Saudi Cultural Missions Theses & Dissertations
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Item Restricted Defining The Role of PRPF8 in Congenital Heart Diseases(The University of Manchester, 2025) Althali, Nouf; Ray O’Keefe, Kathryn Hentges andCongenital heart disease (CHD) is the most common birth abnormality, impacting over 1% of live births worldwide and representing a major cause of infant mortality and morbidity. Despite advances in genetic studies identifying several causal variants, the molecular mechanisms behind many congenital heart defects (CHDs) remain insufficiently elucidated. We have discovered a mutant mouse line with laterality and cilia defects accompanied by cardiac abnormalities caused by a missense mutation in the spliceosome gene Prpf8. The role of PRPF8 in congenital heart defects has not been investigated prior to this thesis research. In this project four PRPF8 missense variants: V250M, P372L, T589M and M1730T, that have been found in CHD patients, were studied to determine if PRPF8 function is altered by these variants. These PRPF8 variants were predicted that may cause aberrant splicing and/or altered biochemical interactions between PRPF8 and the other spliceosomal proteins, leading to developmental heart malformations likely due to aberrant splicing or expression of cardiac genes. PRPF8 variants were investigated using multiple bioinformatics prediction tools to predict the impact of PRPF8 variants found in human CHD patients on protein function. Moreover, PRPF8 variant protein-protein interactions and protein stability were assessed. We also generated the analogous missense variants in yeast Prp8 to study their effect during splicing. Moreover, a Prpf8 mouse model was used to evaluate the expression of Prpf8 in cardiac and neural crest cell markers. Our bioinformatics analysis revealed some of these variants were predicted to be damaging and affect protein functions while others were not. Significantly, although the most highly predicted damaging variant, PRPF8 T589M, did not interfere with PRPF8 interactions with its binding partners, EFTUD2 and PPIL2, it, and other PRPF8 variants, adversely impacted PRPF8 protein stability. This suggests a specific mechanism in which decreased protein stability, rather than a loss of physical interactions, accounts for the impact of these mutations. Our splicing reporter assay findings indicate that all examined mutations impair PRP8's splicing efficiency, causing remarkable gene expression alteration and splicing defects. The Prpf8 mutant mouse displays reduced neural crest marker expression, suggesting a vital role for PRPF8 in cardiac development via the regulation of neural crest cell development. Novel insights gained from this work may aid our ability to understand the role of PRPF8 in CHD and its links with cardiac defects, informing diagnostic and therapeutic strategies.20 0Item Restricted Defining The Role of PRPF8 in Congenital Heart Diseases(The University of Manchester, 2025) Althali ,Nouf; Ray O’Keefe, Kathryn Hentges andCongenital heart disease (CHD) is the most common birth abnormality, impacting over 1% of live births worldwide and representing a major cause of infant mortality and morbidity. Despite advances in genetic studies identifying several causal variants, the molecular mechanisms behind many congenital heart defects (CHDs) remain insufficiently elucidated. We have discovered a mutant mouse line with laterality and cilia defects accompanied by cardiac abnormalities caused by a missense mutation in the spliceosome gene Prpf8. The role of PRPF8 in congenital heart defects has not been investigated prior to this thesis research. In this project four PRPF8 missense variants: V250M, P372L, T589M and M1730T, that have been found in CHD patients, were studied to determine if PRPF8 function is altered by these variants. These PRPF8 variants were predicted that may cause aberrant splicing and/or altered biochemical interactions between PRPF8 and the other spliceosomal proteins, leading to developmental heart malformations likely due to aberrant splicing or expression of cardiac genes. PRPF8 variants were investigated using multiple bioinformatics prediction tools to predict the impact of PRPF8 variants found in human CHD patients on protein function. Moreover, PRPF8 variant protein-protein interactions and protein stability were assessed. We also generated the analogous missense variants in yeast Prp8 to study their effect during splicing. Moreover, a Prpf8 mouse model was used to evaluate the expression of Prpf8 in cardiac and neural crest cell markers. Our bioinformatics analysis revealed some of these variants were predicted to be damaging and affect protein functions while others were not. Significantly, although the most highly predicted damaging variant, PRPF8 T589M, did not interfere with PRPF8 interactions with its binding partners, EFTUD2 and PPIL2, it, and other PRPF8 variants, adversely impacted PRPF8 protein stability. This suggests a specific mechanism in which decreased protein stability, rather than a loss of physical interactions, accounts for the impact of these mutations. Our splicing reporter assay findings indicate that all examined mutations impair PRP8's splicing efficiency, causing remarkable gene expression alteration and splicing defects. The Prpf8 mutant mouse displays reduced neural crest marker expression, suggesting a vital role for PRPF8 in cardiac development via the regulation of neural crest cell development. Novel insights gained from this work may aid our ability to understand the role of PRPF8 in CHD and its links with cardiac defects, informing diagnostic and therapeutic strategies.17 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 Development of a Patient-Specific Streamlined Workflow for a Predictive Tool for Coronary Artery Bypass Graft Outcomes(University College London, 2024-12-01) Alsaleh, Abdullah; Torii, RyoCoronary artery disease is one of the leading causes of mortality worldwide and is treatable by only a few procedures, such as coronary artery bypass grafting (CABG). However, predicting the long-term success of CABG, particularly in terms of graft patency and disease progression, remains a challenge. This thesis aims to develop a patient-specific streamlined workflow for a predictive tool that integrates peri-operative Coronary Computed Tomography Angiography (CCTA) with Computational Fluid Dynamics (CFD) simulations to predict post-CABG outcomes. The patient-specific streamlined workflow for a predictive tool for CABG outcomes is designed to simulate hemodynamic conditions within grafts, providing personalized predictions to guide surgical decisions and improve patient outcomes. The methodology includes creating anatomically accurate 3D models, simulating hemodynamic conditions, and validating the results against clinical data. While the patient-specific streamlined workflow for the predictive tool shows promise, significant challenges remain in terms of boundary condition setup, data integration, and discrepancies between simulation results and hospital data. The overestimation of flow rates and high wall shear stress observed in the simulations indicate the need for refinements in model assumptions, including incorporating non-Newtonian blood properties, vessel wall compliance, and pulsatile blood flow. Despite these limitations, this study demonstrates the potential of a patient-specific streamlined workflow for a predictive tool for CABG outcomes and offers a path forward for personalized cardiovascular treatment. Future work will focus on refining the model to enhance its accuracy, and clinical applicability, and commercializing the method.30 0