by Kwan, Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 108-116). / Abstract --- p.i / Achnowledgment --- p.ii / List of abbreviations --- p.iii / List of table and figures --- p.v / Table of contents --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- THE DISCOVERY OF TUMOUR NECROSIS FACTOR (TNF) --- p.1 / Chapter 1.2 --- THE MOLECULE AND ITS RECEPTORS --- p.1 / Chapter 1.3 --- THE BIOLOGICAL ACTIVITIES OF TNF --- p.3 / Chapter 1.4 --- STUDIES ON THE CYTOTOXIC MECHANISM OF TNF --- p.4 / Chapter 1.5 --- A TENTATIVE MECHANISM OF TNF CYTOTOXICITY --- p.11 / Chapter 1.6 --- THE GLUTATHIONE SYSTEM : A CELLULAR PROTECTIVE MECHANISM AGAINST OXIDATIVE STRESS …… --- p.12 / Chapter 1.7 --- OBJECTIVE AND STRATEGY OF THIS STUDY --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND APPARATI / Chapter 2.1 --- CELL LINES --- p.19 / Chapter 2.2 --- "ISOLATION, MAINTENANCE AND SUBCULTURE OF CELL LINES" --- p.19 / Chapter 1. --- Plain RPMI-1640 medium / Chapter 2. --- Penicillin-streptomycin solution / Chapter 3. --- Foetal bovine serum / Chapter 4. --- Complete RPMI-1640 medium / Chapter 5. --- Trypsin-ethylenediaminetetraacetate solution / Chapter 6. --- Phosphate buffered saline / Chapter 7. --- Cycloheximide / Chapter 8. --- Actinomycin D / Chapter 9. --- Trypan blue stain / Chapter 10. --- Neutral red stain / Chapter 11. --- Recombinant human tumour necrosis factor / Chapter 12. --- Cell culture plates and flasks / Chapter 2.3 --- GROWTH CHARACTERISTIC --- p.22 / Chapter 1. --- Tritiated Thymidine / Chapter 2. --- Tritiated Leucine / Chapter 3. --- Trichloroacetic acid / Chapter 4. --- Scintillation cocktail / Chapter 2.4 --- "RESPONSE TOWARDS ANTICANCER DRUGS, CYTOTOXIC AGENTS, AND ENZYME MODULATORS" --- p.23 / Chapter 1. --- N-acetyl-DL-homocysteinethiolactone / Chapter 2. --- Diethyldithiocarbamic acid / Chapter 3. --- Doxorubicin / Chapter 4. --- Acivicin / Chapter 5. --- Ethacrynic acid / Chapter 6. --- "L'Buthionine-[S,R]-sulfoximine" / Chapter 7. --- Hydrogen peroxide / Chapter 8. --- Methotrexate / Chapter 9. --- Menadione / Chapter 2.5 --- CULTURE OF BACTERIAL CELLS --- p.27 / Chapter 1. --- Ampicillin stock solution / Chapter 2. --- Chloramphenicol stock solution / Chapter 3. --- Tetracycline stock solution / Chapter 4. --- Luria-Bertani medium / Chapter 5. --- LB with ampicillin / Chapter 6. --- SOB medium / Chapter 7. --- SOB medium with ampicillin / Chapter 8. --- SOC medium / Chapter 9. --- SB medium / Chapter 10. --- SB medium with ampicillin / Chapter 11. --- Agar plates / Chapter 2.6 --- PREPARATION OF DNA PROBES FROM BACTERIAL CLONES --- p.29 / Chapter 1. --- FlexiPrep Kit / Chapter 2. --- Restriction endonucleases / Chapter 3. --- GeneClean® II Kit / Chapter 4. --- cDNA clones for making DNA probes / Chapter 5. --- TrisHCl EDTA buffer / Chapter 2.7. --- ELECTROPHORESIS OF DNA --- p.31 / Chapter 1. --- EDTA stock solution / Chapter 2. --- Tris acetate EDTA electrophoresis buffer / Chapter 3. --- Tris borate EDTA electrophoresis buffer / Chapter 4. --- Ethidium bromide / Chapter 5. --- DNA molecular size markers / Chapter 6. --- TAE/TBE agarose gel slab / Chapter 2.8 --- CONSTRUCTION OF MURINE TNFR1 PARTIAL cDNA CLONE --- p.33 / Chapter 1. --- Frist strand cDNA synthesis Kit / Chapter 2. --- Murine TNFR1 forward and reverse primers / Chapter 3. --- Polymerase chain reaction reagents / Chapter 4. --- Cloning vector / Chapter 5. --- Modifing enzymes / Chapter 6. --- T7 SequencingTM Kit / Chapter 7. --- Acrylamide/bis gel stock solution / Chapter 8. --- Urea / Chapter 9. --- TEMED and ammonium persulphate / Chapter 10. --- β-Galactosidase colour test reagents / Chapter 11. --- TFB solution / Chapter 12. --- DnD solution / Chapter 2.9. --- RADIOLABELLING OF DNA PROBES --- p.35 / Chapter 1. --- Oligolabelling kit / Chapter 2. --- Redivue [α-32P] dCTP / Chapter 3. --- PUSH column / Chapter 2.10 --- EXTRACTION OF TOTAL RNA FROM CELL LINES --- p.36 / Chapter 1. --- N-Lauroylsarcosine / Chapter 2. --- 2M sodium acetate (pH48) / Chapter 3. --- Phenol / Chapter 4. --- Isopropanol / Chapter 5. --- Ethanol / Chapter 6. --- Extraction buffer / Chapter 7. --- Chloroform / Chapter 8. --- Isoamyl alcohol / Chapter 2.11 --- HYBRIDIZATION AND NORTHERN ANALYSIS --- p.37 / Chapter 1. --- 20XSSC / Chapter 2. --- 5X formaldehyde running buffer / Chapter 3. --- RNA sample buffer / Chapter 4. --- 10X RNA loading buffer / Chapter 5. --- Formaldehyde slab gel / Chapter 6. --- Hybond®-N membrane / Chapter 7. --- Immobilon®-N membrane / Chapter 8. --- Salmon sperm DNA / Chapter 9. --- Sodium dodecyl sulphate / Chapter 10. --- Dextran sulphate / Chapter 11. --- Kodak Biomax MR and X-OMAT films and developing kits / Chapter 2.12 --- APPARATI USED --- p.39 / Chapter CHAPTER 3 --- METHODS / Chapter 3.1 --- ISOLATION AND MAINTENANCE OF TNF RESISTANT L929 CELLS --- p.40 / Chapter 3.1.1 --- Culture of L929 cells / Chapter 3.1.2 --- Trypan blue exclusion test / Chapter 3.1.3 --- Isolation of TNF-resistant variants of L929 / Chapter 3.1.4 --- Verification of the TNF-resistant trait of rL929 / Chapter 3.1.5 --- Neutral red uptake assay / Chapter 3.2 --- COMPARING L929 AND rL929 CELLS IN TERMS OF GROWTH CHARACTERISTICS --- p.43 / Chapter 3.2.1 --- Doubling time / Chapter 3.2.2 --- Rate of protein synthesis / Chapter 3.2.3 --- Rate of DNA synthesis / Chapter 3.3 --- COMPARING L929 AND rL929 CELLS IN TERMS OF RESPONSE TOWARDS DIFFERENT ENZYME INHIBITORS AND CYTOTOXIC AGENTS --- p.44 / Chapter 3.3.1 --- TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.2 --- Effects of inhibitors of gene transcription and protein synthesis on TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.3 --- Cytotoxic effect of hydrogen peroxide and menadione on L929 and rL929 cells --- p.44 / Chapter 3.3.4 --- TNF cytotoxicity on L929 and rL929 cells: effect of N-acetyl homocysteine thiolatone --- p.45 / Chapter 3.3.4.1 --- The tolerant limit of AHCT / Chapter 3.3.4.2 --- Effect of AHCT on TNF cytotoxicity / Chapter 3.3.5 --- TNF cytotoxicity on L929 and rL929 cells: effect of diethyldithiocarbamate --- p.46 / Chapter 3.3.5.1 --- The tolerant limit of DEDTC / Chapter 3.3.5.2 --- Effect of DEDTC on TNF cytotoxicity / Chapter 3.3.6 --- TNF cytotoxicity on L929 and rL929 cells: effect of buthionice sulfoximine --- p.47 / Chapter 3.3.6.1 --- The tolerant limit of BSO / Chapter 3.3.6.2 --- Effect of BSO on TNF cytotoxicity / Chapter 3.3.7 --- TNF cytotoxicity on L929 and rL929 cells: effect of Acivicin --- p.47 / Chapter 3.3.7.1 --- The tolerant limit of acivicin / Chapter 3.3.7.2 --- Effect of acivicin on TNF cytotoxicity / Chapter 3.3.8 --- TNF cytotoxicity on L929 and rL929 cells: effect of ethacrynic acid --- p.48 / Chapter 3.3.8.1 --- The tolerant limit of ethacrynic acid / Chapter 3.3.8.2 --- Effect of ethacrynic acid on TNF cytotoxicity / Chapter 3.3.9 --- Cytotoxic effect of doxorubicin on L929 and rL929 cells --- p.49 / Chapter 3.3.10 --- TNF cytotoxicity of L929 cells: effect of N-acetyl cysteine --- p.49 / Chapter 3.3.11 --- Cytotoxic effect of methotrexate on L929 and rL929 cells --- p.50 / Chapter 3.3.12 --- Cytotoxic effect of hyperthermia on L929 and rL929 cells --- p.50 / Chapter 3.4 --- NORTHERN ANALYSIS AND HYBRIDIZATION --- p.51 / Chapter 3.4.1. --- Preparing RNA blots --- p.51 / Chapter 3.4.1.1 --- Extraction of total RNA from cells / Chapter 3.4.1.2 --- Making equal loading of RNA samples in formaldehyde gel electrophoresis / Chapter 3.4.1.3 --- Northern blotting of RNA / Chapter 3.4.2. --- Preparation of cDNA probes --- p.53 / Chapter 3.4.2.1 --- Preparing plasmids from A TCC clones / Chapter 3.4.2.2 --- Preparing TNFR1 probe from first-strand cDNA of L929 cells / Chapter 1. --- Construction of recombinant clone from the PCR product of TNFRl fragment / Chapter 2. --- Transforming the recombinant vector into JM109 host / Chapter 3. --- Sequencing of PCR product for identity confirmation / Chapter 3.4.2.3 --- Preparing DNA inserts from plasmids / Chapter 3.4.3 --- Radiolabelling of cDNA probes --- p.56 / Chapter 3.4.4 --- Hybridization of radioactive probes to RNA blots --- p.57 / Chapter CHAPTER 4 --- RESULTS AND DISCUSSIONS / Chapter 4.1 --- ISOLATION OF TNF-RESISTANT VARIANTS OF L929 CELLS --- p.58 / Chapter 4.1.1 --- Single cell subcloning of TNF-resistant L929 variants / Chapter 4.1.2 --- Growth rates of L929 and rL929 cells / Chapter 4.1.3 --- Rate of protein synthesis in L929 and rL929 cells / Chapter 4.1.4 --- Rate of DNA synthesis in L929and rL929 cells / Chapter 4.2 --- EFFECT OF INHIBITORS OF GENE TRANSCRIPTION AND PROTEIN SYNTHESIS ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.67 / Chapter 4.3 --- RESPONSE OF L929 AND rL929 CELLS TOWARDS VARIOUS CYTOTOXIC AGENTS --- p.70 / Chapter 4.3.1 --- "Response towards methotrexate, an anti-metabolite used in cancer treatment" / Chapter 4.3.2 --- "Response towards doxorubicin, an chemotherapeutic agent used in cancer treatment" / Chapter 4.3.3 --- "Response towards menadione, a cytotoxic agent known to generate free radicals inside cells" / Chapter 4.3.4 --- Response towards hydrogen peroxide: a highly oxidative agent / Chapter 4.3.5 --- "Response towards hyperthermia, a treatment known to exert oxidative stress on cells" / Chapter 4.4 --- EFFECTS OF MODULATORS OF CYTOSOLIC SUPEROXIDE DISMUTASE ON TNF CYTOTOXICITY ON L929 and rL929 CELLS --- p.77 / Chapter 4.5 --- EFFECT OF MODULATORS OF GLUTATHIONE METABOLISM ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.82 / Chapter 4.5.1 --- "Effects of L-buthionine [S,R] sulfoximine, an inhibitor of glutathione synthesis" --- p.82 / Chapter 4.5.2 --- "Effect of N-acetyl cysteine, a cysteine derivative" --- p.84 / Chapter 4.5.3 --- "Effects of acivicin , an inhibitor of GSH reuptake and recycle" --- p.85 / Chapter 4.5.4 --- "Effect of ethacrynic acid, an inhibitor of glutathione S- transferase" --- p.87 / Chapter 4.6 --- GENE EXPRESSION IN L929 AND rL929 CELLS IN THE COURSE OF TNF CHALLENGE --- p.89 / Chapter 4.6.1 --- Isolation of total RNA from L929 and rL929 cells --- p.89 / Chapter 4.6.2 --- Preparation of DNA probes for hybridization --- p.89 / Chapter 4.6.3 --- Hybridization of specific probes on RNA blots --- p.90 / Chapter 4.6.3.1 --- Expression of heat shock protein --- p.70 / Chapter 4.6.3.2 --- Expression of the p55 TNF receptor / Chapter 4.6.3.3 --- Expression of glutathione reductase / Chapter 4.6.3.4 --- Expression of glutathione S-transferase pi / Chapter 4.7 --- DISCUSSIONS OF THE EXPERIMENTAL RESULTS --- p.97 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.104 / APPENDIX / Generation of the TNF receptor 1 cDNA probe --- p.106 / REFERENCES --- p.108

by Suk-Fung Tsang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 101-112). / Abstract --- p.i / Acknowledgement --- p.iii / Abbreviation --- p.iv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- TNF molecule / Chapter 1.2 --- Molecular biosynthesis of TNF / Chapter 1.3 --- Antitumor activity of TNF / Chapter 1.4 --- Macrophage-mediated immunity / Chapter 1.5 --- Endogenous production of TNF / Chapter 1.6 --- LPS : the potent inducer for TNF release / Chapter 1.7 --- Natural product: as primer or inducer / Chapter 1.8 --- Aim of this project / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials / Chapter 2.2 --- Animals / Chapter 2.3 --- Cell line / Chapter 2.4 --- Transformed cell line : EAT cells invivo / Chapter 2.5 --- Reagents / Chapter 2.6 --- Methods / Chapter Chapter 3 --- Preparation of sample --- p.33 / Chapter 3.1 --- Alcohol precipitaion of Bupleuri radix / Chapter 3.2 --- Endogenous TNF production by BR fractions / Chapter Chapter 4 --- Purification of BRI --- p.38 / Chapter 4.1 --- Gel filtration chromatography of BRI / Chapter 4.2 --- Anion exchange chromatography / Chapter Chapter 5 --- Purification of PQI --- p.52 / Chapter 5.1 --- Gel filtration chromatography of PQI / Chapter 5.2 --- Anion exchange chromatography / Chapter Chapter 6 --- Capacity of BR and PQ as eliciting agent for endogenous TNF production --- p.62 / Chapter 6.1 --- Time course of endogenous TNF production by BRI subfractions / Chapter 6.2 --- Time course of endogenous TNF production by PQI subfractions / Chapter 6.3 --- BRI subfractions as eliciting agents / Chapter 6.4 --- PQI subfractions as eliciting agents / Chapter Chapter 7 --- Are BR and PQ priming agents in endogenous TNF production ？ --- p.71 / Chapter 7.1 --- Priming by intraperitoneal route / Chapter 7.2 --- Priming by intravenous route / Chapter Chapter 8 --- Removal of LPS by acetic acid treatment --- p.79 / Chapter Chapter 9 --- Antitumor activities of BRI subfractionsin relationship with TNF production --- p.86 / Chapter 9.1 --- BRI subfraction as eliciting agent / Chapter 9.2 --- Pretreatment with BRIA subfractions followed by LPS treatment / Chapter Chapter 10 --- Conclusion --- p.95 / Bibliography --- p.101

"July 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, December 2001. / Includes bibliographical references. Also available on the Internet.

The human melanocortin receptors (hMCRs) play a fundamental role in human behavior such as satiety, feeding, sexual and more. A set of SHU9119 peptide derivatives were studied for their structure-activity relationships. These peptides contained a sequential a-N-methylation amino acid scan.A second set of peptide derivatives intended to be used to create TNF-a; inhibition, via the melanocortin receptors. These peptides were shown to bind to all of the hMCR receptors, and only exhibit cAMP stimulation at hMC1R/hMC5R.The data from both of the sets of compounds illustrate that small changes in the stereochemistry of the SH9119 and TNF-a; derivatives cause drastic changes in the binding and the agonistic/antagonist properties of the compounds.This thesis determined the effect that hMC1R mutations have on the binding and cAMP response of well characterized ligands. This study ruled out 9 different residues for being the required for the cAMP response of the hMC1R.

Due to its escalating prevalence, obesity is becoming a leading cause of morbidity and mortality worldwide. Obesity is a complex health problem accompanied by metabolic abnormalities and low-grade inflammation that increases the risk for developing comorbidities including type 2 diabetes. Recent evidence supports a role for fat (adipose) tissue derived factors, called adipokines, in the development of obesity and obesity-related metabolic pathologies. Chemerin is an adipokine that mediates immune and metabolic effects through the chemokine-like receptor 1 (CMKLR1). Chemerin is secreted as an inactive proform, prochemerin, which subsequently undergoes enzymatic cleavage into multiple chemerin products that differentially activate CMKLR1. Multiple studies have reported elevated total chemerin (a combination of prochemerin and various chemerin products) in obese humans suggesting chemerin involvement in obesity pathophysiology. However, the observational nature of these human studies have restricted them from identifying specific forms of chemerin that are elevated in obesity and the mechanisms that govern them. Herein, I have reported that the levels of both serum total chemerin and chemerin products capable of activating CMKLR1 are elevated in obese mice and in wild type mice following treatment with an obesity-associated inflammatory mediator tumor necrosis factor-? (TNF?). Likewise, cultured adipocytes produced active chemerin under basal conditions and highly active chemerin following TNF? treatment as measured by CMKLR1 activation. The current belief is that prochemerin circulates through blood primed for activation by immune and fibrinolytic enzymes present within injured tissues. My results challenge this theory, identifying adipocytes as cells alone produce and proteolytically activate chemerin. Under basal conditions, a balance between activating serine proteases and deactivating aminopeptidases governed the amount of CMKLR1-activating chemerin formed by adipocytes. Treatment of adipocytes with TNF? elevated the levels of serine proteases elastase and tryptase, which cumulatively shifted the proteolytic balance toward the production of chemerin products that highly activate CMKLR1. Taken together, my results are the first to identify that local TNF? triggers increased adipocyte production of chemerin providing an explanation for the elevated concentrations of chemerin in obese animals and humans. Furthermore, adipocyte processing represents a novel mechanism that likely governs the amount and type of circulating chemerin in obesity.

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Antigen induced activation of Th1 cells in the peripheral blood leads to elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that have been directly linked to disease induction and neuropathic pain. It was hypothesized that following antigenic induction, cytokines gain access to the spinal cord and participate in direct cellular interaction with dorsal horn neurons. Using an animal model of MS, we show that TNF-α gene and protein expression in the dorsal root ganglia (DRG) and spinal cord tissue is increased in the active group. In addition, our findings show TNF-α mRNA expression in the dorsal root entry point. Therefore, our results support the hypothesis that antigen induced DRG derived TNF-α can transport to the spinal cord via the dorsal roots and is involved in the underlying pathogenesis of MS induced neuropathic pain.

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Antigen induced activation of Th1 cells in the peripheral blood leads to elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that have been directly linked to disease induction and neuropathic pain. It was hypothesized that following antigenic induction, cytokines gain access to the spinal cord and participate in direct cellular interaction with dorsal horn neurons. Using an animal model of MS, we show that TNF-α gene and protein expression in the dorsal root ganglia (DRG) and spinal cord tissue is increased in the active group. In addition, our findings show TNF-α mRNA expression in the dorsal root entry point. Therefore, our results support the hypothesis that antigen induced DRG derived TNF-α can transport to the spinal cord via the dorsal roots and is involved in the underlying pathogenesis of MS induced neuropathic pain.

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