https://www.um.edu.mt/library/oar/handle/123456789/49413 2026-06-06T16:41:46Z 2026-06-06T16:41:46Z https://www.um.edu.mt/library/oar/handle/123456789/121349 2024-04-26T04:50:06Z 2019-01-01T00:00:00Z

Title: Real-time PCR assay for the quantification of RNA transcripts involved in the control of globin gene switching Abstract: In this project, the globin gene switch was studied using an innovative combination of small molecule screening and a transcriptomic approach using real time qPCR. This allowed the exciting possibility of directly correlate and assessing the role of small molecules in HbF induction. An in vitro cell culture model system was developed using K562 cell lines and a real time qPCR method optimized on globin transcripts. Induction of γ-globin reduces symptoms in β-thalassaemia and sickle cell disease patients but there is a clear need for drugs with a better efficacy, tolerability and patient response. At present, treatment involves lifelong monthly blood transfusions and iron chelation therapy, both of which impinge significantly on the quality of life and may lead to death prematurely. To the K562 cell line, three novel compounds provided by the European Molecular Biology Laboratory, Heidelberg were added. The correct cell number and drug concentrations were calculated after conducting cell viability assays, and subsequently ribonucleic acid was extracted from this cell line for quantification by real-time PCR to see whether these drugs have an effect on both proliferating and haemin-induced differentiating K562 cells. It was concluded that the drugs do have an effect on the K562 cells in vitro. This study can be beneficial for the therapeutic targeting in the treatment of haemoglobinopathies. Uncovering the molecular basis of γ-globin gene control might serve as a model for other clinically significant gene switch mechanisms. Description: B.SC.(HONS)APPLIED BIOMED.SCI.

2019-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/121002 2024-04-18T13:35:31Z 2019-01-01T00:00:00Z

Title: Protein based approach to identify key players in the control of globin gene switching Abstract: Elevated foetal haemoglobin (HbF) levels are known to lessen the severity of symptoms associated with disorders of β-globin which consequently improves the quality of life of patients affected with such disorders. To date the only treatment for β-thalassaemia remains the life-long blood transfusions which may bring about other complications due to subsequent iron accumulation in the body. In order to develop pharmacological and molecular therapies aimed at increasing HbF levels, an understanding of the mechanisms responsible for the regulation of the switch from foetal to adult haemoglobin during development is required. KLF1, which is a key regulator in the switching of γ to β-globin, directly drives expression of other transcription molecules which are necessary in the switching process, namely BCL11A and ZBTB7A. These are key repressors of foetal γ-globin gene expression and hence, providing a mechanism by which these key molecules can be silenced, can prove to be a fruitful approach in the development of new strategies targeted to increase HbF levels in patients. Other approaches towards increasing the HbF level in patients suffering from haemoglobinopathies, is by the pharmacological induction of the γ-globin gene. However, the detrimental health side-effects associated with these pharmacological inducing agents leaves their role questionable. Throughout this study, the effects of 3 novel compounds and their ability to alleviate HbF levels were investigated. Quantification of the different haemoglobin levels present in K562 cell lysates after exposure to these compounds was then performed using Western blotting. It was identified that in fact, these compounds exerted an effect on the γ-globin gene and hence, this data strongly suggested that these novel compounds under study, could play a role in increasing HbF levels in adults with β-haemoglobinopathies, with the possibility of being associated with less toxic side-effects. Description: B.SC.(HONS)APPLIED BIOMED.SCI.

2019-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/52548 2020-04-26T05:09:35Z 2019-01-01T00:00:00Z

Title: Effect of the COL1A1 G-1997T variant on osteoporosis and fracture susceptibility in Malta Abstract: Osteoporosis is a multifactorial metabolic skeletal disorder characterised by low bone mass and microarchitectural deterioration, predisposing to increased fracture risk. Genetic studies have identified several gene variants associated with osteoporosis and fracture risk, particularly those in the collagen type I alpha I (COL1A1) gene. COL1A1 encodes the pro alpha-1(I) chain of type I collagen which is the most abundant protein in bone and found in a 2:1 ratio with pro-alpha-2(I) collagen. The promoter COL1A1 G-1997T (rs1107946) variant is hypothesised to affect transcription factor binding resulting in altered gene expression and protein production, that in turn creates an imbalance in the 2:1 ratio leading to reduced bone strength and increased porosity culminating in higher fracture risk. The aim of the study was to determine whether the COL1A1 G-1997T variant affects spine and hip bone mineral density (BMD), and fracture susceptibility in Maltese postmenopausal women. The variant was genotyped in a case-control collection of 1,045 Maltese postmenopausal women using the TaqMan® fluorogenic 5’ nuclease allelic discrimination assay. The Mann-Whitney and Kruskal Wallis tests were performed to assess genotype phenotype associations, whilst odds ratios (OR) with 95% confidence intervals (CI) were computed using logistic regression analysis adjusted for age computed to determine the effect of the genotyped variant in relation to osteoporosis and fracture risk. Allele frequencies for the wild-type (G) and alternative (T) allele were of 80.2% and 19.8% respectively, and the population was in Hardy-Weinberg equilibrium. Women with the homozygous alternative genotype had a 2-fold (95% CI 1.0-4.5) increased risk of osteoporosis at the lumbar spine relative to women with the homozygous wild-type genotype. No associations were observed with BMD at the femoral neck and total hip, or with fracture risk. Results indicate that the COL1A1 G-1997T variant predisposes to osteoporosis, particularly in trabecular-rich bone, such as the vertebrae. Description: B.SC.(HONS)APPLIED BIOMED.SCI.

2019-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/52540 2020-04-26T05:09:32Z 2019-01-01T00:00:00Z

Title: Idiopathic hypogonadotropic hypogonadism due to FGFR1 p.Y132C in the Maltese population Abstract: Idiopathic Hypogonadotropic Hypogonadism (IHH) is a rare genetic disorder, characterised by a lack of sexual maturation by the age of 18 years with decreased levels of circulating gonadotropins and testosterone and with no structural abnormalities in the hypothalamic-pituitary axis. Pathogenic mutations in several genes have been identified and are associated with its development, including mutations in FGFR1. Various loss-of-function mutations in this gene are mostly associated with Kallmann Syndrome. Locally, a specific variant in FGFR1 (p.Y132C) was identified in a single patient with IHH and so the likely pathogenicity of this variant was determined through this population study. In the 584 cord blood DNA samples tested using PCR and Restriction Enzyme (RE) digest, the FGFR1 p.Y132C variant was not identified in any of the samples. The rare occurrence of this FGFR1 p.Y132C variant in the general population significantly increases the likelihood that it is pathogenic. However, it does not necessarily mean that individuals with this variant will suffer from IHH since FGFR1 variants tend to exhibit incomplete penetrance which leads to a high variability in phenotypic expression. The individual suffering from IHH was part of the Malta NGS project in which 3 other variants in FGFR1 were identified (p.C650C, p.D630Y and p.K535R) in 3 separate individuals, not known to suffer from IHH, using high throughput sequencing. These variants are not reported in any databases and have not been associated with IHH to date. Description: B.SC.(HONS)APPLIED BIOMED.SCI.

2019-01-01T00:00:00Z