by Yip Mei Chu Pandora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 207-233). / Abstract also in Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER ONE ... --- GENERAL INTRODUCTION / Chapter 1.1 --- the blood cells formation - hematopoiesis --- p.1 / Chapter 1.1.1 --- Hierarchy of hematopoiesis --- p.2 / Chapter 1.1.2 --- Malfunction in the process of hematopoiesis - hematologic neoplasia - Leukemia --- p.6 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Differentiation therapy ´ؤ a new hope in the treatment of leukemia --- p.9 / Chapter 1.2 --- Understanding the pathogenesis of leukemia --- p.12 / Chapter 1.2.1 --- General regulation of hematopoiesis --- p.12 / Chapter 1.2.2 --- Regulation of the differentiation of myeloid lineage --- p.15 / Chapter 1.2.2.1 --- Regulation of myeloid cell differentiation by hematopoietic regulatory protein --- p.16 / Chapter 1.2.2.2 --- Signal transduction pathways in myeloid cell differentiation --- p.20 / Chapter 1.2.2.3 --- Gene regulation of myeloid cell differentiation --- p.22 / Chapter 1.2.2.3.1 --- Transcription factors --- p.23 / Chapter 1.2.2.3.2 --- Myeloid specific genes --- p.31 / Chapter 1.2.2.3.3 --- Protooncogenes and tumor suppressor genes --- p.37 / Chapter 1.2.2.3.4 --- Homeobox genes --- p.42 / Chapter 1.2.2.3.5 --- Cell cycle control in myeloid growth and differentiation --- p.47 / Chapter 1.3 --- Induction of differentiation in myeloid leukemia cell --- p.48 / Chapter 1.3.1 --- Induced myeloid leukemia cell differentiation --- p.48 / Chapter 1.3.2 --- Inducers of myeloid cell differentiation --- p.52 / Chapter 1.3.3 --- Chemical inducers ´ؤ Flavonoids --- p.57 / Chapter 1.3.4 --- Murine myeloid leukemia cell ´ؤ WEHI-3B JCS --- p.60 / Chapter 1.4 --- Aim of study --- p.53 / Chapter CHAPTER TWO ... --- ISOLATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED DURING BIOCHANIN A INDUCED WEHI-3B (JCS) MYELOID LEUKEMIA CELL DIFFERENTIATION / Chapter 2.1 --- Introduction --- p.65 / Chapter 2.1.1 --- Strategy for searching differentially expressed genes - RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP- PCR) --- p.65 / Chapter 2.1.2 --- Reamplification of PCR products by Touchdown PCR --- p.67 / Chapter 2.1.3 --- Methods for eliminating false positives : Dot blot hybridization screening --- p.68 / Chapter 2.2 --- Materials --- p.70 / Chapter 2.2.1 --- "Cell line, Bacterial strain and Vector" --- p.70 / Chapter 2.2.2 --- Chemicals --- p.70 / Chapter 2.2.3 --- Reagents and nucleic acids --- p.71 / Chapter 2.2.4 --- Kits --- p.72 / Chapter 2.2.5 --- Solutions --- p.72 / Chapter 2.2.6 --- Equipments --- p.73 / Chapter 2.3 --- Methods --- p.74 / Chapter 2.3.1 --- Induction of murine myeloid leukemia cell line -WEHI-3B (JCS) cells by biochanin-A --- p.74 / Chapter 2.3.2 --- Isolation of total RNA by guanidium thiocyanate cesium chloride ultracentrifugation --- p.74 / Chapter 2.3.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.75 / Chapter 2.3.3.1 --- Synthesis of first strand cDNA --- p.75 / Chapter 2.3.3.2 --- Normalization of RNA samples --- p.75 / Chapter 2.3.3.3 --- RAP-PCR --- p.76 / Chapter 2.3.3.4 --- Reamplification of differentially amplified fragment --- p.77 / Chapter 2.3.4 --- First round dot blot hybridization screening --- p.78 / Chapter 2.3.4.1 --- Dot blot --- p.78 / Chapter 2.3.4.2 --- Preparation of cDNA probe --- p.79 / Chapter 2.3.4.3 --- 32P-labelling of cDNA probe --- p.79 / Chapter 2.3.4.4 --- Removal of unincorporated probe by NICK´ёØ column --- p.80 / Chapter 2.3.4.5 --- Estimation of 32P labelling efficiency by scintillation counting --- p.80 / Chapter 2.3.4.6 --- Prehybridization and hybridization --- p.81 / Chapter 2.3.4.7 --- Quantitation of hybridization signal by scanning densitometry --- p.81 / Chapter 2.3.5 --- Second round dot blot hybridization screening --- p.81 / Chapter 2.3.5.1 --- Subcloning of differentially amplified fragments --- p.82 / Chapter 2.3.5.1.1 --- Preparation of vector DNA --- p.82 / Chapter 2.3.5.1.2 --- Synthesis of blunt end PCR product --- p.84 / Chapter 2.3.5.1.3 --- Blunt end ligation --- p.34 / Chapter 2.3.5.1.4 --- Transformation --- p.85 / Chapter 2.3.5.1.5 --- Selection and confirmation by polymerase chain reaction --- p.85 / Chapter 2.3.5.2 --- Dot blot hybridization screening --- p.85 / Chapter 2.4 --- Results --- p.87 / Chapter 2.4.1 --- Spectrophotometric analysis of total RNA --- p.87 / Chapter 2.4.2 --- Normalization of RNA samples --- p.88 / Chapter 2.4.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.39 / Chapter 2.4.4 --- Reamplification of isolated RAP-PCR products --- p.91 / Chapter 2.4.5 --- First round of dot blot hybridization screening --- p.92 / Chapter 2.4.6 --- Subcloning of differentially amplified fragments --- p.100 / Chapter 2.4.7 --- Second round of dot blot hybridization screening --- p.102 / Chapter 2.4.8 --- Comparison of the first and second round of dot blot hybridization screening --- p.106 / Chapter 2.5 --- Discussion --- p.108 / Chapter 2.5.1 --- RNA fingerprinting by arbitrarily primed PCR --- p.108 / Chapter 2.5.2 --- Limitation of RAP-PCR --- p.110 / Chapter 2.5.3 --- Two rounds of dot blot hybridization screening --- p.111 / Chapter CHAPTER THREE... --- CHARACTERIZATION OF THE ISOLATED GENE FRAGMENTS / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.1.1 --- Automated DNA sequencing and analysis --- p.113 / Chapter 3.1.2 --- GenBank and the BLAST homology search --- p.115 / Chapter 3.2 --- Materials --- p.118 / Chapter 3.2.1 --- Selected recombinant plasmids --- p.118 / Chapter 3.2.2 --- Chemicals --- p.118 / Chapter 3.2.3 --- Reagents --- p.118 / Chapter 3.2.4 --- Kits --- p.119 / Chapter 3.2.5 --- Solutions --- p.119 / Chapter 3.2.6 --- Equipment --- p.119 / Chapter 3.3 --- Methods --- p.120 / Chapter 3.3.1 --- Preparation of selected recombinant plasmid DNA --- p.120 / Chapter 3.3.2 --- Restriction digestion of recombinant plasmid DNA --- p.120 / Chapter 3.3.3 --- Automated DNA sequencing --- p.120 / Chapter 3.3.3.1 --- Primer annealing to template --- p.120 / Chapter 3.3.3.2 --- Sequencing reactions --- p.121 / Chapter 3.3.3.3 --- Polyacrylamide gel electrophoresis --- p.121 / Chapter 3.3.3.4 --- Data analysis by ALF manager and DNAsis --- p.122 / Chapter 3.3.4 --- Sequence homology search with databases --- p.122 / Chapter 3.4 --- Results --- p.123 / Chapter 3.4.1 --- Spectrophotometric analysis of selected recombinant plasmid DNAs subcloned with differentially amplified fragments --- p.123 / Chapter 3.4.2 --- Restriction digestion of selected recombinant plasmid DNA --- p.124 / Chapter 3.4.3 --- Sequences of the subcloned differentially amplified fragments --- p.126 / Chapter 3.4.4 --- Sequence analysis of the subcloned differentially amplified fragments --- p.144 / Chapter 3.5 --- Discussion --- p.157 / Chapter 3.5.1 --- Sequence analysis of the isolated gene fragment --- p.157 / Chapter CHAPTER FOUR … --- "EXPRESSION PROFILE OF ISOLATED GENES FRAGMENTS IN MYELOID LEUKEMIA CELL, MOUSE EMBRYO, AND TISSUES" / Chapter 4.1 --- Introduction --- p.162 / Chapter 4.1.1 --- Quantitation of mRNA by Reverse transcription-polymerase chain reaction --- p.162 / Chapter 4.1.2 --- Internal primer design by OLIGO´ёØ ver 34 --- p.167 / Chapter 4.2 --- Materials --- p.168 / Chapter 4.2.1 --- Mice --- p.168 / Chapter 4.2.2 --- Cell lysate --- p.168 / Chapter 4.2.3 --- Total RNAs --- p.168 / Chapter 4.3 --- Methods --- p.169 / Chapter 4.3.1 --- Internal primer design by OLIGO´ёØ ver 34 --- p.169 / Chapter 4.3.2 --- "Isolation of total RNA from biochanin A induced JCS cells, mouse embryos and tissue" --- p.169 / Chapter 4.3.2.1 --- Preparation of cell lysate from mouse embryo and postnatal mouse brain --- p.169 / Chapter 4.3.2.2 --- Isolation of RNA by guanidium thiocyanate cesium chloride method --- p.170 / Chapter 4.3.3 --- Preparation of saggital section of mouse embryo --- p.170 / Chapter 4.3.4 --- Confirmation of differential expression of isolated genes fragments during biochanin A and midazolam induced WEHI 3B (JCS) differentiation and the expression profile in mouse tissues and during mouse embryo development by reverse transcription-polymerase chain reaction --- p.171 / Chapter 4.4 --- Results --- p.173 / Chapter 4.4.1 --- Internal primer design of the sequenced fragments --- p.173 / Chapter 4.4.2 --- Spectrophotometric analysis of total RNA --- p.175 / Chapter 4.4.3 --- Saggital section of mouse embryo --- p.176 / Chapter 4.4.4 --- Normalization of RNA samples --- p.180 / Chapter 4.4.5 --- Analysis of mRNA expression of differentially amplified fragmentsin biochanin A or midazolam induced JCS cells and mouse embryos by RT- PCR --- p.182 / Chapter 4.4.5.1 --- "Genes downregulated at 1 hour, 5 hours and 48 hours after biochanin A induction of JCS cells" --- p.183 / Chapter 4.4.5.2 --- Genes up-regulated at 48 hours after biochanin A induction --- p.183 / Chapter 4.4.5.3 --- Genes constitutively expressed during the course of biochanin A treatment --- p.184 / Chapter 4.4.5.4 --- Genes showing undetectable level of expression in biochanin A induced JCS cells --- p.184 / Chapter 4.4.6 --- Tissue expression of the biochanin A induced-differentially expressed fragments by RT-PCR --- p.188 / Chapter 4.5 --- Discussion --- p.191 / Chapter 4.5.1 --- Expression profiles of isolated differentially amplified fragments --- p.191 / Chapter 4.5.2 --- Comparison of the expression profiles of the isolated gene fragments analyzed by dot blot hybridization screening and RT-PCR --- p.197 / Chapter CHAPTER FIVE ... --- GENERAL DISCUSSION --- p.200 / REFERENCES --- p.207 / APPENDIX --- p.234

by Kong Lai Ping, Ada. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 171-189). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.1 --- Development of Hematopoietic Stem Cells and Sites of Hematopoiesis --- p.1 / Chapter 1.1.2 --- Role of Cytokines in the Control of Hematopoiesis --- p.3 / Chapter 1.2 --- Leukemia and Cell Differentiation --- p.5 / Chapter 1.2.1 --- Leukemia as Abnormalities in Hematopoietic Cell Development --- p.5 / Chapter 1.2.2 --- Classification and Etiology of Leukemia --- p.6 / Chapter 1.2.3 --- Current Modalities for the Treatment of Leukemia --- p.9 / Chapter 1.2.4 --- Leukemia Cell Lines as In Vitro Models for the Study of Myeloid Leukemia --- p.10 / Chapter 1.2.5 --- Cytokines as Inducers of Myeloid Leukemia Cell Differentiation --- p.12 / Chapter 1.2.6 --- The Murine Myeloid Leukemia Cell Line (WEHI- 3B JCS) as an Experimental Cell Model --- p.13 / Chapter 1.3 --- Flavonoids: Properties and Biological Activities --- p.15 / Chapter 1.3.1 --- Chemical Structure and Classification of Flavonoids --- p.15 / Chapter 1.3.2 --- Occurrence and Distribution of Flavonoids --- p.16 / Chapter 1.3.3 --- Biological Properties and Action Mechanisms of Flavonoids --- p.17 / Chapter 1.3.4 --- Effects of Flavonoids on Leukemia --- p.20 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.23 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Cell Lines --- p.26 / Chapter 2.1.2. --- Mice --- p.28 / Chapter 2.1.3 --- Flavonoids --- p.28 / Chapter 2.1.4 --- Recombinant Cytokines --- p.30 / Chapter 2.1.5. --- Physiological Differentiation Inducers ´ؤ Vitamin Analogs --- p.31 / Chapter 2.1.6 --- Monoclonal Antibodies --- p.31 / Chapter 2.1.7 --- "Buffers, Culture Medium and Other Reagents" --- p.33 / Chapter 2.1.8 --- Oligonucleotide Primers and Internal Probes --- p.36 / Chapter 2.1.9 --- Reagents for Cytokine Gene Expression Study --- p.38 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Culture of Tumor Cell Lines --- p.44 / Chapter 2.2.2 --- Determination of Cell Growth and Proliferation --- p.45 / Chapter 2.2.3 --- Colony Assay --- p.46 / Chapter 2.2.4 --- In vivo Tumorigenicity Assay --- p.46 / Chapter 2.2.5 --- Induction of Leukemic Cell Differentiation --- p.47 / Chapter 2.2.6 --- Cell Morphological Study --- p.47 / Chapter 2.2.7 --- Assessment of Differentiation Associated Characteristics --- p.48 / Chapter 2.2.7.1 --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter 2.2.7.2 --- Assay of Plastic Adherence --- p.48 / Chapter 2.2.8 --- Flow Cytometric Analysis --- p.49 / Chapter 2.2.8.1 --- Surface Antigen Immunophenotyping --- p.49 / Chapter 2.2.8.2 --- Assay of Non-specific Esterase Activity --- p.50 / Chapter 2.2.8.3 --- Assay of Phagocytic Activity --- p.50 / Chapter 2.2.8.4 --- Assay of Endocytic Activity --- p.51 / Chapter 2.2.8.5 --- Cell Cycle/DNA Content Evaluation --- p.52 / Chapter 2.2.9 --- Gene Expression Analysis --- p.53 / Chapter 2.2.9.1 --- Cell Lysate Preparation --- p.53 / Chapter 2.2.9.2 --- Total RNA Isolation by cesium chloride isopycnic gradient --- p.53 / Chapter 2.2.9.3 --- Reverse Transcription --- p.54 / Chapter 2.2.9.4 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.2.9.5 --- Agarose Gel Electrophoresis --- p.56 / Chapter 2.2.9.6 --- DIG 3，End Labeling of Oligonucleotide Probes --- p.57 / Chapter 2.2.9.7 --- Dot Blot Hybridization --- p.57 / Chapter 2.2.9.8 --- DIG Chemiluminescent Detection --- p.58 / Chapter 2.2.10 --- DNA Fragmentation Analysis --- p.59 / Chapter 2.2.11 --- Statistical Analysis --- p.60 / Chapter CHAPTER 3: --- EFFECTS OF FLAVONOIDS ON THE PROLIFERATION AND APOPTOSIS OF MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.63 / Chapter 3.2.1 --- Growth-Inhibitory Effects of Flavone on Murine Myeloid Leukemia JCS Cells --- p.63 / Chapter 3.2.2 --- Cytotoxic Effects of Flavone on Murine Lymphocytes and Myeloid Leukemia JCS Cells --- p.67 / Chapter 3.2.3 --- Effects of Different Flavonoids on the Proliferation of Leukemia JCS Cells --- p.70 / Chapter 3.2.4 --- Anti-proliferative Effect of Flavonoids on Different Tumor Cell Lines --- p.74 / Chapter 3.2.5 --- Effects of Flavone and Flavonol on the Cell Cycle Kinetics of JCS Cells --- p.86 / Chapter 3.2.6 --- Induction of DNA Fragmentation of JCS cells by Flavone --- p.89 / Chapter 3.2.7 --- Effect of Flavone on the Clonogenicity of JCS Cells In Vitro and Tumorigenicity In Vivo --- p.92 / Chapter 3.3 --- Discussion --- p.94 / Chapter CHAPTER 4: --- EFFECTS OF FLAVONOIDS ON THE DIFFERENTIATION OF MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Results --- p.100 / Chapter 4.2.1 --- Morphological Changes in Flavonoid-Treated JCS Cells --- p.100 / Chapter 4.2.2 --- Induction of Plastic Adherence in Flavonoid- Treated JCS Cells --- p.106 / Chapter 4.2.3 --- Surface Antigen Immunophenotyping of Differentiating JCS Cells --- p.106 / Chapter 4.2.4 --- NBT-Reducing Activity of Flavonoid-Treated JCS Cells --- p.114 / Chapter 4.2.5 --- Non-specific Esterase Activity of Flavonoid- Treated JCS Cells --- p.115 / Chapter 4.2.6 --- Endocytic Activity of Flavonoid-Treated JCS Cells --- p.116 / Chapter 4.2.7 --- Phagocytic Activity of Flavonoid-Treated JCS Cells --- p.117 / Chapter 4.3 --- Discussion --- p.118 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI- PROLIFERATIVE AND DIFFERENTIAION-INDUCING ACTIVITIES OF FLAVONE ON MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 5.1 --- Introduction --- p.122 / Chapter 5.2 --- Results --- p.125 / Chapter 5.2.1 --- Combinations of Flavone with Physiological Differentiation Inducers on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.1 --- Modulatory Effects of Flavone and All-Trans Retinoic Acid (ATRA) on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.2 --- "Modulatory Effects of Flavone and 1,25- dihydroxyvitamin D3 on the Proliferation and Differentiation of JCS Cells" --- p.130 / Chapter 5.2.2 --- Combinations of Flavone and Cytokines on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.1 --- Modulatory Effects of Flavone and rmlFN-γ on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.2 --- Synergistic Effects of Flavone and rmIL-1 on the Proliferation and Differentiation of JCS Cells --- p.137 / Chapter 5.2.3 --- Modulation of Cytokine Gene Expressionin Flavone-Treated JCS Cells --- p.144 / Chapter 5.3 --- Discussion --- p.159 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.165 / REFERENCES --- p.171

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v

Chiu Lai-Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / Chapter Chapter One --- General introduction / Chapter 1.1 --- Haemopoiesis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Regulation --- p.2 / Chapter 1.1.2.1 --- Stromal cells --- p.2 / Chapter 1.1.2.2 --- Haemopoietic regulator --- p.3 / Chapter 1.1.2.3 --- Haemopoietic regulator receptors and signal transduction --- p.5 / Chapter 1.2 --- Disorder of haemopoiesis --- p.9 / Chapter 1.2.1 --- Causes --- p.9 / Chapter 1.2.2 --- Types of leukemia --- p.9 / Chapter 1.2.3 --- Treatment of leukemia --- p.10 / Chapter 1.3 --- Prostaglandins --- p.13 / Chapter 1.3.1 --- Introduction --- p.13 / Chapter 1.3.2 --- Types and biosynthesis --- p.14 / Chapter 1.3.3 --- Prostaglandin receptors --- p.15 / Chapter 1.3.4 --- Prostaglandins and cell differentiation --- p.17 / Chapter 1.3.4.1 --- PGD2 and cell differentiation --- p.19 / Chapter 1.3.4.2 --- PGE2 and cell differentiation --- p.20 / Chapter 1.3.4.3 --- PGJ2 and cell differentiation --- p.22 / Chapter 1.4 --- WEHI-3B JCS cells --- p.25 / Chapter 1.5 --- Aims of study --- p.27 / Chapter Chapter Two --- Roles of Prostaglandin D2，E2 and J2 in WEHI-3B JCS myeloid leukemia cell differentiation / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Morphological studies of JCS cells --- p.28 / Chapter 2.1.2 --- Methods in determining cell proliferation --- p.29 / Chapter 2.1.3 --- Methods in determining differentiated cells --- p.31 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Cell line --- p.33 / Chapter 2.2.2 --- Chemicals --- p.33 / Chapter 2.2.3 --- Solutions and buffers --- p.34 / Chapter 2.3 --- Methods --- p.36 / Chapter 2.3.1 --- Microscopic studies of the JCS cells --- p.36 / Chapter 2.3.1.1 --- Histochemical staining of JCS --- p.36 / Chapter 2.3.1.2 --- Transmission electronic microscopic --- p.36 / Chapter 2.3.2 --- [3H]-thymidine incorporation assay --- p.37 / Chapter 2.3.3 --- MTT assay --- p.37 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- Histochemical staining of JCS cells --- p.38 / Chapter 2.4.2 --- Electron microscopy --- p.40 / Chapter 2.4.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.44 / Chapter 2.4.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.48 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- Morphological differentiation of JCS cells --- p.53 / Chapter 2.5.2 --- The ultra-structures of JCS cells --- p.53 / Chapter 2.5.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.54 / Chapter 2.5.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.55 / Chapter Chapter Three --- Roles of Prostaglandin E2 in normal haemopoiesis and the detection of PGE2 receptors expression in JCS and bone marrow cells / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.1.1 --- Colony assay --- p.57 / Chapter 3.1.2 --- The use of RT-PCR --- p.58 / Chapter 3.1.3 --- Prostaglandin E receptors --- p.59 / Chapter 3.2 --- Materials --- p.62 / Chapter 3.2.1 --- Bone marrow cells --- p.62 / Chapter 3.2.2 --- Cell line --- p.62 / Chapter 3.2.3 --- Chemicals --- p.62 / Chapter 3.2.4 --- Primers --- p.63 / Chapter 3.2.5 --- Solutions and buffers --- p.64 / Chapter 3.2.6 --- Enzymes and reagents --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Titration of mouse IL-3 --- p.66 / Chapter 3.3.2 --- Determination of suitable IL-3 concentration for growth of bone marrow cells in colony assay --- p.66 / Chapter 3.3.2.1 --- Preparation of bone marrow cells --- p.66 / Chapter 3.3.2.2 --- Preparation of culture medium for colony assay --- p.67 / Chapter 3.3.3 --- Investigation of the effect of PGE2 on normal haemopoiesis by colony assay --- p.68 / Chapter 3.3.4 --- Detection of PGE2 receptors expression on JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.1 --- Preparation of cell lysates --- p.68 / Chapter 3.3.4.2 --- Preparation of total RNA of JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.3 --- RT-PCR --- p.69 / Chapter 3.4 --- Results --- p.71 / Chapter 3.4.1 --- Titration of mouse IL-3 --- p.71 / Chapter 3.4.2 --- Effect of mouse IL-3 on normal haemopoiesis --- p.73 / Chapter 3.4.3 --- Effect of PGE2 on mouse IL-3 driven normal bone marrow cell differentiation --- p.76 / Chapter 3.4.4 --- Analysis of total RNA prepared from uninduced JCS cells and bone marrow cells --- p.79 / Chapter 3.4.5 --- "Expression of gapdh in heart, liver, spleen, JCS and bone marrow cells" --- p.81 / Chapter 3.4.6 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.82 / Chapter 3.5 --- Discussion --- p.84 / Chapter 3.5.1 --- Effect of PGE2 on IL-3 driven normal bone marrow cells differentiation --- p.84 / Chapter 3.5.2 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.85 / Chapter Chapter Four --- Gene expression profile of JCS cells under 5 hours of PGE2 induction / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.1.1 --- Review of methods studying differential gene expression --- p.88 / Chapter 4.1.2 --- The choice of method studying differential gene expression --- p.92 / Chapter 4.1.3 --- The microarray --- p.93 / Chapter 4.2 --- Materials --- p.95 / Chapter 4.2.1 --- Cell line --- p.95 / Chapter 4.2.2 --- Kits --- p.95 / Chapter 4.2.3 --- Chemicals --- p.95 / Chapter 4.2.4 --- Solutions and buffers --- p.96 / Chapter 4.2.5 --- Reagents --- p.97 / Chapter 4.3 --- Methods --- p.98 / Chapter 4.3.1 --- Preparation of total RNA from PGE2 induced JCS cells --- p.98 / Chapter 4.3.2 --- Preparation of cDNA probes --- p.98 / Chapter 4.3.2.1 --- Probe synthesis from total RNA --- p.98 / Chapter 4.3.2.2 --- Column chromatography --- p.99 / Chapter 4.3.3 --- Hybridizing cDNA probes to the Atlas Array --- p.99 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Spectrophotometric analysis of total RNA after ethanol precipitation --- p.101 / Chapter 4.4.2 --- Hybridization of cDNA probes to Atlas Array --- p.102 / Chapter 4.5 --- Discussion --- p.121 / Chapter 4.5.1 --- Genes with increased expression --- p.121 / Chapter 4.5.2 --- Genes with decrease expression --- p.127 / Chapter 4.5.3 --- Study of gene expression profile by microarray --- p.128 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.2 --- Roles of PGE2 in JCS cells differentiation --- p.131 / Chapter 5.3 --- Roles of PGE2 in normal haemopoiesis --- p.134 / Chapter 5.4 --- Further studies --- p.135 / References --- p.137

Chan, Hoi Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 206-220). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.viii / 撮要 --- p.xii / PUBLICATIONS --- p.xiv / TABLE OF CONTENTS --- p.xv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis Development --- p.1 / Chapter 1.1.2 --- Leukemia --- p.6 / Chapter 1.1.2.1 --- General Symptoms of Leukemia --- p.7 / Chapter 1.1.2.2 --- Classification of Leukemia --- p.8 / Chapter 1.1.2.3 --- Conventional Treatment against Leukemia --- p.15 / Chapter 1.1.2.4 --- Novel Approaches --- p.19 / Chapter 1.2 --- The Chinese Medicinal Herb-Banlangen (板藍根） --- p.24 / Chapter 1.2.1 --- An Overview on Natural Indigo Compounds Derived from Banlangen --- p.24 / Chapter 1.2.2 --- Tryptanthrin --- p.29 / Chapter 1.2.2.1 --- Anti-bacterial Activity of Tryptanthrin --- p.29 / Chapter 1.2.2.2 --- Anti-tumor Activity of Tryptanthrin --- p.31 / Chapter 1.2.2.3 --- Anti-inflammatory Activity of Tryptanthrin --- p.33 / Chapter 1.2.2.4 --- Cutting Edges of Tryptanthrin as a Drug --- p.34 / Chapter 1.2.2.5 --- Metabolism of Tryptanthrin --- p.35 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.37 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Animals --- p.39 / Chapter 2.1.2 --- Cell Lines --- p.39 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.41 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.45 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.46 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.49 / Chapter 2.1.7 --- Reagents for Measuring Caspase Activity --- p.50 / Chapter 2.1.8 --- Reagents for Total RNA Isolation --- p.53 / Chapter 2.1.9 --- Reagents and Buffers for Reversed Transcription-PCR --- p.54 / Chapter 2.1.10 --- Reagents and Buffers for Real Time-PCR --- p.59 / Chapter 2.1.11 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Acids --- p.59 / Chapter 2.1.12 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.61 / Chapter 2.2 --- Methods --- p.70 / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.70 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.70 / Chapter 2.2.3 --- Determination of Cell Viability --- p.71 / Chapter 2.2.4 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.72 / Chapter 2.2.5 --- Determination of Anti-leukemia Activity In Vivo --- p.73 / Chapter 2.2.6 --- Analysis of Cell Cycle Profile/DNA Content by Flow Cytometry --- p.74 / Chapter 2.2.7 --- Measurement of Apoptosis --- p.75 / Chapter 2.2.8 --- Determination of the Mitochondrial Membrane Potential --- p.77 / Chapter 2.2.9 --- Measurement of Caspase Activity --- p.78 / Chapter 2.2.10 --- Study of Intracellular Accumulation of Reactive Oxygen Species --- p.79 / Chapter 2.2.11 --- Gene Expression Study --- p.80 / Chapter 2.2.12 --- Protein Expression Study --- p.83 / Chapter 2.2.13 --- Measurement of Cell Differentiation --- p.87 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-PROLIFERATIVE EFFECT OF TRYPTANTHRIN AND INDIRUBIN-3'-OXIME ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.90 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Effects of Indirubin-3'-oxime and Tryptanthrin on the Proliferation of Myeloid Leukemia Cell Lines of Human and Murine Origins In Vitro --- p.94 / Chapter 3.2.2 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Tryptanthrin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.108 / Chapter 3.2.3 --- Cytotoxic Effect of Tryptanthrin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.113 / Chapter 3.2.4 --- Cytotoxicity of Tryptanthrin on Non-Cancer Cell Line and Primary Myeloid Cells In Vitro --- p.115 / Chapter 3.2.5 --- Effects of Tryptanthrin on the Cell Cycle Profile of the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.118 / Chapter 3.2.6 --- Effects of Tryptanthrin on the Expression of Cell Cycle Related Genes in Murine Myelomonocytic Leukemia WEHI-3B JCS Cells In Vitro --- p.123 / Chapter 3.2.7 --- Expression of CDK-inhibitors in Tryptanthrin-treated Murine Myeloid Leukemia WEHI-3B JCS Cells --- p.126 / Chapter 3.2.8 --- Effects of Tryptanthrin on the In Vivo Tumorigenicity of the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.128 / Chapter 3.2.9 --- In Vivo Anti-tumor Effect of Tryptanthrin on Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.130 / Chapter 3.3 --- Discussion --- p.132 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF TRYPTANTHRIN ON MURINE MYELOMONOCYTIC LEUKEMIA WEHI-3B JCS CELLS / Chapter 4.1 --- Introduction --- p.139 / Chapter 4.2 --- Results --- p.143 / Chapter 4.2.1 --- Induction of DNA Fragmentation by Tryptanthrin in the Murine Myelomonocytic Leukemia WEHI-3B Cells In Vitro --- p.143 / Chapter 4.2.2 --- Induction of Phosphatidylserine Externalization by Tryptanthrin in Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.145 / Chapter 4.2.3 --- Change of Mitochondrial Membrane Potential of Tryptanthrin- treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.147 / Chapter 4.2.4 --- Induction of Caspase Activity in Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.150 / Chapter 4.2.5 --- Induction of Reactive Oxygen Species in Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.155 / Chapter 4.2.6 --- Expression of Bcl-2 Family Proteins in the Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.160 / Chapter 4.2.7 --- Effects of Tryptanthrin on the mRNA Expression of Bcl-2 Family Proteins in Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.163 / Chapter 4.2.8 --- Expression of Fas and Fas Ligand Proteins in Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.167 / Chapter 4.2.9 --- Expression of Pro-Apoptotic Protein in Tryptanthrin- treated Murine Myelomonocytic Leukemia WEHI-3B JCS cells --- p.170 / Chapter 4.2 --- Discussion --- p.173 / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ABILITY OF TRYPTANTHRIN ON MURINE MYELOMONOCYTIC LEUKEMIA WEHI-3B JCS CELLS / Chapter 5.1 --- Introduction --- p.184 / Chapter 5.2 --- Results --- p.186 / Chapter 5.2.1 --- Morphological Studies on Tryptanthrin-treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.186 / Chapter 5.2.2 --- Effects of Tryptanthrin on the Cell Size and Granularity of the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.189 / Chapter 5.2.3 --- Effects of Tryptanthrin on Induction of NBT-reducing Activity in the Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.191 / Chapter 5.3 --- Discussion --- p.195 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.198 / REFERENCES --- p.206

Yip, Wai Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 203-228). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.x / 撮要 --- p.xiv / TABLE OF CONTENTS --- p.xvii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis and Leukemia / Chapter 1.1.1 --- An Overview on Hematopoiesis Development --- p.1 / Chapter 1.1.1.1 --- Hematopoietic Growth Factors --- p.4 / Chapter 1.1.1.2 --- Site Switching of Hematopoiesis --- p.5 / Chapter 1.1.2 --- An Overview on Leukemia --- p.7 / Chapter 1.1.2.1 --- Classification of Leukemia --- p.7 / Chapter 1.1.2.2 --- Conventional Therapy of Leukemia --- p.10 / Chapter 1.1.2.3 --- Novel Approaches to Leukemia Therapy: Apoptosis and Differentiation Induction --- p.13 / Chapter 1.2 --- Polysaturated Fatty Acids / Chapter 1.2.1 --- An Overview on Polyunsaturated Fatty Acids --- p.16 / Chapter 1.2.2 --- An Overview on Essential Fatty Acids --- p.17 / Chapter 1.2.2.1 --- Alpha Linolenic Acids (ALA) --- p.17 / Chapter 1.2.2.2 --- Gamma Linolenic Acid (GLA) --- p.18 / Chapter 1.2.3 --- "An Overview on Conjugated Fatty Acids: Conjugated Linoleic Acid (CLA), Conjugated EPA and Conjugated DHA" --- p.20 / Chapter 1.2.4 --- Conjugated Linolenic Acid (CLN) --- p.24 / Chapter 1.2.4.1 --- Identification and Production of CLN --- p.28 / Chapter 1.2.4.2. --- Metabolism of CLN --- p.29 / Chapter 1.2.4.3 --- Anti-Obese and Hypolipidemic Effect of CLN --- p.30 / Chapter 1.2.4.4 --- Anti-Proliferative Effect of CLN --- p.30 / Chapter 1.2.4.5 --- Other Novel Effects of CLN --- p.32 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.34 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Animals --- p.36 / Chapter 2.1.2 --- Human Cell Lines --- p.36 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.38 / Chapter 2.1.4 --- Reagents and Buffer for Flow Cytometry --- p.44 / Chapter 2.1.5 --- Reagents for DNA Extraction --- p.47 / Chapter 2.1.6 --- Cell Death Detection ELISApLus --- p.48 / Chapter 2.1.7 --- Reagents for Measuring Caspase Activity --- p.50 / Chapter 2.1.8 --- Reagents for FACE´ёØ ELISA Kit --- p.53 / Chapter 2.1.9 --- Reagents for Western Blotting --- p.55 / Chapter 2.2 --- Methods --- p.65 / Chapter 2.2.1 --- Culturing the Tumor Cell Lines --- p.65 / Chapter 2.2.2 --- "Isolation, Preparation and Culturing of Murine Peritoneal Macrophages and Bone Marrow Cells" --- p.66 / Chapter 2.2.3 --- Anti-proliferation Assays --- p.67 / Chapter 2.2.4 --- Cell Viability Determination --- p.68 / Chapter 2.2.5 --- Determination of Anti-leukemia Activity In Vivo (In Vivo Tumorigenicity Assay) --- p.69 / Chapter 2.2.6 --- Cell Cycle Analysis by Flow Cytometry --- p.69 / Chapter 2.2.7 --- Detection of Apoptosis --- p.70 / Chapter 2.2.8 --- Assessment of Differentiation-associated Characteristics --- p.74 / Chapter 2.2.9 --- Measurement of Caspase Activities --- p.76 / Chapter 2.2.10 --- Protein Expression Study --- p.78 / Chapter 2.2.11 --- Detection of Phosphorylation of JNK by FACE´ёØ JNK ELISA Kit --- p.83 / Chapter 2.2.12 --- Detection of Phosphorylation of NF-kB by FACE´ёØ NF-kB p65 Profiler --- p.85 / Chapter 2.2.13 --- Statistical Analysis --- p.85 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI PROLIFERATIVE EFFECTS OF CONJUGATED LINOLENIC ACIDS ON THE HUMAN MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Anti-proliferative Activity of CLN Isomers on Various Myeloid Leukemia and Lymphoma Cell Lines In Vitro --- p.88 / Chapter 3.2.2 --- Direct Cytotoxic Effect of Jacaric Acid on HL-60 Cells In Vitro --- p.95 / Chapter 3.2.3 --- Cytotoxic Effect of Jacaric Acid on Primary Murine Cells and Human Normal Cell Lines In Vitro --- p.98 / Chapter 3.2.4 --- Kinetics and Reversibility Studies of the Anti-proliferative Effect of Four CLN Isomers on the Human Promyelocytic Leukemia HL-60 Cells --- p.101 / Chapter 3.2.5 --- Synergistic Anti-proliferative Effect of Jacaric Acid with Vitamin D3 and All Trans-Retinoic Acid (ATRA) on the Human Promyelocytic Leukemia HL-60 Cells In Vitro --- p.114 / Chapter 3.2.6 --- Effect of Jacaric Acid on the Cell Cycle Profile of the HL-60 Cells In Vitro --- p.116 / Chapter 3.2.7 --- Effect of Jacaric Acid on the In Vivo Tumorigenicity of the HL-60 Cells --- p.119 / Chapter 3.3 --- Discussion --- p.121 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING AND DIFFERENTIATION-INDUCING EFFECTS OF CONJUGATED LINOLENIC ACIDS ON THE HUMAN MYELOID LEUKEMIA CELLS / Chapter 4.1.1 --- Introduction --- p.128 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Induction of Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells by Jacaric Acid --- p.134 / Chapter 4.2.2 --- Apoptosis-Inducing Effect of Jacaric Acid on the Human Promyelocytic Leukemia HL-60 Cells as Detected by Annexin V-GFP PI Double Staining Method --- p.138 / Chapter 4.2.3 --- Effect of Jacaric Acid on the Mitochondrial Membrane Potential in the Human Promyelocytic Leukemia HL-60 Cells --- p.140 / Chapter 4.2.4 --- Effects of Jacaric Acid on the Caspase Activities in the Human Promyelocytic Leukemia HL-60 Cells --- p.142 / Chapter 4.2.5 --- Effects of Jacaric Acid and Antioxidants on the ROS Induction in the Human Promyelocyic Leukemia hl-6 Cells --- p.147 / Chapter 4.2.6 --- Effect of N-acetyl-L-Cysteine on the Apoptosis-Inducing Activity of Jacaric Acid in the Human Promyelocytic Leukemia HL-60 Cells --- p.149 / Chapter 4.2.7 --- Morphological Studies on the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.151 / Chapter 4.2.8 --- Cell Size and Granularity of the Human Promyelocytic Leukemia HL-60 Cells after Treatment with Different CLN Isomers --- p.153 / Chapter 4.2.9 --- Expression of Differentiation-Related Cell Surface Markers in the Human Promyelocytic Leukemia HL-60 Cells after Treatment with Jacaric Acid --- p.155 / Chapter 4.3 --- Discussion --- p.158 / Chapter CHAPTER 5: --- STUDIES ON THE APOPTOSIS-ASSOCIATED PROTEINS AND SIGNALING PATHWAYS IN CONJUGATED LINOLENIC ACID-INDUCED APOPTOSIS OF THE HUMAN MYELOID LEUKEMIA CELLS / Chapter 5.1 --- Introduction --- p.165 / Chapter 5.2 --- Results / Chapter 5.2.1 --- Expression of Fas and Fas Ligand Proteins in the Jacaric Acid- treated Human Promyelocytic Leukemia HL-60 Cells --- p.171 / Chapter 5.2.2 --- Expression of Bcl-2 Family Member Proteins in the Jacaric Acid- treated Human Promyelocytic Leukemia HL-60 Cells --- p.173 / Chapter 5.2.3 --- Cytochrome c Release in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.175 / Chapter 5.2.4 --- Cleavage of Poly(ADP-ribose) Polymerase (PARP) in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.177 / Chapter 5.2.5 --- Phosphorylation of ERK in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.179 / Chapter 5.2.6 --- Phosphorylation of JNK in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.181 / Chapter 5.2.7 --- Phosphorylation of NF-kB Protein in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.183 / Chapter 5.3 --- Discussion --- p.185 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.195 / REFERENCES --- p.203

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 85-105).

Bcr-abl expression being associated with anti-apoptotic signaling and expression of DNA repair enzymes, we surmised that single molecules capable of blocking abl tyrosine kinase (TK) function and damaging DNA should lead to compounds with potency superior to that of GleevecRTM. To this end, we designed novel agents termed "combi-molecules" programmed to not only behave as bcr-abl inhibitors on their own, but also to further degrade to another inhibitor and a DNA damaging species. The released inhibitor was designed to sustain bcr-abl inhibition following degradation of the combi-molecule and the DNA damaging species to activate pathways leading to apoptosis. To model this strategy termed "combi-targeting", we synthesized ZRCM5 (a monoalkyltriazene) that showed antiproliferative activity superior to that of the classical DNA damaging agent TemodalRTM, but not to that of Gleevec RTM. This result was imputed to the rather weak bcr-abl inhibitory activity of ZRCM5 and its strong DNA damaging property. Another prototype designed to contain an aniline mustard moiety (AK04) was a strong bcr-abl inhibitor but a poor DNA alkylating agent. Its cytotoxic activity was again stronger than that of the clinical alkylating agent chlorambucil but inferior to that of GleevecRTM. Further chemical studies directed at structural modification of the benzamide moiety led to the synthesis of ZRF1 with strong potency against bcr-abl TK and strong DNA damaging property. This novel optimized combi-molecule showed a 1.6-3-fold greater potency than GleevecRTM against bcr-abl expressing cells. Further investigation with ZRF1, showed that its cytotoxic potency was dependent on the p53 wild-type status of the cells. In cells expressing wild-type p53, p21 transactivation was associated with cell cycle arrest and that of Bax with apoptosis. In addition to, the pro-apoptotic effect of bcr-abl inhibition, these multiple mechanisms of action may synergistically enhance the cytotoxic potency of ZRF1 in p53 wild-type cells. The study conclusively demonstrated that p53 is a major determinant for the cytotoxic advantage of the novel combi-molecular approach in chronic myelogenous leukemia (CML), a disease in which 70-85% of all cases express wild-type p53.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.