Chan Ping Kei. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 135-143). / Abstracts in English and Chinese. / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTECTS --- p.v / LIST OF FIGURE --- p.viii / LIST OF ABBREVIATIONS --- p.xii / CHAPTER / Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Epidemilogy --- p.1 / Chapter 1.2 --- Malaria disease --- p.1 / Chapter 1.3 --- Life cycle of Malaria --- p.1 / Chapter 1.4 --- Current measure to control Malaria --- p.6 / Chapter 1.5 --- Anti-malaria vaccine candidate --- p.7 / Chapter 1.6 --- Anti-erythrocytic malaria vaccine MSP-1 --- p.10 / Chapter 1.7 --- Baculovirus Expression System --- p.20 / Chapter 1.8 --- hexa-histidine tagged fusion protein --- p.25 / Chapter 1.9 --- IMAC --- p.26 / Chapter 1.10 --- Aim of study --- p.26 / Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.29 / Chapter 2.2 --- Methods --- p.40 / Chapter 3 --- CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT BmNPV CARRYING PfMSP-l42 / Chapter 3.1 --- Cloning of C-HisPfMSP-l42 into pBM030 --- p.71 / Chapter 3.2 --- Construction of Recombinant BmNPV Carrying PfMSP-l42 --- p.72 / Chapter 3.3 --- Purification of Recombinant BmNPVs --- p.78 / Chapter 3.4 --- In vitro expression of Recombinant --- p.80 / Chapter 3.5 --- In Vivo Expression of Recombinant PfMSP-l42 Protein --- p.80 / Chapter 4 --- PURIFICATION OF BmNPV-EXPRESSED RECOMBINANT C- TERMIAL HEXA-HIS-TAGGED PfMSP-l42 PROTEIN / Chapter 4.1 --- Nickel ion charged Chelating Sepharose Fast Flow (immobilized metal affinity chromatography) --- p.88 / Chapter 4.2 --- POROS HS/M (Strong Cation Exchanger) --- p.105 / Chapter 4.3 --- Combination of chromatographic separations --- p.107 / Chapter 5 --- CHARACTERIZATION OF RECOMBINANT C-HISPfMSP-l42 PROTEIN / Chapter 5.1 --- Proper formation of disulphide bridges in epidermal growth factor (EGF) like domains --- p.115 / Chapter 5.2 --- Characterization of the integrity of hexa-histidines residue on recombinant PfMSP-142 protein --- p.117 / Chapter 5.3 --- Immunogenicity of Recombinant C-HisPfMSP-l42 Protein --- p.117 / Chapter 6 --- DISCUSSION / Chapter 6.1 --- Construction of recombinant BmNPV carrying HisPfMSP-l42 --- p.122 / Chapter 6.2 --- Expression of recombinant HisPfMSP-l42 proteins --- p.123 / Chapter 6.3 --- Purification of recombinant C-HisPfMSP-l42 protein --- p.125 / Chapter 6.4 --- Characterization of recombinant C-HisPfMSP-l42 protein --- p.128 / Chapter 6.5 --- Future prospects --- p.130 / REFERENCE --- p.135 / APPENDICES / Chapter 1. --- Appearance of Mulberry leaves / Chapter 2. --- Biomark 2000 (Beckman) program for sandwich ELISA protocol / Chapter 3. --- Nucleotide Sequence of PfMSP-l42 3D7 Isolate / Chapter 4. --- Nucleotide sequence of PfMSP-l42 FVO isolate / Chapter 5. --- Efficiency of the mAb5.2 immunoaffinity column in purifying the recombinant PfMSP-l42 protein

Yu Yun Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 101-114). / Abstracts in English and Chinese. / Acknowledgments / Abstract / Table of Contents / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- General Structure of Lectins --- p.1 / Chapter 1.1.1 --- Metal Binding Sites --- p.2 / Chapter 1.1.2. --- Hydrophobic Sites --- p.3 / Chapter 1.1.3. --- Glycosylation Sites --- p.3 / Chapter 1.2 --- Carbohydrate Specificities of Lectins --- p.4 / Chapter 1.3 --- Plant Lectins --- p.4 / Chapter 1.3.1 --- Localization of lectins in plants --- p.4 / Chapter 1.3.1.1 --- Localization in seeds --- p.4 / Chapter 1.3.1.2 --- Localization in vegetative parts --- p.5 / Chapter 1.3.1.3 --- Biosynthesis of plant lectins --- p.6 / Chapter 1.3.2 --- Functions of plant lectins in plants --- p.7 / Chapter 1.3.2.1 --- In cell growth --- p.7 / Chapter 1.3.2.2 --- In storage --- p.8 / Chapter 1.3.2.3 --- In plant defence --- p.8 / Chapter 1.3.2.4 --- In nitrogen cycle --- p.10 / Chapter 1.3.3 --- Biological activities of plant lectins in other organisms --- p.13 / Chapter 1.3.3.1 --- Immunomodulatory activity --- p.13 / Chapter 1.3.3.2 --- Antitumor and antiproliferative activities --- p.14 / Chapter 1.3.3.3 --- Mitogenic activity --- p.14 / Chapter 1.3.3.4 --- Antiviral activity --- p.14 / Chapter 1.3.4 --- Relationship between lectins and ribosome inactivating proteins: family of ricin-related proteins --- p.16 / Chapter 1.3.5 --- Applications of plant lectins --- p.18 / Chapter 1.3.5.1 --- In scientific research --- p.18 / Chapter 1.3.5.2 --- In medical research --- p.19 / Chapter 1.4 --- Animal Lectins --- p.20 / Chapter 1.4.1 --- Some properties of animal lectins --- p.20 / Chapter 1.4.2 --- Functions of animal lectins --- p.22 / Chapter 1.4.2.1 --- In protein metabolism --- p.22 / Chapter 1.4.2.2 --- As a mediator of binding and phagocytosis of microorganisms --- p.22 / Chapter 1.4.2.3 --- Control of differentiation and organ formation --- p.23 / Chapter 1.4.2.4 --- Lectins and migration of lymphocytes --- p.23 / Chapter 1.4.2.5 --- Lectins and metastasis --- p.24 / Chapter 1.5 --- Mushroom lectins --- p.25 / Chapter 1.6 --- Regulation of lectins --- p.29 / Chapter 1.7 --- Isolation and purification of lectins --- p.31 / Chapter 1.8 --- Objectives of the present study --- p.33 / Chapter CHAPTER 2 --- "SCREENING FOR HEMAGGLUTINATING ACTIVITY IN EXTRACTS OF SEEDS, FRUITS, VEGETABLES AND CHINESE MEDICINAL HERBS" --- p.35 / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials and methods --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.4 --- Discussion --- p.38 / Chapter CHAPTER 3 --- ISOLATION OF LECTIN FROM RHIZOMES OF SMILAX GLABRA (FAMILY LILIACEAE) --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Introduction about Smilax glabra and its chemical constituents --- p.43 / Chapter 3.1.2 --- Introduction about monocot lectins including Liliaceae lectins --- p.45 / Chapter 3.2 --- Materials and methods --- p.50 / Chapter 3.2.1 --- Isolation of lectins from Smilax glabra rhizomes --- p.50 / Chapter 3.2.2 --- Assay for hemagglutinating activity --- p.55 / Chapter 3.2.3 --- Test of inhibition of lectin-induced hemagglutination by various carbohydrates --- p.55 / Chapter 3.2.4 --- "Effects of acid, alkali, temperature and cations on hemagglutinationg activity of lectin" --- p.56 / Chapter 3.2.5 --- Determination of protein concentration --- p.56 / Chapter 3.2.6 --- Molecular mass determination by SDS-PAGE --- p.56 / Chapter 3.2.7 --- Molecular mass determination by gel filtration --- p.56 / Chapter 3.2.8 --- Amino acid sequence analysis --- p.57 / Chapter 3.3 --- Results --- p.57 / Chapter CHAPTER 4 --- ISOLATION OF LECTIN FROM SEEDS OF THE CHINESE CHESTNUT CASTANEA MOLLISIMA (FAMILY FAGACEAE) --- p.74 / Chapter 4.1 --- Introduction to Castanea mollisima and its chemical constituents --- p.74 / Chapter 4.2 --- Materials and Methods --- p.78 / Chapter 4.2.1 --- Isolation of lectin from Chinese chestnuts --- p.78 / Chapter 4.2.2 --- Assay for hemagglutinating activity --- p.83 / Chapter 4.2.3 --- Test of inhibition of lectin-induced hemagglutination by various carbohydrates --- p.83 / Chapter 4.2.4 --- "Effects of acid, alkali, temperature and cations on hemagglutinationg activity of lectin" --- p.83 / Chapter 4.2.5 --- Determination of protein concentration --- p.83 / Chapter 4.2.6 --- Molecular mass determination by SDS-PAGE --- p.83 / Chapter 4.2.7 --- Molecular mass determination by gel filtration --- p.83 / Chapter 4.2.8 --- Amino acid sequence analysis --- p.83 / Chapter 4.3 --- Results --- p.84 / Chapter 4.4 --- Discussion --- p.96 / Chapter CHAPTER 5 --- GENERAL DISCUSSION AND CONCLUSION --- p.98 / REFERENCES: --- p.101

Ng Kai-on. / "July 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 162-180). / 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.

Chan, Pui Ting. / Thesis submitted in: May 2007. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 110-118). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Peroxisome proliferater-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR isoforms --- p.1 / Chapter 1.1.3 --- PPARα ligands --- p.2 / Chapter 1.2 --- Biological role of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.5 / Chapter 1.2.3 --- Oxidative stress and carcinogenesis --- p.6 / Chapter 1.3 --- Discovery of PPARα-regulated and starvation-induced gene (PPSIG) --- p.7 / Chapter 1.4 --- Objectives of the present study --- p.9 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.10 / Chapter 2.1 --- Cloning of PPSIG cDNA into a pCMV-Tag epitope tagging mammalian expression vector --- p.10 / Chapter 2.1.1 --- Materials --- p.10 / Chapter 2.1.2 --- Methods --- p.10 / Chapter 2.2 --- Transient transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.16 / Chapter 2.2.1 --- Cell culture and transfection --- p.16 / Chapter 2.2.1.1 --- Materials --- p.16 / Chapter 2.2.1.2 --- Methods --- p.19 / Chapter 2.2.2 --- Western blot analysis --- p.20 / Chapter 2.2.2.1 --- Materials --- p.20 / Chapter 2.2.2.2 --- Methods --- p.20 / Chapter 2.3 --- Stable transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.22 / Chapter 2.3.1 --- Linearization of the pCMVT4B-PPSIG construct --- p.22 / Chapter 2.3.1.1 --- Materials --- p.22 / Chapter 2.3.1.2 --- Methods --- p.22 / Chapter 2.3.2 --- Cell culture and stable transfection --- p.23 / Chapter 2.3.2.1 --- Materials --- p.23 / Chapter 2.3.2.2 --- Methods --- p.23 / Chapter 2.3.3 --- Selection of the G418-resistant clones --- p.26 / Chapter 2.3.3.1 --- Materials --- p.26 / Chapter 2.3.3.2 --- Methods --- p.29 / Chapter 2.3.4 --- Picking and expanding the G418-resistant clones --- p.30 / Chapter 2.3.4.1 --- Materials --- p.30 / Chapter 2.3.4.2 --- Methods --- p.30 / Chapter 2.3.5 --- Screening and confirmation of the stable transfectants --- p.31 / Chapter 2.3.5.1 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.31 / Chapter 2.3.5.1.1 --- Materials --- p.31 / Chapter 2.3.5.1.2 --- Methods --- p.31 / Chapter 2.3.5.2 --- Northern blot analysis --- p.35 / Chapter 2.3.5.2.1 --- Materials --- p.35 / Chapter 2.3.5.2.2 --- Methods --- p.35 / Chapter 2.3.5.3 --- Western blot analysis --- p.37 / Chapter 2.3.5.3.1 --- Materials --- p.37 / Chapter 2.3.5.3.2 --- Methods --- p.37 / Chapter 2.3.5.4 --- Immunoprecipitation --- p.37 / Chapter 2.3.5.4.1 --- Materials --- p.37 / Chapter 2.3.5.4.2 --- Methods --- p.38 / Chapter 2.3.5.5 --- Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry analysis --- p.39 / Chapter 2.3.5.5.1 --- Materials --- p.39 / Chapter 2.3.5.5.2 --- Methods --- p.39 / Chapter 2.4 --- "Analysis of the all-trans-13,14-dihydroretinol saturase (RetSat) activity by high-performance liquid chromatography (HPLC) analysis" --- p.41 / Chapter 2.4.1 --- Materials --- p.41 / Chapter 2.4.2 --- Methods --- p.42 / Chapter 2.4.2.1 --- Preparation of all-trans-retinol --- p.42 / Chapter 2.4.2.2 --- Treatment of PPSIG-transfected cells with all-trans-retinol --- p.42 / Chapter 2.4.2.3 --- Retinoid analysis --- p.43 / Chapter 2.5 --- Analysis of fatty acid compositions by gas chromatography-mass spectrometry (GC-MS) --- p.43 / Chapter 2.5.1 --- Materials --- p.43 / Chapter 2.5.2 --- Methods --- p.44 / Chapter 2.5.2.1 --- Preparation of fatty acid-BSA complex --- p.44 / Chapter 2.5.2.2 --- Treatment of PPSIG-transfected cells with fatty acid-BSA complex --- p.44 / Chapter 2.5.2.3 --- Extraction of fatty acids --- p.45 / Chapter 2.5.2.4 --- Methylation of the fatty acids --- p.45 / Chapter 2.5.2.5 --- GC-MS analysis --- p.46 / Chapter 2.5.2.6 --- Statistical analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.48 / Chapter 3.1 --- The PPSIG cDNA was subcloned into a pCMV-Tag epitope tagging mammalian expression vector --- p.48 / Chapter 3.2 --- The pCMVT4B-PPSIG expression construct was transiently transfected into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3 --- Stable transfection of the pCMVT4B-PPSIG expression construct into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3.1 --- PPSIG-transfected CHO-K1 and AML-12 cells were obtained after G418 selection --- p.54 / Chapter 3.3.2 --- PPSIG-transfected CHO-K1 and AML-12 cells had high PPSIG mRNA expression --- p.58 / Chapter 3.3.3 --- PPSIG-FLAG fusion protein was over-expressed in the PPSIG- transfected CHO-K1 and AML-12 cells --- p.61 / Chapter 3.3.4 --- The stable transfectants were immunoprecipitated and identified as PPSIG protein by the mass spectrometry analysis --- p.64 / Chapter 3.4 --- PPSIG protein posseses saturase activity towards all-trans-retinol --- p.66 / Chapter 3.5 --- PPSIG protein is not a fatty acid transporter --- p.78 / Chapter CHAPTER 4 --- DISCUSSION --- p.101 / FUTURE STUDIES --- p.107 / REFERENCES --- p.110 / Appendix A: Prediction of the molecular weight of pCMVT4B- PPSIG protein --- p.119 / Appendix B: Theoretical tryptic peptides of PPSIG --- p.120 / Appendix C: Protein-peptide mass reports --- p.122 / Chapter C1. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone L2H4B18 --- p.122 / Chapter C2. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone AL2L7 --- p.123 / Appendix D: HPLC spectrum of the RetSat activity towards all- trans retinol --- p.124 / Chapter D1. --- RetSat activity towards all-trans retinol according to the Moise's group study ((Moise et al. 2004) --- p.124

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144

The hemopoietic system Is organized as a hierarchy of hemopoietic cell populations distinguished by differences in their proliferation and differentiation potential. Studies using short-term in vitro and in vivo assays based on colony formation in semi-solid medium, or in the spleens of lethally irradiated mice, respectively, have shown that these procedures detect primarily lineage-restricted progenitor types and have provided much information about the characteristics and regulation of such cells. Assessment of lymphoid and myeloid tissue reconstitution after more prolonged periods following transplantation has established the existence of a more primitive stem cell type; however, the retrospective nature of these complex analyses has Impeded characterization and purification of these cells. My first objective was to develop a procedure for the selective isolation of stem cells with short-term in vitro and in vivo multilineage differentiation potential. For this I devised a single-step, four-parameter fluorescence activated cell sorting procedure In which cells were selected according to their forward and orthogonal light-scattering properties, and their surface expression of the Thy-1 and H-2K antigens. Application of this procedure to marrow cells from mice treated 4 days previously with 150 mg/kg of 5-fluorouracil showed that it could be used to sort a subpopulation that was enriched 100-fold in CFU-GEMM and in which 1 in 4 cells was a day 12 CFU-S. To determine the extent to which stem cells with long-term lympho-myeloid repopulating potential had been copurified, I undertook to develop a quantitative procedure that might allow this primitive cell population to be measured and hence characterized on a routine basis. This required an assay that would detect donor-derived hemopoiesis exclusively, and that was sensitive enough for the detection of limiting numbers of cells with long-term lympho-myeloid repopulating potential. This was shown to be possible using a competitive repopulation assay in which lethally irradiated female recipients were transplanted with male "test" cells together with a second suspension of female cells with adequate short-term repopulating activity but greatly diminished long-term repopulating potential. These sex differences were then used to specifically identify the 5 week progeny of stem cells in the test suspension. Assessment of the sorted day 4 5-FU marrow population revealed that it was capable of repopulating all hemopoietic organs after transplantation and that an enrichment of 30-fold over unseparated, 5-FU-treated marrow had been achieved. My second objective was to determine whether the competitive long-term lymphoid and myeloid repopulation obtained with these sorted cells was due to the activity of Individual stem cells with a dual potential for lymphopoiesis and myelopoiesis. For this I used retroviral-infection to uniquely mark sorted cells In vitro, and then transplanted them in sufficiently low numbers to allow individual regenerated clones to be detected and analyzed. In some mice, distribution of cells with the same unique integration marker in different lymphoid and myeloid cell populations established the presence of lympho-myeloid stem cells in the original sorted population. In addition, clones with restricted tissue distributions were also documented. My final objective was to investigate whether the competitive repopulation assay was in fact able to serve as a procedure for the exclusive quantitation of long-term lympho-myeloid repopulating stem cells. A limiting dilution approach was used to compare the frequency of hemopoietic stem cells (competitive repopulating units, CRU) in marrow obtained from a variety of sources, using >20% repopulation by male cells at 5 or 10 weeks post-transplantation as the end point. The results obtained were largely independent of the time of analysis, and whether repopulation of recipient marrow or thymus was evaluated, suggesting that either can be used in this assay to quantitate a hemopoietic stem cell with the potential to regenerate both lymphoid and myeloid systems. These studies have provided procedures for the detection, quantitation and selective enrichment of the most primitive stem cells in the murine hemopoietic system which have competitive long-term lympho-myeloid repopulating ability. The availability of these procedures should facilitate the development of additional purification steps leading to the isolation of these cells as homogeneous suspensions, and their further use as targets for retrovirus-medilated gene transfer to determine the genetic basis of their activation, determination and neoplastic transformation. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In the first part of this study, a non-ferric method for selective elimination of β-lactoglobulin from cheese whey was investigated. A new method was developed based on hexametaphosphate treatment of cheese whey. When Cheddar cheese whey was treated under the optimized conditions, i.e., 1.33 mg/mL sodium hexametaphosphate at 22°C and pH 4.07 for 1 hr, more than 80% of β-lactoglobulin was removed by precipitation. Almost all of the immunoglobulins and the major portion of α-lactalbumin were retained in the supernatant as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemical assays. By dialysis against distilled water 72.2% of the phosphorus was removed from the supernatant. In the second and the third part of the thesis, chromatographic methods were used for isolation of immunoglobulins and lactoferrin from whey proteins. By using gel filtration on Sephacryl S-300, 99, 83.3 and 92.1% biologically active immunoglobulin G were obtained for colostral whey, acid and Cheddar cheese whey, respectively. Lactoferrin, selectively adsorbed to the heparin-attached Sepharose, was eluted with 5 mM Veronal-HC1 containing 0.5M NaC1, at pH 7.2. 1,4-Butanediol diglycidyl ether-iminodiacetic acid on Sepharose 6B, or so-called metal chelate-interaction chromatography (MCIC), was loaded with copper ion and used for the same purpose. Of the two peaks obtained, the first yellowish peak was rich in lactoferrin, while the second peak was rich in immunoglobulins. Some of the physical and chemical properties of the proteins in these peaks, including immunochemical properties, isoelectric points, binding to bacterial lipopolysaccharides, and the mechanism of protein-metal interaction via histidine modification, and the capacity of the method were studied. The possibility of isolating immunoglobulins and lactoferrin from electrodialyzed whey was also investigated. In the fourth, fifth and sixth parts of the thesis, the method developed for isolation of immunoglobulins and lactoferrin from whey protein was applied to isolate these biologically important proteins directly from skimmilk, blood and egg white. The casein in skimmilk was found to compete with immunoglobulins for binding to copper ion in MCIC column when skimmilk was loaded in presence of 0.05 M Tris-acetate buffer containing 0.5 M NaC1, pH 8.2; however, this problem was solved by changing the equilibrating buffer to 0.02 M phosphate buffer containing 0.5 M NaC1, pH 7.0. When blood was directly applied to MCIC column, the yield of biologically active IgG was more than 95%. Ovotransferrin, strongly adsorbed to the MCIC column, was eluted with two-step elution protocols which suggests it exists in two forms. The histidine residues in immunoglobulins, caseins, transferrin and ovotransferrin were found to be involved in the mechanism of the interaction with the MCIC column. / Land and Food Systems, Faculty of / Graduate

Choline acetyltransferase (ChAc), the enzyme responsible for the synthesis of acetylcholine (ACh), has been extensively purified from chicken brains. Purification procedures included ammonium sulfate fractionation, DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150 column chromatography, and affinity chromatography on agarose-hexane-Coenzyme A column. ChAc activity was measured radiochemically. Due to the instability of the enzyme in the course of purification, the most active fraction obtained after agarose-hexane-Coenzyme A chromatography showed a specific activity of only 0.34 μmoles ACh formed/ min./mg protein which corresponded to a 773 fold purification from homogenate. However, on non-denaturing polyacrylamide gel electrophoresis at pH 8.8, the highly purified ChAc preparation showed two distinct bands, and ChAc activity was recovered by slicing and assaying the gel. ChAc activity corresponded to the position of the faster moving band. The same preparation showed one major band and two minor bands on SDS gel electrophoresis. The estimated MW of chicken brain ChAc by gel filtration and SDS gel electrophoresis was 42,500 daltons and no subunit was observed. Two forms of chicken brain ChAc with different Km values for the substrates were eluted from agarose-hexane-CoA column. The pH optimum was estimated to be between pH 7.6-8.0. NaCl, KC1, Ca²⁺ and EDTA stimulated, while Cu²⁺, N-ethylmaleimide and CoA inhibited the enzyme preparation. The apparent Km values for acetyl-CoA and choline were, studied and were found to be similar to those of other mammalian species. The ChAc preparation also showed species specificity by the Ouchterlony double immuno diffusion test. Effect and mechanisms of salt and EDTA activation of ChAc activity are discussed. / Medicine, Faculty of / Graduate

A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms.

Cheuk Shing Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 144-161). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.I / 內容摘要 --- p.II / ABSTRACT --- p.IV / CONTENTS --- p.XI / LIST OF TABLES --- p.XI / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Taxonomy of Streptococcus agalactiae --- p.1 / Chapter 1.2 --- Characteristics of Streptococcus agalactiae --- p.1 / Chapter 1.3 --- Epidemiology of GBS --- p.3 / Chapter 1.3.1 --- Risk groups --- p.3 / Chapter 1.3.1.1 --- Neonates --- p.3 / Chapter 1.3.1.2 --- Pregnant women --- p.5 / Chapter 1.3.1.3 --- Non-pregnant adult --- p.6 / Chapter 1.3.2 --- World wide distribution --- p.7 / Chapter 1.3.2.1 --- Serotypes --- p.7 / Chapter 1.3.2.2 --- Antibiotic susceptibility --- p.8 / Chapter 1.3.3 --- GBS diseases in Hong Kong --- p.10 / Chapter 1.4 --- Putative virulence factors and pathogenesis --- p.10 / Chapter 1.4.1 --- Capsular polysaccharide --- p.10 / Chapter 1.4.2 --- C5a peptidase --- p.11 / Chapter 1.4.3 --- β-haemolysin/cytolysin --- p.12 / Chapter 1.4.4 --- C protein and C a-like protein --- p.12 / Chapter 1.4.4.1 --- C protein --- p.12 / Chapter 1.4.4.1.1 --- C α protein --- p.13 / Chapter 1.4.4.1.2 --- Cβ protein --- p.14 / Chapter 1.4.4.2 --- C α-like protein --- p.15 / Chapter 1.4.5 --- Hyaluronate lyase --- p.16 / Chapter 1.4.6 --- CAMP factor --- p.17 / Chapter 1.4.7 --- Others --- p.17 / Chapter 1.5 --- Antibiotic resistance and resistance genes --- p.18 / Chapter 1.5.1 --- Macrolides --- p.18 / Chapter 1.5.2 --- Tetracyclines --- p.18 / Chapter 1.5.3 --- Aminoglycosides --- p.19 / Chapter 1.5.4 --- Fluoroquniolones --- p.20 / Chapter 1.5.5 --- Others --- p.20 / Chapter 1.6 --- Mobile genetic elements --- p.21 / Chapter 1.7 --- Typing methods --- p.22 / Chapter 1.7.1 --- Phenotypic methods --- p.23 / Chapter 1.7.1.1 --- Serotyping --- p.23 / Chapter 1.7.1.2 --- Multilocus enzyme electrophoresis (MLEE) --- p.23 / Chapter 1.7.2 --- Genotypic methods --- p.24 / Chapter 1.7.2.1 --- Restriction endonuclease analysis (REA) / restriction fragment-length polymorphism (RFLP) --- p.24 / Chapter 1.7.2.2 --- Pulsed-field gel electrophoresis (PFGE) --- p.25 / Chapter 1.7.2.3 --- Random amplified polymorphic DNA (RAPD) --- p.26 / Chapter 1.7.2.4 --- Sequencing --- p.26 / Chapter 1.8 --- Prevention --- p.29 / Chapter 1.8.1 --- Intrapartum antibiotic prophylaxis (IAP) --- p.29 / Chapter 1.8.2 --- GBS Vaccine --- p.33 / Chapter 1.9 --- Objectives --- p.34 / Chapter CHAPTER 2 --- METHODS AND MATERIALS --- p.35 / Chapter 2.1 --- Bacterial isolates --- p.35 / Chapter 2.2 --- Antibiotic susceptibility test --- p.37 / Chapter 2.2.1 --- Antibiotic preparation --- p.37 / Chapter 2.2.2 --- Microbroth dilution method --- p.39 / Chapter 2.2.2.1 --- Microtitre plate preparation --- p.39 / Chapter 2.2.2.2 --- Suspension preparation and inoculation --- p.39 / Chapter 2.2.2.3 --- End points determination --- p.40 / Chapter 2.2.3 --- Inducible lincomycin resistance determination --- p.40 / Chapter 2.3 --- Serotyping --- p.41 / Chapter 2.3.1 --- Preparation of antigens --- p.41 / Chapter 2.3.2 --- Typing of isolates --- p.42 / Chapter 2.4 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.42 / Chapter 2.4.1 --- Preparation of DNA plug for PFGE --- p.43 / Chapter 2.4.2 --- Restriction enzyme digestion of GBS DNA --- p.43 / Chapter 2.4.3 --- Running of PFGE gel --- p.44 / Chapter 2.5 --- Molecular characterization --- p.44 / Chapter 2.5.1 --- Target genes --- p.44 / Chapter 2.5.2 --- DNA preparation --- p.51 / Chapter 2.5.3 --- Master mix preparation --- p.51 / Chapter 2.5.4 --- Polymerase chain reaction --- p.51 / Chapter 2.5.5 --- PCR product analysis by agarose gel electrophoresis --- p.52 / Chapter 2.5.6 --- DNA sequencing --- p.52 / Chapter 2.6 --- Data analysis --- p.53 / Chapter 2.6.1 --- PFGE and molecular characters analysis --- p.53 / Chapter 2.6.2 --- Sequences analysis --- p.53 / Chapter 2.7 --- Molecular identification by real-time PCR --- p.54 / Chapter 2.7.1 --- Bacterial strains --- p.54 / Chapter 2.7.2 --- DNA isolation for specimens --- p.56 / Chapter 2.7.3 --- Design of TaqMan primers and probes --- p.56 / Chapter 2.7.4 --- Cloning of target sequences --- p.59 / Chapter 2.7.5 --- Master mix of real-time PCR --- p.59 / Chapter 2.7.6 --- Specificity and detection limit --- p.60 / Chapter CHAPTER 3 --- RESULTS --- p.62 / Chapter 3.1 --- Serotype distribution of GBS --- p.62 / Chapter 3.1.1 --- Serotyping using antisera --- p.62 / Chapter 3.1.2 --- Serotyping by molecular method --- p.64 / Chapter 3.1.3 --- Molecular subtype of GBS serotype III --- p.66 / Chapter 3.1.4 --- Correlation of serotypes with diseases --- p.69 / Chapter 3.2 --- Antimicrobial susceptibility --- p.71 / Chapter 3.2.1 --- Phenotypic method --- p.71 / Chapter 3.2.2 --- Detection and distribution of resistance genes --- p.76 / Chapter 3.2.2.1 --- Tetracycline resistance --- p.76 / Chapter 3.2.2.2 --- Macrolide and lincosamide resistance --- p.77 / Chapter 3.2.2.3 --- Aminoglycoside resistance --- p.78 / Chapter 3.3 --- Molecular typing --- p.83 / Chapter 3.3.1 --- Pulsed-field gel electrophoresis (PFGE) --- p.83 / Chapter 3.3.2 --- Distribution of GBS surface protein genes profiles --- p.89 / Chapter 3.3.3 --- Distribution of mobile genetic elements --- p.92 / Chapter 3.4 --- "Analysis based on PFGE, surface protein genes, mobile genetic elements and antibiotic resistance genes" --- p.95 / Chapter 3.4.1 --- Intra-molecular serotype --- p.95 / Chapter 3.4.1.1 --- Molecular serotype Ia --- p.95 / Chapter 3.4.1.2 --- Molecular serotype Ib --- p.99 / Chapter 3.4.1.3 --- Molecular serotype II --- p.101 / Chapter 3.4.1.4 --- Molecular serotype III --- p.103 / Chapter 3.4.1.5 --- Molecular serotype V --- p.107 / Chapter 3.4.1.6 --- Molecular serotype VI --- p.110 / Chapter 3.4.1.7 --- "Molecular serotype IV, VII and VIII" --- p.110 / Chapter 3.4.1.8 --- Non-typeable isolate (NT) --- p.111 / Chapter 3.4.2 --- Analysis of Maternal and neonatal strains --- p.115 / Chapter 3.4.3 --- Comparison of GBS strains from Hong Kong to Australia and Korea --- p.118 / Chapter 3.5 --- Molecular identification of GBS by real-time PCR --- p.120 / Chapter 3.5.1 --- Specificity --- p.120 / Chapter 3.5.2 --- Detection limits --- p.122 / Chapter CHAPTER 4 --- DISCUSSION --- p.125 / Chapter 4.1 --- Laboratory methods for typing and characterization of GBS --- p.125 / Chapter 4.1.1 --- Serotyping by agglutination and molecular method --- p.125 / Chapter 4.1.2 --- Antibiotic susceptibility testing and resistance genes --- p.129 / Chapter 4.1.3 --- PFGE --- p.130 / Chapter 4.1.4 --- Surface protein genes --- p.131 / Chapter 4.1.5 --- Mobile genetic elements --- p.132 / Chapter 4.1.6 --- Real-time PCR --- p.133 / Chapter 4.2 --- Characterization of GBS in Hong Kong --- p.135 / Chapter 4.2.1 --- GBS in Hong Kong --- p.135 / Chapter 4.2.2 --- GBS from Australia and Korea --- p.141 / Chapter 4.3 --- Future research --- p.142 / Chapter 4.4 --- Conclusions --- p.143 / REFERENCES --- p.144 / APPENDIX I: MATERIALS AND REAGENTS --- p.162 / APPENDIX II: DENDROGRAMS --- p.168