Saudi Cultural Missions Theses & Dissertations
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Item Restricted Timekeeper: Advancing Circadian Research with GST-Tagged Per3 Protein(The University of Edinburgh, 2024) Alanezi, Sarah Abdullah; Arribas, RaquelThe Per3 gene plays a crucial role in regulating circadian rhythms, profoundly impacting sleep-wake cycles and metabolic processes. Despite its biological significance, the expression and purification of Per3 have presented substantial challenges, often resulting in low yields and poor solubility. This study tackles these obstacles by employing innovative cloning and expression techniques, particularly utilizing a GST-Per3long to enhance solubility and purification efficiency. We designed primers incorporating homology regions complementary to restriction enzyme-digested ends and employed the In-Fusion cloning method to integrate the Per3 gene into the p3E plasmid vector with high precision. The transformation efficiencies were remarkable, with colony counts reaching 2.292 x 10^8 colonies per μg of plasmid DNA. PCR amplification confirmed the successful integration of the Per3 gene, with distinct bands observed at the expected size of 1143 bp, which was further confirmed by DNA sequencing. Protein expression trials identified 25°C as the optimal temperature, significantly improving the yield and solubility of the GST-tagged Per3 protein. Subsequent purification through GST Affinity chromatography and gel filtration chromatography yielded a highly pure protein, as confirmed by SDS-PAGE and native gel electrophoresis. Although the initial yield was modest, the high purity of the purified protein provides a robust foundation for future functional and structural studies. This study not only establishes a reliable protocol for Per3 expression and purification but also opens avenues for investigating the interactions of Per3 with other circadian proteins, such as CRY and BMAL1, to determine their mutual exclusivity and relative affinity. These interactions are crucial for understanding the biological role of Per3 in fine-tuning the circadian clock. Future work should focus on optimizing expression conditions to further increase yield and investigate the intricate biological activity of the Per3 protein within the circadian network.19 0Item Restricted Timekeeper: Advancing Circadian Research with GST-Tagged Per3 Protein(The University of Edinburgh, 2024) Alanezi, Sarah Abdullah; Arribas, RaquelThe Per3 gene plays a crucial role in regulating circadian rhythms, profoundly impacting sleep-wake cycles and metabolic processes. Despite its biological significance, the expression and purification of Per3 have presented substantial challenges, often resulting in low yields and poor solubility. This study tackles these obstacles by employing innovative cloning and expression techniques, particularly utilizing a GST-Per3long to enhance solubility and purification efficiency. We designed primers incorporating homology regions complementary to restriction enzyme-digested ends and employed the In-Fusion cloning method to integrate the Per3 gene into the p3E plasmid vector with high precision. The transformation efficiencies were remarkable, with colony counts reaching 2.292 x 108 colonies per µg of plasmid DNA. PCR amplification confirmed the successful integration of the Per3 gene, with distinct bands observed at the expected size of 1143 bp, which was further confirmed by DNA sequencing. Protein expression trials identified 25°C as the optimal temperature, significantly improving the yield and solubility of the GST-tagged Per3 protein. Subsequent purification through GST Affinity chromatography and gel filtration chromatography yielded a highly pure protein, as confirmed by SDS-PAGE and native gel electrophoresis. Although the initial yield was modest, the high purity of the purified protein provides a robust foundation for future functional and structural studies. This study not only establishes a reliable protocol for Per3 expression and purification but also opens avenues for investigating the interactions of Per3 with other circadian proteins, such as CRY and BMAL1, to determine their mutual exclusivity and relative affinity. These interactions are crucial for understanding the biological role of Per3 in fine-tuning the circadian clock. Future work should focus on optimizing expression conditions to further increase yield and investigate the intricate biological activity of the Per3 protein within the circadian network.12 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 Probing Metabolic Pathways using an RccR Based Genetically Encoded Biosensor(Saudi Digital Library, 2023-11-24) Babtain, Ahmad; Dixon, NeilThe study of metabolic pathways is of utmost importance to contemporary biotechnology. However, mapping metabolic pathways is a burdensome process due to the entangled and complex nature of biological systems. Due to the direct involvement of transcription factors in metabolism, biosensors based on them are a powerful tool for researching metabolism. In this study, we used a biosensor vector where the RccR transcription factor regulates the expression of eGFP in response to the metabolite KDPG to investigate the central metabolism of two bacterial species of high relevancy to biotechnology: Pseudomonas Putida and Escherichia Coli, with an experimental focus on the first. Our research demonstrates that acetate, aromatic acids, and fatty acids enter the central metabolism of P. Putida through the TCA cycle. We also show among the aromatic acid, PCA and benzoic acid flux into central metabolism through one metabolic intermediate through a pathway with moderately low metabolic leakage. On E. Coli’s end, we show that acetate and the fatty acid C18:2 enter metabolism through the TCA cycle as well. We also employ bioinformatic databases to show that HexR is closely related to RccR and that the differences between the HexR proteins in P. Putida and E. Coli might show differing interference with RccR’s function. Our results also indicate that P. Putida preferentially consumes glucose, then acetate, and then aromatic acids when presented with mixtures of them. Finally, we saw strong evidence of the presence of a metabolic pathway that leads from acetate into the central metabolism of E. Coli. through acetyl-CoA.24 0Item Restricted Intragenic suppression of RNase-defective point mutation of the catalytic aspartate in the protein kinase domain of Ire1(Saudi Digital Library, 2023-12-07) Obidan, Amnah; Schroder, MartinIn eukaryotic cells, proper folding of secretory and transmembrane proteins occurs within the endoplasmic reticulum (ER) before their exit the ER. The accumulation of unfolded proteins activates a response known as the unfolded protein response (UPR), mediated by Ire1. In Saccharomyces cerevisiae, Ire1 activation leads to the splicing of HAC1 mRNA, which encodes a transcription factor involved in the UPR. In this study, we focused on specific mutations within the protein kinase domain of Ire1. Specifically, the protein kinase domain was subjected to mutations to alter the catalytic aspartate D797 and lysine K799, which interacts with the terminal phosphate group of ATP, to alanine. Also, point mutations in the Mg2+ coordinating loop converted asparagine N802 and aspartic acid D828 to alanine. To investigate the impact of these mutations, we performed several experiments. Northern blot analysis was employed to detect the splicing of HAC1 mRNA, as it serves as an indicator of Ire1 activity and UPR induction. Additionally, β-galactosidase reporter assays were conducted to assess the expression of a UPRE-lacZ reporter gene, which is also regulated by the Ire1-Hac1 signalling pathway. The results demonstrated that single mutations in the catalytic domain and Mg2+ coordinating loop (K799A, D797A, N802A, and D828A) led to decreased levels of HAC1 mRNA and reduced expression of the UPRE-lacZ reporter gene compared to the WT Ire1. Furthermore, the D797A mutant strain exhibited decreased survival under ER stress conditions when compared to other mutants within the Mg2+coordinating loop and catalytic domain. Interestingly, the D797A mutation resulted in lower levels of HAC1 mRNA species and β-galactosidase activity. However, introducing additional mutations such as D797A N802A or D797A K799A N802A led to significant increases in βgalactosidase activity, the percentage of HAC1 mRNA, and restored growth compared to the single D797A Ire1 mutant. Notably, the expression levels of WT and protein kinase mutants were similar. In conclusion, the findings suggest that introducing specific additional mutations, such as K799A Ire1, D828A Ire1, or N802A Ire1, to the single D797A Ire1 mutant can restore the signalling activity of Ire1.15 0