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Biochemical study of recombinant human tumor necrosis factor mediated cytotoxicity on murine L-929 cells.

by Chan Po-cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 218-244). / Acknowledgement --- p.i / Abbreviations --- p.ii / Abstract --- p.iv / Table of content --- p.ix / Chapter Chapter 1. --- Biochemistry of Tumor Necrosis Factor --- p.1 / Chapter I. --- Introduction --- p.1 / Chapter 1.1 --- The discovery of tumor necrosis factor (TNF) --- p.1 / Chapter 1.2 --- TNF as an antitumor agent --- p.3 / Chapter 1.3 --- Production of TNF --- p.4 / Chapter 1.4 --- Structure of TNF --- p.5 / Chapter 1.5 --- TNF receptor --- p.6 / Chapter 1.6 --- Biological activities of TNF --- p.10 / Chapter 1.7 --- Anti-tumor activity of TNF --- p.14 / Chapter 1.7.1 --- In vitro studies --- p.14 / Chapter 1.7.1.1 --- Synergistic effect of other cytokines --- p.14 / Chapter 1.7.1.2 --- DNA damages --- p.15 / Chapter 1.7.1.3 --- Free radical generation --- p.15 / Chapter 1.7.1.4 --- Utilization of ATP --- p.16 / Chapter 1.7.1.5 --- Phospholipase A2 activation --- p.17 / Chapter 1.7.2 --- In vivo studies --- p.17 / Chapter 1.8 --- Clinical trials --- p.18 / Chapter Chapter 2. --- Materials and Methods --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Solutions commonly used --- p.21 / Chapter 2.3 --- Methods and procedure --- p.23 / Chapter 2.3.1 --- Culture of L-929 cells --- p.23 / Chapter 2.3.2 --- Trypan Blue exclusion test --- p.23 / Chapter 2.3.3 --- Determination of viability of L-929 cells upon rhTNF treatment --- p.24 / Chapter 2.3.4 --- Determination of cellular cAMP level --- p.25 / Chapter 2.3.5 --- Determination of inositol phosphate turnover --- p.26 / Chapter 2.3.6 --- Use of fluorescence probe in the study of rhTNF mediated killing --- p.28 / Chapter 2.3.6.1 --- Determination of changes in internal pH of L-929 cells --- p.29 / Chapter 2.3.6.2 --- Determination of intracellular calcium level in L-929 cells --- p.30 / Chapter 2.3.6.3 --- Determination of membrane potential by fluorescence probes --- p.32 / Chapter 2.3.6.4 --- "Translocation of nucleolar protein, nucleophosmin (B23)in L-929 cells" --- p.32 / Chapter 2.3.6.5 --- Determination of calcium mobilization in L-929 cells by confocal microscopy --- p.34 / Chapter 2.3.6.6 --- Determination of protein kinase C and phospho-tyrosine kinase in L-929 cells --- p.34 / Chapter 2.3.7 --- Uptake of 45Ca2+ in L-929 cells --- p.35 / Chapter 2.3.8 --- Measurement of membrane potential by Patch-clamp assay --- p.36 / Chapter 2.3.9 --- Determination of tyrosine kinase activation by Western blotting --- p.36 / Chapter 2.3.10 --- Statistical analysis --- p.38 / Chapter Chanter 3. --- Effect of rhTNF treatment on nucleophosmin in L-929 cells --- p.39 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Results --- p.43 / Chapter 3.2.1 --- Effect of TNF (in the presence or absence of actinomycin D) on the nucleophosmin translocation in L-929 cells --- p.43 / Chapter 3.2.2 --- Effect of actinomycin D on the TNF-mediated cytotoxicity on L-929 cells --- p.51 / Chapter 3.3 --- Discussion --- p.57 / Chapter Chapter 4. --- Changes in membrane potential and intracellular pH in rhTNF-mediated cytotoxicity in L-929 cells --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.2 --- Results --- p.61 / Chapter 4.2.1 --- Effect of rhTNF on the membrane potential of L-929 cells determined by fluorescence method --- p.61 / Chapter 4.2.2 --- Effect of rhTNF on the membrane potential of L-929 cells determined by patch clamp technique --- p.64 / Chapter 4.2.3 --- "Effect of K+, Na+ and pH on the rhTNF-mediated cytotoxicity on L-929 cells" --- p.67 / Chapter 4.3 --- Discussion --- p.90 / Chapter Chapter 5. --- Effect of intracellular cAMP and cAMP-dependent protein kinase (PKA) on the rhTNF-mediated cytotoxicity on L-929 cells --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.1.1 --- "GTP-binding protein (G protein), cAMP and protein kinase A" --- p.92 / Chapter 2.1.2 --- Role of cAMP as second messenger --- p.96 / Chapter 5.1.3 --- Bacterial toxin used for study of G-protein --- p.98 / Chapter 5.1.4 --- Effect of cAMP on rhTNF cytotoxicity --- p.99 / Chapter 5.1.5 --- Effect of cAMP-dependent protein kinase (PICA) on rhTNF cytotoxicity --- p.101 / Chapter 5.2 --- Results --- p.102 / Chapter 5.2.1 --- Cyclic-AMP (cAMP) level in rhTNF-treated L-929 cells --- p.102 / Chapter 5.2.2 --- Effect of intracellular cAMP level on rhTNF-mediated cytotoxicity on L-929 cells --- p.104 / Chapter 5.2.3 --- Effect of agonist and inhibitor of cAMP dependent protein kinase (protein kinase A) on rhTNF-mediated cytotoxicity on L-929 cells --- p.107 / Chapter 5.2.4 --- Effect of protein kinase A inhibitors on rhTNF-mediated cytotoxicity on L-929 cells --- p.111 / Chapter 5.3 --- Discussion --- p.118 / Chapter Chapter 6. --- "Role of intracellular free calcium, ions and calcium dependent response in rhTNF-mediated cytotoxicity on L-929 cells" --- p.121 / Chapter 6.1 --- Introduction --- p.121 / Chapter 6.1.1 --- Inositol triphosphate and intracellular free calcium --- p.121 / Chapter 6.1.2 --- Diacylglycerol --- p.131 / Chapter 6.1.3 --- Protein kinase C (PKC) --- p.131 / Chapter 6.1.4 --- Intracellular free calcium ions and protein kinase C --- p.134 / Chapter 6.1.5 --- Effect of intracellular free calcium ions and protein kinase C on TNF-mediated cytotoxicity --- p.135 / Chapter 6.1.6 --- Tyrosine kinase induced release of IP3 --- p.136 / Chapter 6.1.7 --- Calcium channels --- p.136 / Chapter 6.2 --- Result --- p.139 / Chapter 6.2.1 --- Effect of rhTNF on intracellular free [Ca2+] of L-929 cells --- p.141 / Chapter 6.2.2 --- Effect of calcium ion channel blockers on rhTNF-mediated cytotoxicity on L-929 cells --- p.148 / Chapter 6.2.3 --- Effect of protein kinase C (PKC) on rhTNF-mediated cytotoxicity on L-929 cells --- p.158 / Chapter 6.2.4 --- Immunofluorescence staining of PKC in rhTNF-treated L-929 cells --- p.162 / Chapter 6.2.5 --- Effect of calmodulin and calmodulin sensitive calcium ATPase on rhTNF-mediated cytotoxicity on L-929 cells --- p.165 / Chapter 6.2.6 --- Role of inositol triphosphate in rhTNF-mediated cytotoxicity on L-929 cells --- p.167 / Chapter 6.2.7 --- Role of tyrosine kinase activity in the rhTNF-mediated cytotoxicity on L-929 cells --- p.185 / Chapter 6.3 --- Discussion --- p.191 / Chapter Chapter 7. --- Effect of antioxidants on rhTNF-mediated cytotoxicity on L-929 cells --- p.195 / Chapter 7.1 --- Introduction: Oxygen free radicals as mediators of rhTNF-induced tumor cell necrosis --- p.195 / Chapter 7.2 --- Results --- p.199 / Chapter 7.3 --- Discussion --- p.203 / Chapter Chapter 8. --- General Discussion --- p.205 / Bibliography --- p.217

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_318260
Date January 1994
ContributorsChan, Po-cheung., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xi, 244 leaves : ill. (some col.) ; 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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