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

by Wong Kin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 348-373). / Abstracts in English and Chinese. / Abstract --- p.ii / 撮要 --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xviii / List of Tables --- p.xxi / List of Figures --- p.xxii / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1. --- Events happened in brain injury --- p.1 / Chapter 1.2. --- An alternate approach based on neuronal regeneration --- p.3 / Chapter 1.3. --- Fate of astrocytes after brain injury --- p.4 / Chapter 1.3.1. --- General information of astrocytes --- p.4 / Chapter 1.3.2. --- Functions of astrocytes --- p.5 / Chapter 1.4. --- Factors relate to astrocytes proliferation --- p.7 / Chapter 1.4.1. --- TNF-α --- p.8 / Chapter 1.4.2. --- β adrenergic mechanism and astrocyte proliferation --- p.11 / Chapter 1.5. --- Cell cycle-related proteins --- p.13 / Chapter 1.5.1. --- Maturation promoting factor (MPF) --- p.15 / Chapter 1.5.2. --- Early G1 phase --- p.16 / Chapter 1.5.3. --- Retinoblastoma protein (pRb) --- p.18 / Chapter 1.5.4. --- Cyclin-dependent kinase (cdk) activating kinase (Cak) --- p.19 / Chapter 1.5.5. --- "Cyclin, cdks, cki" --- p.20 / Chapter 1.5.5.1. --- Cyclins --- p.20 / Chapter 1.5.5.1.1. --- Cyclin D --- p.21 / Chapter 1.5.5.1.2. --- Cyclin E --- p.22 / Chapter 1.5.5.1.3. --- Cyclin A --- p.23 / Chapter 1.5.5.1.4. --- Cyclin B --- p.23 / Chapter 1.5.5.2. --- Cyclin-dependent kinases (cdks) --- p.24 / Chapter 1.5.5.3. --- Cyclin-dependent kinase inhibitor (cki) --- p.24 / Chapter 1.5.5.3.1. --- INK4 proteins (inhibitors of cdk-4 and cdk-6) --- p.25 / Chapter 1.5.5.3.2. --- p21 family proteins --- p.25 / Chapter 1.5.5.3.2.1. --- p21 --- p.25 / Chapter 1.5.5.3.2.2. --- p27 --- p.25 / Chapter 1.6. --- Apoptosis related proteins --- p.26 / Chapter 1.6.1. --- bcl-2 family --- p.26 / Chapter 1.6.1.1. --- bcl-2 --- p.26 / Chapter 1.6.1.2. --- bcl-x --- p.27 / Chapter 1.6.1.3. --- bcl-xα --- p.27 / Chapter 1.6.1.4. --- bcl-w --- p.28 / Chapter 1.6.1.5. --- Myeloid cell leukemia factor 1 (Mcl-1) --- p.28 / Chapter 1.7. --- C6 glioma cell line --- p.28 / Chapter 1.8. --- Aim of this project --- p.30 / Chapter CHAPTER 2. --- MATERIALS & METHODS / Chapter 2.1. --- Materials / Chapter 2.1.1. --- Rat C6 glioma cell line --- p.32 / Chapter 2.1.2. --- Cell culture materials preparation / Chapter 2.1.2.1. --- Complete Dulbecco's Modified Medium (cDMEM) --- p.32 / Chapter 2.1.2.2. --- Serum-free Dulbecco's Modified Medium (sDMEM) --- p.33 / Chapter 2.1.2.3. --- Phosphate buffered saline (PBS) --- p.33 / Chapter 2.1.3. --- Drug preparation / Chapter 2.1.3.1. --- Recombinant cytokines --- p.34 / Chapter 2.1.3.2. --- Antibodies / Chapter 2.1.3.2.1. --- Antibodies used in expression analysis --- p.34 / Chapter 2.1.4. --- Antibodies used in Western blotting --- p.34 / Chapter 2.1.5. --- Reagents for RNA isolation --- p.36 / Chapter 2.1.6. --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.36 / Chapter 2.1.7. --- Reagents for Electrophoresis --- p.38 / Chapter 2.1.8. --- Reagents and buffers for Western blotting --- p.38 / Chapter 2.1.9. --- Other chemicals and reagents --- p.39 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Maintenance of C6 cells --- p.39 / Chapter 2.2.2. --- Preparation of cells for assays --- p.40 / Chapter 2.2.3. --- Drugs preparation --- p.40 / Chapter 2.2.4. --- Determination of RNA expression by RT-PCR analysis / Chapter 2.2.4.1. --- RNA extraction --- p.41 / Chapter 2.2.4.2. --- Spectrophotometric Quantitation of DNA and RNA --- p.43 / Chapter 2.2.4.3. --- RNA gel electrophoresis --- p.43 / Chapter 2.2.4.4. --- Reverse transcription-polymerase chain reaction (RT- PCR) --- p.43 / Chapter 2.2.4.5. --- Separation of PCR products by agarose gel electrophoresis --- p.43 / Chapter 2.2.4.6. --- Quantification of band density --- p.45 / Chapter 2.2.4.7. --- Restriction enzyme (RE) digestion --- p.45 / Chapter 2.2.5. --- Determination of protein expression by Western blotting / Chapter 2.2.5.1. --- Total protein extraction --- p.46 / Chapter 2.2.5.2. --- Western blotting analysis --- p.46 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1. --- Effects of TNF-α on cell cycle related genes and proteins expression --- p.49 / Chapter 3.1.1. --- Effects of TNF-α on the time courses of cyclin D1 gene and protein expression --- p.49 / Chapter 3.1.2. --- Effect of TNF-α on the time course of cyclin D2 gene expression --- p.50 / Chapter 3.1.3. --- Effects of TNF-α on the time courses of cyclin D3 gene and protein expression --- p.53 / Chapter 3.1.4. --- Effects of TNF-α on the time courses of cdk-4 gene and protein expression --- p.55 / Chapter 3.1.5. --- Effects of TNF-α on the time courses of cyclin E gene and protein expression --- p.55 / Chapter 3.1.6. --- Effects of TNF-α on the time courses of cdk-2 gene and protein expression --- p.58 / Chapter 3.1.7. --- Effects of TNF-α on the time courses of p15 gene and protein expression --- p.61 / Chapter 3.1.8. --- Effects of TNF-α on the time courses of p27 gene and protein expression --- p.61 / Chapter 3.1.9. --- Effects of TNF-α on the time courses of p21 gene and protein expression --- p.64 / Chapter 3.1.10. --- Effects of TNF-α on the time courses of p130 gene and protein expression --- p.66 / Chapter 3.1.11. --- Effects of TNF-α on the time courses of Cak gene and protein expression --- p.66 / Chapter 3.1.12. --- Effects of TNF-α on the time courses of cyclin H gene and protein expression --- p.68 / Chapter 3.1.13. --- Effects of TNF-α on the time courses of cyclin B gene and protein expression- --- p.71 / Chapter 3.1.14. --- Effect of TNF-α on the time course of bcl-2 protein expression --- p.71 / Chapter 3.1.15. --- Effects of TNF-α on the time courses of bcl-XL gene and protein expression --- p.73 / Chapter 3.1.16. --- Effect of TNF-α on the time course of bcl-xα gene expression --- p.73 / Chapter 3.1.17. --- Effects of TNF-α on the time courses of bcl-w gene and protein expression --- p.76 / Chapter 3.1.18. --- Effects of TNF-α on the time courses of Mcl-1 gene expression --- p.76 / Chapter 3.2. --- Effects of TNF-R1 and -R2 on cell cycle related genes and proteins expression --- p.81 / Chapter 3.2.1. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin D1 gene and protein expression --- p.81 / Chapter 3.2.2. --- Effect of blocking TNF-R1/ -R2 on the time course of cyclin D2 gene expression --- p.82 / Chapter 3.2.3. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin D3 gene and protein expression --- p.85 / Chapter 3.2.4. --- Effects of blocking TNF-R1/ -R2 on the time courses of cdk-4 gene and protein expression --- p.90 / Chapter 3.2.5. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin E gene and protein expression --- p.93 / Chapter 3.2.6. --- Effects of blocking TNF-R1/ -R2 on the time courses of cdk-2 gene and protein expression --- p.93 / Chapter 3.2.7. --- Effects of blocking TNF-R1/ -R2 on the time courses of p15 gene and protein expression --- p.96 / Chapter 3.2.8. --- Effects of blocking TNF-R1/ -R2 on the time courses of p27 gene and protein expression --- p.99 / Chapter 3.2.9. --- Effects of blocking TNF-R1/ -R2 on the time courses of p21 gene and protein expression --- p.103 / Chapter 3.2.10. --- Effects of blocking TNF-R1/ -R2 on the time courses of pl30 gene and protein expression --- p.106 / Chapter 3.2.11. --- Effect of blocking TNF-R1/ -R2 on the time course of Cak gene expression --- p.110 / Chapter 3.2.12. --- Effects of blocking TNP-R1/ -R2 on the time courses of cyclin H gene and protein expression --- p.110 / Chapter 3.2.13. --- Effects of blocking TNF-R1/ -R2 on the time courses of cyclin B gene and protein expression --- p.112 / Chapter 3.2.14. --- Effect of blocking TNF-R1/ -R2 on the time course of bcl-2 protein expression --- p.116 / Chapter 3.2.15. --- Effects of blocking TNF-R1/ -R2 on the time courses of bcl-xL gene and protein expression --- p.119 / Chapter 3.2.16. --- Effect of blocking TNF-R1/ -R2 on the time course of bcl-xα gene expression --- p.122 / Chapter 3.2.17. --- Effects of blocking TNF-R1/ -R2 on the time courses of bcl-w gene and protein expression --- p.124 / Chapter 3.2.18. --- Effect of blocking TNF-R1/ -R2 on the time course of Mcl-1 gene expression --- p.124 / Chapter 3.3. --- "Effects of other cytokines (IL-6, IL-lα, IL-lβ, IFγ) on cell cycle related genes and proteins expression" --- p.129 / Chapter 3.3.1. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on cyclin D1 gene and protein expression" --- p.129 / Chapter 3.3.2. --- "Effects of TNF-a, IL-6, IL-lα, IL-lβ, IFγ on cyclin D2 gene and protein expression" --- p.132 / Chapter 3.3.3. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin D3 gene and protein expression" --- p.136 / Chapter 3.3.4. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cdk-4 gene and protein expression" --- p.140 / Chapter 3.3.5. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin E gene and protein expression" --- p.144 / Chapter 3.3.6. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cdk-2 gene and protein expression" --- p.148 / Chapter 3.3.7. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on pl5 gene and protein expression" --- p.152 / Chapter 3.3.8. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on p27 gene and protein expression" --- p.152 / Chapter 3.3.9. --- "Effects of TNF-α, IL-6, IL-lα, IL-ip, IFγ on p21 gene and protein expression" --- p.159 / Chapter 3.3.10. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on pl30 gene and protein expression" --- p.162 / Chapter 3.3.11. --- "Effects of TNF-α, IL-6, IL-lα, IL-lp, IFγ on Cak gene expression" --- p.166 / Chapter 3.3.12. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFy on cyclin H gene and protein expression -" --- p.170 / Chapter 3.3.13. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on cyclin B gene and protein expression" --- p.174 / Chapter 3.3.14. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on bcl-2 gene and protein expression" --- p.178 / Chapter 3.3.15. --- "Effects of TNF-a, IL-6, IL-lα, IL-1β, IFγ on bcl-xL gene and protein expression" --- p.178 / Chapter 3.3.16. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on bcl-xα gene expression" --- p.184 / Chapter 3.3.17. --- "Effects of TNF-α, IL-6, IL-lα, IL-lβ, IFγ on bcl-w gene and protein expression" --- p.187 / Chapter 3.3.18. --- "Effects of TNF-α, IL-6, IL-lα, IL-1β, IFγ on Mcl-1 gene expression" --- p.191 / Chapter 3.4. --- Effects of P-ARs on cell cycle related genes expression --- p.194 / Chapter 3.4.1. --- Effects of β-AR agonists and antagonists on cyclin D1 gene expression --- p.195 / Chapter 3.4.2. --- Effects of β-AR agonists and antagonists on cyclin D2 gene expression --- p.198 / Chapter 3.4.3. --- Effects of β-AR agonists and antagonists on cyclin D3 gene expression --- p.201 / Chapter 3.4.4. --- Effects of β-AR agonists and antagonists on cdk-4 gene expression --- p.204 / Chapter 3.4.5. --- Effects of β-AR agonists and antagonists on cyclin E gene expression --- p.207 / Chapter 3.4.6. --- Effects of β-AR agonists and antagonists on cdk-2 gene expression - --- p.210 / Chapter 3.4.7. --- Effects of β-AR agonists and antagonists on p15 gene expression --- p.213 / Chapter 3.4.8. --- Effects of β-AR agonists and antagonists on p27 gene expression --- p.216 / Chapter 3.4.9. --- Effects of β-AR agonists and antagonists on p21 gene expression --- p.219 / Chapter 3.4.10. --- Effects of β-AR agonists and antagonists on p130 gene expression --- p.222 / Chapter 3.4.11. --- Effects of β-AR agonists and antagonists on Cak gene expression --- p.225 / Chapter 3.4.12. --- Effects of β-AR agonists and antagonists on cyclin H gene expression --- p.228 / Chapter 3.4.13. --- Effects of β-AR agonists and antagonists on cyclin B gene expression --- p.231 / Chapter 3.4.14. --- Effects of β-AR agonists and antagonists on bcl-XL gene expression --- p.233 / Chapter 3.4.15. --- Effects of β-AR agonists and antagonists on bcl-xα gene expression --- p.236 / Chapter 3.4.16. --- Effects of β-AR agonists and antagonists on bcl-w gene expression --- p.239 / Chapter 3.4.17. --- Effects of β-AR agonists and antagonists on Mcl-1 gene expression --- p.243 / Chapter CHAPTER 4. --- DISCUSSION & CONCLUSION --- p.247 / Chapter 4.1. --- Effects of TNF-α on the induction of cell cycle regulatory genes/proteins expression --- p.248 / Chapter 4.2. --- Effects of TNF-α on bcl-2 family apoptotic inhibitor genes expression --- p.250 / Chapter 4.3. --- The TNF-R subtype(s) responsible for the TNF-a-induced cell cycle regulatory genes and proteins expression --- p.251 / Chapter 4.4. --- Is the TNF-α-induced cell cycle regulatory genes and proteins expression cytokine specific? --- p.253 / Chapter 4.5. --- The relationship between TNF-α and β-adrenergic mechanism in C6 cell proliferation --- p.254 / Chapter 4.6. --- General Discussion --- p.256 / Chapter 4.7. --- Possible treatments for brain injury --- p.258 / APPENDIX --- p.259 / REFERENCES --- p.348

by Lam Kai Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 156-165). / Abstract also in Chinese. / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Tumor Necrosis Factor-α in Cancer Treatment --- p.1 / Chapter 1.1.1 --- Historical Background --- p.1 / Chapter 1.1.2 --- Mechanisms of Action --- p.2 / Chapter 1.1.2.1 --- Production of Reactive oxidative Species / Chapter 1.1.2.2 --- Increase of Intracellular Free Calcium Concentration / Chapter 1.1.2.3 --- Activation of Ca2+/Mg2+-dependent Endonuclease / Chapter 1.1.2.4 --- Decrease of glucose uptake and Protein Synthesis / Chapter 1.1.2.5 --- Formation of Ion-permeable Channel / Chapter 1.1.2.6 --- Activation of Phospholipase / Chapter 1.1.2.7 --- Increase of S-phase Cells / Chapter 1.1.2.8 --- Immunomodulatory Effects / Chapter 1.1.3 --- Resistance of Cells to TNF-α --- p.7 / Chapter 1.1.4 --- Clinical Studies --- p.11 / Chapter 1.1.5 --- Side Effects --- p.12 / Chapter 1.2 --- Hyperthermia and Cancer Treatment --- p.14 / Chapter 1.2.1 --- Hyperthermic Agents --- p.15 / Chapter 1.2.2 --- Intrinsic Heat Sensitivity --- p.15 / Chapter 1.2.3 --- Mechanisms of Action --- p.17 / Chapter 1.2.3.1 --- Depolarization of Membrane Potential / Chapter 1.2.3.2 --- "Reduction of glucose transport and DNA, mRNA and Protein Synthesis" / Chapter 1.2.3.3 --- Decrease of Intracellular pH / Chapter 1.2.3.4 --- Calcium Imbalance / Chapter 1.2.3.5 --- Effect on Nucleolar Protein / Chapter 1.2.3.6 --- Apoptosis / Chapter 1.2.3.7 --- Induction of Autologous Tumor Killing / Chapter 1.2.3.8 --- "Blood Flow, Tumor Oxygenation and Vascular Damage" / Chapter 1.2.4 --- Clinical Studies --- p.20 / Chapter 1.3 --- Combined Treatment --- p.21 / Chapter 1.3.1 --- Combined Treatment with TNF-α and Fixed-temperature Hyperthermia --- p.22 / Chapter 1.3.2 --- Combined Treatment with TNF + Step-down Hyperthermia --- p.22 / Chapter 1.3.3 --- In Vivo Study --- p.23 / Chapter 1.3.4 --- Sequence of Treatment --- p.24 / Chapter 1.3.5 --- Proposed Mechanism of Synergism --- p.24 / Chapter 1.4 --- Objective of Study --- p.26 / Chapter 1.4.1 --- Sequence of Treatments --- p.26 / Chapter 1.4.2 --- Comparison of Treatments' Effectiveness --- p.27 / Chapter 1.4.3 --- Effect on Normal Cell --- p.27 / Chapter 1.4.4 --- Effect on Distribution of Cells in Cell Cycle Phases --- p.28 / Chapter 1.4.5 --- In Vivo Study --- p.28 / Chapter Chapter Two: --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.30 / Chapter 2.1.1 --- For Cell Culture --- p.30 / Chapter 2.1.2 --- In vitro Treatments --- p.31 / Chapter 2.1.3 --- DNA Electrophoresis --- p.31 / Chapter 2.1.4 --- Flow Cytometry --- p.32 / Chapter 2.2. --- Reagent Preparation --- p.33 / Chapter 2.2.1 --- Culture Media --- p.33 / Chapter 2.2.2 --- Human Recombinant Tumor Necrosis Factor alpha (rhTNF-α) --- p.33 / Chapter 2.2.3 --- Phosphate Buffered Saline (PBS) --- p.33 / Chapter 2.2.4 --- Lysis Buffer --- p.34 / Chapter 2.2.5 --- TE Buffer --- p.34 / Chapter 2.2.6 --- Proteinase K and Ribonuclease A (RNase A) --- p.34 / Chapter 2.2.7 --- 100 Base-Pair DNA Marker --- p.34 / Chapter 2.2.8 --- Propidium Iodide (PI) --- p.35 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Cell Culture --- p.35 / Chapter 2.3.1.1 --- Ehrlich Ascitic Tumor (EAT) and Human Leukemia (HL-60) / Chapter 2.3.1.2 --- Human Coronary Artery Endothelial Cells (HCAEC) / Chapter 2.3.2 --- In vitro Experiments --- p.36 / Chapter 2.3.3 --- Tumor Necrosis Factor Treatment --- p.37 / Chapter 2.3.4 --- Hyperthermia Treatments --- p.37 / Chapter 2.3.5 --- Cell Counting --- p.38 / Chapter 2.3.5.1 --- Trypan Blue Exclusion Assay / Chapter 2.3.5.2 --- Neutral Red Assay / Chapter 2.3.6 --- Determination of Additive or Synergistic Effect --- p.39 / Chapter 2.3.7 --- DNA Electrophoresis --- p.40 / Chapter 2.3.8 --- Flow Cytometry --- p.42 / Chapter 2.3.7.1 --- Preparation of Samples / Chapter 2.3.7.2 --- Flow Cytometry Acquisition / Chapter 2.3.7.3 --- Analysis / Chapter 2.3.9 --- In vivo Experiments --- p.44 / Chapter 2.3.8.1 --- Animal Strain / Chapter 2.3.8.2 --- Cell Line / Chapter 2.3.8.3 --- Tumor Necrosis Factor Treatment / Chapter 2.3.8.4 --- Hyperthermia Treatments / Chapter 2.3.8.5 --- Test of Body Temperature / Chapter 2.3.8.6 --- Cell Harvesting / Chapter Chapter Three: --- Result --- p.50 / Chapter 3.1 --- Optimal Sequence of Treatments --- p.50 / Chapter 3.1.1 --- Optimal Sequence of Treatments on Murine Ehrlich Ascitic Tumor (EAT) cells --- p.50 / Chapter 3.1.1.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.1.1.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.1.1.3 --- TNF + Step-down Hyperthermia3 / Chapter 3.1.2 --- Optimal Sequence of Treatments on Human Leukemia cells HL-60 --- p.60 / Chapter 3.1.2.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.1.2.2 --- TNF + Step-Down Hyperthermia2 / Chapter 3.1.2.3 --- TNF + Step-Down Hyperthermia3 / Chapter 3.2 --- Comparison of Effectiveness of Treatments --- p.72 / Chapter 3.2.1 --- Effectiveness of Various treatments on EAT cells --- p.72 / Chapter 3.2.2 --- Synergistic Effect between rhTNF-α and Hyperthermia on EAT cells --- p.74 / Chapter 3.2.3 --- Decrease of Relative Growth and Viability of EAT with Time --- p.79 / Chapter 3.2.3.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.2.3.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.2.3.3 --- TNF + Step-down Hyperthermia3 / Chapter 3.2.4 --- Comparison of Effectiveness of Various Treatments on HL-60 cells --- p.82 / Chapter 3.2.5 --- Synergistic Effect between rhTNF-α and Hyperthermia on HL-60 cells --- p.87 / Chapter 3.2.6 --- Change of Relative Growth and Viability of HL-60 with Time --- p.90 / Chapter 3.2.6.1 --- TNF + Fixed-temperature Hyperthermia / Chapter 3.2.6.2 --- TNF + Step-down Hyperthermia2 / Chapter 3.2.6.3 --- TNF + Step-down hyperthermia3 / Chapter 3.3 --- Cell Death Pathway --- p.96 / Chapter 3.3.1 --- Experiments on Ehrlich Ascitic Tumor (EAT) Cells --- p.96 / Chapter 3.3.2 --- Experiments on Human Leukemia (HL-60) Cells --- p.100 / Chapter 3.4 --- Experiment on Normal Cell --- p.104 / Chapter 3.5 --- Effect of TNF + Fixed-temperature Hyperthermia on the Cell Cycle Progression --- p.107 / Chapter 3.5.1 --- Different Times of TNF Administration and Distribution of EAT cells in Cell cycle --- p.107 / Chapter 3.5.2 --- Different Times of TNF Administration and Distribution of HL-60 cells in Cell Cycle --- p.114 / Chapter 3.5.3 --- Shift of Cells Cycle after TNF Treatment --- p.120 / Chapter 3.5.3.1 --- Response of Ehrlich Ascitic Tumor Cells / Chapter 3.5.3.2 --- Response of Human leukemia Cells / Chapter 3.6 --- Effectiveness of Treatments in vivo: --- p.129 / Chapter 3.6.1 --- Dose-dependent Response --- p.129 / Chapter 3.6.2 --- Change of Body Temperature During Hyperthermia --- p.131 / Chapter 3.6.3 --- Comparison of Effectiveness of Various Treatments in vivo --- p.133 / Chapter 3.6.4 --- Synergistic Effect Between rhTNF-α and Hyperthermia in vivo --- p.135 / Chapter Chapter Four: --- Discussion --- p.138 / Chapter 4.1 --- Optimal Sequence of Treatments --- p.139 / Chapter 4.2 --- Comparison of Various Treatments --- p.143 / Chapter 4.3 --- Distribution of Cells in Cell Cycle Phases --- p.149 / Chapter 4.4 --- In vivo Study --- p.153 / Chapter Chapter Five: --- References --- p.156

Type I diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreas. Destruction of the body's own proteins, cells, and tissues is precipitated by the dysfunction of cytokine production, protein modification, and signaling pathways in immune cell subtypes. Tumor Necrosis Factor α (TNFα) and its receptors Tumor Necrosis Factor 1 (TNFR1) also known as p55 and TNFRSF1A, and Tumor Necrosis Factor 2 (TNFR2) also known as P75 and TNFRSF1B play a crucial role in this autoimmune process. TNFα has been shown to stimulate cell death through TNFR1 signaling by the caspase system, while promoting cell survival through TNFR2 signaling using the Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells (NF-𝜅B) pathway. Recent findings show a defect in immuno-proteasomes found in autoreactive T cells in people with T1D. This defect causes improper signaling transduction when TNFα binds to TNFR2. The inability to save the cell by activating the NF-𝜅B pathway eventually leads instead to apoptosis using the caspase system. A decrease in TNFα or increase in soluble TNFα receptors might be an explanation for these autoreactive T cells to evade the host immune system, and allow them to cause destruction of the pancreas. We hypothesize that patients with T1D will show abnormal distribution of TNFα and its receptors at basal levels, as well as when stimulated with interleukins, cytokines, and bacteria such as interleukin-2 (IL-2), lipotechoic acid (LTA), granulocyte macrophage-colony stimulating factor (GM-CSF), and Bacillus Calmette-Guérin (BCG). To test this hypothesis, we obtained peripheral blood from T1D patients (n=102) and controls (n=89) and performed in vitro stimulation assays. After a 48-hour incubation, tissue culture supernatants were collected and analyzed for TNF and its receptors production by ELISA, as well as densities of cell membrane receptors by flow cytometry. The data from this study showed significant differences in basal levels of TNFα, TNFR1, and TNFR2 on both the membrane and in the serum between patients and controls. Patients contained a greater percentage of CD4, 8, and 14 - TNFR2 and not TNFR1 double positive cells than their healthy control counterparts. Patient's sera also contained higher levels of all three markers, sTNFα, sTNFR1, and sTNFR2 than the controls. However, no significant differences were found between patient and controls when stimulated with the various compounds listed above.

by Li Jian Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 157-181). / 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 (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains x, 189 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

This thesis evaluated supplementation with two components of flaxseed during exercise training on inflammation in older adults.<P>Experiment 1: This experiment assessed secoisolariciresinol diglucoside (SDG) supplementation during aerobic exercise training on inflammation in older adults. Methods: One hundred subjects aged 50y or older were randomized to receive either SDG or placebo before completing a 6-month walking program. Fasting concentrations of interleukin-6 and tumor necrosis factor-á, glucose, triacylglycerol (TAG), high density lipoprotein (HDL), low density lipoprotein, and total cholesterol as well as leukocyte cell count were measured every two months while body composition, resting blood pressure, and a composite Z-score of six metabolic syndrome risk factors were assessed at baseline and 6 months. Results: Men on placebo increased metabolic syndrome composite Z-score (p<0.05). TAG increased (p=0.017) in men on placebo relative to men on SDG and men on SDG decreased (p=0.045) DBP relative to men on placebo. Conclusions: SDG had no effect on inflammation while it is effective in attenuating risk factors associated with metabolic syndrome in older males but not females.<p>Experiment 2: This experiment evaluated alpha-linolenic acid (ALA) supplementation during strength exercise training on inflammation in older adults. Methods: Fifty-one healthy older adults (65.4±0.8y) were randomized to receive ALA or a placebo before completing a 12 wk strength training program. Subjects were evaluated at baseline and 12 weeks for TNF-á and IL-6, muscle strength, body composition, and muscle thickness. Results: Males supplementing with ALA decreased IL-6 concentration (p=0.003). The female placebo and male ALA group had a significant increase in knee flexor thickness (p<0.05). Chest and leg press strength, lean tissue mass, and muscle thickness significantly increased, while percent fat and total body mass decreased with training (p<0.05), with no difference between ALA and placebo. Conclusions: ALA lowers IL-6 in older men, but has minimal effect on muscle mass and strength during resistance training.<p>General Conclusion: A composite score of metabolic syndrome is attenuated in males supplementing with SDG. ALA reduces IL-6 in older men. Older men, but not older women, derive specific health benefits from increased consumption of components of flaxseed consumed during an exercise program.

This thesis evaluated supplementation with two components of flaxseed during exercise training on inflammation in older adults.<P>Experiment 1: This experiment assessed secoisolariciresinol diglucoside (SDG) supplementation during aerobic exercise training on inflammation in older adults. Methods: One hundred subjects aged 50y or older were randomized to receive either SDG or placebo before completing a 6-month walking program. Fasting concentrations of interleukin-6 and tumor necrosis factor-á, glucose, triacylglycerol (TAG), high density lipoprotein (HDL), low density lipoprotein, and total cholesterol as well as leukocyte cell count were measured every two months while body composition, resting blood pressure, and a composite Z-score of six metabolic syndrome risk factors were assessed at baseline and 6 months. Results: Men on placebo increased metabolic syndrome composite Z-score (p<0.05). TAG increased (p=0.017) in men on placebo relative to men on SDG and men on SDG decreased (p=0.045) DBP relative to men on placebo. Conclusions: SDG had no effect on inflammation while it is effective in attenuating risk factors associated with metabolic syndrome in older males but not females.<p>Experiment 2: This experiment evaluated alpha-linolenic acid (ALA) supplementation during strength exercise training on inflammation in older adults. Methods: Fifty-one healthy older adults (65.4±0.8y) were randomized to receive ALA or a placebo before completing a 12 wk strength training program. Subjects were evaluated at baseline and 12 weeks for TNF-á and IL-6, muscle strength, body composition, and muscle thickness. Results: Males supplementing with ALA decreased IL-6 concentration (p=0.003). The female placebo and male ALA group had a significant increase in knee flexor thickness (p<0.05). Chest and leg press strength, lean tissue mass, and muscle thickness significantly increased, while percent fat and total body mass decreased with training (p<0.05), with no difference between ALA and placebo. Conclusions: ALA lowers IL-6 in older men, but has minimal effect on muscle mass and strength during resistance training.<p>General Conclusion: A composite score of metabolic syndrome is attenuated in males supplementing with SDG. ALA reduces IL-6 in older men. Older men, but not older women, derive specific health benefits from increased consumption of components of flaxseed consumed during an exercise program.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 81-96).