Saudi Cultural Missions Theses & Dissertations
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Item Restricted Subcellular Location-Dependent Regulation of Interferon-Induced Transmembrane Protein 1 in Glioblastoma(The University of Edinburgh, 2024) Mubarak, Rawan; Ball, KathrynGlioblastoma multiforme (GBM) is one of the most aggressive and lethal brain cancers, known for its highly invasive nature and resistance to conventional therapies. Central to this resistance is the presence of glioblastoma stem cells (GSCs), which contribute to tumour recurrence and heterogeneity. This thesis investigates the roles of interferon-induced transmembrane proteins (IFITMs), specifically IFITM1 and IFITM3, within GSCs and their potential as therapeutic targets. The study provides a detailed analysis of IFITM1’s expression, subcellular localisation, and interaction with other proteins in response to interferon stimulation, employing advanced molecular techniques such as co-immunoprecipitation, immunofluorescence microscopy, and proteomic analysis. Key findings include the discovery that IFITM3 plays a critical role in regulating IFITM1’s expression and localisation, with significant implications for IFITM1’s function in cancer cell biology. This thesis also contributes to validating a novel interaction between IFITM1 and Lysosomal-associated membrane protein 1, suggesting a potential role for IFITM1 in autophagy, which could be pivotal in GBM’s resistance to treatment. These insights not only advance the understanding of IFITM proteins in GSCs but also highlight their potential as targets for therapeutic intervention in GBM. This work lays the foundation for future studies aimed at manipulating IFITM proteins to develop novel strategies for overcoming GBM treatment resistance.42 0Item Restricted Generating A novel Avp Knockout Mouse Model using CRISPR/Cas9 and Cre/loxP Gene Editing Systems(2023-07-14) Khan, Shaza; Khundmiri, Syed; Knepper, MarkBackground: Arginine vasopressin (AVP) is a nonapeptide hormone coded by the AVP gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Dysregulation of AVP secretion contributes to a variety of human diseases. Previous studies of functional roles of AVP have been largely dependent on the use of Brattleboro rats, which manifest a spontaneous mutation in the Avp gene and lack circulating AVP. Despite their utility, Brattleboro rats were difficult to breed owing largely to the fixed nature of the Avp mutation, resulting in high incidences of fetal and neonatal deaths, behavioral abnormalities in adults, as well as small litter size. Consequently, commercial breeders have ceased production, despite a continued need. Therefore, the main goal of this project is to create an effective experimental Avp knockout mouse model that could be used in renal and neuroendocrine research to study the control of water balance by AVP. Methods: A mixture of CRISPR elements, including active sgRNAs, Cas9 mRNA, single- stranded oligonucleotides (ssODN), and loxP sites was injected into C57BL/6 embryos. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes BamHI or EcoRI were used to confirm correct targeting. For additional confirmation, the amplified PCR products were subsequently cloned into the TA-cloning vector and subjected to sequencing analysis. Mice harboring the floxed allele were mated to B6.Cg- Tg(CAG-cre/Esr1*)5Amc/J mice that globally express a tamoxifen-inducible Cre recombinase. Results: The resultant inducible Avp knockout mice (flox/flox;Cre/wt) show no signs of polydipsia or polyuria prior to induction, indicating that the floxed gene maintains its wild-type function. The administration of an exogenous inducer like tamoxifen to (8-10) week-old mice, induced Cre- mediated recombination that resulted in a decrease in urine osmolality from 2076 ± 138 to 122 ± 6 mOsm/kgH2O on day 31 after induction. Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 band density in (flox/flox;Cre/wt) mice to 27 ±1 4 % of values in Cre- floxed control mice. Conclusion: This inducible Avp knockout mouse model provides researchers with a valuable tool to investigate the consequences of Avp gene deletion in a controlled and inducible manner. By activating the inducible Cre recombinase at specific developmental stages, researchers can study the effects of Avp deletion on various physiological processes, such as water balance, blood pressure regulation, and social behavior.33 0