Saudi Cultural Missions Theses & Dissertations
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Item Restricted Dissecting the structural requirements for Notch/ligand interactions with mutations derived from cancer genome sequencing(The University of Manchester, 2025) Alanazi, Areej; Baron, MartinNotch is a cell surface receptor with critical roles in development and cellular differentiation, and its altered activity is frequently associated with cancer. Despite extensive sequencing efforts identifying numerous cancer-associated mutations in Notch, the functional consequences of many of these mutations remain poorly understood. Notch activation is mediated by its interaction with cell surface ligands, Delta and Serrate/Jagged, triggering proteolytic cleavage events that release the Notch intracellular domain (NICD). The NICD translocates to the nucleus to regulate transcription, and Notch can also be activated through ligand-independent mechanisms following endocytosis. This thesis exploits the high sequence conservation between human and Drosophila Notch to dissect how cancer-associated mutations alter receptor functionality, using an approach comprising in vitro and in vivo analyses. This work focuses on mutations within the ligand-binding region that are associated with cancers where Notch acts as a tumour suppressor. Through cell culture assays, I categorised Notch mutants based on their ligand-binding properties, signalling efficiencies, and ligand-independent activities. Using CRISPR/Cas9, these mutations were introduced into the Drosophila genome, enabling a comprehensive study of their phenotypic consequences. My analyses revealed diverse mutant classes, including those that completely eliminate signalling or retain partial functionality. For example, I identified mutants that discriminated between different Notch ligands, removed ligand-dependent signalling while retaining ligand-independent activity, and one mutant that retained ligand-dependent activation but removed ligand-independent activity. I also found that different cell-based assays could distinguish between levels of ligand associations required for cell adhesion and cell signalling. In vivo genetic interaction studies further refined our understanding of mutant classifications, revealing Notch mutant-specific interactions with a panel of genetic modifiers and uncovering phenotypes that deviated significantly from null-like behaviour. Additionally, I demonstrated that several mutations compromised cis-inhibitory interactions, with heterogeneous impacts on this regulatory mechanism across different developmental contexts. These findings provide valuable insights into the regulatory dynamics of Notch signalling, paving the way for advancing our understanding of the pathway’s role in development and disease.19 0Item Restricted MODELLING CONGENITAL HEART DISEASES BY TBX1 KNOCKOUT IN A CARDIAC ORGANOID(University of Manchester, 2024) Al Majed, Maryam; Keavney, Maryam; Liu, YingJuanCongenital heart diseases (CHDs) have the highest incidence and mortality rate among birth defects worldwide. They arise from abnormal heart development, majority of which are associated with genetic variations present at birth. Tetralogy of Fallot (TOF) is one of the most common cyanotic CHD subtypes, and it is among the typical CHD phenotypes of patients with 22q11.2 Deletion Syndrome (22q11.2DS). TBX1 has been identified as a causal gene for TOF and other CHDs seen in 22q11.2DS. TBX1 is a key cardiac regulator during early heart development, and the abnormal expression of TBX1 has been proven to disrupt heart development, particularly the septation and elongation of the outflow tract (OFT). Therefore, TBX1 knockout (KO) was generated in this study to model CHDs with 3D cardiac organoids, a novel method that can represent early heart formation better than monolayer cardiomyocytes (CM). Following the protocol, human embryonic stem cell (hESC)-derived CMs were achieved, with positive staining of the CM marker, cTnT, at Day 15 of differentiation. The 3D cardiac organoids were successfully composed and maintained for six weeks, whereby consistently increased organoid sizes and regular beating activities were observed. Markers for different cardiac cell lineages other than CMs were observed, indicating the increased cellular complexity in cardiac organoids compared to the monolayer CMs. With the CRISPR/Cas9 ribonucleoprotein (RNP) method, a partial TBX1 KO (pKO) cell population was successfully generated. The H1 cells with TBX1 pKO displayed comparable cell morphology to the wildtype (WT) cells but delayed cell differentiation progress into CMs. On Day 8 of CM differentiation, markers TNNT2, NKX2-5, and ISL1, essential for differentiation, were decreased, indicating the delayed differentiation of the TBX1 pKO CMs. In conclusion, the deletion of TBX1 alters the in vitro hESC- CM differentiation, potentially supporting the notion of modelling complex CHDs with cardiac organoids in the future.20 0Item 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.46 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