https://www.um.edu.mt/library/oar/handle/123456789/145742 Wed, 02 Sep 2026 19:51:08 GMT 2026-09-02T19:51:08Z https://www.um.edu.mt/library/oar/handle/123456789/148763 Title: Comprehensive profiling of lung cancer markers for early screening and detection Abstract: Lung cancer remains the leading cause of cancer-related mortality worldwide, largely due to late-stage diagnosis and the lack of effective non-invasive screening strategies. The stability of extracellular vesicles in biofluids, combined with their capacity to present cell-derived surface proteins, has led to growing interest in their potential as minimally invasive cancer biomarkers. While EV-based assays are not currently implemented for early cancer diagnosis, methodological challenges related to EV isolation, detection sensitivity and assay reproducibility remain key barriers to their clinical translation. The aim of this study was to comprehensively profile EV-associated surface markers relevant to lung cancer and to evaluate their robustness and detectability across different assay formats, isolation methods, and biological matrices. A multi-step analytical workflow was implemented using four non- small-cell lung cancer (NSCLC) cell lines (A549, NCI-H1975, NCI-H460 and NCI-H520). Cell-surface marker expression was first characterised by flow cytometry to establish a biological reference. EVs were subsequently isolated using three approaches: polyethylene glycol (PEG) precipitation, size-exclusion chromatography (Izon qEV1), and polymer-based precipitation (SubXTM), and further characterised by nanoparticle tracking analysis (NTA). EV surface marker detection was performed using antibody-based bead immunoprecipitation coupled to Luminex FLEXMAP 3D® assays in both single-plex and multiplex formats. Plasma spike-in experiments using tumour-derived EVs were performed to assess assay sensitivity in a clinically relevant matrix, and plasma samples from patients with metastatic lung cancer were analysed to explore translational feasibility. Distinct and biologically meaningful EV surface marker profiles were observed across lung cancer models. CD9 and CD63 were consistently detected across cell line-derived EVs, plasma spike-in experiments and patient plasma samples, with CD63 producing the strongest signal. Multiplex immunoprecipitation improved detection of several lower-abundance markers, including CD24, CD90, and CD274, compared with single-plex capture. Both Izon and SubXTM isolation methods enabled detection of tumour-derived EVs in plasma, however, Izon SEC produced a higher marker signal intensities across spike concentrations. In conclusion, this study establishes a robust technical framework for EV surface marker profiling in lung cancer and demonstrates the feasibility of immunoprecipitation-based bead assays for detecting biologically relevant EV markers. These findings support the potential of EV-associated surface proteins as candidates for future non-invasive lung cancer screening and early detection strategies. Description: M.Sc.(Melit.) Thu, 01 Jan 2026 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/148763 2026-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/147101 Title: Characterisation of two clinical mutations of Xanthine oxidoreductase related to hyperuricaemia Abstract: Xanthine oxidoreductase (XOR) is a molybdoflavin enzyme and a homodimer with a mass of 290 kDa. In mammals, XOR exists in two interconvertible forms: Xanthine Dehydrogenase (XDH) and Xanthine Oxidase (XO). XOR catalyses the final steps of purine catabolism, converting hypoxanthine to xanthine and subsequently to uric acid. These two reactions can generate reactive oxygen species (ROS), specifically hydrogen peroxide and superoxide anion radicals. XOR has been associated with various health conditions, including hyperuricemia, oxidative stress, and hypertension. ROS generated by XOR contribute to endothelial dysfunction, with increased ROS levels reacting with nitric oxide (NO) to form peroxynitrite radicals, exacerbating hypertension. Different variants have also been linked with increased XOR activity as well as higher risk of diseases, such as hyperuricemia and hypertension. In this project, the hXOR wildtype (WT) and two known clinical variants identified in a Maltese study by the MAMI study, I646V and I703V, as well as a novel double mutant (I646V-I703V) also identified in the Maltese population, were characterised. Apart from the clinical variants, the I646A mutation was also constructed to characterise to assess the effect of the substitution of a large non-polar branched amino acid such as isoleucine with a smaller one. The aim of this project was to determine the effect these amino acid changes have on the hXOR protein. Transformed TP1000 E.coli cells harbouring either the pTrc-HisXOR WT or the variants were grown under aerobic conditions without and with heat shock. Purification was performed using immobilised metal affinity chromatography. The purified proteins were characterised using SDS-PAGE, Native-PAGE, Blue-Native gel, and Dynamic Light Scattering (DLS) for size and oligomeric state. The enzymatic activity was evaluated using the uric acid spectrophotometric assay and the Nitro Blue Tetrazolium (NBT) assay. Secondary structure, thermal stability (melting temperature), and amyloid fibril formation were assessed using circular dichroism, DLS, and the Thioflavin T (ThT) assay, respectively. The amyloidogenic potential was further investigated using the ZipperDB server. All variants exhibited significantly higher enzymatic activities compared to the WT hXOR sample, with the I646A variant showing the highest activity (9-fold greater than WT). Analysis of DLS data confirmed that the native proteins of all mutants predominantly occur in the active native dimer conformation. Conversely, the WT hXOR exhibited the lowest activity and formed the largest oligomeric structures. The WT hXOR formed amyloid fibrils in the ThT assay, supported by the identification of amyloidogenic stretches using ZipperDB. Most variants did not show fibrillization, specifically I703V, I646A, I646V+HS, I646V-I703V+HS, and I646A+HS. The substitution of Ile646 with Valine or Alanine (I646V, I646A) significantly reduced the amyloidogenic propensity while increased substantially the enzymatic activity. I646V-I703V+HS also exhibited high enzymatic activity and displayed no fibril formation. These findings show that while WT hXOR activity is dampened by fibril formation, the clinical variants remain hyperactive and stable. This high activity of hXOR may result in significant ROS production and hyperuricemia potentially leading to various health conditions, such as hypertension. Description: M.Sc.(Melit.) Thu, 01 Jan 2026 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/147101 2026-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/146312 Title: Diagnostic accuracy of proton magnetic resonance spectroscopy for non-invasive detection of 2-hydroxyglutarate and IDH mutation status in gliomas : a meta-analysis Abstract: Isocitrate dehydrogenase (IDH) mutation status is a central determinant of modern glioma classification, prognosis, and therapeutic decision-making. Mutant IDH enzymes acquire a neomorphic catalytic function that leads to the pathological accumulation of the oncometabolite D-2-hydroxyglutarate (2-HG), a metabolite that is largely absent in IDHwildtype gliomas and normal brain tissue. This mutation-specific metabolic alteration provides a unique opportunity for non-invasive molecular characterisation, as intracellular 2- HG accumulates to concentrations detectable in vivo using proton magnetic resonance spectroscopy (MRS). The recent approval of IDH-targeted therapy following the phase III INDIGO trial has further increased the clinical importance of early and accurate identification of IDH mutation status. This dissertation aimed to evaluate the diagnostic accuracy of in vivo proton MRS for 2-HG detection as a non-invasive biomarker of IDH1/IDH2 mutation in gliomas, and to assess whether diagnostic performance has changed over time in parallel with methodological advances in MRS acquisition and analysis. A systematic review and meta-analysis were conducted in accordance with PRISMADTA guidelines. PubMed, MEDLINE, and Scopus were searched for studies published between 2012 and 2023 that evaluated in vivo proton MRS for 2-HG detection in human glioma cohorts with histopathologically confirmed IDH status. Studies providing sufficient data to construct 2×2 contingency tables were included. Methodological quality was assessed using the QUADAS-2 tool. Pooled sensitivity and specificity were estimated using a bivariate random-effects model, and overall diagnostic performance was summarised using a summary receiver operating characteristic curve. A predefined temporal subgroup analysis compared studies published up to and including 2018 with those published after 2018. Twenty-one studies met the inclusion criteria. Proton MRS demonstrated high diagnostic accuracy for identifying IDH mutation status, with pooled sensitivity of 86.8% and pooled specificity of 84.3%, and an area under the summary receiver operating characteristic curve of 0.91. Diagnostic performance remained stable over time, with a trend toward improved specificity in more recent studies, consistent with increasing methodological standardisation and clinical translation. Risk of bias was low to moderate across studies, predominantly related to patient selection and diagnostic thresholds. In conclusion, proton MRS enables reliable, non-invasive detection of 2- hydroxyglutarate and accurate prediction of IDH mutation status in gliomas. In the context of newly approved IDH-targeted therapies, these findings support the role of 2-HG MRS as a clinically meaningful adjunct to tissue-based molecular diagnostics, particularly in patients for whom surgical sampling is limited or high-risk. Description: M.Sc.(Melit.) Thu, 01 Jan 2026 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/146312 2026-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/146311 Title: Malignant transformation of hypointense nodules seen only on hepatobiliary phase gadoxetic acid-enhanced MR imaging : a systematic review and meta-analysis Abstract: Background: Hypointense nodules detected on the hepatobiliary phase (HBP) of gadoxetic acid–enhanced magnetic resonance imaging (MRI) are frequently seen in patients with chronic liver disease or cirrhosis. Such nodules may represent a spectrum from dysplasia to early hepatocellular carcinoma (HCC), and the development of arterialphase hyperenhancement and subsequent hypervascular transformation of these lesions is indicative of an increased malignant potential. Aim: Through a comprehensive systematic review and meta-analysis, this study seeks to evaluate the outcomes of hypointense nodules observed in the hepatobiliary phase on gadoxetic acid-enhanced MRI and to identify the risk factors associated with their hypervascular transformation. Methods: A computerised search of the Pubmed, Web of Science and Scopus databases was carried out to identify relevant literature reports on hypovascular hypointense nodules in the hepatobiliary phase of gadoxetic acid–enhanced MRI that will eventually undergo hypervascular transformation. Results: 18 studies were included in this meta-analysis, with 1563 patients and a total of 2136 hypointense hypovascular hepatic nodules. The pooled overall rate of hypervascular transformation was 34.4%, (95% CI: [28.7%, 40.2%]) and I2 was 87.4%. The pooled 1-, 2- and 3-year cumulative incidence rates were 19.7% (95% CI: [9.1%, 30.4%]), 27.9%, (95% CI: [16.3%, 39.5%]); and 27.2% (95% CI: [17.7%, 36.7%], respectively. A metaregression analysis revealed that the heterogeneity of malignant transformation was significantly affected by the initial mean nodule size, with a cutoff value of 9 mm. Conclusion: Hypovascular hypointense nodules observed during the hepatobiliary phase of Gadoxetic acid-enhanced MRI present a considerable risk of progressing to hypervascular hepatocellular carcinomas (HCCs). Initial nodule size is an important predictor of hypervascular transformation, with larger nodules being more likely to undergo this process. This highlights the importance of close monitoring, to enable timely intervention to improve patient outcomes. Description: M.Sc.(Melit.) Thu, 01 Jan 2026 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/146311 2026-01-01T00:00:00Z