https://bura.brunel.ac.uk/handle/2438/32874 2026-10-19T05:35:08Z https://bura.brunel.ac.uk/handle/2438/33994 Title: Targeting Acinetobacter baumannii wound infections: From transcriptional profiling to drug repurposing and bioactive hydrogel development Authors: Han, Jie Abstract: Antimicrobial resistance (AMR) is an increasing public health emergency, driven largely by the overuse and misuse of antibiotics. Among multidrug-resistant pathogens, Acinetobacter baumannii is a major clinical concern due to its ability to survive in hospital environments. Recent studies have shown that artificial sweeteners, including acesulfame potassium (Ace-K), sodium saccharin (SACC), and sodium cyclamate (CYC), can inhibit bacterial growth, motility, and biofilm formation, and may enhance antibiotic activity in vitro. However, effective delivery of these compounds to wound infection sites remains a significant challenge. This thesis investigates alternative non-antibiotic strategies for targeting A. baumannii, with a focus on artificial sweetener-loaded polyvinyl alcohol (PVA)-based hydrogels, behaviour changing in wound-mimic environment, 3D-printable hydrogel development, and screening of FDA-approved drugs that modulate virulence-associated pathways. PVA-Borax and freezethaw PVA hydrogels were formulated with Ace-K, SACC, and CYC and demonstrate strong antimicrobial activity. In addition, this thesis explores how A. baumannii behaves in a woundmimic environment in Lubbock chronic wound biofilm (LCWB) medium. Under LCWB conditions, it induced reduced biofilm formation and iron-starvation responses. With iron supplementation, biofilm formation was restored. This highlights that iron is limited in the LCWB medium and is an important regulator of A. baumannii behaviour. Moreover, a 3Dprintable PVA/hydroxypropyl methylcellulose (HPMC) hydrogel was then developed to improve mechanical properties and control antimicrobial release. The optimised PVA/HPMC/SACC formulation demonstrated improved printability, structural integrity, swelling behaviour, rapid SACC release and antimicrobial activity compared with non-printed formulations. Finally, a high-throughput screen of an FDA-approved drug library identified compounds that regulate pilA expression and biofilm formation in A. baumannii. A cancer drug pomalidomide, was identified as a key hit that suppressed pilA expression, reduced twitching-associated behaviour, enhanced biofilm formation, and increased infection severity in Galleria mellonella and murine infection models. Overall, these findings demonstrate that effective control of A. baumannii wound infections requires more than identifying antimicrobial compounds alone. It also requires wound-relevant testing models, rational biomaterial-based delivery systems, and careful evaluation of how nonantibiotic compounds influence bacterial physiology and virulence. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2026-01-01T00:00:00Z https://bura.brunel.ac.uk/handle/2438/33502 Title: Telomere length elongation by epigenetic modifier drugs: A potential mechanism by which cancer develops chemo-resistance Authors: Al-dulaimi, Sarah Abstract: Telomeres are specialised structures localized at the ends of eukaryotic linear chromosomes. Telomeres play an important role in protecting chromosome ends by preventing the activation of DNA damage response (DDR). In normal cells, telomeres become shorter with each cell division, which eventually leads to cellular senescence. Cancer cells, avoid telomere shortening by activating mechanisms that maintain telomere lengths. Most normal cells reach replicative senescence after 90 population doublings and then enter telomere crisis. This suggest that many premalignant cells lose their ability to keep dividing before they can accumulate additional mutations. Two mechanisms are known: telomerase reactivation and alternative lengthening of telomere (ALT). 80% -85% of cancer cells activates telomerase. The telomerase enzyme consists of two subunits, telomerase reverse transcriptase (TERT) and telomerase RNA (TR). ALT is active in around 15-20 % of cancer cells and maintains telomere lengths via alternative lengthening of telomere via homologous recombination. A common feature of ALT-positive human cells is the presence of C-circles and promyelocytic leukemia (PML) nuclear bodies (ALT-associated PML (APBs)). POLD3, a subunit of DNA polymerase has also been shown to be essential for ALT activity. The hypomethylating drug 5-aza-2′-deoxycytidine (5-aza) is widely used in the treatment of haematological malignancies. Moreover, cancer cells rapidly become resistant to the drug, leading to relapse. The study presented in this thesis investigated the short term (72 h) and long term (over one month) effects of 5-aza on telomere biology in breast cancer cells, with a focus on ALT activity and POLD3 expression. Telomere maintenance mechanisms were investigated using qPCR, c-circle and TRAP assays. The TIF (Telomere dysfunction induced foci) was used to detect DNA damage at telomeres and immunofluorescence for the detection of ALTassociated PML. POLD3 gene expression was quantified using qPCR and western blotting. Short-term 5-aza treatment modestly altered telomerase activity while significantly promoting ALT activation, which was accompanied by increased expression of POLD3. Silencing of POLD3 using siRNA in 5-aza treated cells resulted in telomere shortening. Long-term exposure to 5-aza led to the development of drug resistance, with cells acquiring the ability to proliferate at higher concentrations of 5-aza, and reduced sensitivity to doxorubicin, alongside increased migratory capacity. However, resistant cells exhibited enhanced sensitivity to radiotherapy. Collectively, our findings demonstrate that low-dose (10 uM) and prolonged exposure to 5- aza promotes ALT- dependent telomere maintenance through POLD3 upregulation, contributing to cancer cell survival and therapeutic resistance. These findings identify a novel mechanism by which cancer cells develop resistance to epigenetic therapy and also highlight telomere maintenance pathways as promising targets to improve treatment outcomes. Resistance to 5-aza significantly compromises the efficacy of combination therapies, including those involving chemotherapy and radiotherapy Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z https://bura.brunel.ac.uk/handle/2438/33413 Title: Evaluating genetic liability in hypertension and stroke using machine learning and traditional statistical models: Insights from UK biobank studies Authors: MacCarthy, Gideon Abstract: Stroke is one of the leading causes of death and disability worldwide, with hypertension being a major risk factor for stroke. According to the World Stroke Organisation Global Stroke Fact Sheet, hypertension alone is responsible for over half of all stroke-related deaths and disability-adjusted life years (DALYs). While conventional risk factors for both diseases are well established, the added prediction value of genetic liability remains less clear. The traditional risk prediction models for hypertension and stroke, such as the Framingham Hypertension Risk Score (FHRS) and the Framingham Stroke Risk Score (FSRS), typically rely on clinical and demographic factors, often assume linear effects, and typically overlook genetic liability and complex interactions between predictors. In this thesis, three complementary studies were conducted to examine whether genetic liability could enhance the classification of hypertension and the prediction of strokes via both machine learning (ML) and traditional modelling techniques using data from more than 116,000 participants with European ancestry in the UK Biobank. Genetic variants and their effects obtained from genome-wide association studies were used to construct genetic liabilities for selected cardiovascular disease (CVD) risk factors and stroke, respectively. Multiple predictive models, such as Cox proportional hazards, penalized regression models (both logistic and Cox), tree-based algorithms (random forest, gradient boosting, and decision trees), and neural networks, were assessed after participants were randomly divided into training and testing sets. Discrimination (AUC), calibration, and reclassification indices (NRI, IDI, and Brier score) were used to evaluate the models. Incorporating the genetic liabilities resulted in modest but steady improvements across the studies. The genetic liabilities associated with lipids improved the classification of hypertension best with AUC using random forest. Additionally, stroke genetic liability enhanced stroke prediction with the Cox models, outperforming machine learning models. Among hypertensive individuals, the model's predictive performance (AUC) was higher in men and older adults than in women or younger adults. The Cox models outperformed all the machine learning models. Though ML methods allow for the investigation of non-linearities and interactions. Overall, genetic liability slightly enhances classification and risk prediction. Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London 2025-01-01T00:00:00Z https://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