@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{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{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{Geis2023,
  author    = {Geis, Maria},
  title     = {Identifizierung von Zielmolek{\"u}len und Herstellung zweigeteilter trivalenter T-Zell-aktivierender Antik{\"o}rperderivate zur immuntherapeutischen Behandlung von Multiplen Myelom},
  doi       = {10.25972/OPUS-18690},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186906},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {T-Zell-aktivierende Formate, wie BiTE (bispecific T-cell engagers) Antik{\"o}rper und CAR T Zellen haben in den vergangen Jahren die Therapiem{\"o}glichkeiten f{\"u}r Tumorpatienten erweitert. Diese Therapeutika verkn{\"u}pfen T-Zellen mit malignen Zellen {\"u}ber je ein spezifisches Oberfl{\"a}chenmolek{\"u}l und initiieren, {\"u}ber eine T-Zell-vermittelte Immunantwort, die Lyse der Tumorzelle. Tumorspezifische Antigene sind jedoch selten. H{\"a}ufig werden Proteine adressiert, die neben den Tumorzellen auch auf gesunden Zellen exprimiert werden. Die Folgen sind toxische Effekte abseits der Tumorzellen auf Antigen-positiven gesunden Zellen (on target/off tumor), welche nicht nur die Dosis des Therapeutikums und dessen Effektivit{\"a}t limitieren, sondern zu geringen bis letalen Begleiterscheinungen f{\"u}hren k{\"o}nnen. Der Bedarf an effektiven Therapieformen mit geringen Nebenwirkungen ist folglich immer noch sehr hoch. Diese L{\"u}cke soll durch ein neues Antik{\"o}rperformat, sogenannten Hemibodies, geschlossen werden. Hemibodies sind eine neue Klasse von T-Zell-aktivierenden Antik{\"o}rpern, die sich gegen eine Antigenkombination und nicht einzelne Antigene auf Tumorzellen richten. Sie bestehen aus zwei komplement{\"a}ren Molek{\"u}len mit je einer Antigen-bindenden Sequenz, die entweder mit der leichten (VL) oder der schweren (VH) Kette eines T-Zell-aktivierenden anti CD3 Antik{\"o}rpers fusioniert ist. Nur wenn beide Hemibody-Fragmente gleichzeitig in unmittelbarer N{\"a}he an ihr jeweiliges Antigenepitop auf der Tumorzelle binden, komplementieren die beiden Antik{\"o}rperkonstrukte {\"u}ber das geteilte anti-CD3 und bilden einen trivalenten T Zell aktivierenden Komplex aus. Diese funktionale Einheit rekrutiert T-Zellen zur Tumorzelle und induzierte die T-Zell-vermittelte Lyse der malignen Zelle. Im Rahmen der vorliegenden Arbeit wurden geeignete Antigenkombinationen identifiziert und die erste effektive und spezifische Hemibody-basierte Immuntherapie gegen das Multiple Myelom (MM), ohne Nebenwirkungen auf Antigen-einfach-positiven gesunden Zellen, entwickelt. Basierend auf einer umfangreichen Analyse von Kandidaten-Antigenen wurden Kombinationen aus bekannten MM Zielmolek{\"u}len, wie BCMA, CD38, CD138, CD229 und SLAMF7, und f{\"u}r das MM unbekannte Oberfl{\"a}chenmolek{\"u}len, wie CHRM5 und LAX1, untersucht. Gegen die vielversprechendsten Antigene wurden Hemibodies entwickelt und produziert. Im Zusammenhang mit Analysen zur Produzierbarkeit sowie biochemischen und funktionalen Charakterisierungen, konnte aus 75 initialen Hemibody-Kombinationen drei Kombinationen mit geeigneten Eigenschaften identifiziert werden. Die Bindung von zwei Hemibody-Partnern auf der Oberfl{\"a}che der MM Zelle f{\"u}hrte zur Ausbildung eines trivalenten T-Zell-rekrutierenden Komplexes. Dieser initiierte nachfolgend {\"u}ber eine T-Zell-vermittelte Immunantwort die spezifische Lyse der malignen Zellen, ohne die Viabilit{\"a}t von Antigen-einfach-positiven gesunden K{\"o}rper- oder Effektor-Zellen zu beeinflussen. Zus{\"a}tzlich f{\"u}hrte eine Hemibody-Therapie in vivo in einem NOD SCID MM-Mausmodel innerhalb von 7 Tagen zur kompletten Remission der MM Zellen. Diese Daten zeigten Hemibodies als ein neues, sehr vielversprechendes Antik{\"o}rperformat f{\"u}r eine effektive und tumorspezifische Immuntherapie mit potentiell geringen Nebenwirkungen.},
  language  = {de}
}
@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{Schwarz2023,
  author    = {Schwarz, Jessica Denise},
  title     = {Genome-wide reporter screens identify transcriptional regulators of ribosome biogenesis},
  doi       = {10.25972/OPUS-27901},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-279010},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Cellular growth and proliferation are among the most important processes for cells and organisms. One of the major determinants of these processes is the amount of proteins and consequently also the amount of ribosomes. Their synthesis involves several hundred proteins and four different ribosomal RNA species, is highly coordinated and very energy-demanding. However, the molecular mechanims of transcriptional regulation of the protein-coding genes involved, is only poorly understood in mammals. In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming to identify previously unknown transcriptional regulators of ribosome biogenesis factors (RiBis), which are important for the assembly and maturation of ribosomes, and ribosomal proteins (RPs), which are ribosomal components themself. With that approach and follow-up (validation) experiments, ALDOA and RBM8A among others, could be identified as regulators of ribosome biogenesis. Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter driven reporters on protein and transcript level, as well as to a downregulation of ribosome biogenesis gene transcripts and of mRNAs of other genes important for proliferation. Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent downregulation of one of the fluorescent reporters, but this regulation was independent of the promoter driving the expression of the reporter. However, acute protein depletion experiments in combination with nascent RNA sequencing (4sU-Seq) revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of RP genes, but also to other chromatin regions. Total POL II or elongating and initiating POL II levels were not altered upon acute RBM8A depletion. These data provide a starting point for further research on the mechanisms of transcriptional regulation of RP and RiBi genes in mammals.},
  subject      = {Ribosom},
  language  = {en}
}
@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{Muench2023,
  author    = {M{\"u}nch, Luca},
  title     = {Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus},
  doi       = {10.25972/OPUS-28922},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289228},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Der Name der transposablen Elemente beruht auf ihrer F{\"a}higkeit, ihre genomische Position ver{\"a}ndern zu k{\"o}nnen. Durch Chromosomenaberrationen, Insertionen oder Deletionen k{\"o}nnen ihre genomischen Transpositionen genetische Instabilit{\"a}t verursachen. Inwieweit sie dar{\"u}ber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterf{\"u}hrung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der {\"a}ltesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des b{\"o}sartigen schwarzen Hautkrebses, welcher nach wie vor die t{\"o}dlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit besch{\"a}ftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.},
  subject      = {Transposon},
  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{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{Reuter2023,
  author    = {Reuter, Christian Steffen},
  title     = {Development of a tissue-engineered primary human skin infection model to study the pathogenesis of tsetse fly-transmitted African trypanosomes in mammalian skin},
  doi       = {10.25972/OPUS-25114},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251147},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host. Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression. A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts. The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes. In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection. After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host. In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@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{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{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}
}