http://hdl.handle.net/10900/42126 Wed, 22 Jul 2026 12:34:47 GMT 2026-07-22T12:34:47Z http://hdl.handle.net/10900/181807 Design and application of prime editing to target ARSA P426L mutation in a metachromatic leukodystrophy patient Trosien, Paul Luis Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal storage disease caused primarily by mutations in the Arylsulfatase A (ARSA) gene. Loss of functional ARSA leads to sulfatide accumulation, causing demyelination in the peripheral and central nervous system. MLD presents in different clinical forms, which all eventually result in severe neurological decline and death. Currently available treatment options are limited. Enzyme replacement therapy (ERT) and allogeneic hematopoietic stem cell transplantation (allo-HSCT) may slow disease progression but do not offer a cure. Libmeldy was recently approved by the European Medicines Agency (EMA) as the first gene therapy for early-onset forms of MLD. However, the potential long-term risk of insertional mutagenesis remains a concern and requires ongoing evaluation. In this project we targeted the ARSA P426L mutation, one of the most common MLD-causing mutations in Europe. We used prime editing, a CRISPR-Cas9 based mechanism, which allows to introduce precise changes into the genome. The experiments were carried out in K562 cells and hematopoietic stem and progenitor cells (HSPCs). If the ARSA P426L mutation could be corrected ex vivo in patient-derived HSPCs prior to transplantation, this approach could offer a potentially curative treatment for patients who are currently lacking effective therapeutic options. Such a strategy would eliminate the need to find a matching donor for allogeneic HSCT, evade its side effects and avoid the risk of insertional mutagenesis associated with Libmeldy. Instead, it would restore physiological ARSA expression by precisely correcting the mutation at its endogenous locus. In K562 cells, we successfully introduced the ARSA P426L mutation using prime editing, achieving mean editing efficiencies of 1.88% with PE2 and 10.20% with PE3. However, no editing was detected in HSPCs of healthy donors. As a result, this study did not progress to target patient-derived HSPCs. Further investigations are needed to focus on optimizing the prime editing mechanism as well as related components in order to achieve higher editing efficiency. Also research about target site specific limitations for prime editing should be closely monitored and taken into account when planning for future projects. Although editing of the ARSA P426L locus was not successful, the high efficiency to induce the IL2RG c.458T>C mutation in HSPCs, which was used as a proof of concept, supports the general feasibility of prime editing based gene therapy approaches. Wed, 22 Jul 2026 00:00:00 GMT http://hdl.handle.net/10900/181807 2026-07-22T00:00:00Z http://hdl.handle.net/10900/181806 Biochemical Reconstitution of the Budding Yeast Meiotic Chromosomal Axis Chen, Linda Dissertation gesperrt bis zum 30.06.2028!; The production of gametes requires a specialised cell division to divide the genome, thus enabling the faithful transmission of genetic material from one generation to the next. This process is known as meiosis, the hallmark of which is meiotic recombination. A striking feature here is the physical exchange of DNA between homologous chromosomes, also known as crossing over, which is essential for ensuring that chromosomes segregate accurately. Moreover, genetic exchange between homologous chromosomes promotes species diversity – contributing to the overall evolution of Eukarya. It begins with the highly regulated formation of double-stranded DNA breaks (DSBs) throughout the genome, which take place within the context of a DNA-loop and proteinaceous axis structure. Crucially, a sub-set of these breaks must be repaired using the homologous chromosome as a template, as this is a pre-requisite for crossover formation. In Saccharomyces cerevisiae, this inter-homolog biased repair is ensured by a meiosis-specific protein complex: Hop1-Red1-Mek1 (HRM). Hop1 and Red1 form the structural basis of the proteinaceous axis, whereas Mek1 kinase is involved in both mediating inter-homolog bias as well as meiotic progression. Considerable in vivo efforts have been conducted to understand the functions of these proteins in meiosis, however there is limited knowledge about how HRM form a complex. Moreover, the assembly mechanism of the budding yeast axis itself is still poorly understood on the molecular stage. To extend our understanding of the molecular determinants required for the assembly of the HRM complex, I utilised an in vitro structural biochemistry approach. I investigated how the HRM complex is established and activated by designing a series of protein truncations and mutants, and revealed previously unrecognised roles for the N-terminus of Red1 in its interaction with Hop1 and Mek1. To characterise the enzymatic properties of Mek1, I recapitulated the meiotic conditions required for kinase activation in vitro. Finally, I explored the potential roles that the axis plays in the recruitment of key DSB machinery and cross-over formation proteins. Through this work, I propose a novel model whereby the HRM complex is potentially assembled and primed temporally early on to mediate inter-homolog bias at the onset of meiotic DSBs. The results presented here address the long-standing question regarding how Mek1 is recruited to axial sites and provide insight into the importance of the meiotic chromosome axis in mediating multiple branches of meiotic recombination in budding yeast. Fri, 30 Jun 2028 00:00:00 GMT http://hdl.handle.net/10900/181806 2028-06-30T00:00:00Z http://hdl.handle.net/10900/181805 Narrative Comprehension and Inference Processes Across the Lifespan: The Impact of Stress and Aging Varkentin, Ekaterina In today’s media-saturated environment, defined by widespread smartphone use and accelerating globalization, the delivery of content through visual narratives has become the prevailing norm. Individuals encounter narratives in nearly all areas of daily life, from professional settings to recreational activities, whether reading the morning newspaper, following a work presentation, or interpreting airplane safety instructions. The ability to comprehend narratives is a fundamental cognitive skill across the lifespan and has therefore long been a central focus of research. Despite the extensive body of work, a limited understanding remains of how pervasive life factors, such as stress, health conditions, and lifestyle-related influences, shape narrative comprehension. As these factors become increasingly prevalent in contemporary society, understanding how individuals process and navigate narratives is not only theoretically important but also of growing societal relevance. Thus, the central question of this dissertation is how narrative comprehension and inference generation are shaped by long-term and short-term influences, as well as by factors such as age and education. Across three empirical chapters, the studies presented in this dissertation provide insights into the mechanisms underlying narrative comprehension and examine how these mechanisms are modulated by these diverse influences. A central focus throughout is the role of aging. After introducing the theoretical overview of this dissertation, Chapter 2 investigates the role of long-term protective factors (physical and mental activity, social connectedness) and risk factors (depression, anxiety, chronic pain, long-term stress, poor sleep) as well as the role of age, education, and modality (textual vs. pictorial) for narrative comprehension. The results indicated a general advantage for pictorial over textual narratives, although this effect decreased with age. Narrative comprehension was largely resilient to age, education, and long-term protective and risk factors. Performance correlated with memory but not with other cognitive abilities, highlighting its specificity within the broader cognitive domain. Chapter 3 examines the effects of acute, short-term stress on narrative comprehension in different age groups. The results revealed that acute stress negatively affected visual narrative comprehension in younger adults, whereas the comprehension performance of older adults remained stable. Similarly, younger adults reported lower confidence in their responses under stress, while older adults’ confidence levels were unaffected. These findings highlight the interplay between visual narrative comprehension, stress, and aging, suggesting that with advancing age and experience, individuals may develop more differentiated event schemas that render their comprehension processes more resilient to acute stress. Chapter 4 examines how acute stress affects the generative processes involved in narrative comprehension, thereby offering a more comprehensive understanding of how stress shapes this cognitive function. The findings revealed that inference generation, in contrast to inference recognition, remained stable across both age and education groups and was unaffected by acute stress. These results highlight the resilience of generative and creative cognitive processes, even under challenging conditions, and suggest that the ability to construct coherent narrative connections may be particularly robust to short-term stress. Overall, these findings support a view of narrative comprehension as both dynamic and resilient. On the one hand, comprehension appears to be fundamentally stable across the lifespan and largely resistant to chronic influences, indicating that well-integrated cognitive systems help maintain coherent meaning even in the face of age-related changes. On the other hand, comprehension is sensitive to short-term situational factors in specific ways. For instance, inference recognition processes are affected by acute stress in younger adults, whereas inference generation remains largely stable across all age groups under stress. This distinction suggests that different cognitive mechanisms support different components of narrative comprehension. Recognition relies more on immediate retrieval and evaluative processes, making it more vulnerable to external influences, while generative processes involve constructive integration and creative elaboration, rendering them more resilient even under challenging conditions. From an applied standpoint, the findings endorse the use of pictorial materials in diverse contexts and provide important insights for fostering age-inclusive participation and for guiding educational systems. Wed, 22 Jul 2026 00:00:00 GMT http://hdl.handle.net/10900/181805 2026-07-22T00:00:00Z http://hdl.handle.net/10900/181804 Evolution Beyond DNA: Long-Term Experimental Evolution in Pristionchus pacificus Quiobe, Shiela Pearl Evolution is a continuous and historical process that can be studied experimentally in organisms with rapid generation times. Because evolution ultimately depends on the generation and inheritance of phenotypic variation, a key unresolved question is the extent to which environmental factors can produce heritable changes. Therefore, environmental effects on trait inheritance remain debated because it is often difficult to distinguish true transgenerational inheritance from short-term parental influences. While mammalian systems face technical limitations, nematodes provide powerful experimental models. In this work, I investigated transgenerational epigenetic inheritance (TEI) of environmentally induced traits using Pristionchus pacificus, a nematode that exhibits mouth-form plasticity, including predation. I established long- term environmental induction experiments with alternative diets by propagating 110 isogenic lines over 101 generations, together with complementary food-reversal assays. I found that a Novosphingobium diet enriched in vitamin B12 induces the predatory mouth morph, which is subsequently transmitted across generations and occasionally becomes canalized at low frequency. To uncover the underlying mechanisms, I performed large-scale genetic screening and identified the target- directed microRNA degradation (TDMD) factor EBAX-1/ZSWIM8. I showed that Ppa- EBAX-1 specifically destabilizes the expanded miR-35/miR-2235a microRNA cluster in P. pacificus, revealing an unexpected repressive role of microRNAs in regulating TEI of the predatory morph. By defining the dietary trigger landscape, I further demonstrated that vitamin B12 is sufficient to induce TEI in a concentration-dependent manner and uncovered a complex regulatory network of potential RNA triggers linked to TDMD-like processes. Overall, my work over the past five years provides conceptual and experimental advances on nematode TEI. This expeirmental framework leverages the natural organismal readout of plasticity in P. pacificus and offers key insights into developmental plasticity, its transgenerational inheritance, and low-frequency canalization of traits. Together, these findings establish a foundation for addressing broader evolutionary questions and provide a mechanistic link between environmental responsiveness and heritable phenotypic change. Tue, 21 Jul 2026 00:00:00 GMT http://hdl.handle.net/10900/181804 2026-07-21T00:00:00Z