The passage of fluid, ions and macromolecules across the epithelium is controlled primarily by epithelial tight junctions. Altered epithelial permeability is associated with lung disease, and barrier function is impaired by the Th2 cytokine IL-13. This thesis investigates the signalling pathways involved in the modulation of the epithelial barrier by IL-13 stimulation. Initial experiments demonstrated that the human sub-bronchial epithelial cell line Calu-3 could be easily manipulated when grown using an air-liquid culture system. Expression of various key tight junction proteins was demonstrated, as well as a high trans-epithelial resistance (TER) for up to 7 days. Stimulation with IL-13 resulted in a decrease in TER compared with controls and this decrease was shown to be prevented with the PI3K inhibitor ZSTK474. IL-13 did not increase paracellular permeability of the epithelial monolayer to FITC-dextran from the apical to the basolateral chamber and ZSTK474 did not influence FITC-dextran flux. Immunocytochemistry showed that the expression of the tight junction protein claudin 2 was increased by IL-13 stimulation and this change in expression was shown to be PI3K dependent with the PI3K inhibitor ZSKT474 preventing the increase. Further studies were carried out in an attempt to uncover the PI3K isoform responsible for the effects seen on both the TER and the TJ expression. It was shown that inhibition of the p110α isoform with PIK75 mimicked the result observed with the pan-PI3K inhibitor ZSTK474 and prevented the IL-13-induced claudin 2 upregulation. However none of the PI3K isoform inhibitiors showed the prevention of TER, as shown by the pan PI3K inhibitor ZSTK474. The role of STAT6 in TJ modulation was shown to be similar to that of PI3K, in that inhibition of STAT6 had a positive effect on the epithelial barrier by preventing the IL-13-induced TER decrease and the increase in the expression of claudin 2. In addition, both PI3K inhibition and STAT6 inhibition demonstrated effects on basal TER and claudin 2 expression, indicating that both pathways are involved in maintenance of epithelial barrier integrity.

Human embryonic stem cells (hESCs) are thought to have enormous potential for use in regenerative medicine, whilst simultaneously allowing us insights into human embryonic development, disease modelling and drug discovery. Differentiation to mesodermal lineages, such as cardiomyocytes and blood, may allow for improved treatment of cardiac and haematopoietic diseases. hESC-derived immune cell types may also allow the circumnavigation of the immune barrier. This thesis aims to test the hypothesis that formation of hESC derivatives is regulated by the same mechanisms and ontology as in vivo embryo development. Therefore, by identifying and facilitating the mechanisms of mesoderm induction, hESC differentiation can be optimised to maximise the production of mesoderm, and, ultimately, mesoderm derivatives. Using a Xenopus laevis animal cap model with simultaneous treatment with activin B or fgf4, together with tall, Im02 and gatal mRNA, resulted in substantial increases in mesodermal, haemangioblast and erythropoietic cell markers. One of the most successful methods for hESC differentiation is by the formation of human embryoid bodies (hEBs). To reduce first the number of variables in current mass culture protocols for hEB formation, such as hEB size, a forced aggregation system was established that produced homogeneous hEBs from defined numbers of cells. This system was then optimised to enhance production beating cardiomyocytes by varying the number of hESCs used for hEB formation and also the number of days in culture. This system was assessed in four hESC lines and demonstrated substantial inter-line variability in cardiomyocyte production (1.6± 1.0% to 9.5±0.9°0). Differentiation was also performed using chemically defined media (CDM) with the addition of actiyin A and FGF2 and resulted in 23.6±3.6% of hESs producing beating cardiomyocytcs. In addition immunohistochemistry was performed to assess the relationship of cells expressing markers for mesoderm, pluripotency, ectoderm, and endoderm to establish a standard spatial and temporal map of hEB differentiation.

14-3-3 proteins are a ubiquitous family known for their ability to regulate myriad cellular processes including lifespan, neuronal signalling and transduction, protein trafficking and transmitter production and release. Previous work has shown that 14-3-3 proteins are linked to a variety of pathological neurodegenerative diseases although their role in healthy brain ageing is currently unclear. This study utilised the pond snail, Lymnaea stagnalis to examine the contribution made by 14-3-3 to the decrease in feeding rate that is seen in this model system with age. Interrogation of a Lymnaea CNS cDNA library identified four putative 14-3-3 isoforms, one highly similar to the mammalian 14-3-3ε (14-3-3Lyml), while the other three (14-3- 3 Lym2, 14-3-3Lym3 and 14-3-3Lym3var) were more distinct. Expression, localisation and function of these proteins were studied using a range of biochemical and analytical techniques, in three different animal age groups (3, 6-7 and 10-12 months). Western blot (W8) showed a significant decrease in the overall level of 14-3-3Lym3 in the cerebro-buccal ganglia that correlated with feeding rate. There was no overall change in the expression of 14-3-3Lyml and 2. 14-3-3Lym3var was not detected. CNS 14-3-3 expression was seen in all 11 ganglia including the cerebral and buccal ganglia which are important for regulating feeding. 14-3-3Lyml expression was limited to the neuronal cell cytoplasm and plasma membrane, whereas the remaining isoforms appeared to be distributed throughout the cell, including the nucleus. Expression was shown in key neurones that regulate feeding including identified dopaminergic and serotonergic neurones. 14-3-3 proteins have previously been shown to regulate the synthesis of both dopamine (DA) and serotonin (S-HT) through actions on tyrosine and tryptophan hydroxylase (TH and TPH respectively), the rate-limiting enzymes in their production. HPLC analysis demonstrated that antagonism of 14-3-3 proteins with R18 significantly reduced the production of L-DOPA and S-HTP in the cerebral and buccal ganglia, suggesting that 14-3-3 proteins can regulate DA and S-HT production in these areas. In summary, the 14-3-3Lym proteins are capable of regulating the activity of TH and TPH and the change in expression pattern of these proteins with age may explain the noted age-related changes in S-HT and DA signalling in the cerebro-buccal ganglia and the consequential decrease in feeding rate seen with age.

The ESCRT machinery has a well established role within the endocytic pathway. Studies conducted in Drosophila have identified ESCRT proteins as important regulators of epithelial cell polarity and growth. Consequently ESCRTs have been classified as potential tumour suppressors. Alterations in the expression of various ESCRT components have been observed in human cancers. However, the possible link between ESCRT proteins, mammalian epithelial cell polarity and tumourigenesis has not been investigated. This thesis demonstrates for the first time that the ESCRT-I protein, Tsg101, is required for maintenance of mammalian epithelial cell organisation and polarity. siRNA knockdown of Tsg101 in the human Caco-2 cell line results in the formation of a multilayered epithelium with compromised apicobasal polarity. In addition, Tsg101 depletion impairs differentiation of the epithelial sheet and formation of polarised 3D Caco-2 cysts. Depletion of Tsg101 also results in intracellular accumulation of the tight junction protein, claudin-1. This is shown to be constitutively endocytosed and recycled in Caco-2 epithelial monolayers, suggesting that ESCRT-I is required for claudin-1 recycling to tight junctions. Tsg101 knockdown also impairs epithelial barrier formation and enhances Caco-2 migratory ability. This suggests that tight junction integrity is impaired and may contribute to the loss of Caco-2 cell organisation and polarity observed upon Tsg101 depletion. Finally, Tsg101 depleted Caco-2 cells appear to overproliferate, forming multilayered regions of the epithelial sheet. However, multilayered cells are eventually eliminated via apoptosis. Preliminary results suggest that inhibition of this apoptotic response enhances the aberrant epithelial phenotype, suggesting that the ability to evade apoptosis may be an important factor in determining the tumourigenic potential of ESCRT-I depletion. Therefore, results presented in this thesis suggest that the role of ESCRT-I as a tumour suppressor is conserved from Drosophila to mammals.

E-cadherin (Cdh1), is a major component of epithelial adherens junctions, binds the Wnt pathway effector β-catenin and is lost at the invasive edge of colon cancers. Crypt stem cells give rise to 4 cell lineages that, with the exception of Paneth cells, travel along the crypt-villus axis over 3 days and are shed by anoikis. Gain of function of the Wnt pathway by a mutation in Adenomatous Polyposis Coli (Apc) disrupts enterocyte turnover to result in adenoma formation. Homozygote null Cdh1 is embryonic lethal, and heterozygote Cdh1 can promote Apc^1638N induced adenoma formation, but we lack models that assess additional functions of Cdh1 in the adenoma to carcinoma transition. The aim of this thesis was to evaluate the effect of Cdh1 loss of function in the murine intestine, including in the setting of Wnt pathway activation. To do this, germline and intestinal specific conditional Cdh1 loss of function models were generated. Conditional homozygous deletion of Cdh1 resulted in embryonic lethality using Villin-Cre. In adults, homozygous Cdh1 loss using the tamoxifen inducible Villin-CreER^T2 led to intestinal inflammation, bacteraemia and disrupted crypt-villus architecture. Combined conditional homozygous deletion of Cdh1 and Apc resulted in Wnt pathway upregulation assessed by β-catenin immunolabelling. Strain dependent effects of Cdh1 heterozygosity were apparent on the Apc heterozygote background: Apc^Min/+ Cdh1^+/- (C57BL/6J) had no effect on survival or adenoma phenotype compared to littermate Apc^Min/+; Cdh1^+/fl increased adenoma burden in Apc^+/fl Vil-Cre animals (B6D2/C57BL/6J). Low frequency recombination of Apc^fl/fl using Lgr5-EGFP-IRES-CreER^T2 bypasses the loss of heterozygosity event relied on in heterozygous Apc tumour models achieving a large adenoma burden within 4 weeks. Cdh1 loss did not alter survival or adenoma phenotype in this model, including the development of large caecal tumours. Immunolabelling of tumours from Apc^fl/fl Cdh1^fl/fl animals showed persistent E-cadherin protein expression, suggesting incomplete recombination or that double homozygote enterocytes failed to survive. In vitro adenoma culture was used to test whether E-cadherin loss was incompatible with enterocyte survival in the setting of Wnt activation. Apc^fl/fl Cdh1^{+/+} and Apc^fl/fl Cdh1^fl/fl adenoma were cultured in matrigel and treated with an adenovirus expressing Cre recombinase under a CMV promoter (Ad-Cre). Ad-Cre had no effect on Apc^fl/fl Cdh1^+/+ adenoma growth. Ad-Cre treated Apc^fl/fl Cdh1^fl/fl adenoma organoids showed cells where E‑cadherin loss resulted in Wnt pathway upregulation as assessed by nuclear β-catenin and Axin2 expression, and epithelial mesenchymal transition shown by upregulation of fibronectin, twist and vimentin. This work supports a role for E-cadherin in modulation of the Wnt pathway. Further investigation is required to define the cell adhesion versus Wnt regulatory functions of E-cadherin.