http://bura.brunel.ac.uk/handle/2438/166 2026-05-12T08:24:43Z http://bura.brunel.ac.uk/handle/2438/33186 Title: Investigating replication fork blocks, replication-transcription conflicts and replication restart dynamics in Escherichia coli Authors: Peros, Stelinda Abstract: DNA replication is essential for genome stability, but it is constantly jeopardized by various obstacles such as nucleoprotein complexes and transcription–replication conflicts. If not properly resolved, these impediments lead to replication fork collapse, genomic instability, and even cell death. This thesis investigates how Escherichia coli preserves its replication integrity using three experimental systems: site-specific protein– DNA blockades, engineered replication–transcription conflicts, and chemical stress induced by saccharin exposure. These studies were supported by the development of an automated bioimage informatics pipeline, utilizing deep-learning segmentation to enable high-throughput quantitative analysis of cellular morphology and SOS-induced stress phenotypes in live-cell time-lapse microscopy. Using a novel inducible fork-block model, I demonstrate that the PriA–PriB– DnaT pathway is the primary restart mechanism at nucleoprotein obstacles, with PriA helicase activity being essential for efficient replication restart. Surprisingly, when large tandem repeats were placed on the opposite replichore, PriC rather than PriB played the dominant role, raising the possibility that restart pathway usage is influenced by obstacle size or chromosomal context. Replication–transcription conflicts, generated by introducing an ectopic origin of replication (oriZ), similarly required PriA helicase and PriB for efficient fork restart. In their absence, cells displayed severe filamentation, heterogeneous stress phenotypes, and elevated Cas1–Cas2 foci. To further define the nature of these collisions, I utilized an alternative origin (oriX); the comparison between head-on and co-directional orientations confirmed that cellular pathology was specifically conflict- dependent. Genetic suppression with an RNA polymerase–destabilizing mutation confirmed that these defects stem directly from transcriptional collisions rather than indirect effects. Finally, I show that saccharin, a widely used artificial sweetener, induces replication stress in E. coli, with PriB-deficient cells exhibiting pronounced defects and loss of viability. These findings highlight how dietary compounds may disrupt gut microbial physiology. Collectively, this work establishes PriA helicase as a central player in replication restart and a promising antibacterial target. Since stalled fork rescue is also critical in cancer cells, these results also provide conceptual bridges between bacterial DNA replication and oncogene-induced replication stress, opening avenues for both antimicrobial and cancer therapeutic development. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z http://bura.brunel.ac.uk/handle/2438/32769 Title: Novel memory phenotype Tfh cells arise without overt antigen stimulation and are important for adaptive immune responses against viral infection Authors: Busharat, Zabreen Abstract: Pathogen-induced memory Tfh cells exert a Tfh effector response during reinfection, regulating the generation of high-affinity antibodies. Here, we define novel memory-phenotype Tfh cells which are generated from naïve T cells under homeostatic conditions. These MP Tfh cells are phenotypically and functionally similar to pathogen-induced Tfh cells. MP Tfh cells can be defined by Tfh cell specific markers, CXCR5, BCL6, and PD-1, and markers of pathogen-induced long lived Tfh cells, FR4. T-bethigh MP T cells exert an innate-like Th1 response against viral infections. The transcription factor EGR2 is a repressor of T-bet function, and we found that MP Tfh cells are distinct from T-bethigh MP T cells but express EGR2 highly. Previously, we found Egr2 is required for MP T cell homeostasis and inflammation. Here, we observed that, in Egr2/3-/- CD4+ MP T cells, MP Tfh cell development is impaired. FR4+ EGR2 + MP T cells upregulate genes related to homeostatic proliferation, Tfh cell development and metabolic pathways of pathogen-induced memory Tfh cells. MP Tfh cells can exert an adaptive function by regulating B cell-mediated IgG production in vitro whereas MP Tfr cells are involved in suppressing MP Tfh cell function, thereby preventing excessive inflammation. In vivo, MP Tfh cells support germinal centre formation and induce neutralising antibody production after infection with vaccinia virus. Thus, MP Tfh cells with similar characteristics to pathogen-induced memory Tfh cells are developed in absence of environmental antigens and to date are the only CD4+ MP T cell subset associated with an adaptive immune response against viral infection. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z http://bura.brunel.ac.uk/handle/2438/32731 Title: A comprehensive proteomics analysis of Friedreich's ataxia (FRDA) Authors: Ulukütük Gözlügöl, Zeynep Abstract: First and foremost, I would like to express my deepest gratitude to my Principal Supervisor, Dr. Sara Anjomani Virmouni, for her invaluable guidance, constructive feedback, and unwavering support throughout this journey. Her mentorship has been instrumental in shaping both my research and academic growth. I am also sincerely grateful to members of my supervisor team, Supervisor, Prof. Michael Themis. Additionally, I extend my thanks to Dr. Victor Hernandez, my Research Development Advisor, for his guidance especially during the first year of my PhD. I extend my sincere appreciation to Ministry of National Education, TURKEY, for their financial support as well as to Brunel University of London for providing access to the facilities and resources required for this research. My heartfelt appreciation goes to my collaborators, whose contributions have been vital to my research. I appreciate Associate Professor Faraz Mardakheh from the Barts Cancer Institute (Queen Mary University of London) for conducting proteomics analysis in human FRDA cells. I am also thankful to Professor Richard Wade-Martins' team at Oxford University for providing Human iPS-derived cardiomyocytes and to Professor Marek Napierala’s laboratory at the University of Texas Southwestern Medical Centre for supplying Human iPSCs. I would like to acknowledge Dr. Raha Pazoki from Brunel University London for her support in bioinformatics, particularly in creating the transcriptomics analysis heat map. I am also grateful to Dr. Saqlain Suleman, a postdoctoral researcher in our group, for his academical support and help. Additionally, I extend my thanks to Dr. Zenouska Ramchunder, another postdoctoral researcher in our group, for her academical help and endless support. I want to thank my beloved parents for their endless love, trust, and support throughout my journey. Their encouragement and sacrifices have given me the strength to overcome challenges and reach this milestone. I am forever grateful for everything they have done to help me achieve my dreams.I also want to thank my dear soulmate and husband, Haci, for his patience, love, and support. Even when we were far apart, his constant encouragement and understanding made our bond stronger. I am deeply grateful for his steady presence and for our love that has grown even through tough times. I wish to thank the team members of the Brunel technician staff for their assistance, which greatly facilitated the experimental aspects of this study. Their expertise and support have been invaluable in ensuring the smooth execution of laboratory work. I am also grateful to my colleagues in the Ataxia lab for their encouragement, collaboration, and insightful discussions. Their support has made this journey both intellectually enriching and personally rewarding. Finally, I extend my deepest appreciation to everyone who has contributed, directly or indirectly, to the successful completion of this thesis. Your support has been invaluable, and I am truly grateful. This thesis would not have been possible without the support and guidance of many individuals and organizations. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z http://bura.brunel.ac.uk/handle/2438/32643 Title: Genetic insights into the epidemiology of cataracts, prevention and alternative treatment Authors: Hashimi, Munisa Abstract: Cataract, a leading cause of visual impairment and blindness, remains a significant global health challenge, particularly in the context of an aging population. As the global population continues to age, the burden of cataract on healthcare systems, especially in developing countries, is expected to increase. Cataract involves the clouding of the lens, and surgical extraction remains the sole treatment option. Understanding the genetic mechanisms underlying cataract, identifying preventive measures, and exploring alternative treatments are critical to reducing this burden. This thesis utilised UK Biobank generated genome-wide association study (GWAS), and publicly available GWAS data to investigate the shared genetic mechanisms between cataract subtypes and cataract-associated risk factors. It also assessed alcohol consumption, vitamin D levels and deficiency, and lanosterol as potential modifiable risk factors or alternative treatment options. Genetic correlations were identified between overall cataract and type 2 diabetes (T2D), asthma and diabetic cataract, senile and diabetic cataract, and asthma and overall cataract. Co-localisation analysis highlighted genes of interest, including WWP2 and CDKN2B-AS1 between overall cataract and T2D, and HLA-DQB1 between asthma and overall cataract. Mendelian randomisation analyses found no evidence of a causal relationship between vitamin D levels, vitamin D deficiency, or alcohol consumption and cataract. Similarly, lanosterol was not supported as a viable alternative treatment option. In summary, while this study identified genetic links between cataracts and associated risk factors; it did not provide supporting evidence for vitamin D, alcohol, and lanosterol as effective preventive measures or alternative treatment options. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z