/library/oar/handle/123456789/31724 2025-12-21T23:08:02Z /library/oar/handle/123456789/141610 Title: Genetic, behavioural and molecular studies of Gemin3 using drosophila melanogaster Abstract: N/A Description: Ph.D.(Melit.) 2023-01-01T00:00:00Z /library/oar/handle/123456789/141609 Title: Development of an innate immune cell tolerance model Abstract: Sepsis has been defined as the ‘life-threatening organ dysfunction caused by a dysregulated host response to infection’ carrying a poor prognosis and high mortality rates, both during its progression, as well as after hospital discharge, rendering it quite the adversary to modern medicine. One contributory aspect to this high mortality rates is sepsis-induced immune suppression, where cells of the innate immune system enter a state of tolerance and functional reprogramming, dampening any subsequent immune reaction to secondary infections. The work done in this dissertation aimed to develop an in vitro model of immune tolerance that serves as a standard on which future experimentation may be carried out. This was done by isolating and purifying human primary monocytes from young blood donors, characterising them using CD14, HLA-DR and FACS flowcytometry. Subsequently, a re-stimulation protocol was applied using bacterial LPS in order to induce endotoxin tolerance. The success in generating the tolerant state was assessed following the generation of significant differences in the mean cytokine secretion levels of TNF-α, IL-1β and IL-10 using ELISA and statistical computation, as well as through the confirmation of a retention in overall cell viability throughout using 7-AAD. It was found that a tolerant state was induced within the monocytes and macrophages, with TNF-α producing the most significant difference in mean secretion concentrations between the first and second stimulation, generating a pvalue of 7.824x10-4 . The differences in IL-1β and IL-10 secretion levels were 0.000542794 and 0.018214324, respectively. Following 7-AAD analysis, the overall cell viability was retained, indicating the successful acquisition of a tolerant state within the CD14+ , HLADR+ cell population. Description: B.Sc. (Hons) Med. Biocem.(Melit.) 2024-01-01T00:00:00Z /library/oar/handle/123456789/141359 Title: Unravelling the mechanisms of xanthine oxidoreductase chain formation : insights into protein structure and function Abstract: Xanthine oxidoreductase (XOR) catalyses the last two steps of purine catabolism, converting hypoxanthine to xanthine and subsequently to uric acid. From these reactions, hydrogen peroxide and superoxide anion radicals can be generated. Increased activity of XOR may result in hyperuricemia due to higher production of uric acid and oxidative stress from increased generation of reactive oxygen species (ROS). Hyperuricemia is associated with cardiovascular diseases, including stroke, and hypertension. Hypertension is linked to the depletion of nitric oxide by ROS and the subsequent production of peroxynitrite radicals. Various mutations have also been associated with high XOR activity as well as a higher risk of different conditions, such as hypertension, hyperuricemia, and xanthinuria. From previous studies, it was also shown that XOR can form filaments that may have a role in its enzymatic activity. In this project, the hXOR wildtype (WT) and one clinical mutation identified in a Maltese study, I646V, were characterised. The aim of the project was to determine of wild type hXOR and the I646V assembled into filaments formation and whether the filaments are enzymatically active. Transformed TP1000 E. coli cells harbouring either the pTrcHis-hXOR WT or I646V variant were grown under aerobic and anaerobic conditions in order to compare the filament formation and enzymatic activity. This approach aimed to elucidate the enzymatic role in physiological ischaemic conditions. Some samples were also subjected to heat shock to enhance the folding and activity of hXOR. Purification was carried out using immobilized metal affinity chromatography, with purity and yield assessed by SDS-PAGE. Filament presence was confirmed via Native-PAGE. Additionally, hXOR activity was evaluated using the uric acid spectrophotometric assay and the Nitro Blue Tetrazolium (NBT) assay. The secondary structure and melting temperature were analysed using circular dichroism. Production of XOR under anaerobic conditions resulted in a higher enzymatic activity for both the WT and I646V variant. For the samples produced under aerobic conditions, the I646V variant had the highest activity but had the lowest yield and stability. On the other hand, hXOR WT grown under anaerobic conditions was the most active out of all conditions tested and had the highest stability. Filament formation was observed in most conditions, with anaerobic growth conditions seemingly favouring filament formation for both WT and mutant. Moreover, the filaments formed retained activity as shown in the NBT assays. These findings are particularly important as filament formation might modulate the enzymatic activity of hXOR. Additionally, the observed increase in hXOR activity under anaerobic conditions is noteworthy, as physiologically, hXOR can be produced under hypoxic and ischemic conditions. The high activity of hXOR produced in such environments may result in significant ROS production, potentially leading to various adverse health conditions. Description: B.Sc. (Hons) Med. Biocem.(Melit.) 2024-01-01T00:00:00Z /library/oar/handle/123456789/141358 Title: Characterisation of exosomes using a flow cytometry multiplex assay Abstract: Lung cancer is an extremely morbid condition, with an estimated 2.48 million new diagnoses occurring globally each year. Alarmingly, 70% of these cases are identified at stages III and IV, when the disease is significantly advanced and treatment options become limited and less effective (Aktary et al., 2022). This late-stage diagnosis underscores the urgent need for the development of newer and more effective diagnostic methods that can detect lung cancer at earlier stages, thereby improving prognosis and survival rates. One promising avenue for early diagnosis lies in the study of extracellular vesicles (ECVs). ECVs are nanoscale particles shed by cells, which carry a rich cargo of biomolecules including proteins, cell-free DNA (cfDNA), and RNA (Shedden et al., 2003; Cohen et al., 2017; Okudela et al., 2012). These vesicles play a crucial role in cell-to-cell communication, tumour growth, prognosis, metastasis, and more as such the characterisation of theses vesicles would provide a plethora of information for diagnosis and treatment planning (Kong et al., 2019; Kim, J. et al., 2016; Hasan et al., 2022). In this thesis, lung cancer cell lines were utilized to isolate and characterize ECVs, with the aim of identifying potential biomarkers for early detection of the disease. ECVs were isolated from the cell lines using polyethylene glycol (PEG) precipitation and size exclusion chromatography. These methods are widely used for their efficiency and reliability in purifying ECVs from complex biological samples. Following isolation, the ECVs were characterised using the Luminex xMap® technology, which allows for the simultaneous detection and quantification of multiple biomarkers in a single sample. Multiplexing approaches were employed for the simultaneous detection of multiple cell surface markers, hence increasing specificity. Through the characterisation of ECVs using the methods found in this study. Further developments are liquid biopsy via identifying specific protein markers on the surface of ECVs, it may be possible to develop non-invasive tests for early detection of lung cancer, which could be implemented in routine clinical practice. Description: B.Sc. (Hons) Med. Biocem.(Melit.) 2024-01-01T00:00:00Z