faeces of Orthoptera changed, which is a novel finding. This complex restructuring and reassembly of communities, coupled with the nonlinear specialisation of trophic interactions and a high diversity of associated bacteria, emphasize our currently incomplete comprehension of how ecosystems will develop under future climatic conditions, demanding caution in making simplified predictions for biodiversity change under climate warming. Since these predictions may benefit from including biotic interactions and both, micro- and macroclimate based on our findings, conservation authorities and practitioners must not neglect improving microclimatic conditions to ensure local survival of a diverse set of threatened and demanding species. In this context, mountains can play a pivotal role for biodiversity conservation since these offer heterogeneous microclimatic conditions in proximity that can be utilized by species with distinct niches.},
  subject      = {Heuschrecken},
  language  = {en}
}
@phdthesis{Nirchal2024,
  author    = {Nirchal, Naveen Kumar},
  title     = {Mechanistische Regulierung des gastro{\"o}sophagealen {\"U}bergangs und die Rolle der Retins{\"a}ure bei der Entwicklung des Barrett-{\"O}sophagus},
  doi       = {10.25972/OPUS-31155},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311556},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Der gastro{\"o}sophageale {\"U}bergang (GEJ), der die Region abgrenzt, in der der distale {\"O}sophagus auf die proximale Magenregion trifft, ist bekannt f{\"u}r die Entwicklung pathologischer Zust{\"a}nde, wie Metaplasie und Adenokarzinom des {\"O}sophagus (EAC). Es ist wichtig, die Mechanismen der Entwicklungsstadien zu verstehen, die zu EAC f{\"u}hren, da die Inzidenzrate von EAC in den letzten 4 Jahrzehnten um das 7-fache gestiegen ist und die Gesamt{\"u}berlebensrate von 5 Jahren 18,4 \% betr{\"a}gt. In den meisten F{\"a}llenwird die Diagnose im fortgeschrittenen Stadium ohne vorherige Symptome erstellt. Der Hauptvorl{\"a}ufer f{\"u}r die Entwicklung von EAC ist eine pr{\"a}maligne Vorstufe namens Barrett-{\"O}sophagus (BE). BE ist der metaplastische Zustand, bei dem das mehrschichtige Plattenepithel des nativen {\"O}sophagus durch ein spezialisiertes einschichtiges S{\"a}ulenepithel ersetzt wird, das die molekularen Eigenschaften des Magen- sowie des Darmepithels aufweist. Zu den wichtigsten Risikofaktoren f{\"u}r die Entwicklung von BE geh{\"o}ren die chronische gastro{\"o}sophageale Refluxkrankheit (GERD), eine ver{\"a}nderte Mikrobiota und ver{\"a}nderte Retins{\"a}ure-Signalwege (RA). Es ist unklar, welche Zelle der Ursprung f{\"u}r BE ist, da es keine eindeutigen Beweisen f{\"u}r den Prozess der BE-Initiation gibt. In dieser Arbeit habe ich untersucht, wie die GEJ-Hom{\"o}ostase in gesundem Gewebe durch stammzellregulatorische Morphogene aufrechterhalten wird, welche Rolle der Vitamin-A (RA-Signal{\"u}bertragung) spieltund wie ihre Ver{\"a}nderung zur BE-Entwicklung beitr{\"a}gt. Im ersten Teil meiner Dissertation habe ich anhand von Einzelmolek{\"u}l-RNA in situ-Hybridisierung und Immunhistochemie eindeutig das Vorhandensein von zwei Arten von Epithelzellen nachweisen k{\"o}nnen, dem Plattenepithel in der Speiser{\"o}hre und dem S{\"a}ulenepithel imMagenbereich des GEJ. Mittels Abstammungsanalysen im Mausmodell konnte ich zeigen, dass die Epithelzellen des {\"O}sophagus und des Magens von zwei verschiedenen epithelialen Stammzelllinien imGEJ abstammen. Die Grenze zwischen Plattenepithel und S{\"a}ulenepithelzellen im SCJ des GEJ wirddurch gegens{\"a}tzliche Wnt-Mikroumgebungen streng reguliert. Plattenepithelstammzellen des {\"O}sophagus werden durch das Wnt-hemmende Mikroumgebungssignal aufrechterhalten, w{\"a}hrend Magens{\"a}ulenepithelzellen durch das Wnt-aktivierende Signal aus dem Stromakompartiment erhalten werden. Ich habe die in vivo Erhaltung der Epithelstammzellen des GEJ mit Hilfe eines in vitro Epithel-3D-Organoidkulturmodells rekonstruiert. Das Wachstum und die Vermehrung von Magens{\"a}ulenepithel-Organoiden h{\"a}ngen von Wnt-Wachstumsfaktoren ab, w{\"a}hrend das Wachstum von Plattenepithel-Organoiden von Wnt-defizienten Kulturbedingungen abh{\"a}ngt. Dar{\"u}ber hinaus zeigte die Einzelzell-RNA-Sequenzanalyse (scRNA-seq) der aus Organoiden gewonnenenEpithelzellen, dass der nicht-kanonische Wnt/ planar cell polarity (PCP) Signalweg an der Regulierung der Plattenepithelzellen beteiligt ist. Im Gegensatz dazu werden s{\"a}ulenf{\"o}rmige Magenepithelzellen durch den kanonischen Wnt/beta-Catenin- und den nicht-kanonischen Wnt/Ca2+-Weg reguliert. Meine Daten zeigen, dass die SCJ-Epithelzellen, die am GEJ verschmelzen, durch entgegengesetzte stromale Wnt-Faktoren und unterschiedliche Wnt-Weg-Signalee in den Epithelzellen reguliert werden. Im zweiten Teil der Dissertation untersuchte ich die Rolle der bioaktiven Vitamin A Verbindung RA auf {\"O}sophagus- und Magenepithelstammzellen. Die In-vitro-Behandlung von epithelialen Organoiden der Speiser{\"o}hre und des Magens mitRA oder seinem pharmakologischen Inhibitors BMS 493 zeigte, dass jeder Zelltyp unterschiedlich reguliert wurde. Ich beobachtete, dass eine verst{\"a}rkte RA die Differenzierung von Stammzellen und den Verlust der Schichtung f{\"o}rderte, w{\"a}hrend die RA-Hemmung zu einer verst{\"a}rkten Stammzellbildung und Regeneration im mehrschichtigen Epithel der Speiser{\"o}hre f{\"u}hrte. Im Gegensatz zur Speiser{\"o}hre ist der RA-Signalweg in Magen-Organoiden aktiv, und die Hemmung von RA hat ein reduziertes Wachstum von Magen-Organoiden. Globale transkriptomische Daten und scRNA-seq-Daten zeigten, dass derRA-Signalweg einen Ruheph{\"a}notyp in den {\"O}sophaguszellen induziert. Dagegen f{\"u}hrt das Fehlen von RA in Magenepithelzellen zur Expression von Genen, die mit BE assoziiert sind. Daher isteine r{\"a}umlich definierte Regulation der Wnt- und Retins{\"a}ure-Signalgebung amGEJ entscheidend f{\"u}r eine gesunde Hom{\"o}ostase, und ihre St{\"o}rung f{\"u}hrt zur Entwicklung von Krankheiten.},
  subject      = {Retinoes{\"a}ure},
  language  = {en}
}
@phdthesis{MaierverhHartmann2024,
  author    = {Maier [verh. Hartmann], Carina Ramona},
  title     = {Regulation of the Mevalonate Pathway by the Deubiquitinase USP28 in Squamous Cancer},
  doi       = {10.25972/OPUS-34874},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-348740},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment. De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis. SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis. SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation. It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth. A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation. In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Pitsch2024,
  author    = {Pitsch, Maximilian Jonathan},
  title     = {Zyklisches Adenosinmonophosphat (cAMP) als {\"A}quivalent akkumulierter neuronaler Evidenz},
  doi       = {10.25972/OPUS-35129},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-351292},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Die vier Crz-Neurone des ventralen Nervensystems von Drosophila melanogaster sammeln Evidenz, wann im Rahmen eines Paarungsakts zirka 6 Minuten vergangen sind. Diese Entscheidung ist f{\"u}r die m{\"a}nnliche Fliege von Bedeutung, da das M{\"a}nnchen vor Ablauf dieser ~6 Minuten, welche den Zeitpunkt der Ejakulation darstellen, eher das eigene Leben opfern w{\"u}rde, als dass es die Paarung beenden w{\"u}rde. Nach Ablauf der ~6 Minuten f{\"a}llt die Motivation des M{\"a}nnchens dagegen dramatisch ab. Im Rahmen der vorliegenden Arbeit wurde zun{\"a}chst mittels optogenetischer neuronaler Inhibitionsprotokolle sowie Verhaltensanalysen das Ph{\"a}nomen der Evidenz-akkumulation in den Crz-Neuronen genauer charakterisiert. Dabei zeigte sich, dass die akkumulierte Evidenz auch w{\"a}hrend einer elektrischen Inhibition der Crz-Neurone persistierte. Dieses Ergebnis warf die Hypothese auf, dass das {\"A}quivalent der akkumulierten Evidenz in den Crz-Neuronen biochemischer Natur sein k{\"o}nnte. Es wurde daraufhin ein Hochdurchsatzscreening-Verfahren entwickelt, mittels dessen 1388 genetische Manipulationen der Crz-Neurone durchgef{\"u}hrt und auf eine {\"A}nderung der Evidenzakkumulation getestet wurden. Nur ~30 genetische Manipulationen zeigten eine ver{\"a}nderte Evidenzakkumulation, wobei die meisten dieser Manipulationen den cAMP-Signalweg betrafen. Mittels der optogenetischen Photoadenylatzyklase bPAC, einer Reihe weiterer genetischer Manipulationen des cAMP-Signalwegs sowie der ex vivo Kalzium-Bildgebung und Fluoreszenzlebensdauer-Mikroskopie konnte best{\"a}tigt werden, dass cAMP das {\"A}quivalent der in den Crz-Neuronen spannungsabh{\"a}ngig akkumulierten Evidenz darstellt, wobei die Kombination dieser Methoden nahelegte, dass der Schwellenwert der Evidenzakkumulation durch die cAMP-Bindungsaffinit{\"a}t der regulatorischen PKA-Untereinheiten festgelegt sein k{\"o}nnte. Mittels genetischer Mosaikexperimente sowie bildgebenden Verfahren konnte dar{\"u}ber hinaus gezeigt werden, dass innerhalb des Crz-Netzwerks eine positive R{\"u}ckkopplungsschleife aus rekurrenter Aktivit{\"a}t sowie der cAMP-Akkumulation besteht, welche, sobald die cAMP-Spiegel den Schwellenwert erreichen, zu einem netzwerkweit synchronisierten massiven Kalziumeinstrom f{\"u}hrt, was die Abgabe des Crz-Signals an nachgeschaltete Netzwerke triggert. Dieses Ph{\"a}nomen k{\"o}nnte ein Analogon des Aktionspotenzials auf Netzwerkebene sowie auf Intervallzeitskalen darstellen und wurde als „Eruption" bezeichnet. Genetische, optogenetische sowie Bildgebungsexperimente konnten zeigen, dass die CaMKII derartige Eruptionen durch Niedrighalten der cAMP-Spiegel unterdr{\"u}ckt, was den Zeitmessmechanismus des ersten beschriebenen Intervallzeitmessers CaMKII offenlegt.},
  subject      = {Evidenz},
  language  = {de}
}
@phdthesis{Helmerich2023,
  author    = {Helmerich, Dominic Andreas},
  title     = {Einfl{\"u}sse der Photophysik und Photochemie von Cyaninfarbstoffen auf die Lokalisationsmikroskopie},
  doi       = {10.25972/OPUS-24716},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247161},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {In den letzten Jahren haben sich hochaufl{\"o}sende Fluoreszenzmikroskopiemethoden, basierend auf der Lokalisation einzelner Fluorophore, zu einem leistungsstarken Werkzeug etabliert, um Fluoreszenzbilder weit unterhalb der Aufl{\"o}sungsgrenze zu generieren. Hiermit k{\"o}nnen r{\"a}umliche Aufl{\"o}sungen von ~ 20 nm erzielt werden, was weit unterhalb der Beugungsgrenze liegt. Dabei haben zahlreiche Optimierungen und Entwicklungen neuer Methoden in der Einzelmolek{\"u}l-Lokalisationsmikroskopie die Genauigkeit der orstspezifischen Bestimmung einzelner Fluorophore auf bis zu ~ 1 - 3 nm erh{\"o}ht. Eine Aufl{\"o}sung im molekularen Bereich, weit unterhalb von ~ 10 nm bleibt allerdings herausfordernd, da die Lokalisationsgenauigkeit nur ein Kriterium hierf{\"u}r ist. Allerdings wurde sich in den letzten Jahren {\"u}berwiegend auf die Verbesserung dieses Parameters konzentriert. Weitere Kriterien f{\"u}r die fluoreszenzmikroskopische Aufl{\"o}sung sind dabei unter anderem die Markierungsdichte und die Kopplungseffizienz der Zielstruktur, sowie der Kopplungsfehler (Abstand zur Zielstruktur nach Farbstoffkopplung), die sich herausfordernd f{\"u}r eine molekulare Aufl{\"o}sung darstellen. Auch wenn die Kopplungseffizienz und -dichte hoch und der Kopplungsfehler gering ist, steigt bei Interfluorophordistanzen < 5nm, abh{\"a}ngig von den Farbstoffen, die Wahrscheinlichkeit von starken und schwachen Farbstoffwechselwirkungen und damit von Energie{\"u}bertragungsprozessen zwischen den Farbstoffen, stark an. Daneben sollten Farbstoffe, abh{\"a}nging von der Lokalisationsmikroskopiemethode, spezifische Kriterien, wie beispielsweise die Photoschaltbarkeit bei dSTORM, erf{\"u}llen, was dazu f{\"u}hrt, dass diese Methoden h{\"a}ufig nur auf einzelne Farbstoffe beschr{\"a}nkt sind. In dieser Arbeit konnte mithilfe von definierten DNA-Origami Konstrukten gezeigt werden, dass das Blinkverhalten von Cyaninfarbstoffen unter dSTORM-Bedingungen einer Abstandsabh{\"a}ngigkeit aufgrund von spezifischen Energie{\"u}bertragungsprozessen folgt, womit Farbstoffabst{\"a}nde im sub-10 nm Bereich charakterisiert werden konnten. Dar{\"u}ber hinaus konnte diese Abstandsabh{\"a}ngigkeit an biologischen Proben gezeigt werden. Hierbei konnten verschiedene zellul{\"a}re Rezeptoren effizient und mit geringem Abstandsfehler zur Zielstruktur mit Cyaninfarbstoffen gekoppelt werden. Diese abstandsabh{\"a}nigen Prozesse und damit Charakterisierungen k{\"o}nnten dabei nicht nur spezifisch f{\"u}r die h{\"a}ufig unter dSTORM-Bedingungen verwendeten Cyaninfarbstoffen g{\"u}ltig sein, sondern auch auf andere Farbstoffklassen, die einen Auszustand zeigen, {\"u}bertragbar sein. Dar{\"u}ber hinaus konnte gezeigt werden, dass hochaufl{\"o}sende dSTORM Aufnahmen unabh{\"a}ngig vom Farbstoffkopplungsgrad der Antik{\"o}rpern sind, welche h{\"a}ufig f{\"u}r Standardf{\"a}rbungen von zellul{\"a}ren Strukturen verwendet werden. Dabei konnte durch Photonenkoinzidenzmessungen dargelegt werden, dass aufgrund komplexer Farbstoffwechselwirkungen im Mittel nur ein Farbstoff aktiv ist, wobei h{\"o}here Kopplungsgrade ein komplexes Blinkverhalten zu Beginn der Messung zeigen. Durch die undefinierten Farbstoffabst{\"a}nde an Antik{\"o}rpern konnte hier kein eindeutiger Energie{\"u}bertragungsmechanismus entschl{\"u}sselt werden. Dennoch konnte gezeigt werden, dass Farbstoffaggregate bzw. H-Dimere unter dSTORM-Bedingungen destabilisiert werden. Durch die zuvor erw{\"a}hnten DNA-Origami Konstrukte definierter Interfluorophordistanzen konnten Energie{\"u}bertragungsmechanismen entschl{\"u}sselt werden, die auch f{\"u}r die Antik{\"o}rper diverser Kopplungsgrade g{\"u}ltig sind. Des Weiteren konnten, ausgel{\"o}st durch komplexe Energie{\"u}bertragungsprozesse h{\"o}herer Kopplungsgrade am Antik{\"o}rper, Mehrfarbenaufnahmen zellul{\"a}rer Strukturen generiert werden, die {\"u}ber die spezifische Fluoreszenzlebenszeit separiert werden konnten. Dies stellt hier eine weitere M{\"o}glichkeit dar, unter einfachen Bedingungen, schnelle Mehrfarbenaufnahmen zellul{\"a}rer Strukturen zu generieren. Durch die Verwendung des selben Farbstoffes unterschiedlicher Kopplungsgrade kann hier nur mit einer Anregungswellenl{\"a}nge und frei von chromatischer Aberration gearbeitet werden. Neben den photophysikalischen Untersuchungen der Cyaninfarbstoffe Cy5 und Alexa Fluor 647 wurden diese ebenso photochemisch n{\"a}her betrachtet. Dabei konnte ein neuartiger chemischer Mechanismus entschl{\"u}sselt werden. Dieser Mechanismus f{\"u}hrt, ausgel{\"o}st durch Singulett-Sauerstoff (1O2), zu einer Photozerschneidung des konjugierten Doppelbindungssystems um zwei Kohlenstoffatome, was zu strukturellen und spektroskopischen Ver{\"a}nderungen dieser Farbstoffe f{\"u}hrt. Auf Grundlage dieses Mechanismus konnte eine neue DNA-PAINT Methode entwickelt werden, die zu einer Beschleunigung der Aufnahmezeit f{\"u}hrt.},
  subject      = {Einzelmolek{\"u}lmikroskopie},
  language  = {de}
}
@phdthesis{WasgebHouben2023,
  author    = {Was [geb. Houben], Nina},
  title     = {Die Rolle der nicht-kodierenden RNAs miR-26 und \(Malat1\) bei der \(in\) \(vitro\) Differenzierung zu Neuronen},
  doi       = {10.25972/OPUS-30371},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303714},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {W{\"a}hrend der embryonalen Neurogenese spielt die Repression neuraler Gene in nicht neuralen Zellen, sowie in neuralen Vorl{\"a}uferzellen durch den REST (repressor element silencing transcription factor)-Komplex eine wichtige Rolle. Durch die schrittweise Inaktivierung diese Komplexes im Verlauf der Differenzierung werden neurale Genexpressionsprogramme gesteuert. Zus{\"a}tzlich kommt bei der Kontrolle der r{\"a}umlichen und zeitlichen Regulation der Genexpression w{\"a}hrend der Neurogenese verschiedenen miRNAs eine wichtige Rolle zu. So konnte in vorangegangenen Arbeiten im Zebrafischen gezeigt werden, dass miR-26b die Transkription eines wichtigen Effektorproteins des REST-Komplexes, CTDSP2 (C-terminal domain small phosphatases), w{\"a}hrend der Neurogenese negativ reguliert. Da dar{\"u}ber hinaus die miR-26 Repression zu einer stark verminderten neuronalen Differenzierung f{\"u}hrte, kommt diesem regulatorischen Schaltkreis eine zentrale Rolle bei der Neurogenese im Zebrafisch zu. Die zusammen mit ihren Ctdsp-Wirtsgenen koexprimierte miR-26 Familie liegt in Vertebraten evolution{\"a}r hoch konserviert vor. Analog zum Zebrafisch konnte im murinen in vitro ES-Zell Differenzierungssystem gezeigt werden, dass miR-26 die Expression von Ctdsp2 reprimiert. Weiterhin konnte in diesem System gezeigt werden, dass auch Rest ein miR-26 Zielgen ist und dass der Verlust der miR-26 zu einem Arrest der differenzierenden Zellen im neuronalen Vorl{\"a}uferstadium f{\"u}hrt. Zusammengenommen deuten diese vorangegangenen Arbeiten auf eine zentrale Rolle der miR-26 w{\"a}hrend der Neurogenese hin. Die hier vorgestellte Arbeit zielte zun{\"a}chst darauf ab die Regulation des REST-Komplexes durch die miR-26 auf molekularer Ebene besser zu verstehen. Der Verlust der miR-26 Bindestelle in der Ctdsp2 mRNA f{\"u}hrte zu einer erh{\"o}hten Ctdsp2 Expression, beeinflusste aber nicht die terminale Differenzierung zu Neuronen. Im Gegensatz hierzu f{\"u}hrte der Verlust der miR-26 Bindestelle in der Rest mRNA zu einem Arrest der Differenzierung im neuralen Vorl{\"a}uferzellstadium. Zellen in denen die miR-26 Bindestelle in Rest deletiert war, zeigten zudem, genau wie miR-26 knockout (KO) Zellen, eine erh{\"o}hte Expression von REST-Komplex Komponenten, sowie eine verringerte Expression von REST-regulierten miRNAs. Zusammengenommen weisen diese Daten daraufhin, dass w{\"a}hrend der Neurogenese im S{\"a}ugersystem die Inaktivierung von Rest durch miR-26 f{\"u}r die Maturierung von Neuronen eine zentrale Rolle spielt. Ein weiterer Fokus dieser Arbeit lag auf der Regulation der miR-26 Expression w{\"a}hrend der Neurogenese. Vorangegangene Arbeiten in nicht-neuronalen Zelltypen identifizierten die lnc (long-non-coding) RNA Malat1 als eine ce (competitive endogenous) RNA der miR-26. Um den Einfluss von Malat1 auf die miR-26 Expression w{\"a}hrend der Neurogenese zu untersuchen, wurde zun{\"a}chst mittels CRISPR/Cas9 der vollst{\"a}ndige Malat1-Lokus in ESCs deletiert. Der Verlust von Malat1 f{\"u}hrte zu einer erh{\"o}hten Expression der miR-26 Familienmitglieder sowie deren Ctdsp-Wirtsgene. Weiterhin war die Proliferation von Malat1 KO neuronalen Vorl{\"a}uferzellen stark vermindert, was mit einer Erh{\"o}hung der Frequenz seneszenter Zellen einherging. Durch die Inaktivierung von miR-26 in differenzierenden Malat1 KO ESCs konnte dieser proliferative Ph{\"a}notyp aufgehoben werden. Dar{\"u}ber hinaus konnte eine verst{\"a}rkte neuronale Differenzierung dieser Zellen beobachtet werden. Zusammenfassend zeigen diese Daten, dass neben der Regulation des REST-Komplexes durch miR-26 auch die Kontrolle des Zellzyklus {\"u}ber die Malat1-vermittelte Regulation der miR-26 in neuronalen Vorl{\"a}uferzellen einen kritischen Schritt bei der Differenzierung von neuronalen Vorl{\"a}uferzellen zu maturen Neuronen darstellt.},
  subject      = {Neurogenese},
  language  = {de}
}
@phdthesis{Petrov2023,
  author    = {Petrov, Ivan},
  title     = {Combinational therapy of tumors in syngeneic mouse tumor models with oncolytic Vaccinia virus strains expressing IL-2 and INF-g. Human adipose tissue-derived stem cell mediated delivery of oncolytic Vaccinia virus},
  doi       = {10.25972/OPUS-27355},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-273550},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Cancer is one of the leading causes of death worldwide, with currently assessed chances to develop at least one cancer in a lifetime for about 20\%. High cases rates and mortality require the development of new anticancer therapies and treatment strategies. Another important concern is toxicity normally associated with conventional therapy methods, such as chemo- and radiotherapy. Among many proposed antitumoral agents, oncolytic viruses are still one of the promising and fast-developing fields of research with almost a hundred studies published data on over 3000 patients since the beginning of the new millennia. Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings, and this year we can celebrate an unofficial 100th anniversary since the publication of the fact. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Another advantage of using Vaccinia as an oncolytic agent is its high genome capacity, which allows inserting up to 25 kbps of exogenous genes, thus allowing to additionally arm the virus against the tumor. Oncolytic virus action consists of two major parts: direct oncolysis and immune activation against the tumor, with the latter being the key to successful treatment. To this moment, preclinical research data are mostly generated in immunocompromised xenograft models, which have hurdles to be properly translated for clinical use. In the first part of the current study, fourteen different recombinant Vaccinia virus strains were tested in two different murine tumor cell lines and corresponding immunocompetent animal models. We found, that Copenhagen backbone Vaccinia viruses while being extremely effective in cell culture, do not show significant oncolytic efficacy in animals. In contrast, several of the LIVP backbone viruses tested (specifically, IL-2 expressing ones) have little replication ability when compared to the Copenhagen strain, but are able to significantly delay tumor growth and prolong survival of the treated animals. We have also noted cytokine related toxicity of the animals to be mouse strain specific. We have also tested the virus with the highest therapeutic benefit in combination with romidepsin and cyclophosphamide. While the combination with histone deacetylase inhibitor romidepsin did not result in therapeutic benefit in our settings, the addition of cyclophosphamide significantly improved the efficacy of the treatment, at the same time reducing cytokine-associated toxicity of the IL-2 expressing virus. In the second part of the work, we analyzed the ability of adipose-derived mesenchymal stem cells to serve as a carrier for the oncolytic Vaccinia virus. We showed for the first time that the cells can be infected with the virus and can generate virus progeny. They are also able to survive for a substantially long time and, when injected into the bloodstream of tumor-bearing animals, produce the virus that is colonizing the tumor. Analysis of the systemic distribution of the cells after injection revealed that infected and uninfected cells are not distributed in the same manner, possibly suggesting that infected cells are getting recognized and cleared by an impaired immune system of athymic mice faster than non-infected cells. Despite this, injection of virus-loaded adipose-derived mesenchymal stem cells to human A549 tumor-bearing xenograft mice resulted in rapid tumor regression and reduced virus-related side effects of the treatment when compared to injection of the naked virus. In conclusion, we have tested two different approaches to augmenting oncolytic Vaccinia virus therapy. First, the combination of recombinant Vaccinia virus expressing IL-2 and cyclophosphamide showed promising results in a syngeneic mouse model, despite the low permissivity of murine cells to the virus. Second, we loaded the oncolytic Vaccinia virus into mesenchymal stem cells and have proven that they can potentially serve as a vehicle for the virus.},
  subject      = {Vaccinia-virus},
  language  = {en}
}
@phdthesis{Nguyen2023,
  author    = {Nguyen, Tu Anh Thi},
  title     = {Neural coding of different visual cues in the monarch butterfly sun compass},
  doi       = {10.25972/OPUS-30380},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303807},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Monarch butterflies are famous for their annual long-distance migration. Decreasing temperatures and reduced daylight induce the migratory state in the autumn generation of monarch butterflies. Not only are they in a reproductive diapause, they also produce fat deposits to be prepared for the upcoming journey: Driven by their instinct to migrate, they depart from their eclosion grounds in the northern regions of the North American continent and start their southern journey to their hibernation spots in Central Mexico. The butterflies cover a distance of up to 4000 km across the United States. In the next spring, the same butterflies invert their preferred heading direction due to seasonal changes and start their northward spring migration. The spring migration is continued by three consecutive butterfly generations, until the animals repopulate the northern regions in North America as non-migratory monarch butterflies. The monarch butterflies' migratory state is genetically and epigenetically regulated, including the directed flight behavior. Therefore, the insect's internal compass system does not only have to encode the butterflies preferred, but also its current heading direction. However, the butterfly's internal heading representation has to be matched to external cues, to avoid departing from its initial flight path and increasing its risk of missing its desired destination. During the migratory flight, visual cues provide the butterflies with reliable orientation information. The butterflies refer to the sun as their main orientation cue. In addition to the sun, the butterflies likely use the polarization pattern of the sky for orientation. The sky compass signals are processed within a region in the brain, termed the central complex (CX). Previous research on the CX neural circuitry of the monarch butterflies demonstrated that tangential central complex neurons (TL) carry the visual input information into the CX and respond to a simulated sun and polarized light. However, whether these cells process additional visual cues like the panoramic skyline is still unknown. Furthermore, little is known about how the migratory state affects visual cue processing. In addition to this, most experiments studying the monarch butterfly CX focused on how neurons process single visual cues. However, how combined visual stimuli are processed in the CX is still unknown. This thesis is investigating the following questions: 1) How does the migratory state affect visual cue processing in the TL cells within the monarch butterfly brain? 2) How are multiple visual cues integrated in the TL cells? 3) How is compass information modulated in the CX? To study these questions, TL neurons from both animal groups (migratory and non-migratory) were electrophysiologically characterized using intracellular recordings while presenting different simulated celestial cues and visual sceneries. I showed that the TL neurons of migratory butterflies are more narrowly tuned to the sun, possibly helping them in keeping a directed flight course during migration. Furthermore, I found that TL cells encode a panoramic skyline, suggesting that the CX network combines celestial and terrestrial information. Experiments with combined celestial stimuli revealed that the TL cells combine both cue information linearly. However, if exposing the animals to a simulated visual scenery containing a panoramic skyline and a simulated sun, the single visual cues are weighted differently. These results indicate that the CX's input region can flexibly adapt to different visual cue conditions. Furthermore, I characterize a previously unknown neuron in the monarch butterfly CX which responds to celestial stimuli and connects the CX with other brain neuropiles. How this cell type affects heading direction encoding has yet to be determined.},
  subject      = {Monarchfalter},
  language  = {en}
}
@phdthesis{Solvie2023,
  author    = {Solvie, Daniel Alexander},
  title     = {Molecular Mechanisms of MYC as Stress Resilience Factor},
  doi       = {10.25972/OPUS-30539},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-305398},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Cancer is one of the leading causes of death worldwide. The underlying tumorigenesis is driven by the accumulation of alterations in the genome, eventually disabling tumor suppressors and activating proto-oncogenes. The MYC family of proto-oncogenes shows a strong deregulation in the majority of tumor entities. However, the exact mechanisms that contribute to MYC-driven oncogenesis remain largely unknown. Over the past decades, the influence of the MYC protein on transcription became increasingly apparent and was thoroughly investigated. Additionally, in recent years several publications provided evidence for so far unreported functions of MYC that are independent of a mere regulation of target genes. These findings suggest an additional role of MYC in the maintenance of genomic stability and this role is strengthened by key findings presented in this thesis. In the first part, I present data revealing a pathway that allows MYC to couple transcription elongation and DNA double-strand break repair, preventing genomic instability of MYC-driven tumor cells. This pathway is driven by a rapid transfer of the PAF1 complex from MYC onto RNAPII, a process that is mediated by HUWE1. The transfer controls MYC-dependent transcription elongation and, simultaneously, the remodeling of chromatin structure by ubiquitylation of histone H2B. These regions of open chromatin favor not only elongation but also DNA double-strand break repair. In the second part, I analyze the ability of MYC proteins to form multimeric structures in response to perturbation of transcription and replication. The process of multimerization is also referred to as phase transition. The observed multimeric structures are located proximal to stalled replication forks and recruit factors of the DNA-damage response and transcription termination machinery. Further, I identified the HUWE1-dependent ubiquitylation of MYC as an essential step in this phase transition. Cells lacking the ability to form multimers display genomic instability and ultimately undergo apoptosis in response to replication stress. Both mechanisms present MYC as a stress resilience factor under conditions that are characterized by a high level of transcriptional and replicational stress. This increased resilience ensures oncogenic proliferation. Therefore, targeting MYC's ability to limit genomic instability by uncoupling transcription elongation and DNA repair or disrupting its ability to multimerize presents a therapeutic window in MYC-dependent tumors.},
  subject      = {MYC},
  language  = {en}
}
@phdthesis{Sauerwein2023,
  author    = {Sauerwein, Till},
  title     = {Implementation and application of bioinformatical software for the analysis of dual RNA sequencing data of host and pathogen during infection},
  doi       = {10.25972/OPUS-30307},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303075},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Since the advent of high-throughput sequencing technologies in the mid-2010s, RNA se- quencing (RNA-seq) has been established as the method of choice for studying gene expression. In comparison to microarray-based methods, which have mainly been used to study gene expression before the rise of RNA-seq, RNA-seq is able to profile the entire transcriptome of an organism without the need to predefine genes of interest. Today, a wide variety of RNA-seq methods and protocols exist, including dual RNA sequenc- ing (dual RNA-seq) and multi RNA sequencing (multi RNA-seq). Dual RNA-seq and multi RNA-seq simultaneously investigate the transcriptomes of two or more species, re- spectively. Therefore, the total RNA of all interacting species is sequenced together and only separated in silico. Compared to conventional RNA-seq, which can only investi- gate one species at a time, dual RNA-seq and multi RNA-seq analyses can connect the transcriptome changes of the species being investigated and thus give a clearer picture of the interspecies interactions. Dual RNA-seq and multi RNA-seq have been applied to a variety of host-pathogen, mutualistic and commensal interaction systems. We applied dual RNA-seq to a host-pathogen system of human mast cells and Staphylo- coccus aureus (S. aureus). S. aureus, a commensal gram-positive bacterium, can become an opportunistic pathogen and infect skin lesions of atopic dermatitis (AD) patients. Among the first immune cells S. aureus encounters are mast cells, which have previously been shown to be able to kill the bacteria by discharging antimicrobial products and re- leasing extracellular traps made of protein and deoxyribonucleic acid (DNA). However, S. aureus is known to evade the host's immune response by internalizing within mast cells. Our dual RNA-seq analysis of different infection settings revealed that mast cells and S. aureus need physical contact to influence each other's gene expression. We could show that S. aureus cells internalizing within mast cells undergo profound transcriptome changes to adjust their metabolism to survive in the intracellular niche. On the host side, we found out that infected mast cells elicit a type-I interferon (IFN-I) response in an autocrine manner and in a paracrine manner to non-infected bystander-cells. Our study provides the first evidence that mast cells are capable to produce IFN-I upon infection with a bacterial pathogen.},
  subject      = {Biologie},
  language  = {en}
}
@phdthesis{Schardt2023,
  author    = {Schardt, Simon},
  title     = {Agent-based modeling of cell differentiation in mouse ICM organoids},
  doi       = {10.25972/OPUS-30194},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301940},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Mammalian embryonic development is subject to complex biological relationships that need to be understood. However, before the whole structure of development can be put together, the individual building blocks must first be understood in more detail. One of these building blocks is the second cell fate decision and describes the differentiation of cells of the inner cell mass of the embryo into epiblast and primitive endoderm cells. These cells then spatially segregate and form the subsequent bases for the embryo and yolk sac, respectively. In organoids of the inner cell mass, these two types of progenitor cells are also observed to form, and to some extent to spatially separate. This work has been devoted to these phenomena over the past three years. Plenty of studies already provide some insights into the basic mechanics of this cell differentiation, such that the first signs of epiblast and primitive endoderm differentiation, are the expression levels of transcription factors NANOG and GATA6. Here, cells with low expression of GATA6 and high expression of NANOG adopt the epiblast fate. If the expressions are reversed, a primitive endoderm cell is formed. Regarding the spatial segregation of the two cell types, it is not yet clear what mechanism leads to this. A common hypothesis suggests the differential adhesion of cell as the cause for the spatial rearrangement of cells. In this thesis however, the possibility of a global cell-cell communication is investigated. The approach chosen to study these phenomena follows the motto "mathematics is biology's next microscope". Mathematical modeling is used to transform the central gene regulatory network at the heart of this work into a system of equations that allows us to describe the temporal evolution of NANOG and GATA6 under the influence of an external signal. Special attention is paid to the derivation of new models using methods of statistical mechanics, as well as the comparison with existing models. After a detailed stability analysis the advantages of the derived model become clear by the fact that an exact relationship of the model parameters and the formation of heterogeneous mixtures of two cell types was found. Thus, the model can be easily controlled and the proportions of the resulting cell types can be estimated in advance. This mathematical model is also combined with a mechanism for global cell-cell communication, as well as a model for the growth of an organoid. It is shown that the global cell-cell communication is able to unify the formation of checkerboard patterns as well as engulfing patterns based on differently propagating signals. In addition, the influence of cell division and thus organoid growth on pattern formation is studied in detail. It is shown that this is able to contribute to the formation of clusters and, as a consequence, to breathe some randomness into otherwise perfectly sorted patterns.},
  subject      = {Mathematische Modellierung},
  language  = {en}
}
@phdthesis{FetivaMora2023,
  author    = {Fetiva Mora, Maria Camila},
  title     = {Changes in chromatin accessibility by oncogenic YAP and its relevance for regulation of cell cycle gene expression and cell migration},
  doi       = {10.25972/OPUS-30291},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302910},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes. The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration. Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.},
  subject      = {Chromatin},
  language  = {en}
}
@phdthesis{Fasemore2023,
  author    = {Fasemore, Akinyemi Mandela},
  title     = {Genomic and internet based analysis of \(Coxiella\) \(burnetii\)},
  doi       = {10.25972/OPUS-29663},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296639},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Coxiella burnetii, a Gram negative obligate intracellular bacterium, is the causative agent of Q fever. It has a world wide distribution and has been documented to be capable of causing infections in several domestic animals, livestock species, and human beings. Outbreaks of Q fever are still being observed in livestock across animal farms in Europe, and primary transmission to humans still oc- curs especially in animal handlers. Public health authorities in some countries like Germany are required by law to report human acute cases denoting the significance of the challenge posed by C. burnetii to public health. In this thesis, I have developed a platform alongside methods to address the challenges of genomic analyses of C. burnetii for typing purposes. Identification of C. burnetii isolates is an important task in the laboratory as well as in the clinics and genotyping is a reliable method to identify and characterize known and novel isolates. Therefore, I designed and implemented several methods to facilitate the genotyping analyses of C. burnetii genomes in silico via a web platform. As genotyping is a data intensive process, I also included additional features such as visualization methods and databases for interpretation and storage of obtained results. I also developed a method to profile the resistome of C. burnetii isolates using a machine learning approach. Data about antibiotic resistance in C. burnetii are scarce majorly due to its lifestyle and the difficulty of cultivation in laboratory media. Alternative methods that rely on homology identification of resistance genes are also inefficient in C. burnetii, hence, I opted for a novel approach that has been shown to be promising in other bacteria species. The applied method relied on an artificial neural network as well as amino acid composition of position specific scoring matrix profile for feature extraction. The resulting model achieved an accuracy of ≈ 0.96 on test data and the overall performance was significantly higher in comparison to existing models. Finally, I analyzed two new C. burnetii isolates obtained from an outbreak in Germany, I compared the genome to the RSA 493 reference isolate and found extensive deletions across the genome landscape. This work has provided a new digital infrastructure to analyze and character- ize C. burnetii genomes that was not in existence before and it has also made a significant contribution to the existing information about antibiotic resistance genes in C. burnetii.},
  language  = {en}
}
@phdthesis{Rutschmann2023,
  author    = {Rutschmann, Benjamin},
  title     = {Occurrence and population density of wild-living honey bees in Europe and the impact of different habitat types on their foraging and overwintering success},
  doi       = {10.25972/OPUS-28673},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286732},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The original habitat of native European honey bees (\(Apis\) \(mellifera\)) is forest, but currently there is a lack of data about the occurrence of wild honey bee populations in Europe. Prior to being kept by humans in hives, honey bees nested as wild species in hollow trees in temperate forests. However, in the 20th century, intensification of silviculture and agriculture with accompanying losses of nesting sites and depletion of food resources caused population declines in Europe. When the varroa mite (Varroa destructor), an invasive ectoparasite from Asia, was introduced in the late 1970s, wild honey bees were thought to be eradicated in Europe. Nevertheless, sporadic, mostly anecdotal, reports from ornithologists or forest ecologists indicated that honey bee colonies still occupy European forest areas. In my thesis I hypothesize that near-natural deciduous forests may provide sufficient large networks of nesting sites representing refugia for wild-living honey bees. Using two special search techniques, i.e. the tracking of flight routes of honey bee foragers (the "beelining" method) and the inspection of known cavity trees, I collected for the first time data on the occurrence and density of wild-living honey bees in forest areas in Germany (CHAPTER 3). I found wild-living honey bee colonies in the Hainich national park at low densities in two succeeding years. In another forest region, I checked known habitat trees containing black woodpecker cavities for occupation by wild-living honey bee colonies. It turned out that honey bees regularly use these cavities and occur in similar densities in both studied forest regions, independent of the applied detection method. Extrapolating these densities to all German forest areas, I estimate several thousand wild-living colonies in Germany that potentially interact in different ways with the forest environment. I conclude that honey bees regularly colonize forest areas in Germany and that networks of mapped woodpecker cavities offer unique possibilities to study the ecology of wild-living honey bees over several years. While their population status is ambiguous and the density of colonies low, the fact that honey bees can still be found in forests poses questions about food supply in forest environments. Consequently, I investigated the suitability of woodlands as a honey bee foraging habitat (CHAPTER 4). As their native habitat, forests are assumed to provide important pollen and nectar sources for honey bee colonies. However, resource supply might be spatially and temporally restricted and landscape-scale studies in European forest regions are lacking. Therefore, I set up twelve honey bee colonies in observation hives at locations with varying degree of forest cover. Capitalizing on the unique communication behaviour, the waggle dance, I examined the foraging distances and habitat preferences of honey bees over almost an entire foraging season. Moreover, by connecting this decoded dance information with colony weight recordings, I could draw conclusions about the contribution of the different habitat types to honey yield. Foraging distances generally increased with the amount of forest in the surrounding landscape. Yet, forest cover did not have an effect on colony weight. Compared to expectations based on the proportions of different habitats in the surroundings, colonies foraged more frequently in cropland and grasslands than in deciduous and coniferous forests, especially in late summer when pollen foraging in the forest is most difficult. In contrast, colonies used forests for nectar/honeydew foraging in early summer during times of colony weight gain emphasizing forests as a temporarily significant source of carbohydrates. Importantly, my study shows that the ecological and economic value of managed forest as habitat for honey bees and other wild pollinators can be significantly increased by the continuous provision of floral resources, especially for pollen foraging. The density of these wild-living honey bee colonies and their survival is driven by several factors that vary locally, making it crucial to compare results in different regions. Therefore, I investigated a wild-living honey bee population in Galicia in north-western Spain, where colonies were observed to reside in hollow electric poles (CHAPTER 5). The observed colony density only in these poles was almost twice as high as in German forest areas, suggesting generally more suitable resource conditions for the bees in Galicia. Based on morphometric analyses of their wing venation patterns, I assigned the colonies to the native evolutionary lineage (M-lineage) where the particularly threatened subspecies \(Apis\) \(mellifera\) \(iberiensis\) also belongs to. Averaged over two consecutive years, almost half of the colonies survived winter (23 out of 52). Interestingly, semi-natural areas both increased abundance and subsequent colony survival. Colonies surrounded by more semi-natural habitat (and therefore less intensive cropland) had an elevated overwintering probability, indicating that colonies need a certain amount of semi-natural habitat in the landscape to survive. Due to their ease of access these power poles in Galicia are, ideally suited to assess the population demography of wild-living Galician honey bee colonies through a long-term monitoring. In a nutshell, my thesis indicates that honey bees in Europe always existed in the wild. I performed the first survey of wild-living bee density yet done in Germany and Spain. My thesis identifies the landscape as a major factor that compromises winter survival and reports the first data on overwintering rates of wild-living honey bees in Europe. Besides, I established methods to efficiently detect wild-living honey bees in different habitat. While colonies can be found all over Europe, their survival and viability depend on unpolluted, flower rich habitats. The protection of near-natural habitat and of nesting sites is of paramount importance for the conservation of wild-living honey bees in Europe.  },
  subject      = {Biene},
  language  = {en}
}
@phdthesis{Franzke2023,
  author    = {Franzke, Myriam},
  title     = {Keep on track : The use of visual cues for orientation in monarch butterflies},
  doi       = {10.25972/OPUS-28470},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284709},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The monarch butterfly (Danaus plexippus) performs one of the most astonishing behaviors in the animal kingdom: every fall millions of these butterflies leave their breeding grounds in North Amerika and migrate more than 4.000 km southwards until they reach their overwintering habitat in Central Mexico. To maintain their migratory direction over this enormous distance, the butterflies use a time-compensated sun compass. Beside this, skylight polarization, the Earth's magnetic field and specific mountain ranges seem to guide the butterflies as well the south. In contrast to this fascinating orientation ability, the behavior of the butterflies in their non-migratory state received less attention. Although they do not travel long distances, they still need to orient themselves to find food, mating partners or get away from competitors. The aim of the present doctoral thesis was to investigate use of visual cues for orientation in migrating as well as non-migrating monarch butterflies. For this, field experiments investigating the migration of the butterflies in Texas (USA) were combined with experiments testing the orientation performance of non-migratory butterflies in Germany. In the first project, I recorded the heading directions of tethered butterflies during their annual fall migration. In an outdoor flight simulator, the butterflies maintained a southwards direction as long as they had a view of the sun's position. Relocating the position of the sun by 180° using a mirror, revealed that the sun is the animals' main orientation reference. Furthermore, I demonstrated that when the sun is blocked and a green light stimulus (simulated sun) is introduced, the animals interpreted this stimulus as the 'real' sun. However, this cue was not sufficient to set the migratory direction when simulated as the only visual cue in indoor experiments. When I presented the butterflies a linear polarization pattern additionally to the simulated sun, the animals headed in the correct southerly direction showing that multiple skylight cues are required to guide the butterflies during their migration. In the second project, I, furthermore, demonstrated that non-migrating butterflies are able to maintain a constant direction with respect to a simulated sun. Interestingly, they ignored the spectral component of the stimulus and relied on the intensity instead. When a panoramic skyline was presented as the only orientation reference, the butterflies maintained their direction only for short time windows probably trying to stabilize their flight based on optic-flow information. Next, I investigated whether the butterflies combine celestial with local cues by simulating a sun stimulus together with a panoramic skyline. Under this conditions, the animals' directedness was increased demonstrating that they combine multiple visual cues for spatial orientation. Following up on the observation that a sun stimulus resulted in a different behavior than the panoramic skyline, I investigated in my third project which orientation strategies the butterflies use by presenting different simulated cues to them. While a bright stripe on a dark background elicited a strong attraction of the butterflies steering in the direction of the stimulus, the inverted version of the stimulus was used for flight stabilization. In contrast to this, the butterflies maintained arbitrary directions with a high directedness with respect to a simulated sun. In an ambiguous scenery with two identical stimuli (two bright stripes, two dark stripes, or two sun stimuli) set 180° apart, a constant flight course was only achieved when two sun stimuli were displayed suggesting an involvement of the animals' internal compass. In contrast, the butterflies used two dark stripes for flight stabilization and were alternatingly attracted by two bright stripes. This shows that monarch butterflies use stimulus-dependent orientation strategies and gives the first evidence for different neuronal pathways controlling the output behavior.},
  subject      = {Monarchfalter},
  language  = {en}
}
@phdthesis{Reinhard2023,
  author    = {Reinhard, Sebastian},
  title     = {Improving Super-Resolution Microscopy Data Reconstruction and Evaluation by Developing Advanced Processing Algorithms and Artifcial Neuronal Networks},
  doi       = {10.25972/OPUS-31695},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-316959},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The fusion of methods from several disciplines is a crucial component of scientific development. Artificial Neural Networks, based on the principle of biological neuronal networks, demonstrate how nature provides the best templates for technological advancement. These innovations can then be employed to solve the remaining mysteries of biology, including, in particular, processes that take place on microscopic scales and can only be studied with sophisticated techniques. For instance, direct Stochastic Optical Reconstruction Microscopy combines tools from chemistry, physics, and computer science to visualize biological processes at the molecular level. One of the key components is the computer-aided reconstruction of super-resolved images. Improving the corresponding algorithms increases the quality of the generated data, providing further insights into our biology. It is important, however, to ensure that the heavily processed images are still a reflection of reality and do not originate in random artefacts. Expansion microscopy is expanding the sample by embedding it in a swellable hydrogel. The method can be combined with other super-resolution techniques to gain additional resolution. We tested this approach on microtubules, a well-known filamentous reference structure, to evaluate the performance of different protocols and labelling techniques. We developed LineProfiler an objective tool for data collection. Instead of collecting perpendicular profiles in small areas, the software gathers line profiles from filamentous structures of the entire image. This improves data quantity, quality and prevents a biased choice of the evaluated regions. On the basis of the collected data, we deployed theoretical models of the expected intensity distribution across the filaments. This led to the conclusion that post-expansion labelling significantly reduces the labelling error and thus, improves the data quality. The software was further used to determine the expansion factor and arrangement of synaptonemal complex data. Automated Simple Elastix uses state-of-the-art image alignment to compare pre- and post-expansion images. It corrects linear distortions occurring under isotropic expansion, calculates a structural expansion factor and highlights structural mismatches in a distortion map. We used the software to evaluate expanded fungi and NK cells. We found that the expansion factor differs for the two structures and is lower than the overall expansion of the hydrogel. Assessing the fluorescence lifetime of emitters used for direct Stochastic Optical Reconstruction Microscopy can reveal additional information about the molecular environment or distinguish dyes emitting with a similar wavelength. The corresponding measurements require a confocal scanning of the sample in combination with the fluorescent switching of the underlying emitters. This leads to non-linear, interrupted Point Spread Functions. The software ReCSAI targets this problem by combining the classical algorithm of compressed sensing with modern methods of artificial intelligence. We evaluated several different approaches to combine these components and found, that unrolling compressed sensing into the network architecture yields the best performance in terms of reconstruction speed and accuracy. In addition to a deep insight into the functioning and learning of artificial intelligence in combination with classical algorithms, we were able to reconstruct the described non-linearities with significantly improved resolution, in comparison to other state-of-the-art architectures.},
  subject      = {Mikroskopie},
  language  = {en}
}
@phdthesis{Lippert2023,
  author    = {Lippert, Juliane},
  title     = {Die molekulargenetische Charakterisierung von Nebennierenrindenkarzinomen als Schritt in Richtung personalisierter Medizin},
  doi       = {10.25972/OPUS-24717},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247172},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Nebennierenrindenkarzinome (NNR-Ca; engl. adrenocortical carcinoma (ACC)) z{\"a}hlen zu den sehr seltenen Tumorentit{\"a}ten. Die Prognose f{\"u}r die Patient*innen ist insgesamt eher schlecht, kann aber, im Einzelnen betrachtet, sehr heterogen sein. Eine zuverl{\"a}ssige Prognose anhand klinischer und histopathologischer Marker - wie dem Tumorstadium bei Diagnose, dem Resektionsstatus und dem Proliferationsindex Ki-67 -, die routinem{\"a}ßig erhoben werden, ist nicht f{\"u}r alle Erkrankten m{\"o}glich. Außerdem wird deren Behandlung dadurch erschwert, dass Therapeutika fehlen, von denen ein Großteil der Patient*innen profitiert. Umfassende Multi-Omics-Studien aus den letzten Jahren halfen nicht nur das Wissen {\"u}ber Pathomechanismen in NNR-Cas zu erweitern, es konnte auch gezeigt werden, dass sich Patient*innen anhand molekularer Marker in Subgruppen mit jeweils unterschiedlicher Prognose einteilen lassen. Mit molekulargenetischen Untersuchungen wurden außerdem potentielle neue Therapieziele gefunden. Diese Erkenntnisse finden bisher jedoch keine oder kaum Anwendung, da die Analysen den zeitlichen und finanziellen Rahmen, der f{\"u}r den routinem{\"a}ßigen Einsatz im Klinikalltag zu erf{\"u}llen w{\"a}re, deutlich {\"u}berschreiten. Ziel dieser Arbeit war es, eine Strategie zur verbesserten Patientenversorgung der NNR-CaPatient*innen zu etablieren. Daf{\"u}r sollte gekl{\"a}rt werden, ob ausgew{\"a}hlte molekulare prognostische Marker mit Methoden, die theoretisch einfach in den Klinikalltag zu implementieren w{\"a}ren, gefunden werden k{\"o}nnen. Außerdem sollte nach pr{\"a}diktiven Markern gesucht werden, die helfen, NNR-Ca-Patient*innen zielgerichtet zu therapieren. Statt exom- oder genomweite Analysen durchzuf{\"u}hren wurden gezielt krebs- beziehungsweise NNR-Ca-assoziierte Gene mittels NGS (Next-Generation Sequencing) oder SangerSequenzierung (zusammen 161 Gene) und Pyrosequenzierung (4 Gene) auf somatische Ver{\"a}nderungen hin untersucht. Die Analysen wurden an DNA (Desoxyribonukleins{\"a}ure) durchgef{\"u}hrt, die aus FFPE (mit Formalin fixiert und in Paraffin eingebettet)-Gewebe isoliert worden war, welches standardm{\"a}ßig nach Tumoroperationen in Pathologien f{\"u}r Untersuchungen zur Verf{\"u}gung steht. Durch Analyse der Sequenzierergebnisse von insgesamt 157 Patient*innen aus einem retrospektiven (107 Patient*innen) und einem prospektiven Studienteil (50 Patient*innen) konnten in NNR-Cas bereits beschriebene Ver{\"a}nderungen von Genen und Signalwegen sowie Methylierungsunterschiede gefunden werden. Anhand der Sequenzierdaten der retrospektiven Studie wurden molekulare prognostische Marker (Anzahl an proteinver{\"a}ndernden Varianten pro Tumorprobe, Ver{\"a}nderungen im P53/Rb- und/oder dem Wnt/ß-Catenin-Signalweg und dem Methylierungsstatus von CpG-Inseln von vier 2 Tumorsuppressorgenen (GSTP1, PAX5, PAX6 und PYCARD)) definiert und f{\"u}r jeden einzelnen Marker ein signifikanter Zusammenhang zur L{\"a}nge des progressionsfreien {\"U}berlebens (PFS) der Patient*innen gefunden. Durch die Kombination der molekularen Marker mit den klinischen und histopathologischen Markern war es zudem m{\"o}glich, einen COMBI-Score zu bilden, der, verglichen mit den klinischen und histopathologischen Markern, eine spezifischere und sensitivere Aussage dar{\"u}ber erlaubt, ob Patient*innen innerhalb von 2 Jahren ein Fortschreiten der Tumorerkrankung erfahren. Mit Hilfe der Sequenzierdaten wurden in beiden Kohorten außerdem Ver{\"a}nderungen gefunden, die als pr{\"a}diktive Marker zum Einsatz von zielgerichteten Therapien vewendet werden k{\"o}nnten. Als vielversprechendstes Therapieziel wurde - bei 46 Tumoren in der retrospektiven und 7 Tumoren in der prospektiven Studie - CDK4 identifiziert. CDK4/CDK6-Inhibitoren sind f{\"u}r die Behandlung von fortgeschrittenem und metastasiertem Brustkrebs von der Lebensmittel- {\"u}berwachungs- und Arzneimittelbeh{\"o}rde (FDA; engl. Food and Drug Administration) zugelassene Therapeutika und bei anderen soliden Tumoren Gegenstand von Studien. Im Rahmen der Arbeit konnten außerdem von 12 Patient*innen jeweils zwei Tumoren molekulargenetisch untersucht und die Ergebnisse verglichen werden. Die Analyse zeigte, dass der Methylierungsstatus - im Vergleich zu Ver{\"a}nderungen in der DNA-Sequenz - der stabilere prognostische Marker ist. Mit dieser Arbeit wurde gezeigt, dass molekulare prognostische und pr{\"a}diktive Marker f{\"u}r den Einsatz zielgerichteter Therapien mit Methoden identifiziert werden k{\"o}nnen, die sich im klinischen Alltag bei der Behandlung von NNR-Ca-Patient*innen implementieren lassen. Um einen allgemein anerkannten Leitfaden zu etablieren, fehlen allerdings noch die Ergebnisse weiterer - vor allem prospektiver - Studien zur Validierung der hier pr{\"a}sentierten Ergebnisse. Die gewonnenen Erkenntnisse sind jedoch als wichtiger Schritt in Richtung personalisierter Medizin bei Nebennierenrindenkarzinomen anzusehen.},
  subject      = {Nebennierentumor},
  language  = {de}
}
@phdthesis{DeğirmencineePoelloth2023,
  author    = {Değirmenci [n{\´e}e P{\"o}lloth], Laura},
  title     = {Sugar perception and sugar receptor function in the honeybee (\(Apis\) \(mellifera\))},
  doi       = {10.25972/OPUS-32187},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321873},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {In the eusocial insect honeybee (Apis mellifera), many sterile worker bees live together with a reproductive queen in a colony. All tasks of the colony are performed by the workers, undergoing age-dependent division of labor. Beginning as hive bees, they take on tasks inside the hive such as cleaning or the producing of larval food, later developing into foragers. With that, the perception of sweetness plays a crucial role for all honeybees whether they are sitting on the honey stores in the hive or foraging for food. Their ability to sense sweetness is undoubtedly necessary to develop and evaluate food sources. Many of the behavioral decisions in honeybees are based on sugar perception, either on an individual level for ingestion, or for social behavior such as the impulse to collect or process nectar. In this context, honeybees show a complex spectrum of abilities to perceive sweetness on many levels. They are able to perceive at least seven types of sugars and decide to collect them for the colony. Further, they seem to distinguish between these sugars or at least show clear preferences when collecting them. Additionally, the perception of sugar is not rigid in honeybees. For instance, their responsiveness towards sugar changes during the transition from in-hive bees (e.g. nurses) to foraging and is linked to the division of labor. Other direct or immediate factors changing responsiveness to sugars are stress, starvation or underlying factors, such as genotype. Interestingly, the complexity in their sugar perception is in stark contrast to the fact that honeybees seem to have only three predicted sugar receptors. In this work, we were able to characterize the three known sugar receptors (AmGr1, AmGr2 and AmGr3) of the honeybee fully and comprehensively in oocytes (Manuscript II, Chapter 3 and Manuscript III, Chapter 4). We could show that AmGr1 is a broad sugar receptor reacting to sucrose, glucose, maltose, melezitose and trehalose (which is the honeybees' main blood sugar), but not fructose. AmGr2 acts as its co-receptor altering AmGr1's specificity, AmGr3 is a specific fructose receptor and we proved the heterodimerization of all receptors. With my studies, I was able to reproduce and compare the ligand specificity of the sugar receptors in vivo by generating receptor mutants with CRISPR/Cas9. With this thesis, I was able to define AmGr1 and AmGr3 as the honeybees' basis receptors already capable to detect all sugars of its known taste spectrum. In the expression analysis of my doctoral thesis (Manuscript I, Chapter 2) I demonstrated that both basis receptors are expressed in the antennae and the brain of nurse bees and foragers. This thesis assumes that AmGr3 (like the Drosophila homologue) functions as a sensor for fructose, which might be the satiety signal, while AmGr1 can sense trehalose as the main blood sugar in the brain. Both receptors show a reduced expression in the brain of foragers when compared with nurse bees. These results may reflect the higher concentrated diet of nurse bees in the hive. The higher number of receptors in the brain may allow nurse bees to perceive hunger earlier and to consume the food their sitting on. Forager bees have to be more persistent to hunger, when they are foraging, and food is not so accessible. The findings of reduced expression of the fructose receptor AmGr3 in the antennae of nurse bees are congruent with my other result that nurse bees are also less responsive to fructose at the antennae when compared to foragers (Manuscript I, Chapter 2). This is possible, since nurse bees sit more likely on ripe honey which contains not only higher levels of sugars but also monosaccharides (such as fructose), while foragers have to evaluate less-concentrated nectar. My investigations of the expression of AmGr1 in the antennae of honeybees found no differences between nurse bees and foragers, although foragers are more responsive to the respective sugar sucrose (Manuscript I, Chapter 2). Considering my finding that AmGr2 is the co-receptor of AmGr1, it can be assumed that AmGr1 and the mediated sucrose taste might not be directly controlled by its expression, but indirectly by its co-receptor. My thesis therefore clearly shows that sugar perception is associated with division of labor in honeybees and appears to be directly or indirectly regulated via expression. The comparison with a characterization study using other bee breeds and thus an alternative protein sequence of AmGr1 shows that co-expression of different AmGr1 versions with AmGr2 alters the sugar response differently. Therefore, this thesis provides first important indications that alternative splicing could also represent an important regulatory mechanism for sugar perception in honeybees. Further, I found out that the bitter compound quinine lowers the reward quality in learning experiments for honeybees (Manuscript IV, Chapter 5). So far, no bitter receptor has been found in the genome of honeybees and this thesis strongly assumes that bitter substances such as quinine inhibit sugar receptors in honeybees. With this finding, my work includes other molecules as possible regulatory mechanism in the honeybee sugar perception as well. We showed that the inhibitory effect is lower for fructose compared to sucrose. Considering that sugar signals might be processed as differently attractive in honeybees, this thesis concludes that the sugar receptor inhibition via quinine in honeybees might depend on the receptor (or its co-receptor), is concentration-dependent and based on the salience or attractiveness and concentration of the sugar present. With my thesis, I was able to expand the knowledge on honeybee's sugar perception and formulate a complex, comprehensive overview. Thereby, I demonstrated the multidimensional mechanism that regulates the sugar receptors and thus the sugar perception of honeybees. With this work, I defined AmGr1 and AmGr3 as the basis of sugar perception and enlarged these components to the co-receptor AmGr2 and the possible splice variants of AmGr1. I further demonstrated how those sugar receptor components function, interact and that they are clearly involved in the division of labor in honeybees. In summary, my thesis describes the mechanisms that enable honeybees to perceive sugar in a complex way, even though they inhere a limited number of sugar receptors. My data strongly suggest that honeybees overall might not only differentiate sugars and their diet by their general sweetness (as expected with only one main sugar receptor). The found sugar receptor mechanisms and their interplay further suggest that honeybees might be able to discriminate directly between monosaccharides and disaccharides or sugar molecules and with that their diet (honey and nectar).},
  subject      = {Biene},
  language  = {en}
}