Saudi Cultural Missions Theses & Dissertations
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Item Restricted Using viral infectious clones to study cassava brown streak disease in its native host Manihot esculenta and exploring the role of viral Ham1 proteins(University of Bristol, 2025) Alqahtani, Shekhah; Bailey, Andy; Foster, GaryAbstract disease is caused by virus (CBSV) and Ugandan virus (UCBSV) and it is one of the most devastating viral diseases infecting cassava in sub-Saharan East Africa. This study explored some practices to mitigate the impact of the disease using molecular technologies, including the development of viral infectious clones suitable for cassava infection, functional characterization of viral proteins, and CRISPR-Cas9 editing of targets in Nicotiana benthamiana. The main aim of this study involved the biolistic delivery of CBSV infectious clones into cassava. Methods for biolistic delivery were optimised, revealing Plant Line 60444 to be a very sensitive cassava type. That the growth conditions of plant material suitable for infection were paramount. Moreover, it showed that chimeric clones harboring the Cassava Torrado-like Virus Ham1 domain showed loss of pathogenicity, suggesting that Ham1s were not readily interchangeable, even if they show similar enzymatic properties. The hydrolytic activity of various viral Ham1 proteins was demonstrated, exhibiting its ability to hydrolyze non-canonical nucleotides such as ITP and XTP. Expression and assay of Ham1 proteins from CBSV, UCBSV, Euphorbia ringspot virus , Arracacha Virus A and CsTLV revealed different substrate preferences, but typically with a preference for ribose over deoxyribose noncanonical nucleotides. The role of the Serine-Histidine-Arginine motif in substrate selection was also highlighted. CRISPR-Cas9 technology was successfully employed to edit the Ham1 gene in the model plant, Nicotiana benthamiana. Frameshift as well as non-frameshift mutations were found in T1 and T2 generations, with homozygous lines showing stable Mendelian inheritance. Functional assays indicated that the Ham1 knockout lines were less affected by CBSV, evidenced by less chlorosis and necrosis symptoms. Intriguingly, Ham1 disruption did not impact infection by Turnip mosaic virus, implying a virus-specific activity of Ham1. Phenotypic characterisation suggested the Ham1 mutants were more sensitive to salt and osmotic stress, emphasizing the role of Ham1 in stress physiology and its crosstalk with other pathways. This study enhances the tools to better understand CBSV pathogenesis, the interplay of viruses and hosts, and the multifunctional role of the Ham1 protein. It also highlights CRISPR-Cas9 as a precise method for generating edited plant varieties.26 0Item Restricted Splice up your life: an iron-regulated factor in Toxoplasma gondii!(Saudi Digital Library, 2023) Abuzaid, Aseel; Sloan, MeganToxoplasma gondii is an alveolate belonging to the pathogenically renowned phylum, Apicomplexa and the causative agent of toxoplasmosis. Being an intracellular pathogen, the parasite must contend with the host’s dynamic nutrient availability through transcriptional and post-transcriptional processes. Though not entirely understood, expression of a serine/arginine-rich (SR) factor, dubbed TgSR4i, was discovered to have an abundance of transcripts in tachyzoites cultured in iron chelated environment. This study aims to reveal the genomic and phenotypic influence on parasite survival when TgSR4i is ablated in different iron concentrations. Replication and plaque assays affirmed a stun in the growth of TgSR4i knock out line when cultured in iron chelation, as opposed to iron supplementation. The results of RNA-sequencing analysis marked differentially expression genes associated with the electron transport chain and DNA metabolism under iron deprivation and no pathway enrichment of hetero-lactic fermentation, as opposed to untreated parasites. Additionally, 11 genes were spliced, most exhibiting intron retention. Further investigation into TgSR4i could expand our knowledge about the parasite’s iron metabolism and use it as a potential drug target.21 0Item Restricted Characterising Expression of Muscarinic Acetylcholine Receptors in Human Dental Pulp Stem Cells: An In-Vitro Study(Saudi Digital Library, 2023-08-12) Baradwan, Othman; Hamilton, Andrew; McLean, WilliamDental pulp stem cells (DPSCs) are a promising subgroup of mesenchymal stem cells (MSCs) that have the potential for regenerative applications. They have been reported to express a regenerative response for tooth structure by producing reparative dentine. One of the sources of DPSCs is the pulp of an extracted permanent tooth, which provides a convenient collection method. In addition, there are other groups of MSCs which share similar characteristics among them. Recent studies identified Acetylcholine (ACh) in some of the MSCs and the two major categories of Acetylcholine receptors (AChRs), muscarinic AChRs (m-AChRs) and nicotinic (n-AChRs). Although ACh is a neurotransmitter, there is evidence that it is produced by and influences non-neuronal cells. The involvement of ACh in the non-neuronal cells is called the non-neuronal cholinergic system (NNCS), which includes ACh-synthesizing enzymes, transporters, receptors, and degrading enzymes. Because DPSCs share similar characteristics with other MSCs, it is worth investigating the expression of m-AChRs in DPSCs. Also, to look into the role of m-AChRs in the regenerative function of DPSCs. There is potential for using DPSCs outside the tooth in regenerative applications, such as musculoskeletal regenerative medicine. The first part of the project started with m-AChRs by identifying messenger ribonucleic acid (mRNA) and protein expression. Secondly, to observe the influence of the most prominent m-AChRs in DPSCs, we attempted to generate a knock-out (KO) mutant in DPSCS using the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) genome-editing system. The results showed that m-AChRs are identified on a genetic level through gene expression of mRNA in DPSCs and expressed the following genes CHRM2, CHRM3, and CHRM5. They are functional and can identify the following proteins: M2, M3, and M5. Then, we attempted to generate a CHRM2 KO mutant DPSCs and could only produce a low percentage of the mutant population of DPSCs, as we only managed to generate 13% of CHRM2 KO mutant DPSCs. Although the protocol proved its effectiveness on different types of cells, but it did not work with DPSCs in this experiment. Therefore, the protocol steps can be revisited for further investigation and optimisation to generate a sufficient population of CHRM2 KO mutant DPSCs to allow the exploration of the role of m-AChRs in DPSCs so it can be used in clinical applications in regenerative medicine.28 0