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Expression of Trp gene family in vascular system.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323644
Date January 2001
ContributorsYip, Ham., Chinese University of Hong Kong Graduate School. Division of Physiology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, vi, 141 leaves : ill. (some col., some mounted) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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