https://www.um.edu.mt/library/oar/handle/123456789/31980 Mon, 20 Apr 2026 13:04:56 GMT 2026-04-20T13:04:56Z https://www.um.edu.mt/library/oar/handle/123456789/40423 Title: Genetic analysis of olive (Olea europea L.) varietes in the Maltese islands using DNA markers: method development and technique evaluation Abstract: The Maltese Islands have a long history of association with the cultivation of olive (Olea europaea L.). Being geographically isolated, the Islands host a number of indigenous varieties that show physical and agronomic traits that make the trees highly adapted to the local pedoclimatic conditions. Morphological marker identification of olive varieties is limited because it is subjective and influenced by the environment and farming practices. DNA molecular markers such as Random Polymorphic DNA (RAPD) and microsatellites embedded within the genome remain largely unaffected by the outside environment and therefore provide a more accurate method of identification. Locally, no previous genetic work making use of these markers has been reported for the olive. Therefore, the purpose of this study was to carry out a genetic diversity assessment focusing on three local cultivars of interest ("Bidni", "Bajda" and "Malti") and one form of the Maltese wild olive ("Oleaster") which were compared to two commercial Italian and Tunisian varieties ("Carolea" and "Tunezina" respectively). Development of the methods used for DNA extraction and marker amplification is discussed. The markers were chosen following strict selection criteria. A number of similarity indices, based on binary (presence-absence data) and allele frequencies, were used to provide estimates of the genetic similarity between any two varieties and to evaluate the performance of each. Cluster analysis was also carried out to generate dendrograms representing genetic relationships between the varieties. Additional tests that were carried out to determine the reliability and robustness of the methods are also discussed. A high inter-varietal genetic variability was found with both marker types. The total percentage polymorphic loci for RAPD markers was equal to 84.84% while an average of 4.86 alleles were detected per microsatellite locus. Indeed, one microsatellite locus generated six different allele combinations, allowing identification of all six varieties. Therefore, the local olive genome is composed of a very diverse group of cultivars, with "Bidni" and "Malti" standing out for their genetic uniqueness. This polymorphic content can be commercially useful in varietal identification for breeding programs, in obtaining certification of Maltese Olive Trees and in tracing the authenticity of Maltese olive oil. It has also been further demonstrated that the Maltese cultivars may exhibit significant genetic differences from other 0. europaea varieties because three out of seven microsatellite marker regions may not be conserved in the former. Tile marked differences generated in the two types of markers were attributed to the fact that they check for different parts of the genome. Cluster analysis has shown that similarity in the RAPD profile may not linked to geographic origin or fruit characteristics whereas a clear-cut separation is evident between Maltese varieties and foreign ones. Moreover, since "Oleaster", "Bidni" and "Malti" did not form any close clusters it is implied that perhaps their origins are not directly linked despite the fact that they are Maltese natives. In general, this work represented an important, preliminary step in setting up the methods for using DNA molecular markers to provide the very first insights regarding genetic diversity in local olives. It demonstrates that DNA markers can be used as a financially viable method of identification provided that the unique marker profiles are conserved by clone propagation. Description: M.SC.MEDICINE&SURGERY Tue, 01 Jan 2013 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/40423 2013-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/38800 Title: Metal replacement and structural analysis of iron superoxide dismutase from Escherichia coli Abstract: Superoxide dismutases are claimed to be the most important antioxidant proteins in all living organisms, serving as the first line of defence against superoxide radicals and in essence oxidative stress (Ken et aI., 2005) by catalytically converting the superoxide. radical to oxygen and hydrogen peroxide at near diffusion controlled rates (Abreu & Cabelli, 2010). The structurally homologous iron (FeSOD) and manganese superoxide dismutases (MnSOD) can accommodate either Mn or Fe at their active sites, but they tend to show selectivity toward their 'correct' metal ion and reaction is highly specific, showing inactivity with the 'incorrect' metal ion. The mutant protein FeSOD[Q69G/A141Q/G142D/A68GIY76F] (FeSOD[dblDGFD was previously prepared by Dr. T. Hunter and Prof. G. J. Hunter. This is an FeSOD with five amino acid residues substituted to their corresponding MnSOD amino acids, with the intention oftuming an FeSOD into a MnSOD. This research project therefore aimed at substituting the metal at the active site using in vivo and in vitro techniques and to study the metal substituted protein using physicochemical analysis. In vivo methods were improved so that FeSOD and FeSOD[ dblDGF] incorporated minimal iron at the active site. SOD production in the presence of GroESL, during culturing of E. coli OX326A cells in minimal media, was substantially less. Metal content analysis showed that although this chaperone increased manganese levels in FeSOD, it had the opposite effect on FeSOD[dblDGF]. Activity was retained even at very low quantities of the 'correct' metal. An in vitro substitution method was developed throughout this research that allowed the complete removal of metal from the active site, producing the apo-SOD. This protein was subsequently metallated with the metal of interest, giving reliable results. FeSOD[dblDGF] appear to be very selective towards iron at the active site, similar to FeSOD. Activity of this mutant was observed to be just 25 % when compared to the wild type. When substituted with manganese no detectable activity was present, showing that this combination of mutant residues does not make FeSOD behave as a MnSOD. Although activity with manganese at the active site was not obtained, thermostability results from CD analysis showed that this mutant protein is thermostable. Both the iron containing and the manganese substituted proteins were found to have a similar transition temperature (Tm) but lower than that of the wild type FeSOD. The electron density map of this mutant showed structural similarities to that of MnSOD, as active site residues showed similar conformation, with some bond length measurement more closely related to MnSOD than to FeSOD. EPR spectra comparisons of FeSOD and FeSOD[dblDGF] also confirmed that the electronic configuration of the active site environment is actually different between these two proteins. Description: M.SC. BIOCHEMISTRY; The research work disclosed in this publication is partially funded by the Strategic Educational Pathways Scholarship (Malta), This Scholarship is part-financed by the European Union - European Social Fund (ESF) under Operational Programme 11 - Cohesion Policy 2007-2013, "Empowering People for More Jobs and a Better Quality Of Life" Tue, 01 Jan 2013 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/38800 2013-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/32093 Title: Does summation of alleles account for genetic risk and genotype-phenotype association in Type 2 Diabetes Mellitus? Abstract: Introduction: Type 2 diabetes (T2DM) is a common complex disorder with a high prevalence in the Maltese population. The aim of this study is to further define the genetic interplay between cognate genes from metabolic and inflammatory pathways on the likelihood of developing T2DM in adulthood and to relate the association of certain genetic profiles with defined biological and clinical end points. Method: Four T2DM cohorts, each with 200 cases were recruited. Anthropometric and biochemical parameters, including serum high-sensitivity C-reactive protein (hsCRP) and HOMA-IR levels were determined, and genotyping of 42 cognate genes carried out. Neonatal cord blood samples were used as the control reference population in this study. The gene expression profile of peripheral blood mononuclear cells from T2DM cases was compared to that of healthy non-diabetic controls using an IIlumina HT-12® microarray platform. Results: Ten polymorphisms in metabolic/inflammatory pathways showed significant association with T2DM. Three loci showed significant association with lipid profile, body weight, hsCRP and HOMA-IR levels. hsCRP levels demonstrated a strong positive correlation with body mass index. Genetic score analysis showed that combining multiple genetic markers results in higher relative risks. Transcriptome analysis of peripheral blood mononuclear cells identifies up regulation of diverse mRNAs linked to leukocyte activation, adhesion, migration, inflammation and oxidative damage. Network analysis identified diabetic nephropathy and renal injury as the indirect consequences of the observed gene expression changes in T2DM. Conclusion: A panel of ten candidate genes has consistently demonstrated significant association with type 2 diabetes in the Maltese population. These gene variants serve functional roles in inflammation and adipose tissue function. A recruited cohort of untreated newly-diagnosed T2DM serves to identify and explore genotype-phenotype association. The strong effect sizes of these alleles could be used to develop personal genetic susceptibility profiles for T2DM leading to personalization of care and prevention of chronic complications. Changes in the leukocyte transcriptome, such as overexpression of defensin and versican mRNA in T2DM are potential biomarkers of incipient diabetic nephropathy and microvascular damage. KeyWords: T2DM - type 2 diabetes mellitus hsCRP - high-sensitivity C-Reactive protein HOMA-IR - homeostatic model assessment of insulin resistance mRNA - messenger ribonucleic acid Description: PH.D. Tue, 01 Jan 2013 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/32093 2013-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/31976 Title: A study of the roles of reactive oxygen species and superoxide dismutase in aspirin-induced apoptosis in yeast Abstract: The principal objective of this study was to acquire a better understanding of the mechanism by which aspirin, a promising cancer-preventive agent, induces apoptosis in yeast cells deficient in mitochondrial manganese superoxide dismutase (MnSOD), during aerobic growth in ethanol medium (YPE). In this project, exogenous Escherichia coli iron superoxide dismutase (FeSOD) was targeted to the mitochondria of MnSOD-deficient Saccharomyces cerevlszae EG 11 0 cells, using YEpIPGK-S plasmicl DNA (encoding E. coli FeSOD with the S. cerevisiae MnSOD mitochondrial targeting signal). This was found to rescue the resulting recombinant EGllOF yeast cells (expressing E. coli FeSOD in the mitochondria) from aspirin-induced apoptosis during cultivation in YPE medium. Similarly, wildtypc EGI03 yeast cells, which express endogenous mitochondrial MnSOD, survived in the presence of aspirin during growth in YPE medium. However, MnSOD-ueficienl S. cerevisiae EG 11 OYEp cells (EG 11 0 cells having YEplPGK plasmid DNA and used as a control) underwent apoptosis in YPE medium in the presence of aspirin, as confirmed by dual fluorescence staining of whole yeast cells with FITC-Annexin V and propidium iodide. Subsequent investigation of potential aspirin-induced changes in the levels of different types of reactive oxygen species (ROS) in whole yeast cells grown in YPE medium, was carried out. In parallel to this, a study of potential aspirin-induced changes of yeast cytosolic and mitochondrial SOD activity was also performed. In MnSODdeficient EG 110YEp yeast cells, aspirin-induced apoptosis was accompanied by a sustained and highly significant accumulation of mitochondrial and overall cellular superoxide radicals (02•} This was not prevented by the concomitant aspirin-induced increase of cytosolic copper-zinc superoxide dismutase (CuZnSOD) activity, which failed to compensate for the deficiency of mitochondrial MnSOD and did not provide effective protection against the adverse pro-apoptotic effect of aspirin. In the recombinant EG 11 OF yeast cells (expressing E. coli FeSOD in the mitochondria) growing in YPR l1lodium in the presence of aspirin, an increase in the activity of mitochondrial FeSOD significantly reduced the levels of mitochondrial O2.- and rescued the apoptotic phenotype. In EG 103 wildtype yeast cells (which have endogenous MnSOD) there was a decrease in the level of mitochondrial 02•- throughout growth ill YPE medium in the presence of aspirin. This was due to the highly efficient antioxidant activity of mitochondrial MnSOD, the level of which did not change in response to aspirin. Comparative measurement of near UV-autoflourescence of whole yeast cells grown in YPE medium showed that, in MnSOD-deficient EG 11 OYEp yeast cells, sustained depletion of reduced mitochondrial NA(D)PH in the presence of aspirin took place, thus providing evidence of a positive correlation between aspirin-induced mitochondrial 02•- accumulation and the oxidation of mitochondrial NAD(P)H. In EGllOF yeast cells growing in YPE medium in the presence of aspirin, a significant increase of mitochondrial NAD(P)H was observed, coinciding with the decrease of mitochondrial 02•-. Also, in wildtype EG 103 yeast cells, where the presence of MnSOD reduced the levels of mitochondrial 02•- throughout growth, there was a significant increase of mitochondrial NAD(P)H in the presence of aspirin. IV Therefore, aspirin exerts a pro-oxidant effect on MnSOD-deficient yeast cells grown in YPE medium and causes early accumulation of mitochondrial 02'-, oxidation of mitochondrial NAD(P)H and apoptosis of the yeast cells, which can only be prevented by the presence of active mitochondrial SOD. Keywords: aspirin, apoptosis, yeast, superoxide dismutase, reactive oxygen species, mitchondria, NADPII Description: PH.D. Tue, 01 Jan 2013 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/31976 2013-01-01T00:00:00Z