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Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes.

The extracellular matrix (ECM) is constantly remodelled in healthy tissues; this is essential in normal physiological processes such as wound healing. Remodelling is regulated by the balanced activities of proteases and their inhibitors. An imbalance in activity can result in pathology such as fibrosis and cancer. The proteases mainly responsible for breakdown of the ECM are the matrix metalloproteinases (MMPs). They are important for normal maintenance of the 3M, but also have roles in pathophysiology. The tissue inhibitors of matrix metalloproteinases (TIMPs) are the main specific inhibitors of MM? activity. There are 4 identified human TIMPs, TIMP 1-4. Interestingly, TIMPs have roles that are independent of MMP inhibition, such as promoting cell proliferation and migration. Increased levels of TIMPs have been found in several types of cancers, including gastric cancer.

Epithelial cell-cell adhesion is mediated by tight junctions, adherens junctions and desmosomes. Epithelial cell-matrix adhesion is mediated by hemidesmosomes and focal contacts. These complexes exhibit great plasticity, and each contains molecular components which are able to participate in one or more of the other adhesive complexes. Focal adhesion kinase (FAK/pl25FAK) is a non-receptor tyrosine kinase which transduces signals from integrins at sites of focal contact to promote adhesion, spreading and migration. FAK possesses a central kinase domain which is flanked by large, non-catalytic, amino- and carboxy-terminal domains. Whereas the functions of the carboxy-terminal and kinase domains of FAK are well understood, the role of the amino-terminal domain remains unclear. FAK expression was examined in the human epithelial cell line, HEK 293. Amino-terminal FAK immunoreactivity was noted at sites of cell-cell contacts and in the nucleus, in contrast to carboxy-terminal immunoreactivity, which was largely cytoplasmic and perinuclear. Western blot analysis of endogenous FAK revealed expression of a presumptive proteolytic cleavage fragment corresponding to the amino- terminal domain. A series of FAK constructs was generated to test the hypothesis that the observed amino-terminal FAK localisation was due to this proteolytic fragment. Epitope- tagged Amino-Terminal FAK (ATF) constructs localised primarily at areas of cell-cell contact and in the nucleus in HEK 293 cells. This localisation was independent of Tyrosine 397, the major FAK autophosphorylation site. This sub-cellular distribution was confirmed in another epithelial cell line, MDCK, in which transiently transfected ATF constructs also localised primarily to the nucleus and at cell-cell contacts. HEK 293 cells were characterised with respect to expression of adhesive proteins, and ATF was found to co- localise with the tight junction protein occludin, with cortical actin and with junctional ?1 integrin. Immunoprecipitation data suggests that none of these proteins forms a precipitable complex with ATF. These findings indicate that the amino-terminal domain of FAK is capable of localising at epithelial cell-cell contacts and suggest a novel role for FAK in mediating cross-talk between focal contacts and cell-cell contacts through endogenously expressed amino-terminal FAK fragments.

The genetic basis of the different cancer phenotypes has been a continuous and accelerating subject of investigation. Data accumulated thanks to recently introduced genome-wide scanning technologies have revealed that human diversity and diseases susceptibility is also greatly influenced by structural alterations in the human genome, such as DNA copy number variants (CNVs) and copy number alterations (CNAs), which influence gene expression in both healthy and pathological cells. Our research aims to investigate the influence of structural alterations on gene expression in cancer cells using SNP microarray data. Specifically, we focus on analyzing DNA copy number alternations (CNAs), which can significantly influence gene expression in cancer cells. Several cancer-predisposing mutations affect genes that are responsible for maintaining the integrity of the chromosomes during cell division, which can result in translocations, gains or losses of large parts of chromosome. To our knowledge, there have been no publications that link whole-genome copy number alterations in cancer to gene expression variations using the full range of possibilities offered by SNP arrays. The accurate use of SNP arrays in the analysis of cancer has been difficult due to tumour purity, tumour heterogeneity, aneuploidy/polyploidy and complex patterns of CNA and loss-of-heterozygosity (LOH). In our work, we use and further extend a recently developed novel tool for tumour genome profiling called OncoSNP (Yau, Mouradov et al. 2010), in order to resolve some of those problems and accurately estimate copy number alterations (CNA) and loss-of-heterozygosity (LOH) from SNP array data in cancer cell samples. The methods developed in this thesis tackle the problem of cancer genomic investigation by developing and validating an extension (DPS smoothing) of a new method (OncoSNP). This approach is used in the analysis of global expression versus CNA patterns in experimental systems and large clinical datasets. We analyse various cancer SNP and gene expression arrays of increasing complexity and heterogeneity, starting with a dataset of head and neck squamous cell carcinoma (HNSCC) cell lines, followed by leukaemia samples and finally a large breast cancer dataset. The central findings of our research are multifold. In the HNSCC dataset we find that the level of genetic instability is not indicative of the pathological state; i.e. there are premalignant lesions displaying extensive mutations. However some genetic features are typical of certain lesion type; e.g. we consistently observe copy loss in the short arm of chromosome 3 in carcinoma. The pattern of homozygous deletion in the dataset reveals common deletion of cancer related genes, especially CDK4 (pI6). Furthermore we notice a significant positive correlation between the copy number and the expression on a systematic level. In Leukaemia, we do not observe extended uniparental disomy as previously published (Akagi, Shih et al. 2009) and expected. However large alterations (whole arm amplification) are observed in individual patients: copy loss in chromosome 7 (2 patients), copy gain in chromosome 8 (3 patients) as well as common alterations around the centromeres and telomeres. In the breast cancer dataset significantly different level of mutations were observed in the different subtypes in the cohort. Furthermore 499 genes were identified with significant correlation between their gene expression (GE) and underlying genomic alterations (either copy number (CN) or loss-of-heterozygosity (LOH)). Performing hierarchical clustering on the cohort using the 499 correlated genes enabled us to recover the subtypes' separation previously based on gene expression alone.

The purpose of this thesis is to develop a vertex model framework that can be used to perform computational experiments related to the dynamics of epithelial tissues in developmental systems. We focus on three example systems: the Drosophila wing imaginal disc, the Drosophila epidermis and the visceral endoderm of the mouse embryo. Within these systems, key questions pertaining to size-control mechanisms and coordination of cell migration remain unanswered and are amenable to computational testing. The vertex model presented here builds upon existing frameworks in three key ways. Firstly, we include novel force terms, representing, for example, the reaction of a cell to being compressed and its shape becoming distorted during a highly dynamic process such as cell migration. Secondly, we incorporate a model of diffusing morphogenetic growth factors within the vertex framework, using an arbitrary Lagrangian-Eulerian formulation of the diffusion equation and solving with the finite-element method (FEM). Finally, we implement the vertex model on the surface of an ellipsoid, in order to simulate cell migration in the mouse embryo. Throughout this thesis, we validate our model by running simple simulations. We demonstrate convergence properties of the FEM scheme and discuss how the time taken to solve the system scales with tissue size. The model is applied to biological systems and its utility demonstrated in several contexts. We show that when growth is dependent on morphogen concentration in the Drosophila wing disc, proliferation occurs preferentially in regions of high concentration. In the Drosophila epidermis, we show that a recently proposed mechanism of compartment size-control, in which a growth-factor is released in limited amounts, is viable. Finally, we examine the phenomenon of rosettes in the mouse embryo, which occur when five or more cells meet at a common vertex. We show, by running simulations both with and without rosettes, that they are crucial facilitators of ordered migration, and are thus critical in the patterning of the early embryo.