http://bura.brunel.ac.uk/handle/2438/8615 2026-04-07T02:17:32Z 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 http://bura.brunel.ac.uk/handle/2438/32638 Title: Involvement of matricellular proteins and immune receptors in the dynamic interaction between tumour and immune cells Authors: AL Fashtaki, Reham Adnan Abstract: Background: Osteosarcoma (OS) is the most common primary malignant bone tumour in children and young adults, characterised by high metastatic potential and poor prognosis in advanced stages. Despite recent therapeutic advances, metastatic OS remains challenging to treat. Increasing evidence highlights the tumour microenvironment (TME), particularly immune cell components such as macrophages, as key modulators of tumour progression. Ovarian cancer (OC) is a significant cause of cancer-related deaths among women worldwide. It is a highly metastatic malignancy that shares similar molecular and immunological features with OS, including late-stage diagnosis, immune evasion, and extracellular matrix (ECM) remodelling. Matricellular proteins (MCPs) are non-structural extracellular matrix proteins that regulate cell– matrix interactions and signalling rather than providing structural support. They influence cell adhesion, migration, proliferation, and differentiation, and are often upregulated in cancer, promoting tumour growth, angiogenesis, and metastasis. Comparative analysis of OS and OC may reveal shared immune–metabolic pathways that contribute to tumour aggressiveness. This project investigates how MCPs and the interactions with immune cells influence tumour behaviour, survival, and metastatic progression within the TME. Using OC and OS as model systems, it integrates bioinformatic analyses, experimental validation, and functional assays to elucidate the molecular and immune mechanisms underpinning cancer progression and metastasis. Methods: Publicly available transcriptomic datasets for OS (GSE42352, GSE12865) and ovarian cancer (GSE18520, GSE54388, GSE14407) were analysed to identify differentially expressed genes (DEGs). The UniProt tool was used for functional annotation, with an emphasis on immune receptor (IR), cell adhesion (CAD), and ECM pathways. Survival analyses were performed using the KM Plotter. Candidate genes were validated in vitro using RT-qPCR and Western blotting in OS cell lines (MG63, SaOS-2, MNNG, 143B) and THP-1 monocytes differentiated into M0, M1, and M2 macrophages via PMA and cytokine stimulation. In addition, MNNG and 143B cells were treated with the FPR3 agonist WKYMVm and antagonist WRW4 (10 μM and 50 μM) for 24 h to directly assess the functional impact of FPR3 signalling on OS cell motility. Proliferation and scratch migration assays were performed using tumour- and macrophage-conditioned media. Secretomes were subjected to ultracentrifugation to assess the roles of soluble and vesicle-bound factors. Co-culture experiments were conducted to examine the bidirectional effects of macrophage-tumour signalling. Results: Initial bioinformatics analyses revealed a significant enrichment of DEGs associated with ECM remodelling, immune signalling, and poor survival in both OS and OC datasets. In OC, D24 (Cluster of Differentiation 24), DCN (Decorin), and OGN (Osteoglycin/Mimecan) were associated with adverse prognosis. In OS, SPP1 (osteopontin) and FPR3 (formyl peptide receptor 3) were upregulated, whereas LEPR (leptin receptor) was consistently downregulated across patient tissues, and it was a common gene among OC and OS. Functional assays demonstrated that M2- conditioned media enhanced the migration and proliferation of OS cells. Ultracentrifugation experiments suggested that vesicle-bound factors mediate these effects on the target cells. The reciprocal communication between osteosarcoma cells and macrophages establishes a pro-metastatic feedback loop. In vitro experiments using RT-qPCR and Western blotting revealed the differential expression of key genes involved in tumour progression and immune regulation across various osteosarcoma cell lines. Genes such as FPR3, LEPR, and SPP1 displayed variable mRNA and protein levels, indicating distinct molecular profiles between the cell lines. These differences highlight the heterogeneity of osteosarcoma and suggest that specific gene expression patterns may be linked to variations in tumour behaviour, immune interactions, and metastatic potential. Treatment of MNNG and 143B cells with the FPR3 agonist WKYMVm significantly increased migration velocity at both 10 μM and 50 μM, while the antagonist WRW4 effectively reduced migration, particularly at 50 μM. M2 macrophages showed high expression of CD163, FPR3, LEPR, and SPP1, reflecting their tumour-promoting, anti-inflammatory phenotype.THP-1 cells treated with OS-conditioned media upregulated CD163, consistent with M2 polarisation, whereas OS cell lines treated with macrophage supernatants displayed increased SPP1 and LEPR, particularly under M2 stimulation. Conclusion: This study identified SPP1, FPR3, and LEPR as key immune-metabolic mediators in OS, highlighting their roles in tumour–macrophage crosstalk and metastasis. The combination of in silico and in vitro findings supports a model in which the TME, particularly M2 macrophages, enhances OS progression via dynamic signalling pathways. These findings provide a foundation for targeting tumour–immune interactions in future OS therapies and suggest new directions for precision medicine strategies in metastatic cancers. Notably, LEPR was also consistently downregulated in OC, indicating a shared immunometabolism mechanism that may contribute to metastatic progression across tumour types. 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/2438/32538 Title: Regulation of biofilm formation and desiccation tolerance in the critical priority pathogen Acinetobacter baumannii Authors: Harkova, Lyuboslava G Abstract: The current ’’silent pandemic’’, caused by the increasing levels of antibiotic resistance amongst pathogenic bacteria and fuelled by the lack of new antibiotics, seriously threatens global healthcare systems. Acinetobacter baumannii is the most critical pathogen, for which novel therapeutic interventions are urgently needed. It is highly prevalent in hospitals and more alarmingly in intensive care units, where it can persist on surfaces in the absence of water and nutrients for months. Additionally, the majority of the nosocomial infections caused by this pathogen are biofilm-related. Biofilm formation is a universal bacterial survival strategy that confers protection to cells from harsh environmental conditions. However, majority of the regulatory mechanisms orchestrating biofilm formation and desiccation tolerance in A. baumannii are still elusive. In this thesis we present the screening of the Manoil lab transposon mutant library for differential biofilm biomass levels. This led to uncovering CavA adenylate cyclase (AC) as a novel negative biofilm mediator. Subsequently we characterise the role of CavA as the dominant AC under the conditions tested and reveal the cAMP signalling cascade in A. baumannii AB5075 strain. We show that cAMP inhibits biofilm formation while enhancing motility, which is exerted via its effector protein Vfr, and inhibits quorum sensing (QS). In addition to QS, we elucidate the hierarchy of signalling cascades that cAMP orchestrates, which also involves the other major second messenger c-di-GMP. We demonstrate that contrary to the current dogma, in A. baumannii cAMP increases intracellular levels of c-di-GMP via transcriptional regulation of DgcC diguanylate cyclase (DGC) and PdeE phosphodiesterase (PDE). Furthermore, we unveil that cAMP modulates virulence and transient antibiotic resistance. We also present that AB5075 strain possesses an additional AC, CavB, and a second potential cAMP effector protein Crp. In addition, we investigate regulators governing A. baumannii desiccation tolerance. We show that A. baumannii needs low cAMP levels, via CavB AC downregulation and CpdA PDE upregulation, while c-di-GMP related DGC DgcB negatively impacts desiccation tolerance. Moreover, the protective function of A. baumannii type VI secretion system under desiccation is uncovered. Finally, we demonstrate the central role of phenylacetic acid (PAA) catabolism in the desiccation tolerance of this pathogen. Our data shows that accumulation of intracellular PAA drives A. baumannii cells to enter a viable but non-culturable (VBNC) state, which is a persister strategy used by bacteria to withstand unfavourable conditions. These findings uncover central regulators of fundamental A. baumannii behaviours (formation of biofilms, tolerance to desiccation and VBNC state), which facilitate the survival of this pathogen in patients and hospitals. These new insights uncover targets for the development of novel therapeutics and disinfectants against A. baumannii and will contribute to improved detection and decontamination practices in the future. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z