Xue, Hongmei. / "December 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Liu, Limei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wai Kei Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 155-164). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Sulfonylureas --- p.1 / Chapter 1.2. --- Biological action of sulfonylurea drugs --- p.2 / Chapter 1.2.1 --- Effects on pancreatic β cells --- p.5 / Chapter 1.2.2. --- Effects on cardiac myocytes --- p.7 / Chapter 1.2.3. --- Effects on smooth muscle cells --- p.11 / Chapter 1.2.4. --- Effects on endothelial cells --- p.14 / Chapter 1.3. --- Side effects and toxicity --- p.15 / Chapter 1.4. --- Objectives of the present study --- p.17 / Chapter Chapter 2 --- Methods and Marterials / Chapter 2.1. --- Tissue and Cell Preparation --- p.20 / Chapter 2.1.1. --- Preparation of the isolated rat aorta and mesenteric artery --- p.20 / Chapter 2.1.2. --- Removal of the functional endothelium --- p.20 / Chapter 2.1.3. --- Cell culture --- p.21 / Chapter 2.1.3.1. --- Materials --- p.21 / Chapter 2.1.3.2. --- Aortic smooth muscle cells in primary culture --- p.21 / Chapter 2.1.3.3. --- Aortic endothelial cells in primary culture --- p.23 / Chapter 2.1.3.4. --- Cultured rat aortic smooth muscle cell line (A7r5) --- p.23 / Chapter 2.1.3.5. --- Cultured human umbilical vein endothelial cell line (ECV-304) --- p.24 / Chapter 2.1.3.6. --- Cell subculture --- p.24 / Chapter 2.1.3.7. --- Immunostaining of endothelial cells in primary culture --- p.24 / Chapter 2.2. --- Organ Bath Set-up --- p.25 / Chapter 2.3. --- Force Measurement --- p.28 / Chapter 2.3.1. --- Vascular action of glibenclamide --- p.28 / Chapter 2.3.1.1. --- Antagonistic effect of glibenclamide on relaxation induced by K+ channel openers --- p.28 / Chapter 2.3.1.2. --- Relaxant response of glibenclamide --- p.29 / Chapter 2.3.1.3. --- Role of endothelium-derived vasoactive factors in glibenclamide induced relaxation --- p.29 / Chapter 2.3.1.4. --- Effect of endothelial prostanoids in glibenclamide-induced relaxation --- p.30 / Chapter 2.3.1.5. --- Effects of putative K+ channel blockers on glibenclamide-induced relaxation --- p.30 / Chapter 2.3.1.6. --- Effect of glibenclamide on high K+- and CaCl2-induced contraction --- p.31 / Chapter 2.3.1.7. --- Effect of glibenclamide on prostaglandin F2α-induced contraction --- p.32 / Chapter 2.3.1.8. --- Effect of glibenclamide on protein kinase C-mediated contraction --- p.32 / Chapter 2.3.2. --- Vascular action of glipizide --- p.33 / Chapter 2.3.3. --- Vascular action of tolbutamide --- p.33 / Chapter 2.3.3.1. --- Contractile response of tolbutamide --- p.33 / Chapter 2.3.3.2. --- Effects of inhibitors of endothelium-derived factors --- p.33 / Chapter 2.3.3.3. --- Effects of inhibitors of Ca2+ influx --- p.34 / Chapter 2.3.3.4. --- Effect of protein kinase C inhibitor --- p.34 / Chapter 2.3.3.5. --- Effects of neural factors --- p.34 / Chapter 2.4. --- Cyclic GMP measurement --- p.35 / Chapter 2.4.1. --- Material --- p.35 / Chapter 2.4.2. --- Methods --- p.35 / Chapter 2.4.2.1. --- Tissue preparation --- p.35 / Chapter 2.4.2.2. --- Plasma and tissue according to protocols provided by Amersham --- p.35 / Chapter 2.4.2.3. --- Cyclic GMP content measurement --- p.36 / Chapter 2.4.2.4. --- Protein content measurement --- p.39 / Chapter 2.4.2.5. --- Cyclic GMP measurement protocol --- p.40 / Chapter 2.5. --- Ca2+ measurement --- p.40 / Chapter 2.5.1. --- Materials --- p.40 / Chapter 2.5.1.1. --- PTI RatioMaster Fluorescence System --- p.40 / Chapter 2.5.1.2. --- Confocal Imaging System --- p.42 / Chapter 2.5.2. --- Method --- p.42 / Chapter 2.5.3. --- Protocols for Ca2+ measurement --- p.45 / Chapter 2.5.3.1. --- Effect of glibenclamide in endothelial cells --- p.45 / Chapter 2.5.3.2. --- Effect of glibenclamide in vascular smooth muscle cells --- p.45 / Chapter 2.5.3.3. --- Effect of tolbutamide in vascular smooth muscle cells --- p.46 / Chapter 2.6. --- Cell proliferation --- p.45 / Chapter 2.6.1. --- Materials --- p.45 / Chapter 2.6.2. --- Method --- p.46 / Chapter 2.6.3. --- Protocols for cell proliferation --- p.47 / Chapter 2.6.3.1. --- Effect of glibenclamide on endothelial cell proliferation --- p.47 / Chapter 2.6.3.2. --- Effect of glibenclamide on aortic smooth muscle cell proliferation --- p.47 / Chapter 2.7. --- Chemicals and solutions --- p.48 / Chapter 2.8. --- Statistical analysis --- p.50 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Glibenclamide --- p.51 / Chapter 3.1.1. --- Effect of glibenclamide on the K+ channel activity --- p.51 / Chapter 3.1.2. --- Relaxant response of glibenclamide --- p.55 / Chapter 3.1.3. --- Effects of inhibitors of nitric oxide activity on glibenclamide- induced relaxation --- p.57 / Chapter 3.1.4. --- Role of endothelial relaxing prostanoids in glibenclamide-induced relaxation --- p.69 / Chapter 3.1.5. --- Effect of putative K+ channel blockers on glibenclamide-induced relaxation --- p.73 / Chapter 3.1.6. --- Effect of glibenclamide on high K+-induced arterial contraction --- p.75 / Chapter 3.1.7. --- Effect of glibenclamide on protein kinase C-mediated contraction --- p.83 / Chapter 3.1.8. --- Effect of glibenclamide on prostaglandin F2α-induced contraction --- p.83 / Chapter 3.2 --- Glipizide --- p.85 / Chapter 3.2.1. --- Relaxant response of glipizide --- p.85 / Chapter 3.3. --- Tolbutamide --- p.91 / Chapter 3.3.1. --- Contractile response to tolbutamide --- p.91 / Chapter 3.3.2. --- Effects of endothelium-derived factors --- p.94 / Chapter 3.3.3. --- Effects of inhibitors of Ca2+ influx on tolbutamide-induced contraction --- p.98 / Chapter 3.3.4. --- "Effects of forskolin, sodium nitroprusside, staurosporine on tolbutamide-induced contraction" --- p.102 / Chapter 3.3.5. --- Effect of neural factors --- p.106 / Chapter 3.4. --- Effect of glibenclamide on cGMP levels --- p.112 / Chapter 3.5. --- Effect of glibenclamide on intracellular[Ca2+ ] in cultured endothelial cells --- p.112 / Chapter 3.6. --- Effect of glibenclamide on intracellular [Ca2+] in cultured aortic smooth muscle cells --- p.115 / Chapter 3.7. --- Effect of tolbutamide on intracellular [Ca2+] in cultured aortic smooth muscle cells --- p.121 / Chapter 3.8. --- Effect of glibenclamide on proliferation of cultured endothelial cells --- p.121 / Chapter 3.9. --- Effect of glibenclamide on proliferation of cultured aortic smooth muscle cells --- p.123 / Chapter Chapter 4 --- Discussion / Chapter 4.1. --- Effect of glibenclamide --- p.133 / Chapter 4.2. --- Effect of glipizide --- p.143 / Chapter 4.3. --- Effect of tolbutamide --- p.144 / Chapter 4.4. --- Conclusion --- p.152 / References --- p.155 / Publications --- p.163

Liu, Jian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 127-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Zhang, Yang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

Cyclic nucleotide-gated (CNG) ion channels are Ca2+ permeable nonselective cation channels that are directly gated by binding of cAMP or cGMP, thus providing a linkage between two important signal molecules, cyclic nucleotides and calcium. They are known to play an important role in sensory transduction and in second-messenger modulation of synaptic neurotransmitter release. Previous studies showed that besides in neuronal cells, CNG were found also in non-neuronal tissues including heart, kidney, blood vessels and spleen, they are reported to be involved in a variety of cell function. / Ion channels play an indispensable role in endothelial cells, which is a unique signal-transducting surface in the vascular system that is responsible in altering vascular tone. The present study investigated the expression and functional roles of the cyclic nucleotide gated channels (CNG) in regulating the intracellular calcium level of vascular endothelial cells using molecular and calcium measurement techniques. / The present study provided evidence that the CNG channels, especially that of CNGA2, were expressed in vascular tissues. I used a variety of different methods, including RT-PCR, northern blot, in-situ hybridization, immunohistochemistry and western blot to study the localization of CNGA2 channels. RT-PCR amplify a CNGA2 fragment of 582bp from RNAs isolated from bovine vascular endothelial cell line, rat vascular smooth muscle cell line and rat aorta. Northern blot analysis detected a 2.3-kilobase (kb) CNGA2 transcript in rat aorta mRNA. The cellular distribution of CNGA2 was further studied by in-situ hybridization, which demonstrated expression of CNGA2 mRNA in human vascular endothelial and vascular smooth muscle cells. Immunohistochemistry data also agreed with those generated from in-situ hybridization. Western blot data also demonstrated proteins of CNG2 was expressed in both human vascular endothelial cells and vascular smooth cells layer. Subcellular localization of CNGA2 inside the vascular endothelial cells was also investigated with the use of a GFP linked CNGA2 channel gene. Taken together, the results showed that CNGA2 proteins were expressed on the plasma membrane of the vascular endothelial cells. (Abstract shortened by UMI.) / Cheng Kwong Tai Oscar. / "July 2005." / Adviser: Xiaoqiang Yao. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3531. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 216-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

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

A síndrome dos ovários policísticos (SOP) e a obesidade estão associadas ao aumento do risco cardiovascular, mas não está estabelecido se tal aumento é determinado por estas condições propriamente ditas ou pelos fatores de risco cardiometabólicos a elas associados. Objetivo: determinar, em mulheres jovens e sem fatores de risco cardiometabólicos, a influência da SOP e da obesidade sobre parâmetros vasculares relacionados ao processo de aterogênese. Métodos: foram estudadas pacientes com SOP, subdivididas em portadoras de índice de massa corpórea (IMC) normal e obesas, as quais foram comparadas a mulheres sem SOP (grupo controle) pareadas para o IMC. Foram excluídas participantes tabagistas, com distúrbios do metabolismo da glicose, hipertensão arterial, LDL-C 160 mg/dL e triglicérides 250 mg/dL. Foram avaliados parâmetros clínicos, laboratoriais (perfis hormonal e metabólico) e vasculares [espessura íntimamédia da artéria carótida comum (EIM-ACC), complacência da artéria carótida comum (CP-ACC) e função endotelial da artéria braquial (DMF)], os quais foram avaliados de maneira não-invasiva através de imagens ultrasonográficas de alta-resolução. Para determinar a influência da SOP e da obesidade sobre tais parâmetros, foram formados grupos de acordo com a presença ou ausência de tais condições: grupo SOP vs grupo Controle, independentemente do IMC; grupo IMC normal vs grupo Obesidade, independentemente da presença da SOP. Resultados: Foram selecionadas 25 pacientes com SOP, sendo 10 com IMC normal (34,0 ± 3,2 kg/m2) e 15 obesas (22,4 ± 2,1 kg/m2) e 23 mulheres controles (12 com IMC normal e 11 obesas). As médias de testosterona livre das pacientes com SOP foram significativamente superiores às médias das mulheres controles, independentemente do IMC. As médias do HOMA-IR e da área sob a curva de insulina das pacientes obesas com SOP foram significativamente superiores às observadas nas pacientes com SOP portadoras de IMC normal e mulheres controles. A média da EIM-ACC das pacientes obesas com SOP foi significativamente superior à das mulheres controles com IMC normal (50,0 ± 4,0 vs 47,0 ± 3,0 mm.10-2; p<0,05). As médias da CP-ACC e da DMF foram semelhantes entre pacientes com SOP e mulheres controles, independentemente do IMC. Para avaliar a influência da SOP e da obesidade, as comparações foram, respectivamente: grupo SOP (n=25) vs grupo Controle (n=23); grupo IMC normal (n=22) vs grupo Obesidade (n=26). A faixa etária global foi de 26,0 ± 4,7 anos. Tanto a SOP quanto a obesidade influenciaram os parâmetros de resistência insulínica. A média da EIM-ACC foi maior no grupo SOP do que no grupo Controle (49,1 ± 1,0 vs 47,2 ± 1,0 mm.10-2; p<0,05) e semelhante entre os grupos IMC normal e Obesidade (49,1 ± 1,0 vs 47,3 ± 1,0 mm.10-2; NS). Não foi observada influência da SOP ou da obesidade na CP-ACC e na DMF. Os parâmetros vasculares estudados não se correlacionaram com as outras variáveis analisadas entre as pacientes com SOP e entre as mulheres controles. Conclusão: Em mulheres jovens e sem fatores de risco cardiometabólicos, a presença da SOP teve influência no aumento da EIM-ACC. Assim, a EIMACC pode ser o marcador inicial do processo de aterogênese nesse grupo de pacientes / Polycystic ovary syndrome (PCOS) and obesity are related to the increase in cardiovascular risk, but it is still not known if such risk is due to these conditions themselves or to the cardiometabolic risk factors associated with them. Objective: determine, in young women without cardiometabolic risk factors, the influence of PCOS as well as obesity on vascular parameters related to the process of atherogenesis. Methods: We studied patients with PCOS, subdivided in patients with normal body mass index (BMI) and obeses, who were compared with women without PCOS (control group) pairwise matched for BMI. We excluded smoking subjects, subjects with glucose metabolism disturbances, with arterial hypertension, LDC -L 160 mg/dl and with triglycerides 250 mg/dl. We evaluated clinical, laboratory (hormonal and metabolic profiles) and vascular parameters [common carotidy artery intima-media thickness (CCA-IMT), compliance of commom carotid artery (CP-CCA) and endothelium function of the braquial artery (FMD)], through a non-invasive method using high resolution ultrasound imaging. In order to determine the influence of PCOS and obesity on such parameters, groups were formed according to the presence or absence of such conditions: PCOS group vs Control group, independently of BMI; normal BMI group vs obesity group, independently of PCOS presence. Results: Twenty-five patients with PCOS were selected, being 10 with normal BMI (34.0 ± 3.2 kg/m²), 15 obeses (22.4 ± 2.1 kg/m²) and 23 control women (12 with normal BMI and 11 obeses). The mean values of free testosterone in PCOS patients were significantly higher than the means in controls, independently of BMI. The means of HOMA-IR and the area under the insulin curve in obese PCOS patients were significantly higher than the ones observed in PCOS patients with normal BMI and controls. The means of CCA-IMT in obese PCOS patients was significantly higher than in controls with normal BMI (50.0 ± 4.0 vs 47.0 ± 3.0 mm.10-2; p<0.05). The means of CP-CCA and FMD were similar between PCOS patients and controls, independently of BMI. To evaluate the influence of PCOS and obesity, the comparisons were respectively: PCOS group (n=25) vs Control group (n=23); normal BMI group (n=22) vs Obesity group (n=26). Global age range was 26.0 ± 4.7 years. PCOS as well as obesity influenced the insulin resistance parameters. The means of CCA-IMT was higher in PCOS group than in Control group (49.1 ± 1.0 vs 47.2 ± 1.0 mm.10-2; p<0.05) and similar between normal BMI and Obesity groups (49.1 ± 1.0 vs 47.3 ± 1.0 mm 10-2; NS). It was not observed any influence of PCOS or obesity in CP-CCA and in FMD. The vascular parameters studied did not correlate with the other variables analized between PCOS patients and controls. Conclusions: In young women without cardiometabolic risk factors, the presence of PCOS had influence on the increase of CCA-IMT. Thus, CCA-IMT might be the initial marker of the atherogenic process in this group of patients

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.