N-Glykosylierungen spielen beim Env-Gen eine wichtige Rolle. Sie dienen nicht nur als „Escape-Phänomen“ zur Verhinderung einer Elimination des Virus durch neutralisierende Antikörper. Es hat sich gezeigt, dass bestimmte Menschen sich mit HIV infizieren können, aber es zu keinem Zeitpunkt zu AIDS-typischen Symptomen kommt, ohne die Einnahme antiretroviraler Therapie (ART). Solche Menschen werden als Elite Controller bezeichnet. Ihr Organismus kann selbst die Viruslast in sehr geringen Grenzen halten (< 50 Kopien/ml). Ziel dieser Arbeit ist es, den Einfluss von N-Glykosylierungen in der Entstehung von Elite Controller zu untersuchen und prozentuell eine Tendenz zu schaffen, inwieweit die Glykosylierungsdichte des Env-Proteins entscheidend ist. Es konnte gezeigt werden, dass eine immunologische Kontrolle auch auf der B-Zellebene stattfinden kann. Als Hinweis dient die geringe Glykosylierungsdichte im Bereich CD4bs und MPER, die indirekt über MHC Klasse II zu einer erhöhten Produktion von Antikörpern führen kann. Bisher wurde bei Elite Controller die T-Zellebene als mögliche immunologische Kontrolle beschrieben, jedoch gibt diese Arbeit hinweise, dass auch eine immunologische Kontrolle mittels Antikörper möglich ist. Die glykosylierten Zielepitope können eine große Hilfe sein für das Aussehen eines späteren Impfstoffs.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with an increasing incidence. The majority of MCC cases (approximately 80%) are associated with the Merkel cell polyomavirus (MCPyV). This virus encodes for the MCPyV T antigens (small T (sT) and large T (LT)), which are oncoproteins that drive MCC carcinogenesis. However, the precise cells of the skin that are transformed by the T antigens are not known i.e., the cells of origin of MCC are yet to be discovered. Therefore, the first part of this study involved the generation and evaluation of a vector system that could be used to study MCC oncogenesis. To this end, a set of lentiviral vectors was cloned that allows independent, inducible expression of potential key factors in MCC oncogenesis. In addition, a CRISPR/Cas9 knock in was established that allows the coding sequence for a fluorescent protein to be placed under the control of the promoter of KRT20, one of the most crucial markers of MCC. The functionality of this KRT20 reporter was proven in the MCPyV-positive MCC cell line, WaGa. The different inducible vector systems (doxycycline-inducible MCPyV T antigens or MCPyV sT, RheoSwitch-inducible ATOH1 and IPTG-inducible dnMAML1 and GLI1) were found to have different efficacies in various cellular systems and in particular, a considerable reduction in efficiency was observed at times upon the interaction of several vectors in one cell. In the second and more important part of this study, the role of the well-established anti-malarial drug, artesunate, which possesses additional anti-tumor and anti-viral activity, in the treatment of MCPyV-positive MCC was analyzed. In our study, artesunate was found to be cytotoxic towards MCPyV-positive MCC cell lines in vitro and repressed tumor growth in vivo in a mouse model. Artesunate was also found to downregulate T antigen expression, which is critical for the proliferation of MCPyV-positive MCC cells. The repression of T antigen expression, however, was not the sole mechanism of artesunate’s cytotoxic action; instead, the MCPyV-positive MCC cell line, WaGa, was found to be even less sensitive to artesunate after shRNA knockdown of the T antigens. Since loss of membrane integrity occurred more rapidly than degradation/loss of genomic DNA under the influence of artesunate in four of five MCPyV-positive MCC cell lines examined, apoptosis, although widely described as a modus operandi for artesunate, did not appear to be a determinant of the cytotoxicity of artesunate against MCPyV-positive MCC cells. Instead, we were able to demonstrate that artesunate induced the recently described iron-dependent and lipid peroxide-associated form of cell death known as "ferroptosis". This was achieved primarily through the use of inhibitors that can suppress specific individual steps of the ferroptotic process. Thus, artesunate-induced cell death of MCPyV-positive MCC cells could be suppressed by iron chelators and by the inhibition of lipid peroxidation and lysosomal transport. Surprising results were obtained from the analysis of two proteins associated with the ferroptotic process, namely, ferroptosis suppressor protein 1 (FSP1) and tumor suppressor protein p53. Here, we showed that ectopically- 2 expressed FSP1 cannot suppress artesunate-induced ferroptosis in MCPyV-positive MCC cells and that p53 does not play a pro-ferroptotic role in artesunate-induced cell death of MCPyV-positive MCCs. Since artesunate did not suppress the interferon-γ-induced expression of immune-related molecules such as HLA and PD-L1 on the surface of MCPyV-positive MCCs, our study also provided the first positive evidence for its use in combinatorial immunotherapy. Overall, this study showed that artesunate appears to be an effective drug for the treatment of MCPyV-positive MCC and might also be considered for its use in combinatorial MCC immunotherapy in the future.

Transcription describes the process of converting the information contained in DNA into RNA. Although, tremendous progress has been made in recent decades to uncover this complex mechanism, it is still not fully understood. Given the advances and reduction in cost of high-throughput sequencing experiments, more and more data have been generated to help elucidating this complex process. Importantly, these sequencing experiments produce massive amounts of data that are incomprehensible in their raw form for humans. Further, sequencing techniques are not always 100% accurate and are subject to a certain degree of variability and, in special cases, they might introduce technical artifacts. Thus, computational and statistical methods are indispensable to uncover the information buried in these datasets. In this thesis, I worked with multiple high throughput datasets from herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. During the last decade, it has became clear that a gene might not have a single, but multiple sites at which transcription initiates. These multiple transcription start sites (TiSS) demonstrated to have regulatory effects on the gene itself depending on which TiSS is used. Specialized experimental approaches were developed to help identify TiSS (TiSS-profiling). In order to facilitate the identification of all potential TiSS that are used for cell type- and condition-specific transcription, I developed the tool iTiSS. By using a new general enrichment-based approach to predict TiSS, iTiSS proved to be applicable in integrated studies and made it less prone to false positives compared to other TiSS-calling tools. Another improvement in recent years was made in metabolic labeling experiments such as SLAM-seq. Here, they removed the time consuming and laborious step of physically separating new from old RNA in the samples. This was achieved by inducing specific nucleotide conversions in newly synthesized RNA that are later visible in the data. Consequently, the separation of new and old RNA is now done computationally and, hence, tools are needed that accurately quantify these fold-changes. My second tool that I developed, called GRAND-SLAM proved to be capable to accomplish this task and outperform competing programs. As both of my tools, iTiSS and GRAND-SLAM are not specifically tailored to my own goals, but could also facilitate the research of other groups in this field, I made them publicly available on GitHub. I applied my tools to datasets generated in our lab as well as to publicly available data sets from HSV-1 and HCMV, respectively. For HSV-1, I was able to predict and validate TiSS with nucleotide precision using iTiSS. This has lead to the most comprehensive annotation for HSV-1 to date, which now serves as the fundamental basis of any future transcriptomic research on HSV-1. By combining both my tools, I was further able to uncover parts of the highly complex gene kinetics in HCMV and to resolve the limitations caused by the densely packed genome of HCMV. With the ever-increasing advances in sequencing techniques and their decrease in cost, the amounts of data produced will continue to rise massively in the future. Additionally, more and more specialized omics approaches are appearing, calling for new tools to leverage their full information potential. Consequently, it has become apparent that specialized computational tools such as iTiSS and GRAND-SLAM are needed and will become an essential and indispensable part of the analysis.

Mittels einer klinischen Studie wurden die Furchtgeneralisierung und Aufmerksamkeitslenkung von 44 gesunden Kindern und Jugendlichen im Alter von 9-17 Jahren untersucht. Eine Übergeneralisierung konditionierter Furcht sowie veränderte Aufmerksamkeitsprozesse werden in zahlreichen Arbeiten mit der Entstehung und Aufrechterhaltung von Angsterkrankungen in Verbindung gebracht. Der Hauptteil der Forschung beschränkte sich bislang auf die Untersuchung von erwachsenen Probanden. Da Angsterkrankungen jedoch häufig bereits im Kindes- und Jugendalter entstehen und sich in der Erforschung psychiatrischer Erkrankungen zunehmend eine dimensionale Betrachtungsweise durchsetzt, bestand das Ziel der Studie darin, etwaige Alterseffekte und den Einfluss der Ängstlichkeit auf die genannten Phänomene bei gesunden Probanden zu untersuchen. Darüber hinaus wurde ein potentiell präventiver Ansatz erforscht. Im Ergebnis zeigten sich in den Gruppenvergleichen keine relevanten Differenzen. Interessanterweise deutete sich in der Gruppe der älteren Probanden entgegen der Erwartung eine verstärkte Furchtgeneralisierung an, die womöglich mit einer veränderten Beziehung zu Furcht und Risiko in der Adoleszenz zusammenhängt. Aus den Befunden ergibt sich die Notwendigkeit weiterer, prospektiver Arbeiten, um unser Verständnis der Ätiologie von Angsterkrankungen zu verbessern. Weiterhin ist noch offen, inwiefern es sich bei der Übergeneralisierung und einer veränderten Aufmerksamkeitslenkung um Risikofaktoren für die Entwicklung von Angsterkrankungen oder vielmehr um Epiphänomene handelt, die erst mit Ausbruch der Erkrankung auftreten. Der Einsatz von Methoden der virtuellen Realität erscheint besonders geeignet, diese Prozesse zukünftig noch besser zu erforschen.

In highly polarized neurons, endoplasmic reticulum (ER) forms a dynamic and continuous network in axons that plays important roles in lipid synthesis, Ca2+ homeostasis and the maintenance of synapses. However, the mechanisms underlying the regulation of axonal ER dynamics and its function in regulation of local translation still remain elusive. In the course of my thesis, I investigated the fast dynamic movements of ER and ribosomes in the growth cone of wildtype motoneurons as well as motoneurons from a mouse model of Spinal Muscular Atrophy (SMA), in response to Brain-derived neurotrophic factor (BDNF) stimulation. Live cell imaging data show that ER extends into axonal growth cone filopodia along actin filaments and disruption of actin cytoskeleton by cytochalasin D treatment impairs the dynamic movement of ER in the axonal filopodia. In contrast to filopodia, ER movements in the growth cone core seem to depend on coordinated actions of the actin and microtubule cytoskeleton. Myosin VI is especially required for ER movements into filopodia and drebrin A mediates actin/microtubule coordinated ER dynamics. Furthermore, we found that BDNF/TrkB signaling induces assembly of 80S ribosomes in growth cones on a time scale of seconds. Activated ribosomes relocate to the presynaptic ER and undergo local translation. These findings describe the dynamic interaction between ER and ribosomes during local translation and identify a novel potential function for the presynaptic ER in intra-axonal synthesis of transmembrane proteins such as the α-1β subunit of N-type Ca2+ channels in motoneurons. In addition, we demonstrate that in Smn-deficient motoneurons, ER dynamic movements are impaired in axonal growth cones that seems to be due to impaired actin cytoskeleton. Interestingly, ribosomes fail to undergo rapid structural changes in Smn-deficient growth cones and do not associate to ER in response to BDNF. Thus, aberrant ER dynamics and ribosome response to extracellular stimuli could affect axonal growth and presynaptic function and maintenance, thereby contributing to the pathology of SMA.