Long QT Syndrome 2 (LQTS2) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) are two prevalent cardiac channelopathies. LQTS2 is the second most common form of LQTS and accounts for up to 45% of cases (Schwartz et al, 2012). CPVT1 is the most common form of CPVT and relates to dysfunction of the RYR2, mutations in RYR2 are thought to account for up to 50% of CPVT cases (Nyegaard et al, 2012). Treatment for these conditions is vital as mortality rates for untreated individuals can range from 21% to potentially as high as 50% (Leenhardt et al, 1995; Schwartz et al, 2012). Discovering novel compounds which treat these conditions is beset by suboptimal drug discovery methods which utilise animal models, which can be inaccurate due to inter-species variability in the cardiovascular system (Rajamohan et al, 2013), and heterologous overexpression systems which test the effect of a drug on specific ion channels which are limited by the inability of these systems to model the dynamic interaction between different ion channels in vivo (Kramer et al, 2013). Using human induced pluripotent stem cells (hiPSC) it is possible to generate cardiomyocytes which may enable the creation of humanised models of disease. Such models may enable the discovery of efficacious compounds more readily than current drug development techniques and they may also allow the study of disease pathogenesis in more detail than is currently available in human systems. In this thesis different tissue sample types were investigated for their ability to robustly derive cell lines in vitro for use in reprogramming to hiPSCs. Furthermore in this thesis three different non-integrative methods of hiPSC generation, Sendai virus, episomal reprogramming using nucleofection and episomal reprogramming using GET peptide mediated transduction, were tested to assess which was able to most robustly generate hiPSCs. These hiPSCs were ultimately differentiated into cardiomyocytes (hiPSC-CMs) and used in downstream phenotyping assays. An LQTS2-hiPSC model was used as part of an in vitro drug assay to test the effectiveness of Ikr, Ikatp, PPARδ and Iks agonists in reducing the action potential duration (APD) of the LQTS2-hiPSC-CMs which would thereby indicate a role in enhancing repolarisation. The effects of these new candidate drugs were compared to the effect observed by the traditional LQTS2 therapy, β-blockers. This work was carried out using the CellOPTIQ platform and voltage was analysed by labelling the hiPSC-CMs with voltage sensitive cell dye FluoVolt. Furthermore in this thesis a CPVT-hiPSC model was generated and genetically corrected using CRISPR/Cas9 genome engineering technology. These hiPSCs were then differentiated to cardiomyocytes and analysed for their calcium handling properties using fluorescent labelling dye Fura2-AM. Assessment of the voltage properties of the LQTS2-hiPSC-CMs indicated that the LQTS2-hiPSC-CMs exhibit increased APD in comparison to healthy-hiPSC-CMs and an increase in triangularisation time which is indicative of repolarisation time therefore indicating a faithful recapitulation of the patient’s phenotype in vitro. Moreover the APD of LQT2-hiPSC-CMs was found to decrease in response to treatment with Ikatp, Ikr, PPARδ and Iks agonists. These data indicate that the drugs tested in this thesis may provide a beneficial effect to LQTS2 patients and should be investigated further. Assessment of the calcium handling mechanics of CPVT-hiPSC-CMs and genetically corrected CPVT-WT-hiPSC-CMs indicated that differences exist in the way in which these hiPSC-CMs handle calcium in vitro. The genetically corrected CPVT-WT-hiPSC-CMs were found to show enhanced functionality of RYR2 and SERCA when challenged with relevant pharmacological blockade, potentially indicating a corrected phenotype. However assessment of whether the CPVT-hiPSC-CMs exhibited a “normal” phenotype was inconclusive. There exist differences in the calcium handling properties of CPVT-WT-hiPSC-CMs and unrelated healthy-hiPSC-CMs. To summarise, this thesis assesses the effectiveness of multiple tissue samples to elucidate a robust method to derive starting cell populations from patients. Moreover it goes on to establish a robust protocol for generating hiPSCs from these samples using a non-integrative reprogramming method. The generation of an LQTS2 disease model enabled an in vitro drug assay which identified 9 compounds which were able to reduce the APD in LQTS2-hiPSC-CMs, of which 7 showed utility in reducing the repolarisation time of the LQT2-hiPSC-CMs. These results indicate that these compounds may exert beneficial effects in the treatment of patients with LQTS2. In addition to this, preliminary results in the CPVT disease model created seem to indicate an increase in functionality of both RYR2 and SERCA after CRISPR/Cas9 genetic correction of the S2246L mutation. Attempts to establish if CRISPR/Cas9 genetic correction generated “normal” calcium handling properties were inconclusive. There remains a large amount of variability within genetically distinct hiPSC-CMs with regards to their calcium handling properties, likely owing to the highly spatial and temporal nature of calcium movement in cardiomyocytes and this should be investigated further.

Natural resources and plant extracts with therapeutic properties are considered important sources not only for food products, but also for the treatment of disease. Among edible herbal medicines are the gourd family or Cucurbitaceae, beta glucans, Nigella sativa and lignin. It is worth noting that after many research studies using a variety of screening, characterization and isolation methods that these glycan and glycan-like complexes have therapeutic components which have the ability to reduce the risk of many metabolic diseases. The properties of glycan and glycan-like complexes and the use as anti-diabetics were indicated. Although there have been great efforts made toward revealing the bioactive substances present in these materials, and several research studies have been undertaken, the bioactive components responsible are still not fully explored. The aim of this study is to explore further anti-diabetic properties and the hydrodynamic characterisation of glycan and glycan-like complexes obtained from Analytical Ultracentrifugation (AUC), viscometry, Dynamic Light Scattering (DLS) and Fourier Transform Infrared Spectroscopy (FTIR). Eliminating and analysis of fatty acids was performed in order to separate and purify protein-polysaccharide complexes from the family Cucurbitaceae and the complexes were also the target of investigation in this study. The value of sedimentation coefficient and a corresponding molecular weight yields a value for the frictional ratio ~ 13, suggesting a very high degree of hydration and asymmetry for the complexes in the family of Cucurbitaceae. The results of gas chromatography (GC) analysis indicate that polyunsaturated fatty acids (PUFAs) formed approximately 60% of the total oil from family of Cucurbitaceae which it is of high value in the human diet. The protein-like sedimentation profile of lipase adopts a polysaccharide like broad profile and sedimenting at a significantly faster rate suggesting all the lipase has bound to the beta-glucan. A plate shape structure for all three lignins with aspect ratio ~30:1 seems to be probable.

Studies with APCMin/+ mice and APCMin/+ PPARα-/- mice were undertaken to investigate whether polyp development in the mouse gut was mediated by PPARα. Additionally, the effect of piroxicam treatment dependency on PPARα was assessed. Results showed the number of polyps in the colon was significantly higher in APCMin/+ PPARα-/- mice than in APCMin/+ mice, whilst in the small bowel the difference was not significant. Analysis of gene expression in the colon with Affymetrix® microarrays demonstrated the largest source of variation was between tumour and normal tissue. Deletion of PPARα had little effect on gene expression in normal tissue but appeared to have more effect in tumour tissue. Ingenuity pathway analysis of these data showed the top biological processes were growth & proliferation and colorectal cancer. Collectively, these data may indicate that deletion of PPARα exacerbates the existing APCMin/+ mutation to promote tumorigenesis in the colon. 95 genes from Affymetrix® microarray data were selected for further analysis on Taqman® low density arrays. There was good correlation of expression levels between the two array types. Expression data of two genes proved particularly interesting; Onecut homeobox 2 (Onecut2) and Apolipoprotein B DNA dC  dU - editing enzyme, catalytic polypeptide 3 (Apobec3). Onecut2 was highly up-regulated in tumour tissue. Apobec3 was up-regulated in APCMin/+ PPARα-/- mice only; suggesting expression was mediated via PPARα. There was a striking increase in survival accompanied by a marked reduction in small intestinal polyp numbers in mice of either genotype that received piroxicam. Taqman® low density array analysis of the same 95 genes as previously showed similar expression levels in piroxicam-treated APCMin/+ mice and APCMin/+ PPARα-/- mice. Taken together, these data indicated that the effect of piroxicam treatment was not mediated via PPARα.

No description available.

No description available.

No description available.

ABCG2 is a half-transporter that belongs to the G-subfamily of ABC (ATP- binding cassette) transporters, which are characterised by their unique domain organisation; an N-terminal nucleotide-binding domain (NBD) followed by a transmembrane domain (TMD) to its C-terminal. ABCG2 has been investigated as a multitask transporter widely distributed in normal tissues as well as overexpressed in cancer stem cells and cancer cell lines to confer protection against different xenobiotics and induce multidrug resistance (MDR), respectively. Since the time it was discovered, several studies investigated the effect of certain residues or subdomains within human ABCG2 affecting its substrate specificity, structure, function as well as its dimerization (or oligomerization) status. This study was designed to identify highly co-evolved residues within human cellular localization and/or function. The study also investigated ABCG2 dimerization via a recently developed tool; bimolecular fluorescence complementation (BiFC), through which visualisation of ABCG2 dimerization in live cells was enabled. According to co-evolutionary investigations, eight novel residues were chosen from a wide range of highly correlated coupled residues shared among ABCG- related sequences, and were used to design single mutants that were tested for ABCG2 expression, sub-cellular localization and function, besides control mutants of known effects. All variants were expressed at a comparable level to wild-type ABCG2R482 except for an I573A mutant that showed altered glycosylation level and enhanced intracellular retention. All mutants were functionally capable of extruding Pheophorbide A (PhA), Mitoxantrone and BODIPY-Prazosin, and inhibited by Fumitremorgin C (FTC), as revealed by fluorescent export studies and flow cytometry, except for two mutants, P485A and M549A, which showed altered inhibition profile with FTC. This study revealed that these two residues could participate in the inhibitor binding site or in the communication between the drug and inhibitor binding sites. Bimolecular fluorescence complementation analyses revealed that dimerization of N-terminally YFP-tagged ABCG2 constructs was specifically localized to the plasma membrane of live cells. However, mutating single residues, previously published to participate in ABCG2 dimer formation, did not significantly alter the BiFC signal. BiFC enabled the qualitative investigation of ABCG2 dimerization and function but was insensitive enough to map single residue changes within the dimer formation interface. This study opens the door for future investigations of conserved residues within human ABCG2 that could increase the depth of understanding allosteric interactions between drug and inhibitor binding sites within the transporter via applying a wider range of specific ABCG2 substrates and inhibitors. It also guides further studies to investigate the dimer formation status of ABCG2 and its possible inhibition to overcome multidrug resistance and failure of chemotherapy in cancer cells.

published_or_final_version / Chinese / Doctoral / Doctor of Philosophy

Over-expression of parathyroid hormone-related protein (PTHrP) is commonly described in a number of different forms of cancer and it has been suggested that this over-expression leads to tissue-specific metastasis whereby primary tumours have a propensity to metastasise to one particular organ e.g. breast tumours metastasise to bone whereas gastrointestinal tumours favour the liver. The aim of my PhD was to examine the role PTHrP plays in cancer cell adhesion to the extracellular matrix (ECM), to explore a mechanism of action and to elucidate any tissue-specific differences to explain the apparent partiality during metastasis. In order to do this a small interfering RNA was used to silence PTHrP gene expression and expression vectors containing cDNA for PTHrP were used to create several stable PTHrP over-expressing cell lines. Analysis of cell adhesion revealed that regulating PTHrP expression caused changes in adhesion to the ECM proteins collagen type I, fibronectin and laminin in breast cancer cell lines. However although cell adhesion of gastrointestinal cancer cell lines to collagen type I and fibronectin was similarly affected, adhesion to laminin was unchanged by variations in PTHrP expression. The cell adhesion molecules integrins were subsequently investigated for their role in PTHrP mediated cell adhesion. Integrins are heterodimers composed of an α and β subunit and to date 24 different subunits have been identified. Each unique combination of subunits results in a different ligand specificity, which includes collagens, fibronectin and laminin. Analysis of integrin gene and protein expression in over-expressing cell lines and in cells where PTHrP had been silenced it was possible to demonstrate a link between expression of PTHrP and a number of different integrin subunits. One of the more significant findings was the observation that the integrin subunit α6 changed in parallel with PTHrP in breast but not gastrointestinal cancer cell lines. As laminin is a ligand for this subunit these results correlate with the results previously described regarding this ECM protein. The effects of PTHrP appeared to be mediated at the transcriptional rather than the translational level so integrin transcriptional activity was investigated using a reporter vector containing the coding region for firefly luciferase. The integrin subunit α5 had shown a link to PTHrP expression in both breast and gastrointestinal cell lines so the promoter region for this subunit was inserted into the reporter vector, which was then transiently transfected into PTHrP over-expressing cell lines. Subsequent examination of luciferase activity revealed a significant increase in promoter activity compared with control thus implicating PTHrP as a key factor in integrin gene transcription. The results described here suggest that PTHrP increases cell adhesion by inducing integrin gene transcription and is able to regulate expression of different subunits in different tissues thereby encouraging tissue-specific metastasis.

CD133 is a pentaspan transmembrane glycoprotein of ~120 kDa, which was initially used to identify haematopoietic stem cells and, later on, used for the isolation and study of cancer stem cells in many different types of solid tumour including colorectal cancer. Although CD133 expressing cells are thought to represent cancer stem cells, little is known about the exact role of CD133 and the molecular mechanisms underlying control of CD133 expression. This project sought to investigate these questions in colorectal cancer. Initially the expression of CD133 was tested by immunohistochemistry in a two tissue microarray (TMA) sets consisting of (a) 449 cases of primary colorectal cancer, and (b) 45 cases of primary and matched liver metastases. High CD133 expression was marginally associated with shorter overall survival (OS) (p=0.05, Log-rank test) but no difference in expression was found between primary tumours and corresponding metastases. Next, the functional activity of CD133 was evaluated in colorectal cancer (CRC) cell lines by knockdown in cell lines with high CD133 expression. In order to identify appropriate cell lines, the expression of CD133 was tested by quantitative RT -PCR in a series of 29 CRC cell lines and 10 samples of normal mucosa and, in selected cell lines, validated by testing for protein expression by flow cytometry. CD133 mRNA was expressed in 24/29 colorectal cancer cell lines with a heterogenous level of expression. 10 cell lines were chosen on the basis of CD133 mRNA expression level to assess the protein level. CD133 mRNA and protein expression were generally correlated (rs = 0.831, p= 0.003, spearman rank correlation coefficient test) although, interestingly, CD133 mRNA level was higher in normal samples compared with that in cancer cell lines and was significantly higher in cell lines derived from metastatic sites than those derived from site of primary tumour (p=0.009; Mann-whitney test). In addition, it was noted that many cell lines had a stable biphasic phenotype containing CD133+ and CD133- cell populations. This allowed functional analysis of CD133 by sorting the two populations. HT29 was identified as a high expresser of CD133 (95%) and was used for gene-knockdown studies, SW480 had a biphasic population consisting of 42% CD133+ cells and 58% CD133- cells and each population was isolated by cell sorting before functional analysis. Functional assays included proliferation, migration, colony formation and staurosporine induced apoptosis assays. These showed that CD133 expressing cells had greater cell motility (p= 0.04, and p = 0.03, unpaired t-test, for knocked down cells and sorted populations respectively) , enhanced colony forming abilities (p=0.0001, and p=0.003, unpaired t-test for 2D and 3D colony formation respectively using sorted populations only), and increased resistance to staurosporine induced apoptosis (p=0.01, and p=0.008, unpaired t-test, for knocked down and sorted populations respectively) than CD133 negative counterparts. In addition, sorted monophasic populations reverted to a biphasic state in both CD133+/- populations from SW480. Further studies demonstrated that CD133-induced cell motility was independent of E-cadherin, β-catenin, and suggestive of not being regulated by Cten or Wnt, but further work is warranted to verify these results. In addition, regulation of CD133 was partly dependent on STAT3 signalling and on CD133 promoter methylation. Levels of mRNA of some stem cell related genes such as KLF-4, Musashi-1, OCT4, Nanog, and Lgr5 were higher in CD 133 + compared to CD 133 negative cells (p=0.008, p=0.004, p=0.006, p=0.001, and p=0.11; unpaired t-test, respectively) In conclusion, in CRC, CD133 was found to be a significant prognostic factor which enhances cell motility and is associated with features of "stemness". It is a target of ST AT3 signalling and partly regulated by promoter methylation. More in depth studies are warranted to discover the downstream and upstream targets of CD133 before translating these preclinical and laboratory investigations into clinical management of colorectal cancer.