No description available.

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

In diabetes the body lacks the mechanism for producing insulin. This disease is one of the most prevalent in the world, causing a tremendous loss of health, life and economy. Thus, there is a need for developing novel therapies effective in control of diabetes. In an effort to develop such a therapy we have targeted G-protein coupled receptors (GPCRs) to stimulate 13-cells for insulin secretion. GPCRs are membrane bound receptors which respond to a variety of external signals and mediate intracellular signal Stransduction. GPCRs, therefore, are the targets of many current therapeutic drugs. The objective of this study was to generate chimeric receptors containing portions of two closely related GPCRs to identify domains important in binding various ligands to stimulate increased secretion of insulin by f3-cells of the pancreas. In this collaborative research with Kelly Wilbur of Eli Lilly, domains of receptors GPR40 and GPR41 were exchanged at different regions to construct two chimeric receptors (GPR40.431_41.459 and GPR40.567 41.547) using two separate cloning steps to insert these fragments sequentially into the cloning vector, pcDNA3.1. Construction of the chimeric receptors was carefully planned to include specific amino acid residues important in ligand binding. Priority was given to locate the joining section of the two receptor portions at the transmembrane region and to maintain full length of the receptor. This was to maintain the integrity of external and internal loops of the receptors important in ligand binding and signal transduction. Following transformation of the chimeras into E. coli to obtain sufficient DNA, construction of the desired chimeric receptors was verified by agarose gel electrophoresis for size and by PCR for the presence of the correct portions of each receptor. The two constructs were sent to Eli Lilly for sequencing. One construct was found to be appropriately constructed (GPR40.431_GPR41.459) but the other one was unstable and had undergone recombination as is often seen in cloned membrane proteins which can be toxic to E. coli. In the future, Human Embryonic Kidney cells will be transfected with the chimeric receptor and a FLIPR analysis will be performed to assess the activity of the receptor when stimulated by ligands of interest to Eli Lilly. Construction of additional chimeras will be needed in the future to fully understand the specific regions responsible for ligand binding and activation of GPR40 to aid in the design of drugs to stimulate insulin secretion by 03-cells. / Department of Biology

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize / published_or_final_version / abstract / toc / Microbiology / Doctoral / Doctor of Philosophy

In summary, this thesis has explored the anti-cancer mechanisms of novel medicinal compounds via targeting calpains or S100A4 in the treatment of colon cancer, which could facilitate future establishment of effective medicinal compounds in the treatment of metastatic colon cancers with known molecular targets.;The incidence of colon cancer in Hong Kong and worldwide is on a rising trend, while its metastatic development is the leading cause of cancer-related deaths. Understanding the molecular mechanisms of how tumors progress and metastasize to secondary sites, at both biological and genetic levels, could enable us to identify potential molecular targets in drug development. In the present study, we explored how manipulation of signaling pathways by targeting calpains and S100A4 could facilitate the development of anti-tumor and anti-metastatic drugs.;The study investigating drug targeting on S100A4 in both in vitro and in vivo models had shown that the pharmacological store-operated calcium channel blocker would suppress S100A4-mediated migration by weakening extracellular S100A4-mediated calcium responses. The effects on S100A4-induced metastasis formation were confirmed in vivo with reduced splenic tumor volume and decreased number of liver metastases. These results have provided new insights to correlate between S100A4 and calcium signaling, making an important step forward in characterizing the dependence of calcium homeostasis in the process of metastasis, providing a novel strategy for S100A4-mediated metastasis.;With respect to the targeting on calpains, it was discovered that total Astragalus saponins (AST) and cryptotanshinone (CPT) are effective anti-cancer agents that elicit the endoplasmic reticulum (ER) stress response. They act by upregulating the expression of glucose-regulated protein (GRP) 78, leading to the initiation of apoptosis when the ER recovery process begins to fail. In particular, CPT caused rapid and sustained increase in cytosolic calcium in colon cancer cells that was accompanied by early GRP78 overexpression. The increase in cytosolic calcium was blocked by pre-treatment of BAPTA-AM through depletion of the ER calcium store. In consistent with these, we also confirmed that CPT significantly increased calpain activity, which could be blocked by calcium chelator or calpain inhibitors. Furthermore, a dynamic interaction between GRP78 and calpain under ER stress was unveiled during AST or CPT exposure. The degree of association was increased following prolonged ER stress, and suppressed either as the ER recovery process failed or with the presence of calpain inhibitors. Besides, inhibition of calpain activity suppressed NF-κB activation (a consequence of ER stress) and substantially enhanced the effects of CPT to promote apoptosis. More importantly, it was confirmed that the effects of calpain inhibitors to sensitize colon cancer cells to ER stress-associated apoptosis are p53-dependent. The anti-tumorigenetic effects of CPT were further demonstrated in vivo in xenografted nude mice by trageting calpains and in combination with calpain inhibitors.

No description available.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.