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In vertebrates the cornea must maintain its transparency throughout adult life to ensure sight, and understanding the mechanisms underpinning corneal homeostasis are fundamental to developing new treatments to cure or prevent blindness. This study investigated the role the planar cell polarity pathway plays in the directed migration of adult corneal epithelial cells, in maintaining the homeostatic environment of the eye and during wound healing. RT-PCR confirmed, for the first time, the expression of multiple core PCP genes within human corneal epithelial (HCE) cells. Components of the PCP pathway were pharmacologically and genetically manipulated during wound healing of corneal epithelial cells and the importance of the downstream target JNK, and core PCP gene Vangl2, during wound healing was demonstrated. Manipulation of core PCP components was found also to directly affect the ability of HCE cells to realign and migrate in response to physical topographical cues in vitro. This study therefore indicated that PCP may regulate the directed migration of corneal epithelial cells as they travel over the basement membrane. Using conditional knockout mice the loss of Vangl2, a core PCP gene, and its effect on both planar and the apical-basal polarity of the corneal epithelium was investigated. Severe morphological defects were observed in Vangl2-null mice indicative of underlying problems in apical-basal polarity of the epithelial cells. The basement membrane of Vangl2-null cells was largely absent in vivo, which suggested that at least some of the planar defects were secondary to an unexpected failure of apical-basal polarity. This study has shown for the first time that PCP plays a crucial role in the maintenance of an adult vertebrate tissue, particularly during wound healing and maintenance of the corneal epithelium. It has also indicated a role for the core PCP gene, Vangl2, in setting up apical-basal polarity of these adult cells.

The cancer stem cell (CSC) theory proposes that a minor population in tumor cells with specific features, such as self-renewal and reproducible tumor phenotype could contribute to tumor relapse and chemotherapy resistance. Several studies have convincingly documented the existence of ovarian CSC, but questions related to the biologic behavior and specific biomarkers of ovarian CSC remain to be clarified. In the present study, we firstly established a tumor cell line with capability of regenerating tumors through serial transplantation of ovarian tumor tissue in non-obese/severe combined immunodeficient (SCID) mice. After separation of CD133+ cells with magnetic beads, we compared the phenotype and biologic behavior of CD133+ versus CD133-cells. It was found that the CD133+ cells were much more potent to produce colonies in semi-solid agar culture than CD133-cells. The proportion of the cells in G0/1 cell cycle is much higher in CD133+ cells than in CD133-cells. Furthermore, in vivo experiments demonstrated that the CD133+ cells were capable of repeatedly regenerate tumors in NOD/SCID mice, while the CD133-cells were not. Compared with CD133-cells, the CD133+ cells expressed much higher levels of the stem cell markers Oct4, Sox2, Nanog and Mcl-1. Clinically, among a total of 290 ovarian epithelial cancers, increased level of CD133 expression was positively correlated with a high cancer stage and had a worse 5-year survival rate. Taken together, the results suggest that the CD133+ cells from human ovarian cancer have the characteristics of CSC, which may contribute to ovarian cancer relapse and anti-apoptotic activity. The method of ovarian CSC enrichment we established provides a feasible and practical way of ovarian cancer research in a molecular level. In addition, CD133 may be used as a prognostic marker for ovarian epithelial cancer, which may have a role for future therapeutic effect.

Philosophiae Doctor - PhD / Introduction: Globally, there are over 190,000 new reported cases of ovarian cancers per annum. This comprises 3% to 4% of all cancers in women. Ovarian cancer is one of the leading causes of deaths in women. Ovarian cancer is the second most diagnosed gynaecological malignancy and over all the fifth cause leading to death among all types of cancer in the UK in 2004. More than 70% of epithelial ovarian cancers are diagnosed at an advanced stage. Consequently, the prognosis is poor and the mortality rate high. Thus, the survival rate is affected by how far the disease has progressed or spread. A dire need exists to identify ovarian cancer biomarkers, which could be used as good indicators of expression in ovarian cancer cells in vitro Aim: The aim of this study was to analyse selected cancer biomarkers, which are currently under intense investigation for their suitability to diagnose epithelial ovarian cancer at an early stage. These biomarkers were analysed in terms of their in vitro expression in normal epithelial cells and ovarian cancer cell lines, which allows for their genomic and proteomic classification. The expression analysis of each biomarker is related to the malignancy of a tumour and, therefore, advocates its use for potential future improvement of sensitive tumour markers. Methods: The primary human ovarian surface epithelial cell line (HOSEpiC), SKOV-3 cells and the OAW42 human epithelial ovarian tumour cell lines were used to evaluate the selected cancer biomarkers. Cells were cultured using appropriate media and supplements, and real-time quantitative polymerase chain reaction (RT-PCR) utilized to validate expression levels of the following genes: HDAC1, HDAC2, HDCA3, HDAC5, HDAC6, HDAC7, HDAC8, LPAR1, LPAR2, MUC16 and FOSL1, against normal housekeeping genes GAPDH and HPRT. In addition, immunocytochemistry was also used in the validation process of the aforementioned genes. Significance: ovarian cancer cells express gene signatures, which pose significant challenges for cancer drug development, therapeutics, prevention and management. The present study is an effort to explore ovarian cancer biomarkers to provide a better diagnostic method that may offer translational therapeutic possibilities to increase five- year survival rate. Results: HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 expressed distinctively in ovarian cancers matched to other tissues or cancer types have already been identified by RT-QPCR and confirmed by immunocytochemistry and efforts to generate monoclonal antibodies to the other six genes (HDAC1, HDAC2, HDAC3, HDAC7, HDAC8 and FOSL1) encoded proteins are underway. Conclusions: here we provide strong evidence suggesting that HDAC5, HDAC6, LPAR1, LPAR2, except MUC16 are up regulated in ovarian cancer. These data were confirmed by examining Human Protein Atlas (HPA) databases, in addition to protein expression of HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 in cells cytoplasm. For future prospective, using other techniques that assess the variant expression that could explain the release of these gene candidates into the circulation with serum tumour markers, and protein expression will be strengthened.

Kidney disease has a high incidence across the globe and can be caused by acute and chronic injury. Current methods of treatment range from prevention and management with diet and extend to hemodialysis at End Stage Renal Disease (ESRD). Transmembrane Immunoglobulin Domain Containing-1 (TMIGD1) is mainly expressed in kidney and the intestines and is involved in cell-cell interaction of epithelial cells. This thesis investigated the potential role of TMIGD1 in the development of chronic kidney disease and tubular epithelial cell injury in CRISPR/Cas9-TMIGD1 transgenic mouse. Treatment of wild-type mice with adenine showed that TMIGD1 is downregulated in response to adenine-induced renal cell injury. CRISPR/Cas9-TMIGD1 -/+ mice treated with adenine displayed significantly increased tubular damage compared to wild-type mice. Additionally, expression of TMIGD1 was directly correlated with localization of C/EBPβ to nucleus, a transcription factor that is known to regulate expression of TMIGD1. In conclusion, the loss of TMIGD1 negatively impacts response to renal stress. / 2020-06-17T00:00:00Z

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.