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(2023)ThesisPyoderma Gangrenosum (PG) is an inflammatory cutaneous disease with no standard highly effective treatment. Novel therapeutics with a predictable treatment response are desperately needed, although a major barrier for this is the incomplete understanding of the molecular mechanisms that underpin this disease. Traditionally, PG was thought to be driven by a local dysregulated neutrophilic response. Recent investigations however have identified elevated levels of interleukin (IL) -23 and IL-17 in the serum and tissue of PG patients, suggesting that the Th17 axis may play a role within this disease. This thesis characterises the molecular mechanisms underlying PG and assesses whether the Th17 axis and related cytokines are major drivers of this disease. A systematic review was performed to collate biomarkers that have been associated with PG. Following this, an exploratory analysis of existing and other possible biomarkers of disease activity associated with active and resolving PG was conducted through an open label, single arm clinical trial. Participants diagnosed with active PG underwent lesional and non-lesional biopsies at baseline, with subsequent systemic administration of subcutaneous tildrakizumab (IL-23 antagonist) 200 mg, at dosing intervals of week 0, week 4, and week 8, with repeat lesional and non-lesional biopsies performed at week 12 (trial completion). Clinical markers of disease activity such as ulcer size, physicians’ global assessment, visual analogue scale scores, and quality of life scores were measured throughout the trial. An a priori analysis of biomarkers gathered from the systematic review conducted was performed on biopsied whole skin samples either through RNA sequencing by a nanostring multiple gene expression assay, or through immunohistochemistry, with comparisons made between healthy control (HC), baseline lesional, non-lesional and lesional tissue at week 12 of the trial. Various biomarkers associated with PG including IL-23, IL-17A and IL-17F cytokines were elevated in baseline lesional tissue, and to a lesser extent non-lesional tissue, when compared to HC. These inflammatory mediators had a reduced expression within PG tissue in response to IL-23 antagonism. A statistically significant improvement in quality-of-life scores and VAS scores was identified in participants. A reduction in ulcer size was also noted in ulcerative PG but not peristomal PG. The results of this thesis give valuable insights into the role of the Th17 axis in the development of ulcerative PG, and identifies the IL-23 antagonist tildrakizumab as an effective treatment for ulcerative PG.
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(2023)ThesisBreast cancer (BC) is the leading cause of cancer-related death among women worldwide. Currently, the conventional method for diagnosing BC such as mammogram, is not reliable for detecting small lesions or dense breast tissue. Surgical biopsies cannot provide accurate and real-time information due to the complexity of the tumour. Small extracellular vesicles (sEVs) are one of EV subpopulations and secreted by all cell types, containing various biological cargoes that reflect their cellular origin. sEVs are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Studying sEVs in liquid biopsy for BC diagnosis is a new developing research area. Therefore, disease specific proteins contained in sEVs are considered as a superior choice for non-invasive liquid biopsy biomarker source in BC. In this thesis, I specifically aim to 1) Establish and optimise a method for isolating sEVs from BC cell lines and human BC plasma samples; 2) Find the most effective approach for proteomic analysis; 3) Identify potential sEV protein biomarkers using BC cells and plasma samples by LC-MS/MS proteomics for BC diagnosis and prognosis. sEVs derived from three BC cell lines (MDA-MB-231, MCF-7, and SK-BR-3), one normal breast cell line (MCF-10A), three BC patients' plasma, and three non-cancer controls were isolated using ultracentrifugation (UC), Total Exosome Isolation kits (TEI), and a combined approach of UC and TEI (UCT). In BC cell lines, the UC isolates showed a higher sEV purity and sEV marker expression, as well as a significantly higher number of sEV proteins. UC isolation identified 10 potential sEV protein biomarkers in BC cell lines. In BC plasma samples, the UCT isolates showed the highest proportion of sEV- related proteins and the lowest percentage of lipoprotein-related proteins. UCT isolates demonstrated 9 potential sEV protein biomarkers in BC plasma. In summary, I have demonstrated that the assessment of both quantity and quality of sEV isolation methods is important in selecting the optimal approach for the specific sEV research purpose depending on the sample types and downstream analysis. In addition, future validation of distinct sEV proteins identified in my study holds potential, for developing new diagnostic approaches in BC liquid biopsy and promoting the application of personalised medicine.