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.

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

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.

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

Liang, Fengting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 87-101). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENT --- p.II / ABBREVIATIONS --- p.III / ABSTRACT IN ENGLISH --- p.IV / ABSTRACT IN CHINESE --- p.VI / TABLE OF CONTENTS --- p.VIII / LIST OF FIGURES --- p.XI / LIST OF TABLES --- p.XIII / Chapter CHAPTER I- --- INTRODUCTION / Chapter 1.1 --- Roles of human bronchial epithelial cells --- p.1 / Chapter 1.2 --- Role of epithelium in airway inflammation --- p.3 / Chapter 1.3 --- Pathology of asthma --- p.5 / Chapter 1.4 --- The role of eosinophils in asthma --- p.7 / Chapter 1.5 --- "Effects of poly-L-arginine, a MBP analogue, on airway epithelium" --- p.10 / Chapter 1.6 --- Inflammatory pathways involved in epithelial cytokine production --- p.12 / Chapter 1.7 --- Roles and function of IL-6 and IL-8 in epithelial cells --- p.16 / Chapter 1.8 --- P2 receptors and inflammation --- p.18 / Chapter 1.9 --- Objectives --- p.20 / Chapter CHAPTER II- --- MATERIALS AND METHODS / Chapter 2.1 --- Materials and regents --- p.21 / Chapter 2.2 --- Cell culture --- p.22 / Chapter 2.3 --- RNA extraction and Real-time PCR --- p.23 / Chapter 2.4 --- Measurement of cytokine secretion by antibody array --- p.24 / Chapter 2.5 --- Quantification of IL-6 and IL-8 secretion --- p.27 / Chapter 2.6 --- Western Blotting --- p.28 / Chapter 2.7 --- NF-kB translocation assay --- p.29 / Chapter 2.8 --- Data analysis --- p.30 / Chapter CHAPTER III- --- RESULTS / Chapter 3.1 --- Poly-L-arginine-induced IL-6 and IL-8 release from 16HBE 14o- --- p.31 / Chapter 3.2 --- Signaling pathways involved in poly-L-arginine-induced IL-6 and IL-8 release --- p.34 / Chapter 3.2.1 --- "Effects of p38 MAPK, ERK1/2 and NF-kB inhibitors on poly-L-arginine-induced IL-6 and IL-8 release" --- p.35 / Chapter 3.2.2 --- Poly-L-arginine induces p38 MAPK and ERK1/2 phosphorylation --- p.43 / Chapter 3.2.3 --- Poly-L-arginine activates NF-kB translocation from cytoplasm to nucleus --- p.49 / Chapter 3.3 --- Effects of MAPK and NF-kB inhibitors on IL-6 and IL-8 mRNA expression on poly-L-arginine-challenged 16HBE14o- cells --- p.52 / Chapter 3.4 --- P2 receptors modulate poly-L-arginine-induced IL-6 and IL-8 ^ production --- p.55 / Chapter 3.4.1 --- Extracellular nucleotides modulate IL-6 and IL-8 production --- p.56 / Chapter 3.4.2 --- Effects of P2Y6 antagonist on poly-L-arginine-induced IL-6 and IL-8 production --- p.61 / Chapter 3.4.3 --- Effects of MAPKs inhibitors on UDP-induced IL-6 and IL-8 secretion --- p.64 / Chapter 3.4.4 --- UDP induces NF-kB translocation in 16HBE14o- cells --- p.67 / Chapter CHAPTER IV- --- DISCUSSION / Chapter 4.1 --- Involvement of p3 8 MAPK and NF-kB in poly-L-arginine-induced IL-6 and IL-8 secretion --- p.70 / Chapter 4.2 --- Involvement of p38 MAPK and NF-kB in poly-L-arginine-induced IL-6 and IL-8 mRNA elevation --- p.72 / Chapter 4.2.1 --- Regulation of NF-kB on IL-6 and IL-8 mRNA --- p.73 / Chapter 4.2.2 --- Regulation of p38 MAPK on IL-6 and IL-8 mRNA --- p.75 / Chapter 4.2.3 --- Crosstalk between NF-kB and p38 MAPK --- p.77 / Chapter 4.3 --- Extracellular nucleotides mediate IL-6 and IL-8 production in 16HBE14o- --- p.79 / Chapter 4.3.1 --- P2Y6 receptor is linked to poly-L-arginine-induced IL-6 and IL-8 release --- p.80 / Chapter 4.3.2 --- P2Y6 receptor regulates IL-6 and IL-8 secretion via p38 MAPK and NF-kB --- p.83 / Chapter 4.4 --- Summary --- p.86 / Chapter CHAPTER V- --- References --- p.87 / Publications --- p.102

Zhang, Siwei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 87-98). / Abstracts in English and Chinese. / Chapter 1. --- General introduction --- p.1 / Chapter 1.1. --- "Types, structure and function of human hemoglobin" --- p.1 / Chapter 1.1.1. --- Structure and functions of human hemoglobin --- p.1 / Chapter 1.1.2. --- Types of human hemoglobin --- p.2 / Chapter 1.2. --- Regulatory mechanism of human hemoglobin expression --- p.3 / Chapter 1.2.1. --- The human a and β locus --- p.3 / Chapter 1.2.2. --- Development of globin genes switching concept --- p.4 / Chapter 1.2.3. --- Factors that regulate globin gene expression --- p.5 / Chapter 1.2.3.1. --- The locus control region (LCR) --- p.5 / Chapter 1.2.3.2. --- The cis-regulatory elements --- p.5 / Chapter 1.2.3.3. --- The trans-acting factors --- p.6 / Chapter 1.3. --- The human hemoglobinopathies --- p.8 / Chapter 1.3.1. --- α-thalassemia --- p.8 / Chapter 1.3.2. --- β-thalassemia --- p.9 / Chapter 1.3.3. --- Sickle cell anemia --- p.10 / Chapter 1.4. --- Current approaches towards β-thalassemia treatment --- p.11 / Chapter 1.4.1. --- Blood transfusion --- p.11 / Chapter 1.4.2. --- Bone marrow transplantation --- p.12 / Chapter 1.4.3. --- Drug-induced activation of fetal hemoglobin production --- p.12 / Chapter 1.4.3.1. --- Hydroxyurea --- p.12 / Chapter 1.4.3.2. --- Butyrate and short-chain fatty acids --- p.13 / Chapter 1.4.3.3. --- "Mutagens, DNA methyltransferase inhibitors and other HbF inducible agents" --- p.13 / Chapter 1.4.3.4. --- Cucurbitacin D --- p.14 / Chapter 1.4.4. --- Gene therapy --- p.14 / Chapter 1.5. --- Research Objectives --- p.15 / Chapter 2. --- "Analysis of CuD, Hydroxyurea and other inducers on the induction of α, β, γ, δ， ε，ζ BP-1 genes and fetal hemoglobin induction" --- p.16 / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.1.1. --- Properties of human K562 cell line --- p.16 / Chapter 2.1.2. --- Induction and measurement of fetal hemoglobin --- p.16 / Chapter 2.1.3. --- "Induction of α, β, γ, δ, ε , ζ and BP-1 gene and Real-time RT-PCR analysis" --- p.17 / Chapter 2.2. --- Materials --- p.18 / Chapter 2.2.1. --- Chemicals and reagents --- p.18 / Chapter 2.2.2. --- Kits --- p.19 / Chapter 2.2.3. --- Buffers and solutions --- p.19 / Chapter 2.2.4. --- Cell lines --- p.20 / Chapter 2.3. --- Experimental procedures --- p.20 / Chapter 2.3.1. --- Hemoglobin quantity measurement by HbF ELISA --- p.20 / Chapter 2.3.1.1. --- MTT assay --- p.21 / Chapter 2.3.1.2. --- Preparation of capture-antibody coated ELISA plates --- p.21 / Chapter 2.3.1.3. --- Plate blocking --- p.22 / Chapter 2.3.1.4. --- Sample and standard preparation --- p.22 / Chapter 2.3.1.5. --- HRP antibody and colorimetric detection --- p.23 / Chapter 2.3.1.6. --- Statistical analysis --- p.23 / Chapter 2.3.2. --- Preparation of mRNA extract from K562 cells --- p.23 / Chapter 2.3.3. --- Reverse transcription and Real-time PCR analysis --- p.24 / Chapter 2.4. --- Results --- p.25 / Chapter 2.4.1. --- CuD significantly upregulates HbF expression in K562 cells --- p.25 / Chapter 2.4.2. --- "CuD augments α, β, γ, δ, ε , ζ and BP-1 genes at different level in K562 cells" --- p.28 / Chapter 2.4.3. --- Cucurbitacin D-induced γ-globin gene activation requires12-24 hours in K562 cells --- p.31 / Chapter 2.5. --- Discussion --- p.33 / Chapter 2.5.1. --- Enhancement of fetal hemoglobin production using different chemical compounds --- p.33 / Chapter 2.5.2. --- CuD increased HbF synthesis by increasing γ-globin mRNA amount --- p.35 / Chapter 2.5.3. --- CuD and HU down-regulated the BP-1 gene expression --- p.36 / Chapter 3. --- Determination of potential signal transduction pathways during CuD and HU-mediated fetal hemoglobin production --- p.36 / Chapter 3.1. --- Introductions --- p.36 / Chapter 3.1.1. --- The p38 MAPK family --- p.37 / Chapter 3.1.2. --- The JAK2-STAT3 pathway --- p.38 / Chapter 3.1.3. --- Fundamentals on inhibition assay of p38 MAPK and JAK2-STAT3 pathway --- p.39 / Chapter 3.1.4. --- Fundamentals on nuclear translocation of STAT3 --- p.41 / Chapter 3.2. --- Materials --- p.41 / Chapter 3.2.1. --- Chemicals and reagents --- p.41 / Chapter 3.2.2. --- Kits --- p.44 / Chapter 3.2.3. --- Buffers and solutions --- p.44 / Chapter 3.3. --- Experimental procedures --- p.45 / Chapter 3.3.1. --- Detection of p3 8 MAPK phosphorylation status --- p.46 / Chapter 3.3.1.1. --- Preparation of cytosolic protein extracts --- p.46 / Chapter 3.3.1.2. --- Quantitative measurement of phospho-p38 and pan-p38 by ELIS A method --- p.46 / Chapter 3.3.1.2.1. --- Antigen adsorption and establishment of standard curves --- p.46 / Chapter 3.3.1.2.2. --- Plate washing and application of detection antibody --- p.47 / Chapter 3.3.1.2.3. --- Plate washing and application of secondary antibody --- p.47 / Chapter 3.3.1.2.4. --- Plate washing and chromogen detection --- p.48 / Chapter 3.3.2. --- Detection of signal cascade on JAK2-STAT3 pathway --- p.48 / Chapter 3.3.2.1. --- Preparation of cytosolic protein extracts for Western Blot detection --- p.48 / Chapter 3.3.2.2. --- Gel running and Western Blot detection --- p.48 / Chapter 3.3.3. --- Quantitative measurement of phospho-STAT3-Tyr705 using ELISA method --- p.50 / Chapter 3.3.3.1. --- Preparation of cytosolic protein extracts --- p.50 / Chapter 3.3.3.2. --- Reconstitution and Dilution of STAT3 [pY705] Standard --- p.50 / Chapter 3.3.3.3. --- Measurement of STAT3 [pY705] concentration in cell lysates --- p.51 / Chapter 3.3.4. --- Inhibitor assay of JAK2-STAT3 and p38 MAPK pathway --- p.52 / Chapter 3.3.4.1. --- Establishment of inhibitor assay --- p.52 / Chapter 3.3.4.2. --- HbF ELISA detection --- p.53 / Chapter 3.3.5. --- Detection of STAT3 nuclear translocation and DNA binding affinity --- p.53 / Chapter 3.3.5.1. --- Preparation of nuclear extract from K562 cells --- p.53 / Chapter 3.3.5.2. --- EMS A detection of transcriptional factors binding to γ-promoter region --- p.54 / Chapter 3.3.5.2.1. --- 3´ة end-labeling of EMS A probes --- p.54 / Chapter 3.3.5.2.2. --- Dot blotting for labeling efficiency estimation --- p.56 / Chapter 3.3.5.2.3. --- EMSA binding reaction and non-denaturing gel electrophoresis --- p.57 / Chapter 3.3.5.2.4. --- Membrane development and chemiluminescence detection --- p.58 / Chapter 3.3.5.3. --- Preparation of K562 samples for immunofluorescence detection --- p.60 / Chapter 3.3.5.3.1. --- Slide coating for cell capture --- p.60 / Chapter 3.3.5.3.2. --- Preparation of cell slide --- p.60 / Chapter 3.3.5.3.3. --- Sample fixation and antibody probing treatment --- p.60 / Chapter 3.3.5.3.4. --- Sample imaging and immunofluorescence detection --- p.61 / Chapter 3.4 --- Results --- p.62 / Chapter 3.4.1. --- Activation of p38 MAPK pathway and STAT3 phosphorylation by hydroxyurea --- p.62 / Chapter 3.4.1.1. --- "The p38 MAPK pathway is activated by hydroxyurea, but not activated by Cucurbitacin D" --- p.62 / Chapter 3.4.1.2. --- Increased p38 phosphorylation level elicits STAT3 phosphorylation at Ser727 site --- p.64 / Chapter 3.4.2. --- Activation of JAK2 and STAT3 phosphorylation by Cucurbitacin D --- p.66 / Chapter 3.4.2.1. --- Cucurbitacin D promotes JAK2 activation --- p.66 / Chapter 3.4.2.2. --- Cucurbitacin D and hydroxyurea promote STAT3 phosphorylation at Tyr705 site --- p.66 / Chapter 3.4.3. --- Basal activity of signal transduction pathways is essential for HbF induction --- p.69 / Chapter 3.4.3.1. --- Activation of γ-globin gene requires presence of basal phosphorylation level of p38 MAPK --- p.69 / Chapter 3.4.3.2. --- Inhibition on JAK2-STAT3 pathway results in reduced fetal hemoglobin production --- p.71 / Chapter 3.4.4. --- Translocation and DNA binding of STAT under Cucurbitacin D induction --- p.72 / Chapter 3.4.4.1. --- Cucurbitacin D and hydroxyurea both enhance binding affinity of transcriptional factors to the Gγ/Aγ promoter --- p.72 / Chapter 3.4.4.2. --- Cucurbitacin D and hydroxyurea induces nuclear translocation of STAT3 --- p.75 / Chapter 3.5. --- Discussion --- p.77 / Chapter 3.5.1. --- The role of p38 MAPK activation during γ-globin gene activation --- p.77 / Chapter 3.5.2. --- STAT3 phosphorylation at Ser727 site promotes transcription factor activity and γ-globin gene expression --- p.77 / Chapter 3.5.3. --- The role of JAK2-STAT3 activation during γ-globin gene activation --- p.78 / Chapter 3.5.4. --- Inhibitor assay --- p.79 / Chapter 3.5.5. --- Relations between STAT3 nuclear translocation and enhanced fetal hemoglobin production --- p.82 / Chapter 4. --- Summery and Prospect --- p.83 / Chapter 5. --- References --- p.87

Zhong, Lan. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 122-154). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Zhong, Lan.

Lam, Kwok Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 149-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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