Saudi Cultural Missions Theses & Dissertations
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Item Restricted The Modulation Effect of Inflammatory Cytokines on T cell Proliferation in Hypertension(University of Glasgow, 2024-05) Alsheikh, Eman; Marta, Czesnikiewicz-Guzik; Tomasz, GuzikHypertension is a common medical condition with very serious target organ consequences, increasing the risk of heart disease, stroke, and severe health complications. Despite the identification of various mechanisms (vascular, renal, and central mechanism) contributing to the pathogenesis of hypertension, the majority of cases lack a clear aetiology. Emerging evidence has established a significant association between hypertension and immune responses, particularly involving adaptive immune cells and inflammatory cytokines. Immunosuppressive drugs and cytokine inhibitors have shown potential in mitigating hypertension, suggesting a crucial role of the immune system in this condition. Given the central role of T lymphocytes in the adaptive immune response, this study hypothesises that, in the context of hypertension, inflammatory cytokines can modulate T cell activation independently of antigen stimulation. To test this hypothesis, total T cells were isolated from the spleens and PBMCs of normotensive and hypertensive mice and exposed to a range of cytokines, including TNF-α, IL-6, IL-15, IFN-γ, IL-7, IL-1β, IL-17A, IL-2, and IL-12, using different stimulation protocols. Aiming to understand the effects of these cytokines on T cell proliferation, differentiation, and the expression of activation markers such as CD69. Our findings highlight the varying abilities of cytokines to sustain T cell viability, with IL 7, IL-15, and IL-6 demonstrating a tendency for greater efficacy compared to other cytokines. In addition, IL-7 and IL-15 significantly impact T cell proliferation, notably affecting the CD8+ T cell population. However, despite these effects, no significant difference was detected between normotensive and hypertensive T cells in response to IL-7 and IL-15. This suggests that while these cytokines are potent in driving T cell proliferation, their influence is not specifically heightened in the context of hypertension. In GSEA and KEGG analyses, the Ca2+ signalling pathway was distinctively activated in response to IL-7 and IL-15 in Ang II induced hypertension. Conclusion: These data imply that most studied cytokines linked to hypertension pathology do not substantially affect normotensive or hypertensive T cells in a murine model. However, T cell proliferation was elevated in both Sham and Ang II mice in response to IL-15 and IL-7. Together, the data presented in this thesis warrant further investigations into the role of cytokines in hypertension and may point to IL-15 or IL-7 as biological targets for antihypertension therapy.43 0Item Restricted Pharmacology of novel free fatty acid receptor 4 ligands and their potential use in metabolic studies(University of Glasgow, 2024-05) Alharbi, Abdulrahman Ghali; Tobin, AndrewFree fatty acids serve as both dietary nutrients and signalling molecules by activating the G protein-coupled receptors of the free fatty acid family. After being deorphanized in 2005, free fatty acid receptor 4 (FFAR4) was shown to be highly expressed in the pancreas, where it was proposed to have a function in the production of insulin. The development of synthetic FFAR4 agonists as a potential therapy for type-2 diabetes mellitus has been founded on this. Recently, there has been research on the function of FFAR4 in the pancreas, specifically in δ-cells, which have high levels of FFAR4 expression. FFAR4 has been shown to be expressed in various types of islet cells, such as α, β, δ, and γ cells, inside the pancreas. FFAR4 plays a crucial role in regulating insulin and glucagon synthesis, as well as suppressing the release of somatostatin in the islets of Langerhans. Thus, FFAR4 serves as a compelling pharmaceutical target for metabolic disorders. Nevertheless, the lack of FFAR4 agonists in clinical trials is primarily attributed to a smaller number of available agonists, challenges with drug selectivity, and suboptimal pharmacokinetic and pharmacodynamic characteristics. This underscores the necessity for increased attention and scientific investigation into the development of these receptor agonists. The objective of this thesis was to conduct a pharmacological characterization of new FFAR4 ligands and evaluate their potency and efficacy in comparison to the reference agonist, TUG-891. The research aimed to determine the specific location of FFAR4 in pancreatic islets and examine the effects of FFAR4 expression on the function of these cells. Moreover, the objective of the thesis was also to assess the capacity of FFAR4 ligands to induce insulin secretion from pancreatic islets. This research aims to enhance the understanding of FFAR4's involvement in glucose regulation and its potential as a target for treating metabolic diseases. Moreover, this study aims to enhance the knowledge of FFAR4 pharmacology and its physiological roles in the pancreas. By doing so, it will provide vital insights for the creation of new treatment approaches that specifically target this receptor. In order to analyse the signalling processes of the mouse FFAR4 receptor, functional tests were conducted on cell lines that constitutively express the mouse ortholog of FFAR4. It has been verified that FFAR4 mostly associates with Gαq/11 G proteins, and there is little or no indication of coupling with Gαs or Gαi in cell lines. Out of the ligands tested, FFAR4 Agonist II showed greater efficacy, whereas Merck cpd A and GSK137647A revealed similar or lower efficacy compared to TUG-891, which was used as the standard ligand. Based on the analysis, it was shown that the FFAR4 Agonist II is a superior ligand compared to TUG-891. FFAR4 Agonist II has the potential to be a useful tool to conduct experiments both ex vivo and in vivo to validate FFAR4 as a viable target for treating metabolic diseases. Functional tests have shown that FFAR4 plays a pivotal function in the regulation of hormone production in the pancreas. Although the FFAR4 ligand TUG-891 has a minor impact on the release of insulin from β-cells, FFAR4 is crucial for enhancing insulin secretion caused by the M3 agonist oxotremorine. This effect was not observed in FFAR4- KO islets. Interestingly, the combination of TUG-891 with oxotremorine and FFAR4 antagonist AH7614 resulted in a 2.5-fold reduction in the impact of oxotremorine. In addition, the phosphorylation of the FFAR4 receptor seems to have a role in insulin release. This was shown by comparing the response of islets from a mutant mouse line expressing an FFAR4 variation that is defective in phosphorylation (PD mouse) to wild-type islets when exposed to oxotremorine. In addition, somatostatin release was 2-3 times higher in FFAR4- KO islets than in wild-type islets, demonstrating that FFAR4 regulates somatostatin secretion independently of ligand activation. The results highlight the potential of FFAR4 as a target for treating metabolic diseases. These findings confirm that FFAR4 is a new and promising target for therapeutic development in the treatment of metabolic diseases, namely T2DM. Existing treatments for T2DM, such as metformin, may lead to adverse effects and may not be successful for specific patient groups. This emphasises the need for new and safer medications in clinical practice. The capacity of FFAR4 to regulate the production of insulin and somatostatin in the pancreas, together with its ability to control glucose homeostasis, emphasises its therapeutic promise. Additional investigation into the precise mechanisms that control FFAR4 activation and signalling pathways has the potential to result in the creation of targeted medications that successfully regulate glucose metabolism and enhance patient outcomes while reducing the adverse effects associated with current medicines.31 0Item Restricted Zinc as a potential therapy for Burkitt’s lymphoma(Saudi Digital Library, 2022-06-30) Alhazmi, Bader Ahmed H; Khanim, Farhat; Bunce, ChrisBurkitts lymphoma (BL) is a form of non-Hodgkin lymphoma (NHL) that arises from germinal center B cells. BL is characterized by translocations of the C-MYC oncogene to immunoglobulin light and heavy chain loci resulting in its constitutive deregulated expression. BL shows a rapid and aggressive growth pattern. There are three different forms of BL; sporadic BL (sBL), immunodeficiency-associated BL and endemic BL (eBL) which accounts for ~50% of all paediatric cancers in Sub-Saharan Africa. Due to financial restrictions, treatment and supportive care options are limited resulting in poorer outcomes in low - middle income countries (LMICs). Thus, there is a need to develop new affordable effective low toxicity treatments for eBL. Prior to this study, a panel of BL cell lines were tested against an in-house custom drug repurposing library developed in our lab (FMC Library) that contains ~100 approved and commonly used drug. This screen identified the nutritional supplement zinc acetate as an effective anti-BL candidate. Dose response studies showed that all BL cell lines tested had little/no response to zinc at 50 μM whereas 100 μM zinc killed all BL cell lines. In contrast, 100 μM zinc acetate induced no killing against a panel of non-BL cell lines including acute myeloid leukaemia (AML) which is a non BL cell tumour, diffuse large B cell lymphoma (DLBCL) which represent a B cell lymphoma that arise from germinal centre B cells and EBV infected lymphoblastoid cell lines (LCL) as a control cells. The latter were used as karyotypically normal B cell controls. Cell death in BL cells was associated with positive flow cytometry staining for propidium iodide and annexin V and activation of caspase 3 and 9 (western blotting) indicating cell death by apoptosis. The proto-oncogene C-MYC is mutated or deregulated in >50% of cancers. In BL, deregulated expression occurs as a consequence of translocation of C-MYC on chromosome 8q24 to either the immunoglobulin heavy chain enhancer region on 14q32 (85% of cases) or the immunoglobulin kappa light chain or lambda loci on 2p12 or 22q11, respectively (15% of cases). Thus, the effect of zinc on C-MYC protein levels were studied. Western blot analysis showed that 100 μM zinc was able to reduce C MYC protein levels rapidly and sustainably in BL cell lines whereas no change in C MYC protein levels was observed in non-BL cell lines. Zinc-induced reduction of C-MYC protein levels was time-dependent, reducing by approximately 20% after 6 hours with little/no protein detectable after 24 hours. C-MYC protein levels were not reduced following treatment with 50 μM zinc. Quantitative real time PCR (qRT-PCR) also showed a rapid reduction in C-MYC mRNA levels in BL cell lines after 6 hours exposure to 100 μM but not upon exposure to 50 μM. Again, no reduction in C-MYC mRNA levels was seen in non-BL cell lines. Translocations of other genes to the immunoglobulin loci occur in other forms of NHL. The DLBCL cell line SU-DHL-4 has a t(14;18) translocation resulting in deregulated expression of the protooncogene BCL2. Western blotting showed no decrease in BCL 2 protein levels in SU-DHL-4 in response to either 100 μM or 50 μM zinc acetate after 6 or 24 hours indicating a selectivity of zinc action against C-MYC protein in BL cells. To further investigate the role of altered C-MYC expression in zinc-mediated killing of BL cells, the eBL cell lines Raji and Namalwa were stably transfected with C-MYC using a piggyBac transposon system that allows gene expression under a constitutively active promoter. However, overexpression of C-MYC from an alternative promoter did not rescue BL cells from killing by 100µM zinc. Although western blotting showed that C MYC protein levels were protected after 6 hours, protein reduction and loss of viability was again observed after 24 hours indicating that loss of C-MYC is important in zinc mediated killing of BL cells. In a second approach to rescue C-MYC expression, the proteasome inhibitor Bortezomib was used to inhibit C-MYC protein degradation via the ubiquitin-proteasome system (UPS). Whilst increases were observed in overall ubiquitinated proteins indicating bortezomib was working, western blotting and flow cytometry showed no rescue of C-MYC protein levels. Furthermore, bortezomib did not rescue cells from zinc-mediated killing after 24 hours. In conclusion, findings from this study have identified that 100 mM zinc is effective at killing BL cell lines selectively, and that this killing is associated with activation of apoptotic markers. Treatment with zinc resulted in a rapid and sustained reduction in C-MYC mRNA and protein levels that could not be rescued through constitutive overexpression or the use of proteasome inhibitors. Given that zinc deficiency is common in sub-Saharan Africa and that zinc supplementation is safely used to treat diarrhoeal episodes in children, the studies proposed here indicate that zinc may safely be used as an adjunctive therapy to target C-MYC in BL.38 0