http://theses.ncl.ac.uk/jspui/handle/10443/100 2026-02-04T11:41:25Z http://theses.ncl.ac.uk/jspui/handle/10443/2839 Title: Investigating a Tbx1 and Pax9 genetic interaction during cardiovascular development Authors: Briones Leon, Jose Alberto Abstract: Congenital cardiovascular malformations (CCVM) are the most common type of birth defect in humans and can be life threatening for the newborn. 22q11 deletion syndrome (22q11DS) is one of the most common CCVM in humans, with patients presenting a wide variety of abnormalities including craniofacial dysmorphology, immune deficiency, mental retardation and cardiovascular defects, including ventricular septal defects, abnormal right subclavian artery and interrupted aortic arch type B. TBX1 is considered the main gene underlying the cardiovascular phenotype in 22q11DS patients, however, the great phenotypic variability among 22q11DS patients suggests genetic modifiers define the presentation of the phenotype. The transcription factor Pax9, was found significantly down-regulated in Tbx1-null embryos (a mouse model of 22q11DS). The aim of this project was to determine whether Pax9 is involved in cardiovascular development and to study a potential genetic interaction between Pax9 and Tbx1 during cardiovascular development. The results show that Pax9 is required for cardiovascular development as all Pax9-null embryos have severe cardiovascular abnormalities including IAA, VSD, BAV, DORV, and abnormal or completely absent common carotids. Furthermore, a strong genetic interaction between Pax9 and Tbx1 was found, since double heterozygosity leads to lack of formation of the 4th pharyngeal arch arteries consequently leading to a significant increase in the incidence of IAA. The molecular mechanism of this interaction between Pax9 and Tbx1 was investigated. The results show that Tbx1 does not bind to any region within the Pax9 locus in vitro and in vivo. A physical interaction between Pax9 and Tbx1 proteins was also ruled out by co-immunoprecipitation. qPCR analysis revealed a significant downregulation of Gbx2 in double heterozygous embryos, and luciferase experiments revealed Pax9 is able to promote luciferase expression of a conserved regulatory region within the Gbx2 locus, whereas Tbx1 repressed luciferase expression of this Gbx2 cloned regions. The results in this dissertation suggest Pax9 and Tbx1 regulate cardiovascular development, at least in part, through regulating Gbx2. Description: PHD Thesis 2015-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/1547 Title: The role of endoglin in cell-mediated heart repair Authors: Redgrave, Rachael Elizabeth Abstract: Despite substantial advances in pharmacological and interventional strategies for the treatment of ischaemic heart disease and acute myocardial infarction (MI) many patients go on to develop dilated heart failure with high morbidity and mortality rates. Increasing evidence suggests that stem cells can augment re-vascularisation of the infarcted myocardium leading to improved cardiac function. However, the precise mechanisms involved are not completely understood and further work is required to realise optimised cell based therapy. The aim of this project was to investigate the role of endoglin, a pro-angiogenic TGF co-receptor, in bone marrow (BM)-derived endothelial progenitor cells (EPCs) and in cardiac-resident cardiosphere-derived cells (CDCs) using mouse models. I hypothesised that endoglin acts as a key marker and functional regulator of the pro-angiogenic repair properties of these stem cells and that manipulating TGF signalling by controlling endoglin levels could provide a better understanding of the properties of these cells, with the longer term goal of developing more effective revascularisation therapies. My data show that inhibiting TGF /Alk5 signalling in vitro increases EPC yield from both endoglin heterozygous and wild-type BM cells. In addition, although endothelial specific knock-down of endoglin in vivo led to increased endoglin expression in cells from the BM, these cells did not incorporate into endoglin depleted heart or lung vasculature. I also showed that endoglin deficiency results in a significant reduction in EPC yield from BM cells and inducible endoglin depletion in CDCs (using a conditional endoglin knock-out mouse model) impairs cellular proliferation and migration in vitro alongside up-regulated TGF /Alk5 signalling. Furthermore, injection of wild-type CDCs into the ischaemic border zone of the heart, following coronary artery occlusion in a mouse MI model, led to an increased angiogenic response at 4 weeks. This response was significantly reduced when endoglin depleted cells were transplanted. However both wild-type and endoglin depleted CDCs were able to rescue heart function, as measured using cardiac MRI. In conclusion, the results of this thesis provide evidence of the important roles that endoglin and TGF signalling play in promoting angiogenesis in tissue repair. Further advances in this field could aid in the development of more effective cell therapies for MI patients. Description: PhD Thesis 2012-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/1278 Title: Unravelling cylindromas : a molecular dissection of CYLD defective tumours Authors: Rajan, Neil Abstract: Patients with germline mutations in the tumour suppressor gene CYLD develop multiple cutaneous tumours on the head and neck; historically this has been termed “turban tumour” syndrome. Cylindromas and spiradenomas, hair follicle related tumours seen in this syndrome, cause significant clinical morbidity. Here we characterise the clinical phenotype of these patients, utilising tumour mapping to determine the location of tumours in mutation carriers from two large pedigrees. We demonstrate the disease often affects sites outwith the head and neck, and that androgen stimulated hair follicles are particularly vulnerable to tumour formation. The impact of this disease is severe, with 1 in 4 carriers of this gene undergoing complete scalp removal. To improve this outcome, we performed whole genome profiling of CYLD defective tumours, characterising genomic and transcriptomic changes to determine targetable signalling pathways. High resolution analysis using whole genome array based comparative genomic hybridisation and single nucleotide polymorphism analysis suggest that loss of heterozygosity at the CYLD locus may be the only change required for tumour phenotype. Gene expression profiling highlighted transcriptomic similarity between cylindromas and spiradenomas. Threedimensional reconstruction in silico from serial sections of tumours demonstrated contiguous growth between cylindromas and spiradenomas, in support of this finding. In both tumour types, dysregulated tropomyosin receptor kinase (TRK) signalling was found. Consistent with this, was the finding that TRKB and TRKC protein was overexpressed selectively in the tumour samples, demonstrated on a tissue microarray. Therapeutic utility of targeting this pathway was demonstrated by reduced viability of CYLD defective primary cell cultures in the presence of TRK inhibitors. These preliminary data support the use of TRK inhibitors as a therapeutic strategy in severely affected CYLD mutation carriers. Description: Ph. D. 2011-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/1277 Title: Neuronal potential of umbilical cord blood non-hematopoietic multipotent stem cells Authors: Ali, Hamad Abstract: The human central nervous system, one of the most complex organ systems anatomically and physiologically in the human body, is the body control center which manages and coordinates functions of all different organ systems. The lack of effective treatments and therapeutic intervention make injuries and disorders associated with the central nervous system one of the most dangerous and fatal health conditions worldwide. The adult nervous system has a limited capability of self-repair and regeneration following either a neurological disorder or injury. Despite being limited and ineffective in initiating recovery from injuries and disorders, this property opened the doors for a new direction of research aimed at investigating the possibility of developing therapies and treatments for central nervous system injuries and disorders based on the concept of regeneration and cell transplantations. Stem cells have gained significant public attention over the past decade due to their differentiation capabilities and potential utilization in clinical applications. The ability to differentiate stem cells into neural lineages including neurons and glial cells, highlighted their potential role as a therapeutic tool for central nervous system injuries and disorders. The main aim of this thesis is to show that umbilical cord blood stem cells are a potential source of cells that could be used therapeutically in central nervous system injuries and disorders. A distinct population of cells has been purified from human umbilical cord blood. These cells have been characterized and differentiated in-vitro into neuron-like cells using fully defined sequential neuronal induction protocol. The differentiated cells were shown to have similar morphological and functional properties to developing central nervous system endogenous neurons using several different techniques, including immunocytochemistry, real-time PCR, cDNA arrays and calcium imaging. The results highlight the potential role of umbilical cord blood stem cells as a therapeutic tool for central nervous system injuries and disorders for which current mode of therapy is inadequate. In addition, they might provide an in-vitro model of neural cells for toxicology and drugs testing research. Description: Ph. D. 2011-01-01T00:00:00Z