/library/oar/handle/123456789/87511 2025-12-23T12:47:57Z 2025-12-23T12:47:57Z /library/oar/handle/123456789/88440 2022-02-08T05:55:56Z 2021-01-01T00:00:00Z

Title: Expression of the KLF1 interactome in an erythrocytic differentiation model Abstract: Erythroid Krüppel-like factor 1 or KLF1 is a DNA binding transcription factor that has a very important role in the foetal to adult haemoglobin switching. For this project the expression of this transcription factor was explored in human immortalised myelogenous leukaemia K562 cell lines. The expression of both beta and gamma globin genes was also investigated by qPCR analysis, which was essential for studying the effect of K562 on the expression of these genes. K562 cells were grown in culture for a few days before their differentiation was initiated by the addition of hemin. The differentiation of the K562 cells led to the expression of KLF1 which was confirmed by flow cytometry analysis done on both hemin induced and non-induced cells. In addition to the flow cytometry analysis, RNA extraction and qPCR analysis was done to measure the expression of the KLF1, Gamma globin and Beta globin genes. It was concluded, that the KLF1 had an effect on the expression of both the Gamma and Beta globin genes indicating the importance of this transcription factor in haemoglobin switching and regulation. KLF1 is therefore essential to prevent haemoglobinopathies that could be brought about by abnormal levels of haemoglobin, or abnormal types of haemoglobin. Description: B.Sc. (Hons) Med. Biochem.(Melit.)

2021-01-01T00:00:00Z /library/oar/handle/123456789/88432 2022-02-08T05:55:09Z 2021-01-01T00:00:00Z

Title: Characterisation of xanthine oxidoreductase variants associated with heart disease and hypertension Abstract: Hypertension has been linked with hyperuricaemia and polymorphisms of xanthine oxidoreductase, the protein which catalyses the last two steps of uric acid production. In this study, the A932T variant, a variant of xanthine oxidoreductase which is known to increase the risk of hypertension, was produced as part of a project to determine how the mutation affects the activity, structure and stability of the human xanthine oxidoreductase protein. The wildtype and the A932T variant were expressed in TP1000 cells and purified by immobilised metal affinity chromatography. Native-PAGE and an NBT assay were carried out to determine whether the purified protein samples were active. A uric acid assay was then performed to measure the activity of both the wildtype and the A932T variant and to determine the percentage xanthine oxidase and the percentage xanthine dehydrogenase that were present within the samples. The NBT assay showed that both the wildtype and the A932T variant were active, while the native-PAGE showed that only the wildtype was active. The uric acid assay confirmed that the A932T variant was active but it showed that it was less active than the wildtype. Further optimisation of the expression of the variant needs to be carried out since previous studies have shown that this variant is more active than the wildtype. The purification step also needs to undergo optimisation since the protein samples obtained for both the wildtype and the variant were not pure enough to perform circular dichroism. Therefore, the secondary structure and the stability of the A932T variant were not analysed. Description: B.Sc. (Hons) Med. Biochem.(Melit.)

2021-01-01T00:00:00Z /library/oar/handle/123456789/88417 2022-02-08T05:54:21Z 2021-01-01T00:00:00Z

Title: Neural induction of mesenchymal stem cells via small molecule cocktail treatment Abstract: Mesenchymal Stem Cells (MSCs) are capable of transdifferentiation into cells of the neuronal lineage when cultured in spent media obtained from cultures of neuroblastoma cell lines or when treated with a cocktail of small molecules. In this study, the principal aim was to transdifferentiate MSCs via spent medium from cultures of the neuroblastoma cell line SH-SY5Y and small molecule cocktails, and to determine the neuronal stage achieved upon treatment. To fulfil these aims, MSCs were extracted from umbilical cord and cultured aseptically. MSCs were first transdifferentiated by spent medium or a small molecule cocktail and then further differentiated using different small molecule cocktails in two distinct stages. RNA was extracted from differentiating cells and used to check CD marker (CD34, CD45, CD73, CD90, and CD105), and neuronal marker (Nanog, SOX2, Nestin, Tubb3, NeuN, and TH) expression. Additionally, protein lysates were used for Western blotting for SOX2, NeuN, Actin, Tubulin, HSP27 and HSP90. The MSCs were successfully differentiated towards mature neuronal cells, as indicated by (1) a stark change in cellular morphology whereby the resultant cells greatly resembled neurons, and (2) a significant change in early and late neuronal marker expression as the cells progressed through the induction protocol. Thus, it was concluded that the devised neural induction protocol works in producing neurons. However, further experiments are required to determine which neuronal stage these MSCs achieve upon differentiation with this protocol. This study lays the groundwork for generation of a robust population of mature and functional neuronal cells and has many possible applications in regenerative therapy and research. Description: B.Sc. (Hons) Med. Biochem.(Melit.)

2021-01-01T00:00:00Z /library/oar/handle/123456789/88341 2022-02-07T05:59:13Z 2021-01-01T00:00:00Z

Title: Nitric oxide and aglycaemia induced axonal injury Abstract: White matter degradation is known to lead to several neurological disorders. Recent unpublished data from our research group has shown that inducing injury through aglycaemia causes a rise in nitric oxide in white matter. The pathways for the generation of this NO are still unclear, but it is theorised that one possible source is from the overactivation of nNOS attached to NMDA receptors via the PSD95 domain. Aglycaemia-induced excitotoxicity results in a large influx of Ca2+ ions through NMDA receptors and thus results in white matter injury. In this study QNZ-46, a fluorescent drug which localises within the myelin sheets, was tested for its ability to inhibit the specific GluN2C/D-containing NMDA receptors found in the periaxonal space. NO production during aglycaemia was established in acute brain slices with live-imaging using 2-photon microscopy, and further confirmed through immunohistochemistry, where the endproduct of NO – nitrotyrosine – was detected both immediately after the insult, and at higher levels 2 hours after the insult. Application of QNZ-46 significantly lowers the initial spike in the production of NO following aglycaemia, thus confirming our hypothesis that it may offer protection to white matter from excitotoxicity-induced injury. Thus, our results suggests that QNZ-46, a non-toxic dose-dependent drug, may offer protection to neurological conditions in which excitotoxicity may induce white matter injury. Description: B.Sc. (Hons) Med. Biochem.(Melit.)

2021-01-01T00:00:00Z