The discovery of novel members of tumorigenic pathways remains a critical step to fully dissect the molecular biology of cancer. Indeed, because a number of cancer drivers are themselves undruggable, elucidating the signaling apparatuses in which they participate is essential for discovering novel therapeutic targets that will allow the treatment of aggressive neoplastic growth. In the context of oncoproteins and tumor suppressors, novel participants may be upstream regulators, downstream effectors, or physical cognate binding partners. In this work, we develop in silico approaches to more fully elucidate the tumorigenic signaling machinery used by tumor suppressors and oncoproteins. We first report applications of machine-learning algorithms to integrate diverse networkbased information to generate testable hypotheses of proteins involved in canonical oncogenic pathways. We develop the OncoSig algorithm to elucidate novel members of protein-centric maps to elucidate upstream modulators, cognate binding partners, and downstream effectors for any tumor suppressor or oncogene in a tumor-specific fashion. We specifically apply OncoSig to elucidate the oncogenic KRAS regulatory map in Lung adenocarcinoma (LUAD). Oncogenic KRAS is a key driver of aggressive tumor growth in many LUAD patients, yet has no FDA-approved drugs targeting it. Thus, elucidating members of the KRAS protein-centric map is critical for discovering synthetic lethal interactions that may be subject to therapeutic targeting. Critically, 18/22 of novel predicted KRAS interactors elicited synthetic lethality in LUAD organoid cultures that harbored an activating KRAS mutation. We then extend the OncoSig algorithm to 10 oncogenic/tumor suppressor pathways (such as TP53, EGFR, and PI3K), and show that OncoSig is able to recover known regulators and downstream effectors of these critical mediators of tumorigenesis. We then focus specifically on dissecting KRAS’s physical protein-protein interactions. Many cognate binding partners bind to KRAS via a structurally conserved RAS-Binding Domain (RBD), thus propagating KRAS signal transduction. Thus, for example, CRAF, PI3K, and RALGDS, all bind to KRAS via an RBD. To elucidate novel KRAS protein-protein interactors, we use structural and sequence based approaches to discover biophysical properties of known RBDs. We apply the PrePPI algorithm, which predicts novel protein-protein interactions based on structural similarity, and find that PrePPI successfully recovers known RBDs while discriminating from domains structurally similar to the RBD that do not bind to KRAS. Using this information, we develop biophysical features to computationally predict novel KRAS binding partners. Finally, we report computational and experimental work addressing whether KRAS forms a homodimer. The precise mechanism for how KRAS propagates signal transduction after binding to the RBD remains elusive, and KRAS homo-dimerization, for example, may play a key role in KRAS induced tumorigenesis. Using Analytical Utracentrifugation to measure binding affinity, we find that KRAS forms either a weak dimer or a large non-specific multimer. Furthermore, analysis of KRAS protein structures deposited in the Protein Data Bank reveals key regions that have a propensity to form homodimer contacts in the crystal complexes, and may mediate KRAS homo-dimerization in a biological setting as well. These results provide mechanistic insight into how KRAS dimerization may facilitate cellular signal transduction.

Chow Chit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 130-156). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Malignant Lymphoma --- p.1 / Chapter 1.2 --- Non-Hodgkin's Lymphoma --- p.1 / Chapter 1.3 --- NK/T Cell Lymphoma --- p.2 / Chapter 1.3.1 --- General Features of NK/T Cell Lymphoma --- p.2 / Chapter 1.3.2 --- Histology of NK/T Cell Lymphoma --- p.3 / Chapter 1.3.3 --- Subtypes NK/T Cell Lymphoma --- p.4 / Chapter 1.3.4 --- Overview of NK/T Cell Lymphoma Cell Lines --- p.5 / Chapter 1.3.4.1 --- NK/T Cell Lymphoma Cell Lines-NK-92 and SNK-6 --- p.6 / Chapter 1.3.5 --- NK/T Cell Lymphoma and Interleukins --- p.8 / Chapter 1.4 --- Interleukin-2 and Interleukin-15 --- p.9 / Chapter 1.5 --- IL-2 and IL-15 Receptor --- p.10 / Chapter 1.6 --- Cellular Signaling Pathways Regulated by IL-2 and IL-15 --- p.11 / Chapter 1.6.1 --- JAK/STAT Pathway --- p.12 / Chapter 1.6.2 --- PI3K/Akt Pathway --- p.14 / Chapter 1.7 --- Epstein-Barr Virus: an Oncogenic Virus --- p.20 / Chapter 1.7.1 --- Overview of EBV --- p.20 / Chapter 1.7.2 --- Epidemiology --- p.20 / Chapter 1.7.3 --- Life Cycle of EBV --- p.21 / Chapter 1.7.4 --- Latency Infection of EBV --- p.21 / Chapter 1.7.5 --- Role of EBV latent genes in oncogenesis --- p.23 / Chapter 1.7.5.1 --- EBER1 and 2 --- p.23 / Chapter 1.7.5.2 --- EBNAs --- p.24 / Chapter 1.7.5.3 --- LMPs --- p.25 / Chapter 1.7.6 --- Lytic Cycle of EBV --- p.26 / Chapter 1.7.7 --- Signaling Pathways and EBV --- p.27 / Chapter Chapter 2: --- Aim of Study --- p.29 / Chapter Chapter 3: --- Materials and Methods --- p.31 / Chapter 3.1 --- IL-2 and IL-15 on NK/T Cell Lymphoma Cell Lines and patients --- p.31 / Chapter 3.1.1 --- Cell Lines Maintenance --- p.31 / Chapter 3.1.2 --- Patients --- p.32 / Chapter 3.2 --- "Assays of IL-2, IL-15 and IFN-γ in culture supernatants and patient sera" --- p.32 / Chapter 3.2.1 --- IL-2 ELISA --- p.32 / Chapter 3.2.2 --- IL-15 ELISA --- p.33 / Chapter 3.2.3 --- IFN-γ ELISA --- p.34 / Chapter 3.3 --- Effect of IL-2 and IL-15 on NK/T Cell Lymphoma Cell Lines --- p.35 / Chapter 3.3.1 --- Cell Growth and Viability Determination --- p.35 / Chapter 3.3.2 --- Apoptosis Assays on Interleukin-starved NK-92 cells --- p.35 / Chapter 3.3.2.1 --- DNA Laddering Analysis --- p.36 / Chapter 3.3.2.2 --- Cell Cycle and Apoptosis Determination by PI Staining --- p.37 / Chapter 3.3.2.3 --- Caspase 3 Activity Assay --- p.37 / Chapter 3.4 --- PI3K/Akt Pathway Study --- p.39 / Chapter 3.4.1 --- Determination of AKT1 Gene Amplification by Real-Time Quantitative PCR --- p.39 / Chapter 3.4.1.1 --- DNA Extraction for Real-Time Quantitative PCR --- p.39 / Chapter 3.4.1.2 --- AKT1 Real-Time Quantitative PCR --- p.40 / Chapter 3.4.2 --- Determination of Akt Expression --- p.41 / Chapter 3.4.2.1 --- Normal NK Cell Purification from Buffy Coat --- p.41 / Chapter 3.4.2.2 --- Determination of the Purity of Extracted NK Cells --- p.43 / Chapter 3.4.2.3 --- Interleukin Treatment of Normal NK Cells and NK-92 --- p.43 / Chapter 3.4.2.4 --- Protein Extraction and Western Blot Analysis --- p.43 / Chapter 3.4.3 --- Study of PI3 K/Akt pathway using PI3K inhibitor --- p.47 / Chapter 3.4.3.1 --- Cell Growth and Viability Assay --- p.47 / Chapter 3.4.3.2 --- Apoptosis Assay by DNA Laddering and Pi-Staining on NK-92 cells --- p.48 / Chapter 3.4.3.3 --- Determination of activated Akt after LY294002 and Wortmannin treatment --- p.48 / Chapter 3.5 --- Effect of IL-2 and IL-15 on the JAK/STAT pathway and PDK/Akt pathway of NK/T Cell Lymphoma Cell Lines --- p.49 / Chapter 3.5.1 --- Cell Treatment --- p.49 / Chapter 3.5.2 --- Study of JAK/STAT and PI3K/Akt Pathways by Western Blotting --- p.49 / Chapter 3.5.3 --- "Assays of IL-2, IL-15 and IFN-γ in the NK-92 Cell Culture Medium by ELISA" --- p.50 / Chapter 3.5.4 --- Determination of EBV Status after IL-2 and IL-15 Treatment --- p.51 / Chapter 3.5.4.1 --- RNA Extraction --- p.51 / Chapter 3.5.4.2 --- Reverse-transcriptase Reaction --- p.52 / Chapter 3.5.4.3 --- PCR for EBV-related Genes --- p.53 / Chapter 3.5.4.4 --- EBER-ISH --- p.54 / Chapter 3.5.4.5 --- Real-time Quantitative PCR for EBER1 --- p.56 / Chapter 3.5.4.6 --- Western Blot for LMP1 --- p.56 / Chapter 3.6 --- Statistical Analysis --- p.57 / Chapter Chapter 4: --- Results --- p.59 / Chapter 4.1.1 --- "IL-2, IL-15 and IFN-γ Levels in the Serum of Patients with NK/T Cell Lymphoma" --- p.59 / Chapter 4.1.2 --- IL-2 and IL-15 Level in Culture Supernatant of NK-92 --- p.59 / Chapter 4.1.3 --- IFN-γ induction in supernatant of NK-92 --- p.60 / Chapter 4.2 --- Effect of IL-2 and IL-15 on NK/T Cell Lymphoma Cell Lines --- p.61 / Chapter 4.2.1 --- Cell Growth and Viability --- p.61 / Chapter 4.2.2 --- Apoptosis Study of Interleukin-starved NK-92 --- p.62 / Chapter 4.2.2.1 --- DNA fragmentation and Cell Cycle studies --- p.62 / Chapter 4.2.2.2 --- Caspase 3 Activity in NK-92 --- p.62 / Chapter 4.3 --- Akt in NK-92 --- p.63 / Chapter 4.3.1 --- Confirmation ofAKTl Amplification in NK-92 --- p.63 / Chapter 4.3.2 --- Akt Protein Quantification in NK-92 cells --- p.63 / Chapter 4.3.3 --- Activated Akt and STAT proteins in IL-2 or IL-15 stimulated NK-92 and normal NK cells --- p.64 / Chapter 4.4. --- PI3K/Akt Pathway --- p.64 / Chapter 4.4.1 --- Phosphorylation of Components of the PI3K/Akt Pathway --- p.64 / Chapter 4.4.2. --- Role of PI3K/Akt Pathway in NK/T Cell Lymphoma Cell Lines --- p.65 / Chapter 4.4.2.1 --- Cell Growth and Viability Studies --- p.65 / Chapter 4.4.2.2 --- Apoptosis and Cell Cycle Arrest Induction by LY294002 in NK-92 --- p.66 / Chapter 4.4.2.3 --- Confirmation of the effect of LY294002 on the PDK/Akt pathway in NK-92 cells --- p.67 / Chapter 4.5 --- Phosphorylation of STAT family proteins --- p.67 / Chapter 4.6 --- Regulation of EBV-related genes in NK-92 --- p.68 / Chapter Chapter 5: --- Discussion --- p.71 / Chapter 5.1 --- Cytokine level in patient serum --- p.71 / Chapter 5.2 --- Source of IL-2 and IL-15 for NK-92 cells --- p.72 / Chapter 5.3 --- Induction of IFN-γ in NK-92 --- p.73 / Chapter 5.4 --- Role of IL-2 and IL-15 on NK/T cell lymphoma cell lines --- p.75 / Chapter 5.4.1 --- Cell Growth and Viability Maintenance by IL-2 and IL-15 --- p.75 / Chapter 5.4.2 --- Apoptosis induced by interleukin-starving in NK-92 --- p.75 / Chapter 5.5 --- Aberrant activation of signaling pathways by IL-2 or IL-15 in NK-92 --- p.77 / Chapter 5.5.1 --- Hypersensitivity of NK-92 cells to IL-2 or IL-15 --- p.77 / Chapter 5.5.2 --- PI3K/Akt pathway in NK/T cell lymphoma cell lines --- p.78 / Chapter 5.5.2.1 --- Confirmation of AKT1 amplification --- p.78 / Chapter 5.5.2.2 --- Constitutive activation of Akt in NK/T cell lymphoma cell lines --- p.79 / Chapter 5.5.2.3 --- Role of PI3K/Akt in NK/T cell lymphoma cell lines --- p.80 / Chapter 5.5.2.4 --- IL-2 and IL-15 induce differential sensitivity of NK-92 to LY294002 --- p.81 / Chapter 5.5.2.5 --- NK/T cell lymphoma cell lines are wortmannin-insensitive --- p.82 / Chapter 5.6 --- Jak/STAT pathway in NK/T cell lymphoma cell lines --- p.83 / Chapter 5.6.1 --- STAT3 and STAT5 were activated by both IL-2 and IL-15 --- p.83 / Chapter 5.6.2 --- STAT6 was activated in NK/T cell lymphoma cell lines --- p.84 / Chapter 5.6.3 --- "Differential regulation of STAT 1, STAT3 (Ser-727) and STAT6 in NK/T cell lymphoma cell lines" --- p.85 / Chapter 5.6 --- EBV gene regulation in NK-92 --- p.87 / Chapter Chapter 6: --- Conclusion --- p.91 / Tables --- p.93 / Figures --- p.102 / Reference --- p.128

Leung, Pan Cheung Catherine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 105-120). / Abstracts in English and Chinese. / DECLARATION --- p.II / ACKNOWLEDGEMENTS --- p.III / ENGLISH ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VII / Chapter MODULE 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Vascular Endothelium --- p.1 / Chapter 1.1.1. --- Vascular Endothelial Functions --- p.1 / Chapter 1.1.2. --- Calcium Signaling in Vascular Endothelial Cells --- p.2 / Chapter 1.2. --- The Founding Member of TRP Family: Drosophila TRP --- p.3 / Chapter 1.2.1. --- Discovery of Drosophila TRP and TRP-related Proteins --- p.3 / Chapter 1.2.2. --- Drosophila TRPs: Ca2+-permeable Channels? --- p.3 / Chapter 1.3. --- Mammalian TRP Superfamily --- p.5 / Chapter 1.3.1. --- The TRP Subfamily: TRPV --- p.9 / Chapter 1.3.2. --- The TRP Subfamily: TRPM --- p.9 / Chapter 1.3.3. --- The TRP Subfamily: TRPC --- p.11 / Chapter 1.4. --- Functional and Physiological Roles of Mammalian TRPCs --- p.12 / Chapter 1.4.1. --- TRPC1 --- p.15 / Chapter 1.4.2. --- TRPC2 --- p.16 / Chapter 1.4.3. --- "TRPC3, TRPC6 and TRPC7" --- p.17 / Chapter 1.4.4. --- TRPC4 and TRPC5 --- p.19 / Chapter 1.4.5. --- Over-expression of TRPCs: Physiologically Relevant Channels? --- p.20 / Chapter 1.4.6. --- Alternatives to Heterologous Expression Study --- p.21 / Chapter 1.5. --- Aims of the Study --- p.23 / Chapter MODULE 2. --- MATERIALS AND METHODS --- p.24 / Chapter 2.1. --- Functional Characterization of TRPCs by Antisense Technique --- p.24 / Chapter 2.1.1. --- Restriction Enzyme Digestion --- p.26 / Chapter 2.1.2. --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.27 / Chapter 2.1.3. --- "Ligation of TRPC Genes into Mammalian Vector, pcDNA3" --- p.27 / Chapter 2.1.4. --- Transformation for the Desired Clones --- p.28 / Chapter 2.1.5. --- Plasmid DNA Preparation for Transfection --- p.28 / Chapter 2.1.6. --- Confirmation of the Clones］ --- p.29 / Chapter 2.1.6.1. --- Restriction Enzymes Strategy --- p.29 / Chapter 2.1.6.2. --- Polymerase Chain Reaction (PRC) Check --- p.30 / Chapter 2.1.6.3. --- Automated Sequencing --- p.31 / Chapter 2.2. --- Establishing Stable Cell Lines --- p.33 / Chapter 2.2.1. --- Cell Culture --- p.33 / Chapter 2.2.2. --- Transfection Conditions Optimization --- p.33 / Chapter 2.2.3. --- Geneticin Selection --- p.35 / Chapter 2.3. --- Expression Pattern Studies of TRPC Genes in Vascular Tissues --- p.38 / Chapter 2.3.1. --- Immunofluorescence Staining in Cultured CPAE Cells --- p.38 / Chapter 2.3.2. --- Immunolocalization in Human Cerebral and Coronary Arteries --- p.40 / Chapter 2.3.2.1. --- Paraffin Section Preparation --- p.40 / Chapter 2.3.2.2. --- "Immunohistochemistry for TRPC1, 3, 4 and 6 Channels" --- p.40 / Chapter 2.3.2.3. --- Subcellular Localization of hTRPC1 and hTRPC3 Channels in Endothelial Cells --- p.42 / Chapter 2.4. --- Study of Bradykinin-induced Ca2+ Entry by Calcium Imaging --- p.47 / Chapter 2.4.1. --- Primary Aortic Endothelial Cell Culture --- p.47 / Chapter 2.4.2. --- Fura-2 Measurement of [Ca2+］］］ --- p.47 / Chapter 2.5. --- Study of Functional Role of TRPC6 in Stably Transfected H5V Cells … --- p.49 / Chapter 2.5.1. --- Protein Sample Preparation --- p.49 / Chapter 2.5.2. --- Western Blot Analysis --- p.50 / Chapter 2.5.3. --- Confocal Microscopy for Bradykinin-induced Calcium Entry --- p.51 / Chapter 2.6. --- Data Analysis --- p.52 / Chapter MODULE 3. --- RESULTS --- p.53 / Chapter 3.1. --- Bradykinin-induced Calcium Entry in Vascular Endothelial Cells --- p.53 / Chapter 3.1.1. --- Bradykinin-induced Calcium Entry --- p.53 / Chapter 3.1.2. --- Effects of cGMP and PKG on Bradykinin-induced Ca2+ Entry --- p.54 / Chapter 3.1.3. --- Effects of HOEUO on Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.1.4. --- Involvement of Phospholipase C Pathway in Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.2. --- Expression Pattern of TRPC Channels in Vascular Systems --- p.63 / Chapter 3.2.1. --- Immunolocalization of TRPC Homologues in CPAE Cells --- p.63 / Chapter 3.2.2. --- Immunolocalization of TRPC Homologues in Human Cerebral and Coronary Arteries --- p.66 / Chapter 3.2.3. --- Subcellular Localization of TRPC1 and TRPC3 Fused to Enhanced Green Fluorescence Protein (EGFP) --- p.77 / Chapter 3.3. --- Functional Role of TRPC6 Channels in Bradykinin-induced Calcium Entry --- p.81 / Chapter 3.3.1. --- Effect of Antisense TRPC6 Construct on Protein Expression --- p.81 / Chapter 3.3.2. --- Effect of Antisense TRPC6 on Bradykinin-induced Ca2+ Entry --- p.81 / Chapter 3.3.3. --- Effect of Antisense TRPC6 on Thapsigargin-depleted Ca2+ Stores --- p.82 / Chapter MODULE 4. --- DISCUSSION --- p.89 / Chapter 4.1. --- Characterization of Bradykinin-induced Ca2+ Entry in Endothelial Cells --- p.89 / Chapter 4.2. --- The Expression Pattern of TRPC Isoforms in Vascular Tissues --- p.93 / Chapter 4.3. --- Functional Characterization of TRPC6 Homologues in Bradykinin-induced Ca2+ Entry --- p.98 / Chapter 4.4. --- Perspectives --- p.103 / Chapter 4.5. --- Conclusion --- p.104 / Chapter MODULE 5. --- REFERENCES --- p.105

Hao Yanli. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 113-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

"June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 162-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The phosphatidylinositol-3-kinase/AKT (Protein Kinase B)/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway, downstream of tyrosine kinase receptors, is upregulated in human cancers including breast and colon cancers. Glycogen synthase kinase 3 (GSK 3) is a serine/threonine protein kinase plays important role in various cellular processes including glycogen synthesis mediated by insulin signalling pathway. Moreover, 5' adenosine monophosphate activated protein kinase (AMPK), a crucial cellular energy sensor, has regulatory role in cell growth and proliferation through mTOR pathway. Phosphatidylcholine (PtdCho) is the major phospholipid in the mammalian cell membranes and is mainly synthesized by the CDP-choline pathway. Malignant transformation has been reported to be associated with altered choline metabolism. Hyperactivation of the PI3K/AKT signalling pathway upregulates the key enzymes of phospholipid metabolism. The first line antidiabetic drug, metformin, modulates glucose and concomitant lipid metabolism through AMPK activation. Studies suggest phosphatidylcholine biosynthesis and breakdown through CDP-choline pathway are modulated by glucose metabolism and de novo fatty acid synthesis. Cancer cell growth inhibitory effect of PI3K/AKT/mTOR/GSK3 pathway inhibitors and metformin were investigated by cytotoxic assay, western blot and cell cycle analysis in breast and colon cancer cells. IC50 values of anticancer drugs and combination indices between drug combinations were determined. 31P-NMR was carried out on cell extracts after drug treatments. [14C (U)] glucose and [3H] choline incorporation into lipids were also determined. All inhibitors targeting PI3K/AKT/mTOR signaling pathway, GSK3 and metformin have cancer cell growth inhibition. By 31P-NMR, PI3K/AKT/mTOR pathway inhibition induced agent-specific changes in PCho intensity. Increased UDP-sugars observed in breast and colon cancer cell extracts treated with LY294002 and AZD8055, an effect abrogated by inclusion of a GSK3 inhibitor. A link between glycolytic intermediates and phosphatidylcholine biosynthesis was investigated by metformin and GSK3 inhibitor in breast and colon cancer cells.

Phospholipase C enzymes are a class of enzymes that catalyse the cleavage of the membrane phospholipid, phosphatidylinositol bisphosphate (PtdIns(4,5)P₂) into the second messengers, inositol trisphosphate (Ins(4,5)P₃) and diacylglycerol (DAG). Six classes of PLC enzymes have been identified based on their structure and mechanism of activation. PLCηs are the most recently identified family and consist of two isozymes, PLCη1 and PLCη2. The aim of this thesis is to further understand the mechanisms of PLCη activation, the role of PLCη2 in relation to neuritogenesis and their roles in certain disease states. Both isoforms were found to be activated by physiological concentrations of intracellular Ca²⁺. Activation of PLCη2 by Gß₁γ₂ was confirmed using a bacterial 2A co-expression system to allow expression of PLCη2, Gß₁ and Gγ₂ with a single plasmid. Localisation studies show a nuclear distribution for PLCη2, but a cytoplasmic distribution for PLCη1 in a neuroblastoma cells line (Neuro2A). PLCη2 has been implicated in brain development and neurite formation. Building on this, a neuronal differentiation model using RA-treated Neuro2A cells stably expressing mutant forms of PLCη2 was utilised, revealing that PLCη2 activity is essential for neuritogenesis but that this process is independent of the enzymes high sensitivity towards Ca²⁺. Furthermore, the direct interaction of PLCη2 and LIMK-1, a previously identified PLCη2 associated protein, is confirmed in the aforementioned neuronal model. Due to the high sensitivity of PLCη enzymes to Ca²⁺ and because of their presence within neurons, they may be involved in Ca²⁺ dysregulation that occurs in certain diseases such as Alzheimer's disease (AD). The role of PLCη2 was assessed in amyloid-ß (Aß) treated differentiated Neuro2A cells, a cellular model for AD pathogenesis. Also a developmental role for PLCη1 was investigated due to a recently identified PLCη1 polymorphism in patients with holoprosencephaly, an embryonic midline defect.

by Chuen Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 134-166). / Abstracts in English and Chinese. / ACKNOWLDEGEMENT --- p.ii / PUBLICATIONS --- p.iii-iv / ABBREVIATIONS --- p.v-viii / INDEX FOR FIGURES --- p.ix xii / INDEX FOR TABLES --- p.xiii / ABSTRACT (Chinese and English) --- p.xiv-xvi / TABLE OF CONTENT --- p.xvii / Chapter 1. --- INTRODUCTION --- p.1-37 / Chapter 2. --- OBJECTIVES --- p.38-40 / Chapter 3. --- METHODS AND MATERIALS --- p.41 -70 / Chapter 4. --- RESULTS AND DISCUSSION --- p.71-130 / Chapter 5. --- GENERAL DISCUSSION AND CONCLUSION --- p.131-133 / BIBLIOGRAPHY --- p.134-166

Yip Ham. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 132-141). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abbreviations --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Chapter Chapter 1: --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling --- p.5 / Chapter 1.1.1 --- Importance of Calcium to Life Forms --- p.5 / Chapter 1.1.2 --- Calcium Channels in Excitable and Non-excitable Cells --- p.6 / Chapter 1.2 --- Vascular Endothelial Cells --- p.8 / Chapter 1.2.1 --- General Functions --- p.8 / Chapter 1.2.2 --- Calcium signaling in Endothelial Cells --- p.9 / Chapter 1.3 --- Capacitative Calcium Entry (CCE) or Store-operated Calcium Entry (SOC) --- p.10 / Chapter 1.3.1 --- Definition --- p.10 / Chapter 1.3.2 --- Endoplasmic Reticulum (ER) as the Main Intracellular Calcium Stores --- p.10 / Chapter 1.3.3 --- Types of Experiments leading to the Identification of SOCs --- p.11 / Chapter 1.3.4 --- Emptying the Internal Calcium Store --- p.11 / Chapter 1.3.4.1 --- Inhibition of Calcium ATPase --- p.11 / Chapter 1.3.4.2 --- IP3 Triggered Release of Calcium --- p.12 / Chapter 1.3.5 --- "Store-operated Calcium Current, Icrac" --- p.15 / Chapter 1.3.6 --- Different Types of SOCs in Animal Cells --- p.16 / Chapter 1.4 --- Transient Receptor Potential (Trp) Gene & Transient Receptor Potential Like (Trpl) Gene in Drosophila --- p.17 / Chapter 1.4.1 --- Discoverery of Trp and Trpl --- p.17 / Chapter 1.4.2 --- Expression Studies of Drosophila Trp and Trpl --- p.19 / Chapter 1.4.2.1 --- Trp and Trpl form Channels but only Trp is Store Operated --- p.19 / Chapter 1.4.2.2 --- Co-expression Studies of Trp and Trpl --- p.20 / Chapter 1.5 --- Molecular Cloning and Expression of Mammalian Trp Homologues --- p.21 / Chapter 1.5.1 --- Seven Human Homologus of Trp were found --- p.21 / Chapter 1.5.2 --- Expression Pattern of mammalian Trp Homologues in Different Tissues --- p.23 / Chapter 1.5.3 --- Expression Studies of Mammalian Trp Homologues Yields Contradictory Results --- p.27 / Chapter 1.5.3.1 --- Trpl --- p.27 / Chapter 1.5.3.2 --- Trp2 --- p.28 / Chapter 1.5.3.3 --- Trp3 --- p.29 / Chapter 1.5.3.4 --- Trp4 --- p.30 / Chapter 1.5.3.5 --- Trp5 --- p.31 / Chapter 1.5.3.6 --- Trp6 --- p.31 / Chapter 1.5.3.7 --- Trp7 --- p.31 / Chapter 1.5.3.8 --- "Activation of Trp3, Trp6 and Trp7 by Diacylglycerol (DAG)" --- p.32 / Chapter 1.5.3.9 --- Functional Consequence after Co-expression of Trp Homologues --- p.34 / Chapter 1.5.3.10 --- Antisense Strategy to Determine the Functional Subunits of Store-operated Channels --- p.35 / Chapter 1.5.3.11 --- Possible Reasons for the Contradictory Results of Trp Homologues When Expressed in a Heterologous System --- p.36 / Chapter 1.6 --- Aims Of Study --- p.37 / Chapter Chapter 2. --- Materials and Methods --- p.38 / Chapter 2.1 --- Cell Culture --- p.38 / Chapter 2.2 --- Total RNA extraction from HCAEC 5286 --- p.39 / Chapter 2.3 --- Reverse Transcription from Cultured Human Coronary Artery Endothelial Cell Line HCAEC 5286 --- p.40 / Chapter 2.4 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.41 / Chapter 2.5 --- Separation and Purification of PCR Products --- p.43 / Chapter 2.5.1 --- Separation --- p.43 / Chapter 2.5.2 --- Purification --- p.43 / Chapter 2.6 --- Confirmation of PCR Products --- p.44 / Chapter 2.7 --- Molecular Cloning of Trp Gene Family --- p.45 / Chapter 2.7.1 --- "Cloning of HTrpl, HTrp3, HTrp4,HTrp5,HTrp6, HTrp7" --- p.45 / Chapter 2.7.1.1 --- Polishing the Purified PCR Products --- p.47 / Chapter 2.7.1.2 --- Determination of the Amount of Polished PCR Products --- p.47 / Chapter 2.7.1.3 --- Inserting the PCR Products into the pPCR-Script Amp SK(+)Cloning Vector (Ligation) --- p.48 / Chapter 2.7.1.4 --- Transformation --- p.48 / Chapter 2.7.1.5 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.49 / Chapter 2.7.1.6 --- Plasmid DNA Preparation --- p.49 / Chapter 2.8.1.7 --- Clones Confirmation --- p.50 / Chapter 2.8 --- In situ Hybridization --- p.54 / Chapter 2.8.1 --- Probe Preparation --- p.54 / Chapter 2.8.1.1 --- Trp1 Probe --- p.54 / Chapter 2.8.1.2 --- Trp3 Probe --- p.58 / Chapter 2.8.1.3 --- Trp4 Probe --- p.61 / Chapter 2.8.1.4 --- Trp5 Probe --- p.62 / Chapter 2.8.1.5 --- Trp6 Probe --- p.63 / Chapter 2.8.1.6 --- Trp7 Probe --- p.65 / Chapter 2.8.1.7 --- Control Probe --- p.66 / Chapter 2.8.2 --- Testing of DIG-Labeled RNA Probes --- p.66 / Chapter 2.8.3 --- Paraffin Sections Preparation --- p.67 / Chapter 2.8.4 --- In Situ Hybridization: Pretreatment --- p.67 / Chapter 2.8.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.68 / Chapter 2.8.5.1 --- Pre-Hybridization --- p.68 / Chapter 2.8.5.2 --- Hybridization --- p.68 / Chapter 2.8.5.3 --- Post-Hybridization --- p.69 / Chapter 2.8.6 --- Colorimetric Detection of Human Trps mRNA --- p.69 / Chapter 2.9 --- Northern Hybridization --- p.70 / Chapter 2.9.2 --- Labelling of Riboprobe with 32P --- p.70 / Chapter 2.9.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.73 / Chapter Chapter 3. --- Results --- p.74 / Chapter 3.1 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.74 / Chapter 3.2.1 --- Expression of TRPs RNA in Human Coronary Artery --- p.78 / Chapter 3.2.1.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.79 / Chapter 3.2.1.2 --- Expression of Trp Transcripts in the Tunica Adventitia --- p.88 / Chapter 3.2.2 --- Expression of TRPs RNA in Human Cerebral Artery --- p.97 / Chapter 3.2.2.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.97 / Chapter 3.3 --- Northern Blot Analysis of Human Trp5 RNA in Human Multiple Tissue Blot --- p.115 / Chapter Chapter 4: --- Discussion --- p.117 / Chapter 4.1 --- Co-expression of Trps in Vascular Tissues --- p.117 / Chapter 4.1.1 --- Expression of Trps in Endothelia --- p.117 / Chapter 4.1.2 --- In Smooth Muscle Cells --- p.118 / Chapter 4.2 --- Trp Channel and Store-operated Channel in Endothelial Cells --- p.119 / Chapter 4.3 --- Heteromultimerization of Trps Subtypes --- p.120 / Chapter 4.4 --- Northern Blot Analysis --- p.124 / Chapter 4.5 --- Potential Physiological Functions of Trps --- p.125 / Chapter 4.6 --- Trp Channels as a Therapeutic Target? --- p.128 / Chapter 4.7 --- Technical Aspects in the Present Studies --- p.129 / Chapter 4.8 --- Conclusion --- p.131 / Reference --- p.133

Alterações genéticas nos genes RET, RAS e BRAF resultam na ativação constitutiva da sinalização MAPK e estão presentes em aproximadamente 70% dos carcinomas papilífero de tiroide, a forma mais prevalente de câncer de tiroide. Múltiplas vias de sinalização podem atuar em conjunto com a via MAPK na oncogênese tiroidiana. Nesse estudo, testamos a hipótese que a via MAPK regula a sinalização Notch e que o crosstalk entre as vias de sinalizações são importantes na regulação da diferenciação e proliferação celular no câncer de tiroide. A ativação condicional dos oncogenes RET/PTC3 e BRAFT1799A em linhagem de célula folicular normal de tiroide aumentou a atividade da via de sinalização Notch. Por outro lado, o bloqueio farmacológico da sinalização MAPK reduziu a sinalização Notch na linhagem celular TPC-1 derivada de carcinoma papilífero de tiroide. Glândulas tiroide de animais transgênicos expressando BRAFT1799A e amostras de carcinoma papilífero de tiroide apresentaram elevados níveis de NOTCH1. A superexpressão de NOTCH1 em célula folicular normal de tiroide aumentou a expressão proteica de NIS. A inibição farmacológica e por RNA de interferência da sinalização Notch apresentou um efeito anti-proliferativo em linhagem de CPT. Além disso, a combinação do inibidor farmacológico de Notch e MAPK diminuiu a proliferação de células de carcinoma papilífero de tiroide. Esses dados sugerem um importante papel da sinalização Notch na oncogênese do carcinoma papilífero de tiroide induzida pela sinalização MAPK e que a via Notch pode ser uma potencial terapia adjuvante no câncer de tiroide. / Genetic alterations in RET, RAS and BRAF result in constitutive activation of the MAPK signaling and are present in approximately 70% of papillary thyroid carcinomas, the most prevalent form of thyroid cancer. Multiple signaling pathways can act with MAPK pathway in thyroid oncogenesis. In this study, we tested the hypothesis that MAPK pathway control Notch signaling and that the crosstalk between these pathways plays an important role in thyroid cancer cell differentiation and proliferation. The conditional activation of RET/PTC3 and BRAFT1799A enhanced Notch signaling pathway in normal follicular thyroid cell. By contrast, pharmacological inhibition of MAPK reduced Notch signaling in TPC-1 cell line derived from papillary thyroid carcinoma. Transgenic mice expressing BRAFT1799A restrict in thyroid gland and human papillary thyroid carcinoma samples showed higher Notch1 expression. NOTCH1 overexpression in normal thyroid follicular cell increased NIS protein expression. Pharmacological inhibition and RNA interference of Notch signaling showed an anti-proliferative effect in papillary thyroid carcinoma cells. Furthermore, the combination of MAPK and Notch signaling inhibitors reduced papillary thyroid carcinoma proliferation. These data suggest an important role of Notch signaling in papillary thyroid carcinoma induced by MAPK-related oncogenes and that Notch signaling pathway could be a potential adjuvant therapy in thyroid cancer.