by Nathan, Yiu-hung Yam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 139-157). / Acknowledgements --- p.i / Table of Contents --- p.ii / Abstract --- p.viii / List of Abbreviations --- p.x / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Aims of the present study --- p.2 / Chapter 1.3 --- Sequencing of an adult human heart cDNA library --- p.3 / Chapter 1.4 --- Rat/mouse CRIP --- p.5 / Chapter 1.5 --- LIM proteins --- p.13 / Chapter 1.6 --- Zinc-binding proteins --- p.17 / Chapter 1.7 --- Bacterial expression system using the pAED4 vector --- p.24 / Chapter Chapter 2 --- Identification and sequence analysis ofhCRHP --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and methods --- p.29 / Chapter 2.2.1 --- Bacterial strains and vectors --- p.29 / Chapter 2.2.2 --- "Mediums, buffers and solutions" --- p.31 / Chapter 2.2.3 --- Bacteriophage clones preparation --- p.34 / Chapter 2.2.4 --- Amplification of clones by PCR --- p.35 / Chapter 2.2.5 --- Cycle sequencing of PCR products --- p.36 / Chapter 2.2.6 --- DNA sequences analysis --- p.38 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Sequence analysis of hCRHP --- p.39 / Chapter 2.3.2 --- Comparison of hCRHP with CRIP --- p.52 / Chapter 2.3.3 --- Comparison of hCRHP with some LIM proteins --- p.56 / Chapter 2.4 --- Discussions --- p.61 / Chapter Chapter 3 --- Study of hCRHP at the nucleic acid level --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and methods --- p.66 / Chapter 3.2.1 --- Animals --- p.66 / Chapter 3.2.2 --- "Mediums, buffers, enzymes and solutions" --- p.66 / Chapter 3.2.3 --- Preparation of total RNA --- p.70 / Chapter 3.2.3.1 --- Preparation of RNA by the CsCl method --- p.70 / Chapter 3.2.3.2 --- Preparation of RNA by the AGPC method --- p.71 / Chapter 3.2.4 --- Northern hybridization of hCRHP --- p.72 / Chapter 3.2.4.1 --- Formaldehyde agarose gel electrophoresis --- p.72 / Chapter 3.2.4.2 --- Preparation of radioactive probe --- p.73 / Chapter 3.2.4.3 --- RNA transfer and Northern hybridization --- p.74 / Chapter 3.2.5 --- Preparation of human genomic DNA --- p.77 / Chapter 3.2.6 --- Southern hybridization of hCRHP --- p.78 / Chapter 3.2.6.1 --- Restriction cutting and agarose gel electrophoresis of genomic DNA --- p.78 / Chapter 3.2.6.2 --- DNA transfer and Southern hybridization --- p.79 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Southern hybridization of hCRHP --- p.80 / Chapter 3.3.2 --- Identification of hCRHP in neonatal human heart --- p.83 / Chapter 3.3.3 --- Tissue distribution of CRIP mRNA in rat tissues --- p.85 / Chapter 3.3.4 --- Time course of CRIP expression in rat heart --- p.85 / Chapter 3.4 --- Discussions --- p.87 / Chapter Chapter 4 --- Subcloning and expression of hCRHP --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and methods --- p.90 / Chapter 4.2.1 --- Bacterial strains and vectors --- p.90 / Chapter 4.2.2 --- "Mediums, buffers, enzymes and solutions" --- p.92 / Chapter 4.2.3 --- Subcloning of hCRHP into pAED4 --- p.98 / Chapter 4.2.3.1 --- Primers design and PCR --- p.98 / Chapter 4.2.3.2 --- Purification of PCR products by Geneclean II´ёØ (BIO 101 Inc) --- p.99 / Chapter 4.2.3.3 --- Restriction digestion of purified PCR product and pAED4 --- p.100 / Chapter 4.2.3.4 --- Ligation and transformation of hCRHP --- p.101 / Chapter 4.2.3.5 --- Amplification and purification of pAED4-hCRHP --- p.103 / Chapter 4.2.4 --- Expression of hCRHP --- p.105 / Chapter 4.2.4.1 --- Induction of hCRHP expression --- p.105 / Chapter 4.2.4.2 --- SDS-PAGE and protein detection --- p.106 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3.1 --- Subcloning of hCRHP into pAED4 --- p.108 / Chapter 4.3.2 --- Induction and optimization of hCRHP expression --- p.110 / Chapter 4.4 --- Discussions --- p.117 / Chapter Chapter 5 --- Partial purification and isoelectric focusing of hCRHP --- p.120 / Chapter 5.1 --- Introduction --- p.120 / Chapter 5.2 --- Materials and methods --- p.121 / Chapter 5.2.1 --- "Mediums, buffers and mediums" --- p.121 / Chapter 5.2.2 --- Purification of hCRHP by ammonium sulphate precipitation --- p.121 / Chapter 5.2.3 --- Purification of hCRHP by hydrochloric acid extraction --- p.122 / Chapter 5.2.4 --- Purification of hCRHP by ultrafiltration --- p.123 / Chapter 5.2.5 --- Isoelectric focusing of hCRHP --- p.127 / Chapter 5.3 --- Results --- p.128 / Chapter 5.3.1 --- Partial purification of hCRHP by ammonium sulphate precipitation --- p.128 / Chapter 5.3.2 --- Partial purification of hCRHP by hydrochloric acid extraction --- p.128 / Chapter 5.3.3 --- Partial purification of hCRHP by ultrafiltration --- p.131 / Chapter 5.3.4 --- Isoelectric focusing of hCRHP --- p.133 / Chapter 5.4 --- Discussions --- p.133 / Chapter Chapter 6 --- Discussions --- p.136 / Chapter 6.1 --- The possible role(s) of hCRHP/CRIP --- p.136 / Chapter 6.2 --- Future prospects --- p.137 / References --- p.139 / Appendix 1 --- p.158

To Wah Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 118-142). / Acknowledgements --- p.1 / Summary --- p.3 / List of Abbreviations --- p.7 / Chapter Part I --- Alpha2-HS glycoprotein: Identification and Characterization of the Jacalin Binding Properties --- p.8 / Chapter Chapter 1 --- Introduction --- p.9 / Chapter 1.1 --- Jackfruit and jacalin --- p.10 / Chapter 1.2 --- Biochemical and immunological properties of jacalin --- p.11 / Chapter 1.2.1 --- Molecular Weight of Jacalin --- p.11 / Chapter 1.2.2 --- Molecular structure of jacalin --- p.11 / Chapter 1.2.3 --- Specificity of jacalin to Thomsen-Fredenreich- antigen (T-antigen) --- p.13 / Chapter 1.2.4 --- The internal repeated sequence in the jacalin --- p.13 / Chapter 1.2.5 --- Jacalin-Bound Proteins (JBP) --- p.15 / Chapter 1.2.6 --- Interaction of jacalin to JBP --- p.15 / Chapter 1.2.7 --- Immunological properties of jacalin --- p.16 / Chapter 1.3 --- Application of jacalin in medical research --- p.17 / Chapter 1.4 --- Background knowledge of α2HSG --- p.18 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Design of experiment --- p.21 / Chapter 2.2 --- Identification of the Unknown JBP --- p.21 / Chapter 2.2.1 --- Sera --- p.21 / Chapter 2.2.2 --- Isolation of JBP by Affinity Chromatography --- p.22 / Chapter 2.2.3 --- Fast protein liquid chromatography (FPLC) of JBP --- p.22 / Chapter 2.2.4 --- Affinity chromatography with anti-human IgA column --- p.23 / Chapter 2.2.5 --- Preparation of non-IgA JBP fraction --- p.24 / Chapter 2.2.6 --- N-terminal sequencing of the non-IgA JBP fraction --- p.24 / Chapter 2.2.7 --- SDS-PAGE and immunoblot of gel filtration fractions --- p.25 / Chapter 2.2.8 --- ELISA of FPLC fractions of JBP --- p.26 / Chapter 2.2.9 --- Immunochemical analysis --- p.27 / Chapter 2.3 --- α2HSG: the property of jacalin binding --- p.28 / Chapter 2.3.1 --- α2HSG -jacalin binding curve and competitive ELISA --- p.28 / Chapter 2.3.2 --- Purification of jacalin-crude extract (JCE) --- p.28 / Chapter 2.3.3 --- Characterization of JCE and ASJ --- p.29 / Chapter 2.3.4 --- Comparison of jacalin from different sources for binding to α2HSG by competitive ELISA --- p.29 / Chapter Chapter 3 --- Results --- p.31 / Chapter 3.1 --- Identification of the unknown JBP --- p.32 / Chapter 3.1.1 --- Isolation and FPLC of JBP --- p.32 / Chapter 3.1.2 --- Identification of non-IgA JBP by anti-human IgA affinity column --- p.32 / Chapter 3.1.3 --- Identification of the known JBP in the FPLC fractionated JBP --- p.36 / Chapter 3.1.4 --- Characterization and confirmation of non-IgA JBP --- p.36 / Chapter 3.2 --- α2HSG: the property of jacalin binding --- p.42 / Chapter 3.2.1 --- Characterization of α2HSG-jacalin binding --- p.42 / Chapter 3.2.2 --- Characterization of the purified jacalin --- p.45 / Chapter 3.2.3 --- Comparison of different batches of jacalin to interact with α2HSG --- p.45 / Chapter Chapter 4 --- Discussion --- p.53 / Chapter Part II --- Jacalin-α2HSG binding: the Clinical Values --- p.57 / Chapter Chapter 5 --- Introduction --- p.58 / Chapter Chapter 6 --- Materials and Methods --- p.61 / Chapter 6.1 --- Preparation of IgA-specific jacalin (ASJ) by IgA-Sepharose 4B affinity column --- p.62 / Chapter 6.2 --- Preparation of JCE- and ASJ-Sepharose-4B affinity column --- p.62 / Chapter 6.3 --- Factors affecting the yield of α2HSG --- p.62 / Chapter 6.4 --- Miscellaneous methods --- p.63 / Chapter Chapter 7 --- Results and Discussion --- p.65 / Chapter Part III --- Application of Jacalin for Studying the Pathogenesis of IgA Nephropathy --- p.77 / Chapter Chapter 8 --- An Overview of IgA Nephropathy --- p.78 / Chapter 8.1 --- Clinical manifestation of IgA nephropathy --- p.79 / Chapter 8.2 --- Mesangial deposits in IgAN --- p.80 / Chapter 8.3 --- Human IgA system --- p.81 / Chapter 8.4 --- The role of circulating IgA in the pathogenesis of IgA nephropathy --- p.84 / Chapter 8.5 --- Pathogenesis of primary IgA nephropathy --- p.86 / Chapter 8.6 --- Interaction between circulatory IgA and fibronectin (FN) in primary IgAN --- p.87 / Chapter Chapter 9 --- Materials and Methods --- p.90 / Chapter 9.1 --- Design of experiment --- p.91 / Chapter 9.2 --- Sera --- p.91 / Chapter 9.3 --- Analysis of IgAl/IgA ratio in sera and JBP --- p.91 / Chapter 9.4 --- Purification and Fast protein liquid chromatography (FPLC) of jacalin-bound protein (JBP) --- p.92 / Chapter 9.5 --- Analysis of FPLC-fractionated JBP --- p.93 / Chapter 9.5.1 --- ELISA of IgA-containing immune complexes (IgA-IC) --- p.93 / Chapter 9.5.2 --- "Quantitative ELISA of IgA, K-IgAl, and λ-IgAl" --- p.94 / Chapter 9.5.3 --- "Measurement of sIgA,dIgA and IgA containing immune complex (IgA-IC)" --- p.95 / Chapter 9.5.4 --- SDS-PAGE analysis --- p.95 / Chapter 9.6 --- Statistics --- p.95 / Chapter Chapter 10 --- Results --- p.96 / Chapter 10.1 --- IgA1/IgA ratio of serum and JBP --- p.97 / Chapter 10.2 --- Isolation and FPLC of JBP --- p.97 / Chapter 10.3 --- SDS-PAGE analysis of FPLC fractionated JBP --- p.97 / Chapter 10.4 --- ELISA of the FPLC fractionated JBP --- p.99 / Chapter 10.4.1 --- "Distribution of IgA, secretory IgA (sIgA) and dimeric IgA (dIgA) in the FPLC fractions" --- p.99 / Chapter 10.4.2 --- Distribution of IgA containing immune complex (IgA-IC) in the FPLC fractions --- p.99 / Chapter 10.4.3 --- Quantitation of IgA --- p.106 / Chapter 10.4.4 --- "Quantitation of K-IgAl and λ-IgA1, and determination of k/λ ratio of IgAl" --- p.106 / Chapter 10.4.5 --- "Quantitation of sIgA,dIgA, and IgA-containing immune complex (IgA-IC)" --- p.109 / Chapter Chapter 11 --- Discussion --- p.112 / References --- p.118

by Au Lai Yin, Cathy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 50-66). / Chapter I. --- ABSTRACT --- p.i / Chapter II. --- ACKNOWLEDGMENTS --- p.iii / Chapter III. --- TABLE OF CONTENTS --- p.iv / Chapter IV. --- LIST OF TABLES --- p.viii / Chapter V. --- LIST OF FIGURES --- p.x / Chapter VI. --- INTRODUCTION --- p.1 / Chapter VII. --- LITERATURE REVIEW --- p.3 / Chapter A. --- Mycobacterial tuberculosis Infections --- p.3 / Chapter B. --- Diagnostic Criteria forM .tuberculosis Infections --- p.3 / Chapter C. --- Mycobacteriological Laboratory Investigations for M. tuberculosis --- p.4 / Chapter 1. --- Conventional methods --- p.4 / Chapter 2. --- Rapid methods --- p.4 / Chapter D. --- Polymerase chain reaction (PCR) - the Principle --- p.5 / Chapter E. --- Application of PCR for Detection of M. tuberculosis --- p.6 / Chapter 1. --- Choice of target sequences --- p.6 / Chapter 2. --- Choice of method for the detection & identification of PCR-amplified product --- p.7 / Chapter 3. --- Studies on pure cultures --- p.9 / Chapter a. --- Detection limit - target DNA --- p.9 / Chapter b. --- Detection limit - Colony forming units --- p.9 / Chapter c. --- Detection limit - Number of cells --- p.10 / Chapter 4. --- Studies on clinical specimens --- p.10 / Chapter 5. --- Problems --- p.12 / Chapter a. --- Availability of target DNA --- p.13 / Chapter (i) --- Cell breakage efficiency --- p.13 / Chapter (ii) --- Target sequence --- p.14 / Chapter b. --- Inhibitory factors for Taq polymerase --- p.14 / Chapter c. --- Contamination --- p.15 / Chapter VIII. --- MATERIALS AND METHODS --- p.16 / Chapter A. --- Bacterial Strains and Strain Maintenance --- p.16 / Chapter 1. --- Reference Strains --- p.16 / Chapter 2. --- Clinical isolates --- p.16 / Chapter B. --- Growth media and culture conditions --- p.17 / Chapter C. --- Restriction Fragment Length Polymorphism (RFLP) --- p.17 / Chapter 1. --- Extraction of chromosomal DNA from M. tuberculosis --- p.18 / Chapter 2. --- Digestion of chromosomal DNA by PVU II --- p.19 / Chapter 3. --- Separation of digested DNA fragment by electrophoresis --- p.19 / Chapter 4. --- Southern Blotting --- p.19 / Chapter 5. --- Preparation of DNA probes by Polymerase Chain Reaction --- p.20 / Chapter 6. --- Hybridization --- p.21 / Chapter 7. --- Detection --- p.21 / Chapter D. --- Assessment of number of organisms --- p.22 / Chapter 1. --- Viable cell count --- p.22 / Chapter 2. --- Direct cell count --- p.22 / Chapter E. --- Assessment of the presence of IS6110/986 in M. tuberculosis isolates --- p.23 / Chapter F. --- Human leukaemic monocytic cell line (THP-1) --- p.23 / Chapter 1. --- Growth media and maintenance --- p.23 / Chapter 2. --- Culture Conditions --- p.24 / Chapter 3. --- Uptake of M. tuberculosis --- p.24 / Chapter G. --- Cell breakage and DNA extraction methodologies --- p.25 / Chapter H. --- Polymerase chain reaction (PCR) methodologies --- p.28 / Chapter 1. --- Primer and probe --- p.28 / Chapter 2. --- PCR conditions --- p.28 / Chapter 3. --- Detection --- p.29 / Chapter I. --- Patients and Clinical specimens --- p.30 / Chapter 1. --- Patients recruitment --- p.30 / Chapter 2. --- Clinical specimens --- p.30 / Chapter IX. --- RESULTS --- p.32 / Chapter A. --- "Development or Selection of a ""Standardized"" PCR Protocol for the Detection of M. tuberculosis Using Pure Cultures In Vitro" --- p.32 / Chapter 1. --- Selection of organisms for verification of the PCR protocol --- p.32 / Chapter 2. --- Optimization of the PCR conditions --- p.32 / Chapter 3. --- Detection limit of target DNA using the PCR procedure --- p.33 / Chapter B. --- Initial Screening of Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 &22a Based on Detection Limits of Colony Forming Units and Number of Cells --- p.34 / Chapter C. --- Comparison of Method 1 and Method 2 Based on Detection Limits of Colony Forming Units and Number of Cells Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis with variable copies of IS6110/986 --- p.34 / Chapter D. --- Detection of M. tuberculosis Isolates Within Macrophages --- p.35 / Chapter 1. --- Uptake of M. tuberculosis cells by THP-1 --- p.35 / Chapter 2. --- Comparison of the Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 & 22a Phagocytized by Activated THP-1 Macrophages --- p.35 / Chapter 3. --- Comparison of Method 1 and Method 2 Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis Phagocytized by Activated THP-1 Macrophages --- p.36 / Chapter E. --- Analysis of Clinical Specimens Using Method 1 & 2 with the Optimized PCR Protocol --- p.36 / Chapter 1. --- Bronchial Aspirate & Bronchoaveolar Lavage Fluid --- p.36 / Chapter 2. --- Pleural Fluid --- p.37 / Chapter 3. --- Tissue --- p.37 / Chapter 4. --- Sputum --- p.38 / Chapter 5. --- Cerebrospinal Fluid --- p.38 / Chapter X. --- DISCUSSION --- p.39 / Chapter A. --- Selection of IS6110/986 for DNA amplification --- p.39 / Chapter B. --- Optimization of PCR conditions reflected by detection limit of target DNA --- p.40 / Chapter C. --- Selection of cell breakage methods based on detection limits of CFU and/or number of mycobacterial cells --- p.41 / Chapter D. --- Application of Methods 1 & 2 and the optimized PCR protocol for clinical specimens --- p.43 / Chapter 1. --- Bronchial aspirates and bronchoaveolar lavage fluids --- p.43 / Chapter 2. --- Pleural fluids --- p.44 / Chapter 3. --- Tissues --- p.45 / Chapter 4. --- Sputa --- p.46 / Chapter 5. --- Cerebrospinal fluids --- p.46 / Chapter XI. --- CONCLUSION --- p.48 / Chapter XII. --- LITERATURE CITED --- p.50 / Chapter XIII --- TABLES --- p.67 / Chapter XIV. --- FIGURES --- p.85

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

Vehicle occupants are typically exposed to unpleasant whole-body vibration (WBV) for extended period of time. It is well known that the transmission of unwanted vibration to the human body can lead to fatigue and discomfort. Moreover, the unwanted vibration normally distributed in the low-frequency range has been found as the main risk factor for lower back pain and lumbago, which seriously affect the health and working performance of occupants. Thus vibration cancellation on seats has attracted considerable interest in recent years. So far, for most vehicle seats, vibration isolation is achieved passively by using seat cushions and conventional energy absorbers, which have very limited performance in the low-frequency range. The work presented in this thesis forms a successful development and experimental study of an active seat and control algorithm for occupants’ WBV reduction under low frequency excitations. Firstly, a modelling study of the seat human subjects (SHS) and an extensive experimental measurement of the vibration transmissibility of a test dummy and vehicle seat are carried out. The biodynamic responses of SHS exposed to uncoupled vertical and fore-and-aft WBV is modelled. A comparison with the existing models is made and the results show that an improved fit with the aggregated experimental data is achieved. Secondly, an active seat is developed based upon the observations and understanding of the SHS and seat system. The characteristics of the active seat dynamics are identified through experimental tests found suitable for the development of an active seat to attenuate the vibration experienced by vehicle occupants. The vibration cancellation performance of the active seat is initially examined by feedforward plus proportional-integral (PI) control tests. Through these tests, the effectiveness of the actuators control authority is verified, but the limitations are also revealed. Because the active seat system is subject to non-linear and time-varying behaviour, a self-tuning fully adaptive algorithm is a prime requirement. The Filtered-x Least-Mean-Square (FXLMS) algorithm with the Fast-block LMS (FBLMS) system identification technique is found suitable for this application and is investigated through experimental tests. Substantial vibration reductions are achieved for a variety of input vibration profiles. An excellent capability of the active seat and control system for efficiently reducing the vibration level of seated occupants under low-frequency WBV is demonstrated.

The Active Control of Structural Response (ACSR) vibration suppression system, where hydraulic actuators located between the gearbox and the fuselage are used to cancel vibration in large helicopters, has been used successfully for many years. However the power consumed by the actuators can be high, and using hydraulic actuation for smaller rotorcraft has not been seen as practical. In contrast to active vibration reduction systems, passive vibration isolation systems require no external power. Passive vibration isolation systems however have the disadvantage of being limited to working at one specific frequency which will not be acceptable as slowed rotor flight becomes more common for fuel efficiency and noise legislation reasons. In this thesis two electrically powered actuation concepts, one piezoelectric, and one electromagnetic were initially evaluated. An electrically powered actively augmented passive, or hybrid, vibration reduction system based on an electro hydrostatic actuator (EHA) concept was proposed to be developed further. This hybrid actuator will have a wider range of operating frequencies than a purely passive system, and have lower power consumption than a purely active system. The design is termed a “Resonant EHA”; in that the resonant frequency of the coupled fluid, pump and electric motor rotor inertia matches the fundamental vibration frequency. The hydraulic cylinder, fluid and pump act as a single stage gear ratio, and the. brushless electric motor’s inertia is the main resonating mass as in a Dynamic Antiresonant Vibration Isolator (DAVI) passive vibration reduction system. The electrical power is used to compensate for friction in the actuator and other losses, and if needed can shift the operating point away from the resonant frequency. Simulation results indicated that a hydraulic circuit in which the pump leakage is fed back into the low pressure line would introduce unacceptable disturbances in the flows to and from the cylinder. To eliminate the source of the disturbances, a fully integrated electric motor and pump circuit design was chosen in which the electric motor is immersed in hydraulic fluid. An EHA demonstrator was built sized for a 1.5 tonne rotorcraft. For sizing comparison purposes the frameless brushless D.C motor for each strut of 1.5 tonne rotorcraft has a rotor and stator mass of approximately 1 kg, and can produce a continuous stall torque of 2 Nm. The bidirectional pump has a displacement of 1.5 cm3/rev, the mean system pressure was taken as 90 bar, and the double ended hydraulic cylinder has a 32 mm diameter bore, and 18 mm rod. Initial test results for the proof of concept EHA showed highly significant free play with a reversal of torque direction, resulting in unacceptable loss in transmission stiffness. The free play was traced to the gear pump and a hypothesis for the origin of the free play was put forward. To avoid torque reversals the EHA was further tested with a constant offset torque bias which proved successful in restoring a sufficient stiffness to the transmission. The sizing of the electric motor and power consumed with a non-zero offset torque is greater than a torque reversing motor, which limits the immediate application of the device in the present form. Future research investigating the use of other transmission elements, such as a piston pump, to obtain a more linear stiffness is recommended. As a hybrid vibration isolation system a Root Mean Square (RMS) reduction by a factor of four and near elimination of the fundamental frequency vibrations was achieved for the frequency range of 10 to 20 hertz.

Loneliness and social isolation can have a serious impact on one’s mental health, leading to increased stress, lower self-esteem, panic attacks, and drug or alcohol addictions. Older adults and international students are disproportionately affected by loneliness. This thesis investigates Socialoscope, a smartphone app that passively detects loneliness in smartphone users based on the user’s day-to-day social interactions, communication and smartphone activity sensed by the smartphone’s built-in sensors. Statistical analysis is used to determine smartphone features most correlated with loneliness. A previously established relationship between loneliness and personality type is explored. The most correlated features are used to synthesize machine learning classifiers that infer loneliness levels from smartphone sensor features with an accuracy of 90%. These classifiers can be used to make the Socialoscope an intelligent loneliness sensing Android app. The results show that, of the five Big-Five Personality Traits, emotional stability and extraversion personality traits are strongly correlated with the sensor features such as number of messages, number of outgoing calls, number of late night browser searches, number of long incoming or outgoing calls and number of auto-joined trusted Wi-Fi SSIDs. Moreover, the classifier accuracy while classifying loneliness levels is significantly improved to 98% by taking these personality traits into consideration. Socialoscope can be integrated into the healthcare system as an early warning indicator of patients requiring intervention or utilized for personal self-reflection.

Orientador: Samuel Monroe Cohen / Resumo: O aumento da incidência de distúrbios testiculares e a possível influência de substâncias químicas ambientais, como o dibutilftalato (DBP) e a acrilamida (AA), exigem a identificação de modos de ação. A maioria dos estudos de toxicologia reprodutiva utiliza amostras de RNA provenientes de todo o testículo para avaliar a expressão genica; entretanto, análises de tipos celulares isolados poderiam gerar resultados mais específicos. Entre as células germinativas testiculares, as espermatogônias são importantes pois representam o início da espermatogênese. Este estudo objetivou, 1) estabelecer técnica de isolamento de espermatogônias; 2) aplicar esta técnica para verificar possíveis alterações na expressão gênica (Pou5f1, Kitlg, Mki-67, Bak1 e Spry4) em testículos de ratos pré-púberes (DPN24) e púberes (DPN45) após exposição in utero e pós-natal ao DBP ou à AA. A técnica foi eficiente para o isolamento das espermatogônias. A exposição ao DBP levou à redução do peso corporal da ninhada ao nascer, da distância anogenital dos filhotes machos no DPN4 e ao aumento da frequência de retenção de mamilos no DPN14. Os pesos relativos dos testículos expostos ao DBP estavam reduzidos apenas no DPN24. Animais expostos ao DBP mostraram níveis reduzidos de expressão de Pou5f1 e Mki67 no DPN24, e de Pou5f1 e Spry4 no DPN45. A exposição a AA reduziu a expressão de Pou5f1, Mki67 e Spry4, embora não significativamente. Nossos resultados sugerem que DBP atue reduzindo a proliferação celular e prejudi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The increased incidence of testicular disorders in young men and the possible influence of environmental chemicals, such as dibutyl phthalate (DBP) and acrylamide (AA), requires experimental models for identifying modes of action. Most published reproductive toxicologic studies use RNA samples from the total testis to evaluate testicular gene expression; however, analyses of isolated cell types could provide a more specific tool. Among testicular germ cells, spermatogonia are critical since they represent the onset of spermatogenesis. This study aimed, 1) to establish a technique for spermatogonia isolation; 2) to apply this isolation technique to verify possible gene expression alterations (Pou5f1, Kitlg, Mki-67, Bak1 and Spry4) in prepubertal post-natal day, (PND24) and pubertal (PND45) testes after in utero and postnatal exposure to DBP or AA. The technique was efficient for isolation of a majority of spermatogonia. In utero DBP exposure led to reduced litter body weight at birth, reduced anogenital distance of male pups on PND4, and increased frequency of male nipple retention on PND14 compared to controls. DBP-exposed relative testes weights were reduced only at PND24 compared to control but they did not differ at PND45. DBP-exposed animals showed reduced expression levels of Pou5f1 and Mki67 on PND24, and reduced expression of Pou5f1 and Spry4 on PND45. AA exposure reduced expression of Pou5f1, Mki67 and Spry4 at PND45 although not significantly. Our results suggest tha... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2015-03-05T12:06:53Z (GMT). No. of bitstreams: 0 Previous issue date: 8 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A presente tese analisa a experiência com a lepra de doentes do Rio Grande do Sul isolados no Hospital Colônia Itapuã. Procurou-se discutir o processo de isolamento, exclusão e reconstrução da vida na Instituição. Fundado em 1940 o Leprosário isolou doentes de todo o Estado, alguns destes antigos enfermos ainda vivem no Hospital e através da história oral foi possível registrar suas experiências. Do mesmo modo foi possível contatar pessoas que deixaram o Leprosário, assim por meio de duas distintas trajetórias analisou-se o drama médico-social que representava um diagnóstico de lepra há apenas algumas décadas atrás. Lori e GM são os personagens principais desta trama. Ela moradora mais antiga do Itapuã, tem sua história pessoal intimamente ligada à história da Instituição; ele internado nos anos de 1940, vive fora do Hospital há mais de 50 anos, mas sua trajetória foi bastante marcada pela passagem pelo Leprosário e o convívio com esta lembrança. O olhar voltado para estes micro-universos individuais teve a p / The present theses analyses the experience of having leprosy in Rio Grande do Sul and being isolated in the Colony Hospital Itapuã. We aimed at discussing the processes of isolation, exclusion and rebuilding of life inside the institution. Founded in the 1940’s the leprosarium isolated inflicted people from all over the state. Some of them still live in the hospital and through their story we were able to capture their experience. It was also possible to contact people who left the leprosarium and through those two distinct paths we analyzed the medical and social struggle that meant a leprosy diagnosis only a few decades ago. Lori and GM are the main characters of that plot. She is the oldest resident in Itapuã and her personal story is deeply connected to the institution’s; he was admitted in the 1940’s but has lived outside the hospital for over 50 years, and his life has been enormously affected by the time he spent there and the living with that memory. The focus on those two individual micro universes h