by Linda Shiou-Mei Ooi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 118-126). / 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.

by Lam Ying Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 193-209). / 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.

Qi Chang. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 173-192). / 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.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

by Lee Kai Fai, Calvin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 159-167). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iv / ABBREVIATIONS --- p.xi / Chapter CHAPTER ONE --- General Introduction --- p.1 / Chapter 1.1 --- The Phenomenon of Host-controlled Restriction --- p.1 / Chapter 1.2 --- Classification of Restriction and Modification Systems --- p.2 / Chapter 1.2.1 --- Type I Restriction-Modification Systems --- p.2 / Chapter 1.2.2 --- Type II Restriction-Modification Systems --- p.3 / Chapter 1.2.3 --- Type III Restriction-Modification Systems --- p.4 / Chapter 1.2.4 --- Type IV Restriction-Modification Systems --- p.5 / Chapter 1.3 --- Occurrence of Restriction-Modification Systems --- p.6 / Chapter 1.4 --- Effect of Methylation --- p.7 / Chapter 1.5 --- Alternation of Recognition Specificities --- p.7 / Chapter 1.5.1 --- Cross Protection by DNA Methyltransferase --- p.8 / Chapter 1.5.2 --- A-Assisted Restriction Endonuclease (RARE) Cleavage --- p.9 / Chapter 1.5.3 --- Site-specific Cleavage mediated by Triple-helix formation --- p.9 / Chapter 1.5.4 --- Site-specific Cleavage of Duplex DNA with a λ repressor- Staphylococcal Nuclease Hybrid --- p.10 / Chapter 1.5.5 --- Achilles' heel Cleavage --- p.10 / Chapter 1.5.6 --- Chimeric Restriction Endonuclease --- p.11 / Chapter 1.6 --- Cloning of Restriction and Modification Systems --- p.11 / Chapter 1.6.1 --- Selection based on Modification --- p.11 / Chapter 1.6.2 --- Other Cloning Strategies --- p.12 / Chapter 1.6.2.1 --- Sub-Cloning of Plasmids --- p.12 / Chapter 1.6.2.2 --- Selection based on Restriction --- p.13 / Chapter 1.6.2.3 --- Multi-step Cloning --- p.13 / Chapter 1.6.2.4 --- Cloning in AP1-200 and AP1-200-9 strain --- p.13 / Chapter 1.6.2.5 --- Direct Cloning of Restriction gene by 'endo-blue' method --- p.14 / Chapter 1.7 --- Genetic Location of Restriction-Modification Systems --- p.14 / Chapter 1.8 --- Sequences of Restriction-Modification Systems --- p.15 / Chapter 1.9 --- Catalytic Properties of Type II Restriction-Modification Systems --- p.17 / Chapter 1.10 --- Crystallography of Type II Restriction and Modification Enzymes --- p.19 / Chapter 1.11 --- Evolution of Type II Restriction and Modification Enzymes --- p.22 / Chapter 1.12 --- Aim of Study --- p.23 / Chapter CHAPTER TWO --- Materials and Methods --- p.24 / Chapter 2.1 --- Bacterial Strains --- p.24 / Chapter 2.2 --- General Techniques --- p.25 / Chapter 2.2.1 --- Phenol/Chloroform Extraction --- p.25 / Chapter 2.2.2 --- Ethanol Precipitation --- p.25 / Chapter 2.2.3 --- Spectrophotometry --- p.25 / Chapter 2.2.4 --- Restriction digestion of DNA --- p.26 / Chapter 2.2.5 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.2.6 --- Recovery of DNA fragment from Agarose gel --- p.26 / Chapter 2.2.7 --- Minipreparation of Plasmid --- p.27 / Chapter 2.2.8 --- Large-Scale Preparation of Plasmid DNA --- p.28 / Chapter 2.2.8A --- By Equilibrium Centrifugation in Cesium Chloride- Ethidium Bromide Gradient --- p.28 / Chapter 2.2.8B --- By Using Qiagen-tip 100 Cartridge --- p.29 / Chapter 2.2.9 --- Preparation of Competent Cells --- p.30 / Chapter 2.2.10 --- Transformation of Competent Cells --- p.31 / Chapter 2.2.11 --- Screening of Recombinant Plasmids --- p.32 / Chapter 2.2.11A --- Using Selective media --- p.32 / Chapter 2.2.11B --- Rapid Alkaline Lysis Method --- p.32 / Chapter 2.2.12 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.13 --- Size Exclusion Chromatography --- p.34 / Chapter 2.2.14 --- Electroblotting of Protein on Polyvinylidene Difluoride (PVDF) membrane --- p.35 / Chapter 2.2.15 --- Isoelectric Focusing (EEF) --- p.36 / Chapter 2.2.16 --- Protein Assay --- p.37 / Chapter 2.3 --- DNA Sequencing --- p.37 / Chapter 2.3.1 --- Isolation of a template DNA --- p.38 / Chapter 2.3.2 --- DNA Denaturation and Annealing Reaction --- p.38 / Chapter 2.3.3 --- Labeling and Termination Reaction --- p.38 / Chapter 2.3.4 --- DNA Sequencing Electrophoresis --- p.39 / Chapter 2.3.5 --- Autoradiography --- p.40 / Chapter CHAPTER THREE --- Purification and Characterization of Restriction Endonuclease from Escherichia coli HK31 --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Preparation of Crude enzyme Extract --- p.42 / Chapter 3.2.2 --- Purification of R.EcoHK31I --- p.42 / Chapter 3.2.3 --- Characterization of Restriction endonuclease --- p.43 / Chapter 3.2.3.1 --- Enzyme Activity assay --- p.43 / Chapter 3.2.3.2 --- "Optimal pH, Temperature, Metal Ion and Salt concentration of R.EcoHK31I" --- p.43 / Chapter 3.2.3.3 --- Assay for the Purity of R.EcoHK31I --- p.43 / Chapter 3.2.3.4 --- Determination of Recognition Specificity --- p.44 / Chapter 3.2.3.5 --- Determination of the Cleavage Specificity --- p.44 / Chapter 3.3 --- Results and Discussion --- p.45 / Chapter 3.3.1 --- Purification ofR.EcoHK31I from Escherichia coli HK31 --- p.45 / Chapter 3.3.2 --- "Optimal pH,Temperature, Metal ions and Salt concentration of R.EcoHK31I" --- p.46 / Chapter 3.3.3 --- Unit Definition --- p.51 / Chapter 3.3.4 --- Purity of the R.EcoHK31I --- p.51 / Chapter 3.3.5 --- Recognition Site of the R.EcoHK31I --- p.51 / Chapter 3.3.6 --- Sensitivity of the R.EcoHK31I to dcm Methylation --- p.52 / Chapter 3.3.7 --- Cleavage Specificity of R.EcoHK31I --- p.52 / Chapter CHAPTER FOUR --- Cloning of EcoEK31I Restriction and Modification (R-M) System from Escherichia coli HK31 --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and Methods --- p.58 / Chapter 4.2.1 --- Extraction of genomic DNA from E. coli HK31 --- p.58 / Chapter 4.2.2 --- Extraction of Extra-Chromosomal DNA from E. coli HK31 --- p.59 / Chapter 4.2.3 --- Restriction Digestion of the Total DNA --- p.59 / Chapter 4.2.4 --- Preparation of Linearized and Dephosphorylated Vector --- p.60 / Chapter 4.2.5 --- Fill-in Reaction --- p.60 / Chapter 4.2.6 --- Ligation between Vector and Digested Chromosomal DNA --- p.61 / Chapter 4.2.7 --- Selection of Clones Harboring Methyltransferase gene --- p.61 / Chapter 4.2.8 --- Screening of the Survival Clones --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Construction of Genomic Libraries --- p.62 / Chapter 4.3.2 --- Selection of the Methyltransferase Gene --- p.66 / Chapter 4.3.3 --- In vitro Detection of R.EcoHK31I activity --- p.67 / Chapter 4.3.4 --- Functional Localization of EcoHK31I --- p.67 / Chapter 4.3.5 --- Subcloning of the Complete EcoHK31I R-M System --- p.72 / Chapter 4.4 --- Discussion --- p.72 / Chapter 4.4.1 --- Construction of Genomic Libraries --- p.72 / Chapter 4.4.2 --- Cloning of EcoHK31I Restriction and Modification System --- p.75 / Chapter 4.4.2.1 --- Selecting Endonuclease --- p.75 / Chapter 4.4.2.2 --- Detection of Restriction Endonuclease Activity --- p.76 / Chapter 4.4.3 --- Functional Localization of the R-M System --- p.76 / Chapter CHAPTER FIVE --- The Nucleotide Sequences of the EcoHK31I R-M System --- p.78 / Chapter 5.1 --- Introduction --- p.78 / Chapter 5.2 --- Materials and Methods --- p.79 / Chapter 5.2.1 --- Sequencing Strategies --- p.79 / Chapter 5.2.2 --- DNA Sequencing --- p.80 / Chapter 5.2.3 --- Sequence Analysis --- p.80 / Chapter 5.3 --- Results and Discussion --- p.80 / Chapter 5.3.1 --- Nucleotide Sequences and Deduced Amino Acid sequences --- p.80 / Chapter 5.3.2 --- Comparison of Amino Acid Sequences --- p.85 / Chapter CHAPTER SIX --- Purification and Characterization of EcoHK31I Methyltransferase from E. coli K802 [pEcoHK31E] --- p.91 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.92 / Chapter 6.2.1 --- Preparation of Crude enzyme Extract --- p.92 / Chapter 6.2.2 --- Purification of M.EcoHK31I --- p.92 / Chapter 6.2.3 --- Characterization of EcoHK31I Methyltransferase --- p.93 / Chapter 6.2.3.1 --- Enzyme Activity assay --- p.93 / Chapter 6.2.3.2 --- Determination of Methylation specificity --- p.93 / Chapter 6.2.3.3 --- Determination of Molecular weight of M.EcoHK31I --- p.94 / Chapter 6.2.3.4 --- Determination ofM.EcoHK31I Kinetics --- p.94 / Chapter 6.3 --- Results and Discussion --- p.96 / Chapter 6.3.1 --- Purification of EcoHK31I Methyltransferase --- p.96 / Chapter 6.3.2 --- M.EcoHK31I Modification Specificity --- p.99 / Chapter 6.3.3 --- "Determination of Molecular Weight ofM,EcoHK31I" --- p.99 / Chapter 6.3.4 --- Catalytic Properties of EcoHK31I Methyltransferase --- p.103 / Chapter 6.3.5 --- A Novel m5C-MTase M.EcoHK31I --- p.103 / Chapter CHAPTER SEVEN --- Over-expression and Characterization of EcoHK31I Restriction and Modification Enzymes --- p.106 / Chapter 7.1 --- Introduction --- p.106 / Chapter 7.1.1 --- Expression Vector pTrc series --- p.107 / Chapter 7.1.2 --- Expression Vector pET series --- p.107 / Chapter 7.2 --- Materials and Methods --- p.109 / Chapter 7.2.1 --- General technique --- p.109 / Chapter 7.2.2 --- Polymerase Chain Reaction --- p.110 / Chapter 7.2.3 --- Construction of plysSM13 --- p.110 / Chapter 7.2.4 --- Construction of pTrc99A-R36 --- p.110 / Chapter 7.2.5 --- Construction of pET3a-M38 --- p.111 / Chapter 7.2.6 --- Construction of pET3a-C23 --- p.111 / Chapter 7.2.7 --- Expression of Recombinant Proteins in E. coli hosts --- p.115 / Chapter 7.2.8 --- Purification of Recombinant R.EcoHK31I --- p.115 / Chapter 7.2.9 --- Determination of Molecular Weight of Recombinant R. EcoHK31I --- p.115 / Chapter 7.2.10 --- Polyclonal Antibodies against R.EcoHK31I --- p.116 / Chapter 7.2.11 --- Western Blotting --- p.116 / Chapter 7.2.12 --- Purification of Recombinant M.EcoHK31I polypeptide α --- p.117 / Chapter 7.2.13 --- Purification of Recombinant M.EcoHK31I polypeptide β --- p.118 / Chapter 7.2.14 --- In vitro Complementation Methylation Activity --- p.118 / Chapter 7.2.15 --- Incorporation of [3H]-AdoMet to non-methylated Lambda DNA --- p.119 / Chapter 7.3 --- Results and Discussion --- p.119 / Chapter 7.3.1 --- Expression of Recombinant R. EcoHK31I --- p.119 / Chapter 7.3.2 --- Purification and Characterization of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.1 --- Purification of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.2 --- Characterization of Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.1 --- Molecular Weight and Isoelectric point of the Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.2 --- Antibodies to Recombinant R.EcoHK31I --- p.125 / Chapter 7.3.3 --- Expression and Purification of M.EcoHK31Ipolypeptide α --- p.127 / Chapter 7.3.4 --- Expression and Purification of M.EcoHK31I polypeptide β --- p.127 / Chapter 7.3.5 --- Characterization of M.EcoHK31I polypeptides a and β --- p.129 / Chapter 7.3.5.1 --- Molecular Weight Determination --- p.129 / Chapter 7.3.5.2 --- Isoelectric Point Determination --- p.132 / Chapter 7.3.5.3 --- In vivo and in vitro Methylation Activity --- p.132 / Chapter CHAPTER EIGHT --- Generation and Activity Assay of Q193G Mutein of M.EcoHK31I Polypeptide a --- p.138 / Chapter 8.1 --- Introduction --- p.138 / Chapter 8.2 --- Materials and Methods --- p.139 / Chapter 8.2.1 --- Construction of pET3a-M38 (Q193G) --- p.139 / Chapter 8.2.2 --- Expression and Purification of Q193G protein --- p.140 / Chapter 8.2.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.140 / Chapter 8.3 --- Results and Discussion --- p.145 / Chapter 8.3.1 --- "Construction, Expression and Purification of Q193G Mutein" --- p.145 / Chapter 8.3.2 --- Determination of Molecular Weight and Isoelectric point of Q193G --- p.145 / Chapter 8.3.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.145 / Chapter 8.3.4 --- Recognition Specificity of Q193G Mutein --- p.147 / Chapter CHAPTER NINE --- General Discussion --- p.151 / REFERENCES --- p.159 / APPENDIX A --- p.168

by Lap-Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 90-101). / Abstract also in Chinese. / Abstract --- p.i / Contents --- p.iv / List of tables --- p.ix / List of figures --- p.x / Abbreviations --- p.xi / Acknowledgements --- p.xii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- The biology of hepatitis A virus --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- General characteristics of HAV --- p.2 / Chapter 1.1.3 --- Stability and disinfection of HAV --- p.3 / Chapter 1.1.4 --- Molecular biology of HAV --- p.5 / Chapter 1.1.4.1 --- Genomic organization of HAV --- p.5 / Chapter 1.1.4.2 --- Antigenic sites on the capsid of HAV --- p.8 / Chapter 1.1.5 --- Laboratory diagnosis and methods of study for HAV --- p.8 / Chapter 1.1.5.1 --- Cell-culture propagation and antigen detection --- p.8 / Chapter 1.1.5.2 --- Nucleic acid detection --- p.10 / Chapter 1.1.6 --- Epidemiology of HAV --- p.12 / Chapter 1.1.6.1 --- Distribution of HAV infection --- p.12 / Chapter 1.1.6.2 --- Seasonal pattern of HAV infection --- p.13 / Chapter 1.1.6.3 --- Mode of transmission --- p.13 / Chapter 1.1.6.4 --- Molecular epidemiology --- p.15 / Chapter 1.1.7 --- Epidemiology of HAV infection in Hong Kong --- p.15 / Chapter 1.2 --- Transmission of viruses through contaminated food --- p.18 / Chapter 1.2.1 --- Active accumulation of water contaminants by shellfish --- p.20 / Chapter 1.2.2 --- Retention of viruses by shellfish in contaminate water --- p.21 / Chapter 1.2.3 --- Elimination of viruses in contaminated shellfish --- p.21 / Chapter 1.2.4 --- Indicators for contamination by enteric viruses --- p.21 / Chapter 1.3 --- Detection of viruses from foods --- p.23 / Chapter 1.3.1 --- Recovery of viruses from foods --- p.23 / Chapter 1.3.2 --- Detection of viral nucleic acid --- p.23 / Chapter 1.4 --- Objectives of the study --- p.26 / Chapter Chapter 2- --- Materials and methods / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Sera from patients with acute viral hepatitis --- p.27 / Chapter 2.1.2 --- Collection of shellfish samples --- p.28 / Chapter 2.1.3 --- Purified HAV preparations as positive control --- p.30 / Chapter 2.1.4 --- Control samples for the virion capture method --- p.30 / Chapter 2.1.5 --- Preparation of dissecting instruments and processing of shellfish samples --- p.30 / Chapter 2.1.6 --- Plasticwares and glasswares --- p.30 / Chapter 2.1.7 --- "Chemicals, reagents and commercial kits" --- p.31 / Chapter 2.1.7.1 --- Samples processing --- p.31 / Chapter 2.1.7.2 --- Reagents for RNA extractions --- p.31 / Chapter 2.1.7.3 --- Oligonucleotide primers synthesis --- p.33 / Chapter 2.1.7.4 --- Primers purification after synthesis --- p.34 / Chapter 2.1.7.5 --- Gel electrophoresis --- p.34 / Chapter 2.1.7.6 --- Reagents for hybridization --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Samples processing --- p.37 / Chapter 2.2.2 --- Artificially seeded HAV in shellfish --- p.37 / Chapter 2.2.3 --- RNA extraction methods --- p.37 / Chapter 2.2.3.1 --- Acid phenol method --- p.37 / Chapter 2.2.3.2 --- Spin cartridge method --- p.38 / Chapter 2.2.3.3 --- Virion capture method --- p.39 / Chapter 2.2.4 --- "Oligonucleotides used for RT, PCR and hybridization" --- p.40 / Chapter 2.2.4.1 --- Oligonucleotides used in HAV RT-PCR --- p.40 / Chapter 2.2.4.2 --- Primer set used for the evaluation of inhibitors of PCR in shellfish homogenates --- p.41 / Chapter 2.2.4.3 --- Preparation of oligonucleotide primers --- p.41 / Chapter 2.2.4.4 --- Detachment of the primer from the column --- p.42 / Chapter 2.2.4.5 --- Purification of the oligonucleotides --- p.42 / Chapter 2.2.4.6 --- Confirmation of synthesed oligonucleotide --- p.43 / Chapter 2.2.5 --- Reverse transcription of HAV genomic RNA template and PCR --- p.43 / Chapter 2.2.6 --- Human β-actin gene PCR for the evaluation of shellfish homogenates --- p.45 / Chapter 2.2.7 --- Analysis of PCR products --- p.46 / Chapter 2.2.7.1 --- Agarose gel electrophoresis for the analysis of PCR products --- p.46 / Chapter 2.2.7.2 --- Dot blot hybridization for the confirmation of PCR products --- p.46 / Chapter 2.2.7.3 --- Southern blot hybridization for the confirmation of PCR products --- p.47 / Chapter 2.2.7.4 --- 5'-end DNA labelling of oligonucleotide probe --- p.47 / Chapter 2.2.7.5 --- Hybridization in sodium chloride / sodium citrate --- p.48 / Chapter Chapter 3- --- Results / Chapter 3.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.52 / Chapter 3.2 --- Synthesis and yields of oligonucleotide primers --- p.54 / Chapter 3.3 --- Development of reverse-transcription polymerase chain reaction (RT-PCR) for HAV --- p.55 / Chapter 3.4 --- Sampling of shellfish from different markets in Hong Kong --- p.59 / Chapter 3.5 --- Quantitation of HAV RNA in stock virus preparations --- p.62 / Chapter 3.6 --- Comparison of RNA extraction methods and the detection limit of the established RT-PCR method for HAV --- p.62 / Chapter 3.7 --- Specificity of the RT-PCR in combination with virion capture method for the detection of HAV --- p.65 / Chapter 3.8 --- Detection of HAV RNA by RT-PCR in shellfish in Hong Kong / Chapter Chapter 4- --- Discussion / Chapter 4.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.76 / Chapter 4.2 --- Development of RT-PCR method for the detection of HAV --- p.78 / Chapter 4.3 --- Evaluation of RNA extraction methods for the detection of HAV in shellfish sample by RT-PCR --- p.78 / Chapter 4.4 --- Application of the established RT-PCR method for the detection of HAV contamination in locally available shellfish --- p.79 / Chapter Chapter 5- --- References --- p.90 / Appendix I Figures of agarose gel electrophoresis of RT-PCR products for all samples (I1 -I23 ) --- p.101 / Appendix II Figures of dot-blot hybridization assay (II2 -II4 ) --- p.114

by Yuen Wai Keung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 99-112). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Table of Contents --- p.viii / Chapter chapter one --- introduction --- p.1 / Chapter 1.1 --- Glutathione S-transferase --- p.2 / Chapter 1.1.1 --- Introduction --- p.2 / Chapter 1.1.2 --- Classification of mammalian GST --- p.2 / Chapter 1.1.3 --- Classification of insect GST --- p.7 / Chapter 1.1.4 --- Substrate specificity --- p.11 / Chapter 1.2 --- The chironomidae --- p.13 / Chapter 1.2.1 --- Biology and life history of chironomidae --- p.13 / Chapter 1.3 --- Chironomidae larvae --- p.16 / Chapter 1.3.1 --- Bloodworm t --- p.6 / Chapter 1.3.2 --- Sources of chironomidae larvae --- p.17 / Chapter 1.4 --- Aim of research --- p.18 / Chapter chapter two --- materials and methods --- p.20 / Chapter 2.1 --- Screening of GST in different subcellular fractions --- p.21 / Chapter 2.1.1 --- Preparation of mitochondria --- p.21 / Chapter 2.1.2 --- Preparation of microsomes --- p.22 / Chapter 2.1.3 --- Preparation of cytosol --- p.22 / Chapter 2.2 --- Assay for GST activity --- p.23 / Chapter 2.2.1 --- Activity Units --- p.23 / Chapter 2.3 --- Protein assay --- p.23 / Chapter 2.4 --- Preparation of glutathione-affinity column --- p.25 / Chapter 2.5 --- Purification of cytosolic GSTs --- p.26 / Chapter 2.5.1 --- Preparation of cytosol --- p.26 / Chapter 2.5.2 --- Chromatography on Sephadex G25 --- p.26 / Chapter 2.5.3 --- Affinity Chromatography --- p.26 / Chapter 2.5.3.1 --- Specific elution of GSTs --- p.26 / Chapter 2.5.3.2 --- Non-specific elution of GSTs --- p.27 / Chapter 2.5.4 --- Fast Protein Liquid Chromatography with Mono Q --- p.27 / Chapter 2.6 --- Determination of molecular mass --- p.29 / Chapter 2.6.1 --- Subunit molecular mass --- p.29 / Chapter 2.6.2 --- Native molecular mass --- p.31 / Chapter 2.7 --- Isoelectric focusing PAGE --- p.31 / Chapter 2.8 --- Enzyme activities and kinetic studies --- p.34 / Chapter 2.8.1 --- Optimum pH --- p.34 / Chapter 2.8.2 --- Heat inactivation assay --- p.34 / Chapter 2.8.3 --- Km and Vmax --- p.34 / Chapter 2.8.4 --- Substrate specificity --- p.35 / Chapter 2.8.5 --- Glutathione peroxidase activity --- p.38 / Chapter 2.9 --- N-terminal amino acid sequence analysis --- p.39 / Chapter 2.9.1 --- Semidry electroblotting --- p.39 / Chapter 2.9.2 --- Staining of proteins on PVDF membrane --- p.40 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.40 / Chapter 2.9.4 --- On-membrane deblocking of protein --- p.40 / Chapter 2.9.5 --- BLAST search --- p.41 / Chapter chapter three --- results --- p.42 / Chapter 3.1 --- Screening of GST in different subcellular fractions --- p.43 / Chapter 3.2 --- Purification of cytosolic GSTs by chromatography --- p.45 / Chapter 3.2.1 --- Sephadex G25 column --- p.45 / Chapter 3.2.2 --- GSH affinity column --- p.45 / Chapter 3.2.3 --- Mono-Q column --- p.45 / Chapter 3.3 --- Determination of molecular mass --- p.53 / Chapter 3.3.1 --- Subunit molecular mass --- p.53 / Chapter 3.3.2 --- Native molecular mass --- p.53 / Chapter 3.4 --- Isoelectric point determination --- p.53 / Chapter 3.5 --- Enzymes activities and kinetic studies --- p.57 / Chapter 3.5.1 --- Optimum pH --- p.57 / Chapter 3.5.2 --- Thermostability --- p.57 / Chapter 3.5.3 --- Km and Vmax --- p.57 / Chapter 3.5.4 --- Substrate specificity --- p.76 / Chapter 3.5.5 --- Glutathione peroxidase Activity --- p.76 / Chapter 3.6 --- N-terminal amino acid sequence analysis --- p.83 / Chapter chapter four --- discussion --- p.89 / Chapter 4.1 --- GST in different subcellular fractions --- p.90 / Chapter 4.2 --- Purification of cytosolic GST --- p.91 / Chapter 4.3 --- Physical properties --- p.93 / Chapter 4.3.1 --- Subunit molecular mass --- p.93 / Chapter 4.3.2 --- Native molecular mass --- p.93 / Chapter 4.3.3 --- Isoelectric point --- p.95 / Chapter 4.4 --- Kinetic properties --- p.94 / Chapter 4.4.1 --- Optimum pH --- p.94 / Chapter 4.4.2 --- Thermostability --- p.95 / Chapter 4.4.3 --- Km and Vmax --- p.95 / Chapter 4.4.4 --- Substrate specificity --- p.96 / Chapter 4.4.5 --- Glutathione peroxidase activity --- p.96 / Chapter 4.5 --- N-terminal amino acid sequence data --- p.97 / Chapter 4.6 --- Conclusion --- p.98 / references --- p.99

by Tse Man Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 74-81). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.III / TABLE OF CONTENTS --- p.1 / Chapter CHAPTER 1 --- INTRODUCTION --- p.2 / Chapter 1.1 --- rIbosome-inactivatIng proteins (RIPS) --- p.2 / Chapter 1.1.1 --- Classification of RIPs --- p.2 / Chapter 1.1.2 --- Distribution of RIPs --- p.6 / Chapter 1.1.3 --- Molecular biology of RIPs --- p.7 / Chapter 1.1.4 --- Physical and chemical properties of RIPs --- p.9 / Chapter 1.1.5 --- Enzymatic and translation-inhibitory activities --- p.12 / Chapter 1.1.6 --- RIP-based Immunotoxins --- p.16 / Chapter 1.2 --- MOMORDICA CHARANTIA and its RIBoosome-inactivating proteins (RIP) --- p.17 / Chapter 1.2.1 --- Momordica charantia --- p.17 / Chapter 1.2.2 --- Ribosome-inactivating proteins (RIPs) in Momordica charantia --- p.18 / Chapter 1.3 --- Objective of this study --- p.28 / Chapter CHAPTER 2 --- STUDY ON A NEW RIBOSOME-INACTIVATING PROTEIN (RIP) FROM MOMORDICA CHARANTIA SEEDS --- p.30 / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Materials --- p.33 / Chapter 2.2.2 --- RIP isolation --- p.34 / Chapter 2.2.3 --- Characterization --- p.35 / Chapter 2.3 --- Results --- p.42 / Chapter 2.4 --- Discussion --- p.48 / Chapter CHAPTER 3 --- STUDY ON A NEW RIBOSOME-INACTIVATING PROTEIN (RIP) FROM MOMORDICA CHARANTIA FRUITS --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and methods --- p.53 / Chapter 3.2.1 --- Materials --- p.53 / Chapter 3.2.2 --- RIP isolation --- p.54 / Chapter 3.2.3 --- Characterization --- p.56 / Chapter 3.3 --- Results --- p.56 / Chapter 3.4 --- Discussion --- p.62 / Chapter CHAPTER 4 --- GENERAL DISCUSSION AND CONCLUSION --- p.64 / Chapter 4.1 --- General discussion --- p.64 / Chapter 4.2 --- Conclusion --- p.72 / REFERENCES --- p.74

by Yijin Cai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 185-207). / Introduction / Chapter 1.1 --- Biochemistry of cellulose degradation --- p.1 / Chapter 1.1.1 --- "Occurrence, distribution and structure of cellulose" --- p.1 / Chapter 1.1.2 --- Cellulose-degrading microorganisms --- p.3 / Chapter 1.1.2.1 --- Cellulolytic bacteria --- p.4 / Chapter 1.1.2.2 --- Cellulolytic fungi --- p.4 / Chapter 1.1.3 --- An overview of fungal cellulases --- p.4 / Chapter 1.1.3.1 --- Endoglucanase (EG) --- p.7 / Chapter 1.1.3.2 --- Cellobiohydrolase (CBH) --- p.16 / Chapter 1.1.3.3 --- β-Glucosidase (BGL) --- p.25 / Chapter 1.1.4 --- Synergism between the different components of the cellulolytic systems of filamentous fungi --- p.28 / Chapter 1.1.5 --- Molecular genetics of cellulases --- p.31 / Chapter 1.2 --- Secretion of cellulases by filamentous fungi --- p.32 / Chapter 1.2.1. --- Overview of enzyme secretion in filamentous fungi --- p.33 / Chapter 1.2.2 --- Glycosylation --- p.35 / Chapter 1.2.3 --- Protein secretion and the fungal cell wall --- p.37 / Chapter 1.2.4 --- Factors affecting protein secretion --- p.38 / Chapter 1.3 --- Volvariella volvacea --- p.39 / Chapter 1.4 --- Project aims --- p.43 / Materials and methods / Chapter 2.1 --- Organisms and culture conditions --- p.44 / Chapter 2.1.1 --- Basal medium --- p.44 / Chapter 2.1.2 --- Culture conditions for biomass and enzyme production on different carbon sources --- p.45 / Chapter 2.1.3 --- Culture conditions for large-scale enzyme production for purification --- p.45 / Chapter 2.1.4 --- Culture conditions for confocal microscopy --- p.46 / Chapter 2.1.5 --- Culture conditions for electron microscopy --- p.47 / Chapter 2.2 --- Mycelial extracts --- p.47 / Chapter 2.2.1 --- Large scale extraction --- p.47 / Chapter 2.2.2 --- Small scale extraction --- p.48 / Chapter 2.3 --- Enzyme purification --- p.48 / Chapter 2.3.1 --- Cell-associated enzymes (β-glucosidases) --- p.48 / Chapter 2.3.2 --- Extracellular enzymes --- p.50 / Chapter 2.3.2.1 --- Purification of cellulase complex --- p.50 / Chapter 2.3.2.2 --- Purification of CBH --- p.51 / Chapter 2.3.2.3 --- Purification of endoglucanase-III --- p.53 / Chapter 2.3.2.4 --- Partial purification of β-glucosidases --- p.55 / Chapter 2.3.3 --- Other purification methods --- p.56 / Chapter 2.3.3.1 --- FPLC Phenylsuperose hydrophobic interaction chromatography --- p.56 / Chapter 2.3.3.2 --- Affinity gel chromatography --- p.56 / Chapter 2.3.3.3 --- Isoelectric focusing by Rotorfor --- p.57 / Chapter 2.3.3.4 --- Preparative gel electrophoresis --- p.57 / Chapter 2.3.3.5 --- (NH4)2S04 Precipitation --- p.58 / Chapter 2.4 --- Electrophoresis --- p.59 / Chapter 2.4.1 --- Mini Protean-II system (BioRad) --- p.59 / Chapter 2.4.2 --- PhastGel system (Pharmacia) --- p.60 / Chapter 2.5 --- Enzyme assays --- p.61 / Chapter 2.5.1 --- β-Glucosidase --- p.61 / Chapter 2.5.2 --- Endoglucanase --- p.63 / Chapter 2.5.3 --- Cellobiohydrolase --- p.65 / Chapter 2.6 --- β-Glucosidase characterization studies --- p.66 / Chapter 2.6.1 --- pH optimum --- p.66 / Chapter 2.6.2 --- Temperature optimum --- p.66 / Chapter 2.6.3 --- Thermal stability --- p.66 / Chapter 2.6.4 --- Kinetic parameters --- p.67 / Chapter 2.6.5 --- Enzyme inhibitor studies --- p.67 / Chapter 2.6.6 --- Effect of lignin-derived phenolic monomers --- p.67 / Chapter 2.6.7 --- Substrate specificity towards p-nitrophenyl-linked glycosides --- p.67 / Chapter 2.6.8 --- "Substrate specificity towards different cellulosic substrates, mono- and disaccharides, hemicellulose, sugar alcohols and saponins" --- p.68 / Chapter 2.6.9 --- Cellulose-binding assay --- p.68 / Chapter 2.6.10 --- Effect of purified β-glucosidase on the production of glucose from crystalline cellulose and carboxymethylcellulose by Aspergillus niger cellulase --- p.69 / Chapter 2.7 --- Miscellaneous analytical methods --- p.69 / Chapter 2.7.1 --- Protein determination --- p.69 / Chapter 2.7.2 --- Determination of isoelectric points --- p.69 / Chapter 2.7.3 --- Activity staining of gels for cellulolytic enzyme activity --- p.70 / Chapter 2.7.4 --- Staining for glycoprotein --- p.71 / Chapter 2.7.5 --- Molecular weight determination --- p.71 / Chapter 2.8 --- "Production, purification and specificity of antibodies to β-glucosidases and EG-III" --- p.72 / Chapter 2.8.1 --- Antibodies to β-glucosidases --- p.72 / Chapter 2.8.2 --- Antibodies to EG-III --- p.74 / Chapter 2.9 --- Immunocytochemical studies --- p.75 / Chapter 2.9.1 --- Confocal laser scanning microscopy --- p.75 / Chapter 2.9.1.1 --- β-Glucosidases --- p.75 / Chapter 2.9.1.2 --- Endoglucanase-III --- p.75 / Chapter 2.9.2 --- Transmission electron microscopy --- p.76 / Chapter 2.9.3 --- Scanning electron microscopy --- p.77 / Chapter 2.10 --- Chemicals --- p.77 / Results / Chapter 3.1 --- "Effect of culture conditions on the growth of, and the production of cellulolytic enzymes, by V. volvacea" --- p.79 / Chapter 3.1.1 --- Growth --- p.79 / Chapter 3.1.2 --- Endoglucanases --- p.81 / Chapter 3.1.3 --- Cellobiohydrolase --- p.84 / Chapter 3.1.4 --- β-Glucosidase --- p.87 / Chapter 3.2 --- Purification of cellulolytic enzymes from V. volvacea --- p.92 / Chapter 3.2.1 --- Preliminary purification of V. volvacea extracellular cellulolytic enzymes --- p.92 / Chapter 3.2.1.1. --- (NH4)2SO4 precipitation --- p.92 / Chapter 3.2.1.2 --- Ultrafiltration --- p.94 / Chapter 3.2.1.3 --- Batch adsorption by anion exchanger --- p.94 / Chapter 3.2.1.4 --- Separation by column chromatography --- p.96 / Chapter 3.2.2 --- CBH enzymes …… --- p.102 / Chapter 3.2.3 --- Endoglucanase enzymes --- p.106 / Chapter 3.2.4 --- Cell-associated β-glucosidase enzymes --- p.113 / Chapter 3.2.5 --- Extracellular β-glucosidase enzymes --- p.120 / Chapter 3.3 --- Characterization of cell-associated β-glucosidases from V. volvacea --- p.122 / Chapter 3.3.1 --- Influence of pH and temperature --- p.122 / Chapter 3.3.2 --- Enzyme stability --- p.125 / Chapter 3.3.3 --- Kinetic parameters --- p.128 / Chapter 3.3.4 --- Enzyme inhibitors --- p.131 / Chapter 3.3.5 --- Substrate specificity --- p.137 / Chapter 3.3.6 --- Cellulose-binding and hydrolysing properties --- p.139 / Chapter 3.3.7 --- Molecular weights and isoelectric points --- p.142 / Chapter 3.4 --- Immunocytochemical studies on cellulolytic enzymes from V. volvacea --- p.146 / Chapter 3.4.1 --- Cell-associated β-glucosidase --- p.146 / Chapter 3.4.1.1 --- "Production, specificity and purification of polyclonal antibody" --- p.146 / Chapter 3.4.1.2. --- Localization --- p.151 / Chapter 3.4.1.3. --- Localization of cell-associated β-glucosidases by immuno- labelling --- p.151 / Chapter 3.4.2 --- Endoglucanase-III --- p.161 / Discussion / Chapter 4.1 --- Production --- p.163 / Chapter 4.2 --- Composition of cellulolytic enzyme system of V. volvacea --- p.168 / Chapter 4.3 --- Properties --- p.172 / Chapter 4.4 --- Localization --- p.179 / References --- p.185

Lai Chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 93-100). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / ABBREVIATION --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Catalases --- p.2 / Chapter 1.2 --- Classification of catalases --- p.3 / Chapter 1.2.1 --- Catalase peroxidase (HPI) --- p.3 / Chapter 1.2.2 --- Monofunctional catalases (HPII) --- p.6 / Chapter 1.2.2.1 --- NADPH in catalases --- p.9 / Chapter 1.2.3 --- Mn-catalases --- p.11 / Chapter 1.3 --- Sources and cytotoxic effects of hydrogen peroxide --- p.13 / Chapter 1.4 --- The Chironomidae --- p.14 / Chapter 1.4.1 --- Life cycle of Chironomidae --- p.14 / Chapter 1.4.2 --- Bloodworms --- p.18 / Chapter 1.4.3 --- Sources of bloodworms --- p.19 / Chapter 1.5 --- Aim of the project --- p.22 / Chapter 1.6 --- Application of the project --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- Protein determination --- p.25 / Chapter 2.2 --- In vitro activity assays --- p.27 / Chapter 2.2.1 --- Catalase activity assay --- p.27 / Chapter 2.2.2 --- Peroxidase activity assay --- p.27 / Chapter 2.3 --- Screening of catalase in different subcellular fractions --- p.28 / Chapter 2.3.1 --- Preparation of mitochondrial fractions --- p.28 / Chapter 2.3.2 --- Preparation of microsomal fractions --- p.29 / Chapter 2.3.3 --- Preparation of cytosolic fractions --- p.29 / Chapter 2.3.4 --- Preparation of post-mitochondrial fractions --- p.29 / Chapter 2.4 --- Purification of post-mitochondrial catalase --- p.29 / Chapter 2.4.1 --- Preparation of post-mitochondrial fractions --- p.30 / Chapter 2.4.2 --- Ethanol-chloroform precipitation --- p.30 / Chapter 2.4.3 --- Affinity chromatography --- p.30 / Chapter 2.4.4 --- Cation exchange chromatography --- p.31 / Chapter 2.5 --- Molecular mass determination --- p.34 / Chapter 2.6 --- Isoelectric focusing --- p.39 / Chapter 2.7 --- Kinetic studies of the purified enzyme --- p.42 / Chapter 2.7.1 --- Optimal pH --- p.42 / Chapter 2.7.2 --- Thermal stability --- p.42 / Chapter 2.7.3 --- Km and Vmax --- p.42 / Chapter 2.7.4 --- Inhibition studies --- p.43 / Chapter 2.7.4.1 --- "3-amino-1,2,4-triazole" --- p.43 / Chapter 2.7.4.2 --- Potassium cyanide and sodium azide --- p.43 / Chapter 2.8 --- Spectroscopic analysis --- p.44 / Chapter 2.8.1 --- Native enzyme --- p.44 / Chapter 2.8.2 --- Denatured enzyme --- p.44 / Chapter 2.8.3 --- Determination of pyridine hemochrome --- p.44 / Chapter 2.9 --- N-terminal amino acid sequence analysis for blotted protein --- p.45 / Chapter 2.9.1 --- Semi-dry electroblotting --- p.45 / Chapter 2.9.2 --- Protein staining on PVDF membrane --- p.46 / Chapter 2.9.3 --- N-terminal amino acid sequence analysis --- p.46 / Chapter 2.9.4 --- N-terminal deblocking of protein bound on PVDF membrane… --- p.47 / Chapter 2.9.5 --- BLAST® search --- p.48 / Chapter CHAPTER 3 --- RESULTS --- p.49 / Chapter 3.1 --- Catalase in different sub-cellular fractions --- p.50 / Chapter 3.2 --- Purification of post-mitochondrial catalase --- p.51 / Chapter 3.2.1 --- Ethanol-chloroform precipitation --- p.51 / Chapter 3.2.2 --- Affinity chromatography --- p.51 / Chapter 3.2.3 --- Cation exchange chromatography --- p.52 / Chapter 3.3 --- Determination of molecular mass --- p.57 / Chapter 3.4 --- Determination of isoelectric point --- p.57 / Chapter 3.5 --- Kinetic studies of the catalase --- p.62 / Chapter 3.5.1 --- Optimal pH --- p.62 / Chapter 3.5.2 --- Thermal stability --- p.62 / Chapter 3.5.3 --- Km and Vmax --- p.65 / Chapter 3.5.4 --- Inhibition studies --- p.65 / Chapter 3.5.4.1 --- "3-amino-1,2,4-triazole" --- p.65 / Chapter 3.5.4.2 --- Potassium cyanide and sodium azide --- p.65 / Chapter 3.5.5 --- Catalase peroxidase activity --- p.66 / Chapter 3.6 --- Spectroscopic analysis --- p.73 / Chapter 3.6.1 --- Native enzyme --- p.73 / Chapter 3.6.2 --- Denatured enzyme --- p.73 / Chapter 3.6.2.1 --- Potassium cyanide --- p.73 / Chapter 3.6.2.2 --- Sodium azide --- p.73 / Chapter 3.6.3 --- Pyridine hemochrome characterization --- p.73 / Chapter 3.7 --- N-terminal amino acid sequence analysis --- p.79 / Chapter CHAPTER 4 --- DISCUSSION --- p.81 / Chapter 4.1 --- Subcellular locations of catalase in bloodworms --- p.82 / Chapter 4.2 --- Purification of post-mitochondrial catalase --- p.82 / Chapter 4.3 --- Physical properties of the purified enzyme --- p.84 / Chapter 4.3.1 --- Native and subunit molecular mass --- p.84 / Chapter 4.3.2 --- Isoelectric point --- p.85 / Chapter 4.4 --- Kinetic properties of the purified enzyme --- p.85 / Chapter 4.4.1 --- Optimal pH --- p.85 / Chapter 4.4.2 --- Thermal stability --- p.85 / Chapter 4.4.3 --- Km and Vmax --- p.87 / Chapter 4.4.4 --- Inhibition studies --- p.87 / Chapter 4.4.5 --- Catalase peroxidase activity --- p.87 / Chapter 4.5 --- Spectroscopic analysis --- p.88 / Chapter 4.5.1 --- Native and denatured enzyme --- p.88 / Chapter 4.5.2 --- Pyridine hemochrome characterization --- p.88 / Chapter 4.6 --- N-terminal amino acid analysis --- p.89 / Chapter 4.7 --- Conclusions --- p.89 / REFERENCES --- p.93