http://theses.ncl.ac.uk/jspui/handle/10443/5256 Mon, 22 Jun 2026 17:39:01 GMT 2026-06-22T17:39:01Z http://theses.ncl.ac.uk/jspui/handle/10443/6827 Title: New methods for the discovery and characterization of bacterial natural products Authors: Sumang, Felaine Anne Abstract: Actinomycetes are renowned for their ability to produce a wide array of natural products (NPs), with a broad range of applications. NPs are typically encoded by biosynthetic gene clusters (BGCs). Bioinformatic analysis of sequenced Actinomycetes genomes have shown that they possess many BGCs whose potential products are not detected under standard laboratory culture conditions. Advances in genome mining and molecular biology have led to the development of diverse strategies to activate these “cryptic” BGCs, but further improvements in these methods are warranted. This thesis explores multiple approaches for discovering novel bioactive compounds from Actinomycetes and investigating their BGCs. Chapters 3 and 4 focus on the development of a new bacterial artificial chromosome (BAC) vector, designated pJE2, and optimization of a protocol for genomic library construction enabling the isolation of large BGCs. This approach was applied to Actinomadura madurae T576, a producer of unusual sulphated metabolites, resulting in a BAC library from which a clone harbouring the complete 80 kbp BGC responsible for these metabolites was identified. Heterologous expression of this clone in Streptomyces albus successfully led to production of the sulphated metabolites. Screening of additional clones also revealed the successful capture of several previously uncharacterized BGCs. Chapter 5 focuses on a chemical elicitation method, using plant extracts to stimulate NP production in soil-derived Actinomycetes. Notably, hibiscus flower extract induced the production of the antibiotic thiolutin by Streptomyces strain MBN 2-2. Further analysis identified hibiscus acid and hydroxycitric acid as the elicitor compounds. The final chapter centres on associating putative BGCs with the synthesis of three novel antibiotics demurilactone A, persiathiacin, and quinovosamycin. In all three cases, insertional mutagenesis led to the abolition of antibiotic synthesis, thereby validating assignment of the BGCs to their respective antibiotics. Description: Ph. D. Thesis. Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6827 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6825 Title: Investigating the Role of Structural Variation in Male Infertility Authors: Kalyon, Oguzhan Abstract: Infertility affects approximately one in six couples globally, with male factors contributing to roughly half of all cases. Although chromosomal abnormalities such as Klinefelter’s syndrome and Y chromosome microdeletions are well-established causes of male infertility (MI), nearly 40% of cases remain idiopathic. The role of structural variants (SVs) and dominant inheritance pattern has been understudied, primarily due to technical limitations in identifying SVs and the lack of patient-parent trio analyses required to investigate dominant de novo variants. In this thesis, we performed whole-genome sequencing (WGS) on 216 patients with idiopathic azoospermia and their parents to investigate SVs across different inheritance models, with a particular focus on the dominant model. Additionally, whole-exome sequencing (WES) was conducted on 234 additional patients with azoospermia. In these cohorts, we identified several SVs that clearly explained the patients' phenotypes, as well as numerous potentially causative SVs that revealed novel candidate male infertility genes and loci. This study demonstrates that WGS is an effective tool for studying SVs and significantly advances our understanding of the genetic basis of male infertility, particularly regarding the contribution of SVs. Description: PhD Thesis Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6825 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6818 Title: Using systems biology to explore temporal changes in human fibroblast cellular senescence. Authors: Scanlan, Rebekah-Louise Anne Abstract: Cellular senescence is a complex phenotype characterised by permanent cell cycle arrest and a senescence-associated secretory phenotype, which includes growth factors and inflammatory cytokines. While primarily thought of as a tumour suppressive mechanism, senescence also plays roles in wound healing and embryogenesis. Senescent cells are normally transient but accumulate with age due to dysregulated immune clearance, contributing to low-grade chronic inflammation and age-related diseases. Characterising senescent cells is challenging due to phenotype heterogeneity, and the temporal dynamics of senescence remain poorly understood. This thesis employed an integrated approach to investigate human fibroblast senescence at transcriptomic and protein levels. A systematic review identified 119 transcriptomic datasets on human fibroblast senescence, forming the database SenOmic, publicly hosted online and allowing users to filter by variables of interest such as gene and timepoint. Computational modelling of key selected proteins in DNA damage-induced senescence (DDIS) and oncogene-induced senescence (OIS) was also performed, including knockdown interventions. Analysis of SenOmic reinforced the challenges of defining a universal geneset across cell lines and senescence types. However, 28 genes were significantly up- or downregulated across DDIS, OIS, replicative senescence, and bystander senescence compared to proliferating controls, with only one gene present in the KEGG senescence pathway. Distinct phenotypes were also observed, including significantly stronger p53 signalling in DDIS compared to OIS, clustering of samples by time, and significant upregulation of genes involved in protein secretion between days 5-7 in gene set enrichment analysis. Protein level modelling demonstrated the importance of multi-macrolevel analysis, highlighting post-translational modifications and network-wide effects of knockdowns. In conclusion, while unique universal senescence biomarkers remain challenging to identify, conventional senescence markers follow predictable profiles, distinct phenotypic differences exist across timepoints and senescence types, and further interrogation of resources like SenOmic with an established framework provides a valuable means to enhance our understanding of senescence. Description: Ph. D. Thesis. Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6818 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6817 Title: Investigating the role of Asporin in musculoskeletal development Authors: Pearson, Rachel Deborah Abstract: Understanding musculoskeletal development is of high importance, as it forms the foundation for understanding various skeletal disorders and can illuminate pathways involved in other musculoskeletal conditions including osteoarthritis (OA). This thesis focuses on the role of Asporin, a small leucine-rich proteoglycan (SLRP), expressed in the developing periosteum, perichondrium, tendons, and subchondral bone. Asporin possesses a unique ability among SLRPs to mineralise type I collagen fibres. A polymorphism associated with risk of OA and IDD was identified in GWAS studies increases Asporin function, moreover Asporin is significantly upregulated in OA cartilage in comparison to healthy cartilage. The study employs an in vitro approach utilising Asporin overexpression in cartilage and bone cells to replicate the conditions observed in OA. In addition, the research integrates the use of Asporin null mouse models and col2cre conditional knockout of Asporin to elucidate the role of Asporin in musculoskeletal development and specifically in cartilage and periosteal cells. This research shows the effect of Asporin in the mineralisation of the ECM and reduction of sulphated proteoglycan in the overexpression model, interesting for the role of Asporin in OA pathogenicity. Phenotyping of the global mouse model demonstrates that Asporin is not essential for endochondral ossification or cartilage structure, highlighting modulation of Asporin expression as potential therapeutic target of OA with minimal off target effects. Further, studying of the role of Asporin in bone using µCT, histomorphometric analysis and histological techniques to gain a well rounded understanding of its involvement in development showed that the loss of Asporin does not disrupt bone or spinal development nor affect the tendon structure. However, deletion of Asporin in type II collagen positive periosteal skeletal progenitor cells (under Col2Cre) hinders bone remodelling and appositional growth in the col2cre conditional Asporin knockout, suggesting a potential role for Asporin in bone homeostasis and fracture repair. Description: Ph. D. Thesis. Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6817 2025-01-01T00:00:00Z