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Cleft palate is one of the most common birth abnormalities. Figures published in 2006 by the American Centres for Disease Control and Prevention, report the incidence of those born in the United States with a cleft palate without the presence of a cleft lip (CPI) to be 6.39 for every 10000 in the three years between 1999 to 2001 and for cleft lip in association with a cleft palate (CLP) to be even greater - 10.48 per 10000 live births. In 2001, Braybrook and colleagues reported that mutations in the TBX22 gene cause X-linked cleft palate (CPX), a disease characterised by a cleft of the secondary palate and is often seen in association with ankyloglossia (tongue-tie) (Braybrook et al. 2001). A cleft of the secondary palate arises as a consequence of disturbance to correct development during palatogenesis: an anomaly in palatal shelf growth; delayed or failed shelf elevation; defective shelf fusion or a failure of medial edge epithelium cell death. This thesis reveals that the expression of TBX22 during these key developmental events in human embryos is consistent with the phenotype seen in CPX. To enable an investigation for TBX22 target genes, a DNA binding sequence is determined for the TBX22 protein. This sequence is used to generate a generic TBX22 DNA binding site, the presence of which is screened for in promoter regions, defined as 2kb upstream of transcription start sites. 132 genes were selected as candidate TBX22 targets on the basis that they underlie human disorders that include a cleft palate. The screen shows that 28 of these genes have at least one perfect or near perfect match to the generic TBX22 DNA binding site. Of these, only two both contained a perfect TBX22 generic DNA binding site and mouse mutants also had cleft palates: SUMO1 and MSX1. Interaction between SUMO1 and TBX22 has already been shown (Andreou et al. 2007). This study investigated MSX1 as a downstream target of TBX22 using a luciferase reporter gene construct in vitro. The results showed that in the presence of TBX22, the luciferase signal was reduced and support MSX1 being a downstream target gene of TBX22. These findings further the understanding of the molecular networks regulating craniofacial development. Unravelling these complex interactions is crucial to identifying the mechanisms of oro-facial clefting, important steps towards improved methods of counselling, treatment and prevention of these common birth disorders.

Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive disease caused by the mutations in EVC or EVC2 genes. Human patients with EvC syndrome showed clinical features as short limbs, short ribs, postaxial polydactyly and orofacial defects and about 60% of affected individuals showed atrial septum defects. Evc-/- mice showed short limbs and short ribs similar with those observed in human patients. However, the cardiac phenotype of Evc-/- mice had not been studied. In this study, histological analysis was performed in both C57Bl/6J X 129Sv mixed background and inbred C57Bl/6J 25 background mouse embryos. No obvious cardiac development defects were observed. Parallel to this, the Evc mRNA and Evc protein were also analyzed by in situ hybridization and using the lacZ reporter system. X-gal positive cells were observed at the dorsal to atrial wall and primary atrial septum in E11.5 and E12.5, respectively. No Evc mRNA was detected in developmental heart by in situ hybridization by probes located at c.926 to c.1717. Previous study demonstrated that Evc and Evc2 protein are localized to the base of cilia. In this study, two different methods, deciliation and immuno-TEM, were applied to localize the Evc protein more precisely. The deciliation treatment sheared the cilia off at the distal end of transition zone and the immunofluorecent staining results indicated that the localization of the Evc protein was not beyond the distal end of transition zone. In the immuno-TEM experiment, although several factors were modified for proper staining, no informative results were obtained from this experiment. As the mutations of ciliary proteins might result in malformation of cilia, the ciliary structure of chondrocytes were also examined in this study. No structural II difference was observed in Evc-/- cells comparing to wildtype control. The Evc2 protein was undetectable at the base of cilia in MEFs, chondrocytes and osteoblasts when Evc protein was absent. The existence of Evc2 protein was detected in total lysate Evc-/- cells by western blot, indicating rather than affecting the expression of Evc2 protein, Evc plays a role in Evc2 protein correct localization. In human fibroblasts, EVC protein was localized to the base of cilia and the nucleus. The nuclear localization of Evc protein was never been observed in mouse cells by immunofluorescence staining. This inconsistence was examined through subcellular fractionation assay. The western blotting results demonstrated that the Evc protein exists in the nuclear fraction in the mouse MEFs. Several potential Evc associated proteins suggested by yeast two-hybrid study were also examined in subcellular fractionation assay. No distribution differences between wildtype and Evc-/- cells were observed.

Several bioinformatic methods have been developed to aid the identification of novel nuclear-mitochondrial genes involved in disease. Previous research has aimed to increase the sensitivity and specificity of these predictions through a combination of available techniques. This investigation shows the optimum sensitivity and specificity can be achieved by carefully selecting seven specific classifiers in combination. The results also show that increasing the number of classifiers even further can paradoxically decrease the sensitivity and specificity of a prediction. Additionally, text mining applications are playing a huge role in disease candidate gene identification providing resources for interpreting the vast quantities of biomedical literature currently available. A workflow resource was developed identifying a number of genes potentially associated with Lebers Hereditary Optic Neuropathy (LHON). This included specific orthologues in mouse displaying a potential association to LHON not annotated as such in humans. Mitochondrial DNA (mtDNA) fragments have been transferred to the human nuclear genome over evolutionary time. These insertions were compared to an existing database of 263 mtDNA deletions to highlight any associated mechanisms governing DNA loss from mitochondria. Flanking regions were also screened within the nuclear genome that surrounded these insertions for transposable elements, GC content and mitochondrial genes. No obvious association was found relating NUMTs to mtDNA deletions. NUMTs do not appear to be distributed throughout the genome via transposition and integrate predominantly in areas of low %GC with low gene content. These areas also lacked evidence of an elevated number of surrounding nuclear-mitochondrial genes but a further genome-wide study is required.

Steroids have been an indispensable immunosuppressive component since they were first introduced to transplantation. However, very few studies have pursued the correlation between individual steroid sensitivities and their transplant outcomes. In this PhD project, we mainly investigated glucocorticoid receptor (GR) and FKBP5, two fundamental molecules in GC signaling pathways, at the levels of gene polymorphism and gene expression. Associations were sought between genetic variations and in vitro steroid responsiveness in PBMC cell culture, as well as graft function after transplantation. Additionally, polymorphisms of several genes on chromosome 6p, Le. VEGF, FKBP5, HLA-DR and TNF-a, were studied for underlying linkage disequilibrium. The data have shown the GR gene haplotype (e.g. GR33388*A-766*C-3·569*A) may be related to steroid sensitivities in normal and transplant populations, although clinical applications for this genotype-phenotype association are still vague. GR3669*A/G has also been shown to be associated with regulation of the production of the hGRp isoform. The dexamethasone (Dex) dosage vs. inhibition curves established in in vitro PBMC proliferation assays have defined individual variations in response to Dex treatment and may provide a feasible laboratory test candidate to predict the cellular steroid sensitivity of tre.'5plant patients who possibly confront inflammatory challenges. In addie. n, the study of transplant patients for their FKBP5 and GR isoform gene expression has shown dramatic changes induced by steroids and/or inflammatory stimulators. The hGR-P isoform stood out from other GR isoforms and provides a new direction in studying regulation of GR isoforms during immune activition. Finally, two putative haplotypes were identified in association with DR52 and DRI on chromosome 6p, and named as 'high inflammatory' and 'low inflammatory' haplotypes respectively. The uncovering of such haplotypes may help to differentiate individuals according to their risk of acute and chronic rejection after transplantation.

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