The redox state in the cell plays a major role in determining vital functions and its major imbalance can lead to severe cell injury or death. Redox active proteins and cytokines involved in this process includes thioredoxin (Trx), protein disulfide isomerase (PDI), and tumor necrosis factor (TNF) superfamilies. Trx is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system). Trx has gene regulatory activity of several transcription factors. It also controls in a fascinating way redox-sensitive “on-off” decisions for apoptotic or hypertrophic pathways. Trx protects against H2O2 and TNFmediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. TNF is an autocrine growth factor and survival factor in vitro and in vivo for B-type of chronic lymphocytic leukemia (B-CLL) cells. The overall aim of this study was to investigate the importance of redox active proteins and cytokines in inflammation and cancer. We focused on: i) the role of Trx, TrxR, and selenium in carcinogenesis and in resistant cancer cells. ii) the importance of Trx in cancer cells and the redox regulation of TNF and its receptors TNFR1 and TNFR2. iii) the potential role of Trx as a key regulator in cellular redox balance, in the pathogenesis of cardiac dysfunction; its relationship to stress response parameters. iv) whether unmutated CLL (UCLL) responses to PKC and ROS pathways were different from mutated CLL (M-CLL) responses. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy resistant cells and suggest that redox regulation through the Trx system is an important target for cancer therapy. Trx was strikingly elevated in heart failure cases compared with controls signifying an adaptive stress response that is higher the more severe the disease. TNF autocrine release was redox modulated and the TNF receptors interacted at the cell surface membrane with the redox-active PDI, which excerted a stringent redox-control of the TNFR signaling. The proliferative response as well as increase of autocrine TNF and Trx were higher in U-CLL than in M-CLL. The overall conclusion of the four papers included in this thesis is that redox-active proteins and cytokines plays an important role in control and regulation of cancer and inflammation. Furthermore, redox regulation via thioredoxin by selenium, may offer novel treatment possibilities for resistant tumors disease.

The murine cytomegalovirus (MCMV) immediate early 1 (IE1) protein has been described as a trans-activator of viral and host gene expression. However, the precise role that IE1 plays in the viral life cycle, and in particular its effect on the host immune response is not known. This thesis investigates the functional relationship of the IE1 protein and the immune response induced after infection. By using an ie1-deletion mutant MCMV (MCMVdie1) it was demonstrated that, early after infection, tumor necrosis factor (tnf ) gene activation and protein production was significantly induced in infected-primary macrophages (M ) to a much greater extent than its wild type counterpart. In addition, preliminary studies on the signalling pathways activated upon infection were carried out in order to gain information about the pathways that might be involved in MCMVinduced modulation of tnf activation. Initial observations on the MAPK family members Erk1/2, p38 and JNK did not revealed any differential activation in the absence of IE1. However, due to a number of limitations, it was not possible to draw any firm conclusions from this study. Investigation of the role of IE1 in the in vivo production of TNF were also performed in both susceptible (BALB/c) and resistant (C57Bl/6) mice. These experiments confirmed the attenuated phenotype of MCMVdie1 in vivo, whereby the mutant strain grew to much lower titers than wild type. When cytokine production was assessed in relation to PFU levels a significant production of TNF after infection is observed in different organs of both mice strains. This raises the question whether IE1 contributes to MCMV modulation of TNF production in the natural host. Although, because it is still unclear whether the phenotype of MCMVdie1 in vivo is due to a defect in the virus or the result of a immune response, it was not possible to conclude unequivocally that IE1 is responsible for dampening this cytokine response. This thesis also tested whether the attenuated replication of MCMVdie1 in vivo was due to the increased TNF production induced after infection. An initial investigation in tnf depleted mice revealed that the MCMVdie1 growth phenotype is not due to TNF response. Overall, this study has provided insight into a potential immune modulatory function by MCMV associated with IE1 protein and the regulation of TNF in vivo and in vitro.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers exhibit or acquire resistance to TRAIL. We report herein that TRAIL activates a TGF-ß-activated kinase 1→mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6→p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the "death-inducing signaling complex", activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and apoptosome function were dispensable. Even following MOMP, however, mitochondrial-generated reactive oxygen species activated a secondary signaling pathway, involving c-Jun N-terminal kinases, that likewise upregulated MCL-1 expression and partially rescued cells from death. Thus, stress kinases activated at distinct steps in the extrinsic pathway mediate TRAIL resistance through maintenance of MCL-1 expression. / text

Doctor of Philosophy / Department of Animal Sciences / Barry Bradford / The transition into lactation is a period of primary concern to dairy producers because of the tremendous incidence of health disorders observed during this time. Two common disorders that lead to decreases in production and retention within the herd include fatty liver disorder (FL) and ketosis. These two disorders have been commonly associated with negative energy balance, yet recently it has been hypothesized that inflammation is a contributor to the etiology of these disorders. Three individual projects were completed for this dissertation, all involving inflammation. The role of endogenous inflammation was determined by administration of sodium salicylate (SS) to cows for 7 d after parturition, and metabolites and production responses were evaluated. Overall it appears that SS induced hypoglycemic conditions and increased triglyceride accumulation in the liver (while administered), increased lipid mobilization and ketones (2 weeks after administration ended), and increased whole lactation milk production in older cows. A sensitive, specific sandwich ELISA for bovine tumor necrosis factor-[alpha] was developed, which provided the ability to measure “normal” circulating levels of this cytokine. The final study involved inducing inflammation by daily injections of the TNF[alpha] to the early lactation dairy cow. In this model, cows receiving TNF[alpha] had a reduction in dry matter intake, water intake, and decreases in milk production and milk components. Overall, it appears that inflammation is involved in the normal biology of the transition dairy cow and disrupting this can lead to interesting negative effects and some improvements of production; however, when inflammation is much greater it can lead to negative production effects.

by Wong Wah Yau. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 219-228.

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

by Chang Chuen Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 125-140). / Acknowledgement --- p.4 / List of Abbreviations --- p.5 / Abstract --- p.7 / Chapter CHAPTER I --- INTRODUCTION --- p.10 / Chapter 1.1 --- Astrocytes in the Central Nervous System --- p.10 / Chapter 1.1.1 --- Characteristics of astrocytes --- p.10 / Chapter 1.1.2 --- Functional roles of astrocytes --- p.11 / Chapter 1.1.2.1 --- General functions of astrocytes --- p.11 / Chapter 1.1.2.2 --- Volume regulation of astrocytes in CNS injuries --- p.12 / Chapter 1.1.2.3 --- Immunological functions of astrocytes --- p.13 / Chapter 1.2 --- Taurine in the CNS --- p.15 / Chapter 1.2.1 --- The biochemistry and distribution of taurine --- p.15 / Chapter 1.2.2 --- Physiological functions of taurine in the CNS --- p.19 / Chapter 1.2.3 --- Uptake and release of taurine by cultured astrocytes --- p.20 / Chapter 1.2.3.1 --- Taurine uptake in astrocytes --- p.21 / Chapter 1.2.3.2 --- Taurine release in astrocytes --- p.22 / Chapter 1.3 --- Tumor necrosis factor in the CNS --- p.23 / Chapter 1.3.1 --- Characteristics of tumor necrosis factor --- p.23 / Chapter 1.3.2 --- Sources of TNF in the CNS --- p.25 / Chapter 1.3.3 --- Functions of TNF in the CNS --- p.26 / Chapter 1.3.4 --- TNF and signal transduction --- p.27 / Chapter 1.4 --- cGMP second messenger system in astrocyte --- p.29 / Chapter 1.4.1 --- cGMP as second messenger in astrocytes --- p.29 / Chapter 1.4.2 --- Post cGMP cascade effects --- p.30 / Chapter 1.5 --- The aims of this project --- p.30 / Chapter CHAPTER II --- METHODS --- p.34 / Chapter 2.1 --- Primary astrocytes culture --- p.34 / Chapter 2.1.1 --- Primary rat astrocytes culture --- p.34 / Chapter 2.1.2 --- Primary mouse astrocytes culture --- p.36 / Chapter 2.1.3 --- Culture of rat C6 glioma cell line --- p.36 / Chapter 2.1.4 --- Subculture of astrocytes in different media --- p.37 / Chapter 2.2 --- Taurine uptake and release assay --- p.39 / Chapter 2.2.1 --- Taurine uptake assay --- p.39 / Chapter 2.2.2 --- Taurine release assay --- p.41 / Chapter 2.3 --- The effects of TNF on taurine transport --- p.42 / Chapter 2.4 --- The effects of TNF on cell volume in astrocytes --- p.43 / Chapter 2.5 --- "The effects of TNF on amino acids, glucose and neurotransmitters uptake" --- p.43 / Chapter 2.5.1 --- The effects of TNF on amino acids uptake --- p.43 / Chapter 2.5.2 --- The effects of TNF on glucose uptake --- p.44 / Chapter 2.5.3 --- The effects of TNF on neurotransmitters uptake --- p.45 / Chapter 2.6 --- The effects of LPS on taurine uptake in astrocytes --- p.46 / Chapter 2.7 --- The effects of IFN-¡’ on taurine uptake in astrocytes --- p.46 / Chapter 2.8 --- The effects of PMA on taurine uptake in astrocytes --- p.47 / Chapter 2.9 --- "The effects of TNF on thymidine, uridine and leucine incorporation in astrocytes" --- p.47 / Chapter 2.10 --- The effects of TNF on basal level of cGMP in astrocytes --- p.48 / Chapter 2.11 --- The effects of TNF on protein phosphorylation in astrocytes --- p.49 / Chapter 2.12 --- The effects of TNF on calcium uptake in astrocytes --- p.50 / Chapter CHAPTER III --- RESULTS --- p.51 / Chapter 3.1 --- The effects of TNF on taurine transport in cultured rat astrocytes --- p.51 / Chapter 3.1.1 --- The effects of TNF on [3H]-taurine uptake -time course study --- p.52 / Chapter 3.1.2 --- The effects of TNF on the kinetic parameters of the taurine uptake system --- p.54 / Chapter 3.1.3 --- The effects of TNF concentration on taurine uptake --- p.63 / Chapter 3.1.4 --- The effects of TNF exposure time on taurine uptake --- p.65 / Chapter 3.1.5 --- The effects of TNF on cell volume change in astrocytes --- p.67 / Chapter 3.1.6 --- "Comparison of the effects of TNF on taurine uptake amongst cultured primary rat astrocytes, primary mouse astrocytes and C6 glioma cell line" --- p.69 / Chapter 3.1.7 --- The effects of TNF on taurine release --- p.71 / Chapter 3.1.8 --- The specificity of the effects of TNF on taurine uptake --- p.74 / Chapter 3.1.8.1 --- The effects of TNF on the uptake of amino acids and glucose in primary rat astrocytes --- p.79 / Chapter 3.1.8.2 --- The effects of TNF on neurotransmitters uptake --- p.87 / Chapter 3.1.9 --- The effects of LPS on taurine uptake in astrocytes --- p.92 / Chapter 3.1.10 --- The effects of IFN-¡’ on taurine uptake in astrocytes --- p.97 / Chapter 3.1.11 --- The effects of PMA on taurine uptake --- p.99 / Chapter 3.2 --- The effects of TNF on cell metabolism in rat astrocytes --- p.102 / Chapter 3.2.1 --- The effects of TNF on astrocyte proliferation --- p.102 / Chapter 3.2.2 --- The effects of TNF on RNA synthesis --- p.103 / Chapter 3.2.3 --- The effects of TNF on protein synthesis --- p.106 / Chapter 3.2.4 --- The effects of TNF on basal level of cGMP --- p.108 / Chapter 3.2.5 --- The effects of TNF on protein phosphorylation --- p.111 / Chapter 3.2.6 --- The effects of TNF on calcium uptake --- p.113 / Chapter Chapter IV --- DISCUSSION AND CONCLUSION --- p.116 / References --- p.125

by Chan Yick Bun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 145-165). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / List of Figures --- p.XVI / List of Tables --- p.XVII / Chapter Chapter One --- General introduction / Chapter 1.1 --- Leukemia: an overview --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Classification of leukemia --- p.1 / Chapter 1.1.3 --- Origin of leukemia --- p.3 / Chapter 1.1.4 --- Treatment of leukemia --- p.5 / Chapter 1.2 --- Introduction of leukemia cell re-differentiation --- p.8 / Chapter 1.2.1 --- Introduction --- p.8 / Chapter 1.2.2 --- Inducers of cell differentiation --- p.8 / Chapter 1.2.3 --- Genes involved in myeloid leukemia cell differentiation --- p.11 / Chapter 1.2.3.1 --- Transcription factors --- p.11 / Chapter 1.2.3.2 --- Signal transduction cascades --- p.16 / Chapter 1.2.3.3 --- Receptors --- p.18 / Chapter 1.2.3.4 --- Cytokines --- p.19 / Chapter 1.3 --- Tumor necrosis factor alpha induced WEHI 3B JCS cell differentiation --- p.21 / Chapter 1.3.1 --- Introduction --- p.21 / Chapter 1.3.2 --- Tumor necrosis factor alpha --- p.21 / Chapter 1.3.3 --- WEHI 3B JCS cells --- p.23 / Chapter 1.4 --- Aims of study --- p.25 / Chapter Chapter Two --- Isolation of differentially expressed genes during TNF-α induced WEHI 3B JCS cell differentiation / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.1.1 --- Overview of differential genes screening methods --- p.26 / Chapter 2.1.2 --- Differential hybridization for analysis of gene expression profiles --- p.29 / Chapter 2.1.3 --- Factors affect differential hybridization --- p.33 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.2.1 --- Cell line --- p.35 / Chapter 2.2.2 --- Mouse brain cDNA library --- p.35 / Chapter 2.2.3 --- E.coli strains --- p.35 / Chapter 2.2.3 --- Kits --- p.35 / Chapter 2.2.5 --- Chemicals --- p.35 / Chapter 2.2.6 --- Solutions and buffers --- p.36 / Chapter 2.2.7 --- Enzymes and reagents --- p.37 / Chapter 2.3 --- Methods --- p.38 / Chapter 2.3.1 --- Preparation of total RNA from TNF-a induced WEHI 3B JCS cells --- p.38 / Chapter 2.3.1.1 --- Preparation of cell lysates --- p.38 / Chapter 2.3.1.2 --- Extraction of total RNA --- p.38 / Chapter 2.3.2 --- Preparation of cDNA clones from cDNA library --- p.39 / Chapter 2.3.2.1 --- Rescue of phagemids from cDNA library --- p.39 / Chapter 2.3.2.2 --- Preparation of plasmids --- p.39 / Chapter 2.3.3 --- Primary differential hybridization --- p.40 / Chapter 2.3.3.1 --- Preparation of cDNA blots --- p.40 / Chapter 2.3.3.2 --- Preparation of cDNA probes --- p.40 / Chapter 2.3.3.3 --- Primary differential hybridization --- p.41 / Chapter 2.3.4 --- Subcloning of putative differential cDNA clones --- p.42 / Chapter 2.3.4.1 --- Preparation of DH5a competent cells --- p.42 / Chapter 2.3.4.2 --- Transformation of cDNA clones --- p.42 / Chapter 2.3.5 --- Secondary differential hybridization --- p.42 / Chapter 2.3.5.1 --- Preparation ofcDNA blots --- p.42 / Chapter 2.3.5.2 --- Secondary differential hybridization --- p.43 / Chapter 2.4 --- Results --- p.44 / Chapter 2.4.1 --- Analysis of total RNA prepared from TNF-α induced WEHI 3B JCS cells --- p.44 / Chapter 2.4.2 --- Spectrophotometric analysis of plasmid DNA --- p.46 / Chapter 2.4.3 --- Primary differential hybridization --- p.48 / Chapter 2.4.4 --- Secondary differential hybridization --- p.58 / Chapter 2.4.5 --- Comparison of two rounds of differential hybridization --- p.61 / Chapter 2.5 --- Discussions --- p.63 / Chapter 2.5.1 --- Study of gene expression profile by differential hybridization --- p.63 / Chapter 2.5.1.1 --- cDNA library --- p.63 / Chapter 2.5.1.2 --- Blots --- p.64 / Chapter 2.5.2 --- Two rounds of differential hybridization --- p.66 / Chapter 2.5.3 --- Comparison of two rounds of differential hybridization --- p.68 / Chapter Chapter Three --- Sequence analysis of putative differentially expressed genes / Chapter 3.1 --- Introduction --- p.70 / Chapter 3.1.1 --- Basic structure of cDNA clones --- p.70 / Chapter 3.1.2 --- Strategies for DNA sequencing --- p.71 / Chapter 3.1.2.1 --- Primer walking --- p.71 / Chapter 3.1.2.2 --- Restriction digestion and subcloning --- p.71 / Chapter 3.1.2.3 --- Nested deletion sets --- p.72 / Chapter 3.1.2.4 --- Shotgun sequencing --- p.72 / Chapter 3.1.2.5 --- Other sequencing strategies --- p.73 / Chapter 3.1.3 --- Sequence alignment and database search --- p.74 / Chapter 3.1.3.1 --- Sequence database --- p.74 / Chapter 3.1.3.2 --- Sequence alignment --- p.74 / Chapter 3.1.3.3 --- BLAST algorithm --- p.75 / Chapter 3.2 --- Materials --- p.76 / Chapter 3.2.1 --- Kits --- p.76 / Chapter 3.2.2 --- Restriction enzymes --- p.76 / Chapter 3.2.3 --- Solutions and buffers --- p.76 / Chapter 3.2.4 --- Enzymes and reagents --- p.77 / Chapter 3.3 --- Methods --- p.78 / Chapter 3.3.1 --- Restriction digestion --- p.78 / Chapter 3.3.2 --- Subcloning --- p.79 / Chapter 3.3.2.1 --- Gel purification --- p.79 / Chapter 3.3.2.2 --- Ligation --- p.79 / Chapter 3.3.2.3 --- Transformation --- p.80 / Chapter 3.3.3 --- Shotgun sequencing --- p.80 / Chapter 3.3.4 --- Sequencing reaction --- p.81 / Chapter 3.3.4.1 --- Preparation of sequencing gel --- p.81 / Chapter 3.3.4.2 --- Sequencing reaction --- p.81 / Chapter 3.4 --- Results --- p.83 / Chapter 3.4.1 --- Restriction mapping of cDNA inserts --- p.83 / Chapter 3.4.2 --- Sequencing results --- p.85 / Chapter 3.4.3 --- Sequence analysis --- p.90 / Chapter 3.5 --- Discussions --- p.103 / Chapter 3.5.1 --- Sequencing strategies --- p.103 / Chapter 3.5.2 --- Sequence analysis --- p.104 / Chapter Chapter Four --- Characterization of the putative differentially expressed genes / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Midazolam induced WEHI 3B JCS cells differentiation --- p.107 / Chapter 4.1.2 --- Gene expression profiles in embryogenesis --- p.108 / Chapter 4.2 --- Materials --- p.110 / Chapter 4.2.1 --- Mouse embryo multiple tissue Northern (MTN´ёØ) blot --- p.110 / Chapter 4.2.2 --- Megaprime´ёØ DNA labelling system --- p.110 / Chapter 4.2.3 --- Chemicals --- p.110 / Chapter 4.2.3 --- Solutions and buffers --- p.111 / Chapter 4.3 --- Methods --- p.112 / Chapter 4.3.1 --- Preparation of Northern blots --- p.112 / Chapter 4.3.1.1 --- Preparation of total RNA from midazolam induced WEHI 3B JCS cells --- p.112 / Chapter 4.3.1.2 --- Preparation of Northern blots --- p.112 / Chapter 4.3.2 --- Preparation of DNA probes --- p.113 / Chapter 4.3.2.1 --- Preparation of DNA templates --- p.113 / Chapter 4.3.2.2 --- Preparation of 32P labelled probes --- p.114 / Chapter 4.3.3 --- Northern blot analysis --- p.115 / Chapter 4.3.3.1 --- Northern hybridization --- p.115 / Chapter 4.3.3.2 --- Stripping of Northern blot --- p.115 / Chapter 4.4 --- Results --- p.117 / Chapter 4.4.1 --- Analysis of midazolam induced JCS cells total RNA --- p.117 / Chapter 4.4.2 --- Preparation of DNA templates for probe syntheses --- p.119 / Chapter 4.4.3 --- Northern Hybridization --- p.121 / Chapter 4.4.4 --- Comparison of the results of differential hybridization and Northern hybridization --- p.126 / Chapter 4.5 --- Discussions --- p.127 / Chapter 4.5.1 --- Northern hybridization --- p.127 / Chapter 4.5.1.1 --- Gene expression patterns under TNF-α induction --- p.127 / Chapter 4.5.1.2 --- Normalization of Northern hybridization --- p.129 / Chapter 4.5.1.3 --- Gene expression patterns under midazolam induction --- p.130 / Chapter 4.5.1.4 --- Gene expression pattern during embryo development --- p.133 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Identification of differentially expressed genes in TNF-α induced WEHI 3B JCS diffentiation --- p.135 / Chapter 5.2 --- Differentially expressed genes and myeloid leukemia cell differentiation --- p.137 / Chapter 5.3 --- Differentially expressed genes and embryogenesis --- p.142 / Chapter 5.4 --- Further studies --- p.144 / References --- p.145

by Ho Wai Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 207-243). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / Table of Contents --- p.x / List of Abbreviations --- p.xviii / List of Figures --- p.xxiv / List of Tables --- p.xxix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Traumatic brain injury --- p.1 / Chapter 1.2 --- Ceils of the nervous system: glia --- p.1 / Chapter 1.2.1 --- Astroglia - / Chapter 1.2.1.1 --- Molecular markers of astroglia --- p.3 / Chapter 1.2.1.2 --- Functions of astroglia --- p.3 / Chapter 1.2.2 --- Oligodendrocyte --- p.5 / Chapter 1.2.2.1 --- Molecular markers of oligodendrocyte --- p.6 / Chapter 1.2.2.2 --- Functions of oligodendrocyte --- p.6 / Chapter 1.2.3 --- Microglia --- p.7 / Chapter 1.2.3.1 --- Molecular markers of microglia --- p.7 / Chapter 1.2.3.2 --- Functions of microglia --- p.8 / Chapter 1.3 --- Cytokine and brain injury --- p.8 / Chapter 1.4 --- Tumor necrosis factor alpha (TNF-α) --- p.9 / Chapter 1.5 --- TNF-α receptor --- p.10 / Chapter 1.6 --- Biological activities of TNF-α --- p.11 / Chapter 1.7 --- Signaling mechanism --- p.13 / Chapter 1.7.1 --- Protein kinase C --- p.13 / Chapter 1.7.2 --- Protein kinase A --- p.14 / Chapter 1.7.3 --- p38 mitogen-activated protein kinase (p38 MAPK) --- p.15 / Chapter 1.7.3.1 --- Biological activities of p38 MAPK --- p.18 / Chapter 1.7.4 --- Inducible nitric oxide synthase (iNOS) --- p.20 / Chapter 1.7.5 --- cAMP responsive element binding protein (CREB) --- p.21 / Chapter 1.7.6 --- Transcription factor c-fos --- p.23 / Chapter 1.7.7 --- Nuclear factor kappa-B (NF-kB) --- p.24 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.26 / Chapter 1.9 --- β-adrenergic receptor (β-AR) --- p.28 / Chapter 1.9.1 --- Functions of β-AR in astrocytes --- p.29 / Chapter 1.10 --- Why do we use C6 glioma cell? --- p.31 / Chapter 1.11 --- Fluorescent differential display (FDD) --- p.34 / Chapter 1.12 --- Aims and Scopes of this project --- p.36 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Material --- p.40 / Chapter 2.1.1 --- Cell line --- p.40 / Chapter 2.1.2 --- Cell culture reagents --- p.40 / Chapter 2.1.2.1 --- Complete Dulbecco's modified Eagle medium (CDMEM) --- p.40 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.41 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.41 / Chapter 2.1.3 --- Recombinant cytokines --- p.41 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.42 / Chapter 2.1.4.1 --- Modulators of p38 mitogen-activated protein kinase (p38 MAPK) --- p.42 / Chapter 2.1.4.2 --- Modulators of protein kinase C (PKC) --- p.42 / Chapter 2.1.4.3 --- Modulators of protein kinase A (PKA) --- p.42 / Chapter 2.1.4.4 --- β-Adrenergic agonist and antagonist --- p.43 / Chapter 2.1.5 --- Antibodies --- p.44 / Chapter 2.1.5.1 --- Anti-p38 mitogen-activated protein kinase (p38 MAPK) antibody --- p.44 / Chapter 2.1.5.2 --- Anti-phosporylation p38 mitogen-activated protein kinase (p-p38 MAPK) antibody --- p.44 / Chapter 2.1.5.3 --- Antibody conjugates --- p.44 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.45 / Chapter 2.1.7 --- Reagents for DNase I treatment --- p.45 / Chapter 2.1.8 --- Reagents for reverse transcription of mRNA and fluorescent PCR amplification --- p.45 / Chapter 2.1.9 --- Reagents for fluorescent differential display --- p.46 / Chapter 2.1.10 --- Materials for excision of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.11 --- Reagents for reamplification of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.12 --- Reagents for subcloning of reamplified cDNA fragments --- p.47 / Chapter 2.1.13 --- Reagents for purification of plasmid DNA from recombinant clones --- p.47 / Chapter 2.1.14 --- Reagents for DNA sequencing of differentially expressed cDNA fragments --- p.47 / Chapter 2.1.15 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.1.16 --- Reagents for electrophoresis --- p.50 / Chapter 2.1.17 --- Reagents and buffers for Western blot --- p.50 / Chapter 2.1.18 --- Other chemicals and reagents --- p.50 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.51 / Chapter 2.3 --- RNA isolation --- p.52 / Chapter 2.3.1 --- Measurement of RNA yield --- p.53 / Chapter 2.4 --- DNase I treatment --- p.53 / Chapter 2.5 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.54 / Chapter 2.6 --- Fluorescent differentia display --- p.55 / Chapter 2.7 --- Excision of differentially expressed cDNA fragments --- p.59 / Chapter 2.8 --- Reamplification of differentially expressed cDNA fragments --- p.59 / Chapter 2.9 --- Subcloning of reamplified cDNA fragments --- p.60 / Chapter 2.10 --- Purification of plasmid DNA from recombinant clones --- p.63 / Chapter 2.11 --- DNA sequencing of differentially expressed cDNA fragments --- p.64 / Chapter 2.12 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.66 / Chapter 2.13 --- Western bolt analysis --- p.67 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- DNase I treatment --- p.71 / Chapter 3.2 --- FDD RT-PCR and band excision --- p.71 / Chapter 3.3 --- Reamplification of excised cDNA fragments --- p.74 / Chapter 3.4 --- Subcloning of reamplified cDNA fragments --- p.77 / Chapter 3.5 --- DNA sequencing of subcloned cDNA fragments --- p.77 / Chapter 3.6 --- Confirmation of the differentially expressed cDNA fragments by RT-PCR and Western blotting --- p.84 / Chapter 3.6.1 --- Effects of TNF-α on p38a mitogen protein kinase (p38 α MAPK) --- p.84 / Chapter 3.6.2 --- Effects of TNF-α on p38 a MAPK and p-p38 α MAPK protein level --- p.86 / Chapter 3.7 --- Effects of TNF-α on p38 MAPK --- p.88 / Chapter 3.7.1 --- "Effects of TNF-α on p38 α, β,γ andδ MAPK" --- p.88 / Chapter 3.7.2 --- Role of TNF-receptor (TNF-R) subtype in the TNF-α-induced p3 8 MAPK expression in C6 cells --- p.89 / Chapter 3.7.3 --- The signaling system mediating TNF-α-induced p38 a MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.1 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.2 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.98 / Chapter 3.7.4 --- The relationship between p38 MAPK and β-adrenergic mechanisms in C6 cells --- p.99 / Chapter 3.7.4.1 --- Effects of isoproterenol and propanol on p38 MAPK mRNA levels in C6 cells --- p.103 / Chapter 3.7.4.2 --- Effects of β1-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.106 / Chapter 3.7.4.3 --- Effects of β2-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.107 / Chapter 3.8 --- The relationship between p3 8 MAPK and inducible nitric oxide synthase (iNOS) expression --- p.113 / Chapter 3.8.1 --- Effects of TNF-α on the iNOS expression in C6 cells --- p.113 / Chapter 3.8.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3 --- The signaling system mediating TNF-α-induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3.1 --- The involvement of p38 MAPK in the TNF-α-induced iNOS expression in C6 cells --- p.117 / Chapter 3.8.3.2 --- The involvement of PKA in the TNF-α-induced iNOS expression in C6 cells --- p.119 / Chapter 3.9 --- The relationship between p38 MAPK and cAMP-responsive element binding protein (CREB) expression --- p.120 / Chapter 3.9.1 --- Effects of TNF-α on the CREB expression in C6 cells --- p.120 / Chapter 3.9.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced CREB expression in C6 cells --- p.124 / Chapter 3.9.3 --- The signaling system mediating TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.1 --- The involvement of p38 MAPK in the TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.2 --- The involvement of PKC in the TNF-α-induced CREB expression in C6 cells --- p.128 / Chapter 3.9.3.3 --- The involvement of PKA in TNF-α-induced CREB expression in C6 cells --- p.129 / Chapter 3.9.4 --- The relationship between CREB and β-adrenergic mechanisms in C6 cells --- p.136 / Chapter 3.9.4.1 --- Effects of isoproterenol and propanol on CREB mRNA levels in C6 cells --- p.136 / Chapter 3.9.4.2 --- Effects of β1-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.139 / Chapter 3.9.4.3 --- Effects of (32-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.142 / Chapter 3.10 --- The relationship between p38 MAPK and transcription factor c-fos expression --- p.146 / Chapter 3.10.1 --- Effects of TNF-α on the c-fos expression in C6 cells --- p.146 / Chapter 3.10.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced c-fos expression in C6 cells --- p.146 / Chapter 3.10.3 --- The signaling system mediating TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.1 --- The involvement of p38 MAPK in the TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.2 --- The involvement of PKC in the TNF-α-induced c-fos expression in C6 cells --- p.151 / Chapter 3.10.3.3 --- The involvement of PKA in TNF-α-induced c-fos expression in C6 cells --- p.154 / Chapter 3.10.4 --- The relationship between c-fos and β-adrenergic mechanisms in C6 cells --- p.157 / Chapter 3.10.4.1 --- Effects of isoproterenol and propanolol on c-fos mRNA levels in C6 cells --- p.157 / Chapter 3.10.4.2 --- Effects of β1-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.160 / Chapter 3.10.4.3 --- Effects of β2-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.164 / Chapter 3.11 --- The relationship between p38 MAPK and transcription factor NF-kB expression --- p.168 / Chapter 3.11.1 --- Effects of TNF-α on the NF-kB expression in C6 cells --- p.168 / Chapter 3.11.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced NF-kB expression in C6 cells --- p.168 / Chapter 3.11.3 --- The signaling system mediating TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.1 --- The involvement of p38 MAPK in the TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.2 --- The involvement of PKC in the TNF-α-induced NF-kB expression in C6 cells --- p.173 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of tumor-necrosis factor-alpha (TNF-α) on C6 cell proliferations --- p.176 / Chapter 4.2 --- The Signaling System Involved in TNF-α-Induced p38 MAPK Expression in C6 cells --- p.178 / Chapter 4.3 --- The Signaling System Involved in TNF-α-Induced iNOS Expression in C6 cells --- p.184 / Chapter 4.4 --- The Signaling System Involved in TNF-α-Induced CREB Expression in C6 cells --- p.186 / Chapter 4.5 --- The Signaling System Involved in TNF-α-Induced c-fos Expressionin in C6 cells --- p.190 / Chapter 4.6 --- The Signaling System Involved in TNF-α-Induced NF-kB Expression in C6 cells --- p.193 / Chapter 4.7 --- Conclusions --- p.195 / Chapter 4.8 --- Possible application / References