錯誤折疊並聚集的促澱粉樣變蛋白和多肽分子通常以β折疊含量豐富的纖維狀澱粉態存在，這種纖維狀澱粉態被認為與多種神經退行性疾病的發病有關，例如老年癡呆症，多聚穀氨醯胺症以及傳染性海綿狀腦病。澱粉態沉積物作為多種神經退行性疾病的顯著標誌，促澱粉樣變蛋白和多肽發生錯誤折疊並聚集進而導致神經毒性的機理仍未被闡明。在當前的研究中，我們選擇酵母感染性蛋白Sup35作為探索促澱粉樣變蛋白聚集機理的模型。Sup35是一種存在於釀酒酵母細胞中的感染性蛋白，作為一種翻譯終止因子，它可以通過改變自身構象，進而形成不溶的纖維狀澱粉態沉澱。根據位置和功能的不同，Sup35可被劃分為3個結構域，即N，M和C。作為控制其感染性的區域，Sup35-NM被廣泛接受為一種用於研究促澱粉樣變蛋白的模型。研究人員已經針對Sup35的聚集機理開展了很多研究，其中最為廣泛接受的是Lindquist 等人提出的β螺旋模型。在這個模型中，相鄰的氨基酸片段形成了一種頭對頭和尾對尾的構象。我們的研究目的就是要探究這種聚集機理模型是否正確。如果不正確，我們將對聚集機理提出一種新的假設。 / 作為探索促澱粉樣變蛋白聚集過程的重要前提，研究人員必須首先製備出只含有單獨的蛋白單體的樣品溶液。否則，相關的動力學過程研究將被干擾。我們通過動態激光光散射研究發現，使用現有的多種用於溶解促澱粉樣變蛋白和多肽的實驗方法並不能製備出真正的蛋白溶液，得到的樣品中總含有微量的、尺寸大約為10-10² nm的聚集體。這些聚集體會極大地影響聚集的動力學過程。這也可以在一定程度上解釋為什麼在不同的文獻報導中，同一種蛋白在相同的環境中卻表現出差異巨大的動力學過程。在當前的研究中，我們將傳統方法與我們實驗室新進開發的超濾法相結合，發展出了一套可以用於製備真正的、不含有聚集體的促澱粉樣變蛋白或多肽溶液的方法。製備出的溶液可以保持其中的蛋白或多肽處於單體狀態至少一個星期，這為研究在生理條件下蛋白的聚集過程提供了重要的基礎。 / 為了研究Sup35不同亞基之間的相互作用，我們分別在其N結構域的頭，腰和尾做了半胱氨酸點突變，並用兩種相互獨立的方法研究亞基之間的相互作用。第一種方法是在突變位點引入空間位阻，從而減弱所謂的頭對頭尾對尾的相互作用。我們的想法很直接，如果Lindquist等人提出的機理是正確的，那麼突變後的蛋白將無法形成纖維狀澱粉態沉澱。第二種方法是通過形成二硫鍵在不同蛋白的半胱氨酸突變位點之間引入連接分子，共有兩種連接分子，一種長約2 Å，另一種長約11 Å。選擇這兩種連接分子的原因是，聚集體中兩條Sup35蛋白鏈之間的距離通常約4.7 Å，連接分子長於或短于這個距離應會對聚集產生不同影響，從而反映出聚集體的結構資訊。 / 在這篇博士論文中，首先，我將介紹促澱粉樣變蛋白研究的背景和激光光散射測量的原理以及研究中用到的主要實驗方法。然後，我將闡述如何將傳統方法和我們實驗室新進發展出的超濾法相結合，從而製備出真正的、不含聚集體的蛋白溶液。接下來，我還將證明通過動態和靜態激光光散射相結合，我們可以得到更多關於促澱粉樣變蛋白的微觀參數，包括分子量，蛋白單體和聚集體的流體力學半徑等。最後，我將針對不同Sup35突變體的聚集動力學過程來研究其亞基之間的相互作用並提出Sup35的聚集模型。 / Misfolding and aggregation of amyloidogenic protein/peptide are frequently found in a β-sheet-rich fibrillar protein conformation known as amyloids, which are related to the onset of neurodegenerative diseases, ranging from Alzheimer and polyglutamine diseases to transmissible spongiform encephalopathies. While amyloid deposits are hallmarks of many neurodegenerative diseases, the mechanism by which these proteins/peptides gain their neurotoxic function upon misfolding and aggregation remains unclear. In the current study, we choose the yeast prion Sup35 as a model system to investigate the aggregation mechanism of amyloidogenic protein. The Sup35 protein is a yeast (Saccharomyces cerevisiae) prion protein, a translation termination factor that can convert into insoluble amyloid fibril. The structure of Sup35 protein can be divided into three regions; namely, N, M, and C based on their positions and different functions. Being the prion-determining region, Sup35-NM has been widely accepted as a model to study the amyloidogenic proteins. Many studies have been focused on the aggregation mechanism. The β-helix model proposed by Lindquist and her coworkers is mostly accepted. In such a model, Sup35-NM is folded to form a “head and a “tail region in the N region and different Sup35-NM chains aggregate together via a cooperative Head-to-Head and Tail-to-Tail stacking. The aim of our current study is to check whether this proposed mechanism is valid. / To gain insight into the mechanism of aggregation process, one must start with a solution that contains only individual (monomeric) protein chains. Otherwise, the kinetic study would be compromised. Our dynamic laser light scattering (LLS) study shows that the existing protocols of dissolving amyloidogenic protein/peptide do not result in a true solution; namely, there always exist a trace amount of interchain aggregates with an average size of ~10-10² nm, which greatly affect the association kinetics, partially explaining why different kinetics were reported even for a solution with identical protein and solvent. In this thesis study, by using a combination of the conventional dissolution procedure and our newly developed ultra-filtration method, we have developed a novel protocol to prepare a true solution of amyloidogenic protein/peptide without any interchain aggregates. The resultant solutions remain in their monomeric state for more than one week, which is vitally important for further study of the interchain association under the physiological conditions / To investigate the inter-subunit relationship, cysteine variants mutated at “Head, Waist, Tail" of the N region have been constructed. Two independent assessments have been proposed to study the inter-subunit interaction. One is to provide steric hindrance to the mutated sites so that the so called “Head-to-Head and Tail-to-Tail" interaction will be attenuated. Our strategy is quite straightforward, if the mechanism proposed by Lindquist and her coworkers is valid, the modified protein should lose its ability to form amyloid fibrils. The other strategy is to introduce disulfide cross-linkage between different mutation sites. Two types of disulfide cross-linkage have been chosen, one with a bond length of ~2 Å and the other, ~11 Å. The reason for such choices is that Sup35-NM has a characteristic inter-strand distance of ~4.7 Å. The disulfide bond length shorter or longer than this distance is supposed to play a different role in the protein aggregation, shedding light on the structural information. / In this Ph.D. thesis, we first introduce the background of amyloidogenic protein research and present the principle and instrumentation of laser light scattering, the main technique applied in our study. Next we show how to obtain a true solution of amyloidogenic protein with no oligomeric aggregates by combining a conventional dissolution procedure and our newly developed ultra-filtration method. We also show how to combine static and dynamic laser light-scattering measurements in the study of protein solutions, which leads to more microscopic parameters, such as the molar mass and the hydrodynamic sizes of individual protein chains and their aggregates. Our focus is on the aggregation kinetics of modified Sup35-NM variants and on the investigation of the inter-subunit interaction. Finally, we propose a model for the aggregation of Sup35-NM prion protein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Diao, Shu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-101). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT (in Chinese) --- p.i / ABSTRACT --- p.iii / Table of content --- p.v / Acknowledgement --- p.viii / Chapter Chapter 1 --- Introduction and background --- p.1 / Chapter 1.1 --- The biological role of amyloidogenic protein --- p.1 / Chapter 1.1.1 --- The role of amyloidogenic protein in human disease --- p.1 / Chapter 1.1.2 --- The functional role of amyloidogenic protein in living system --- p.2 / Chapter 1.1.3 --- The role of amyloidogenic protein asnonchromosomal genetic elements --- p.3 / Chapter 1.2 --- The structure of amyloid fibrils --- p.4 / Chapter 1.2.1 --- Macromolecular structure of amyloid fibrils --- p.5 / Chapter 1.2.2 --- Structure models for protofilament --- p.6 / Chapter 1.2.3 --- The polymorphism of amyloid fibrils --- p.9 / Chapter 1.3 --- Aggregation mechanism of amyloidogenic protein --- p.10 / Chapter 1.3.1 --- The nucleated polymerization mechanism --- p.11 / Chapter 1.3.2 --- Multiple conformations adopted by amyloidogenic protein chains --- p.13 / Chapter 1.3.3 --- Sequence effect on amyloid formation --- p.15 / Chapter 1.4 --- The pathogenesis of amyloid diseases --- p.16 / Chapter 1.4.1 --- Prefibrillar aggregates may be the real culprits --- p.16 / Chapter 1.4.2 --- Strategies to prevent amyloid diseases --- p.17 / Chapter 1.5 --- References and Notes --- p.19 / Chapter Chapter 2 --- Principle of Laser Light Scattering and Instrumentation --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Static Laser Light Scattering --- p.28 / Chapter 2.2.1 --- Scattering by a small particle --- p.28 / Chapter 2.2.2 --- Scattering by many small-particle system --- p.30 / Chapter 2.2.3 --- Scattering by real systems --- p.31 / Chapter 2.3 --- Dynamic Laser Light Scattering --- p.37 / Chapter 2.3.1 --- Power spectrum of scattered light --- p.37 / Chapter 2.3.2 --- Siegert relation --- p.39 / Chapter 2.3.3 --- Translational diffusions --- p.40 / Chapter 2.3.4 --- Analysis of the correlation function profile --- p.42 / Chapter 2.4 --- Combination of Static and Dynamic Light Scattering --- p.44 / Chapter 2.5 --- Practice of Laser Light Scattering --- p.45 / Chapter 2.5.1 --- Light source --- p.45 / Chapter 2.5.2 --- Optics and cell design --- p.46 / Chapter 2.5.3 --- Detector --- p.47 / Chapter 2.5.4 --- Sample Preparation --- p.47 / Chapter 2.5.5 --- Differential refractometer --- p.48 / Chapter 2.6 --- References and Notes --- p.49 / Chapter Chapter 3 --- How to obtain a true solution of amyloidogenic protein/peptide with no oligomeric aggregates --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Experimental section --- p.53 / Chapter 3.3 --- Results and discussion --- p.59 / Chapter 3.4 --- Conclusion --- p.68 / Chapter 3.5 --- References and Notes --- p.71 / Chapter Chapter 4 --- Aggregation mechanism investigation of the Yeast prion protein Sup35-NM --- p.73 / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Experimental section --- p.75 / Chapter 4.3 --- Results and discussion --- p.82 / Chapter 4.3.1 --- Aggregation kinetics of Sup35-NM protein initiated from monomeric state --- p.82 / Chapter 4.3.2 --- Does Sup35-NM protein aggregate in a head-to-head and tail-to-tail fashion? --- p.87 / Chapter 4.3.2.1 --- The effect of dimerization on Sup35-NM aggregation --- p.88 / Chapter 4.3.2.2 --- Inter-subunit investigation by Pyrene excimer fluorescence assay --- p.92 / Chapter 4.3.2.3 --- The effect of PEGylation on Sup35-NM aggregation --- p.94 / Chapter 4.4 --- Conclusion --- p.98 / Chapter 4.5 --- References --- p.100 / Publications --- p.102

Miu Fei Fei. / On t.p. "42" are subscripts following the word "MSP-1" in the title. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 117-125). / Abstracts in English and Chinese. / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.v / LIST OF FIGURE --- p.vii / LIST OF ABBREVIATIONS --- p.ix / CHAPTERS: / Chapter 1. --- BACKGROUND OF MALARIA / Chapter 1.1 --- Epidemilogy --- p.2 / Chapter 1.2 --- Mode of Infection --- p.4 / Chapter 1.3 --- Conventional Control & Vaccination --- p.9 / Chapter 1.4 --- Vaccine Candidate PfMSV-142 --- p.16 / Chapter 1.5 --- Cloning and Expression of pfMSP-142 --- p.26 / Chapter 1.6 --- Aims of Study --- p.32 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.2 --- Methods --- p.38 / Chapter 3. --- Construction of recombinants N-PfMSP-142 and C- PfMSP-142 / Chapter 3.1 --- Construction of C-PfMSP-l42 --- p.51 / Chapter 3.2 --- Construction ofN-PfMSP-l42 --- p.56 / Chapter 4. --- Purification with IMAC / Chapter 4.1 --- Immobilized Metal Affinity Chromatography (IMAC) --- p.58 / Chapter 4.2 --- Purification ofN-PfMSP-l42 --- p.61 / Chapter 4.3 --- Purification profile of N-PfMSP-142 --- p.68 / Chapter 4.4 --- Purification of C-PfMSP-l42 --- p.70 / Chapter 4.5 --- Purification profile of C-PfMSP-142 --- p.73 / Chapter 4.6 --- Expression pattern of recombinants PfMSP-142 --- p.76 / Chapter 5. --- Purification combined with other chromatography method / Chapter 5.1 --- Affinity chromatography --- p.78 / Chapter 5.2 --- Gel filtration chromatography --- p.80 / Chapter 5.3 --- Ion exchange chromatography --- p.83 / Chapter 5.4 --- Conclusion --- p.93 / Chapter 6. --- Characteristic of IMAC products --- p.94 / Chapter 7. --- Characteristic of N-hisPfMSP-l42 & C-hisPfMSP-l --- p.42 / Chapter 7.1 --- Immunogenitcity of N-PfMSP-l42 and C-PfMSP-142 --- p.100 / Chapter 7.2 --- Competitive ELISA --- p.105 / Chapter 8. --- Discussion --- p.107 / REFERENCE --- p.117

by Chan Wai Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 170-185). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vii / Abbreviations --- p.xii / for Nucleotides --- p.xii / for Amino acids --- p.xii / for Standard genetic codes --- p.xiii / for Units --- p.xiii / for Prefixes of units --- p.xiv / for Terms commonly used in the report --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome --- p.1 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.3 --- Cell Biology of Coronavirus Infection and Replication and the Role of Nucleocapsid Protein --- p.9 / Chapter 1.4 --- Recent Advances in the SARS-CoV Nucleocapsid Protein --- p.16 / Chapter 1.5 --- The Sumoylation System --- p.24 / Chapter 1.6 --- Objectives of the Present Study --- p.28 / Chapter Chapter II --- SARS-CoV N protein and Fragment Purification --- p.29 / Chapter 2.1 --- INTRODUCTION --- p.29 / Chapter 2.2 --- METHODOLOGY --- p.31 / Materials --- p.31 / Methods --- p.39 / Chapter 2.2.1 --- Construction of the pMAL-c2P vector --- p.39 / Chapter 2.2.2 --- Sub-cloning of the N protein into expression vectors --- p.42 / Chapter 2.2.2.1 --- Design of primers for the cloning of N protein --- p.43 / Chapter 2.2.2.2 --- DNA amplification using Polymerase Chain Reaction (PCR) --- p.44 / Chapter 2.2.2.3 --- DNA extraction from agarose gel --- p.45 / Chapter 2.2.2.4 --- Restriction digestion of purified PCR product and vectors --- p.46 / Chapter 2.2.2.5 --- Ligation of N protein into expression vectors --- p.47 / Chapter 2.2.2.6 --- Preparation of competent cells --- p.48 / Chapter 2.2.2.7 --- Transformation of plasmids into competent Escherichia coli --- p.49 / Chapter 2.2.2.8 --- Preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.1 --- Mini-preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.2 --- Midi-preparation of plasmid DNA --- p.51 / Chapter 2.2.3 --- Expression of tagged and untagged N protein --- p.53 / Chapter 2.2.3.1 --- Preparation of E. coli competent cells for protein expression --- p.53 / Chapter 2.2.3.2 --- Expression of N protein --- p.53 / Chapter 2.2.3.3 --- Solubility tests on the fusion proteins expressed --- p.54 / Chapter 2.2.4 --- Purification of N protein Chromatographic methods --- p.55 / Chapter 2.2.4.1 --- Affinity chromatography --- p.55 / Chapter 2.2.4.1.1 --- Ni-NTA affinity chromatography --- p.55 / Chapter 2.2.4.1.2 --- Glutathione affinity chromatography --- p.56 / Chapter 2.2.4.1.3 --- Amylose affinity chromatography --- p.56 / Chapter 2.2.4.2 --- Ion exchange chromatography --- p.57 / Chapter 2.2.4.2.1 --- Cation exchange chromatography --- p.57 / Chapter 2.2.4.2.2 --- Anion exchange chromatography --- p.58 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.58 / Chapter 2.2.4.4 --- Size exclusion chromatography Purification strategies --- p.60 / Chapter 2.2.4.5 --- Purification of His6-tagged N proteins --- p.60 / Chapter 2.2.4.6 --- Purification of MBP-tagged N proteins --- p.60 / Chapter 2.2.4.7 --- Purification of GST-tagged N proteins --- p.61 / Chapter 2.2.4.8 --- Purification of untagged N proteins --- p.61 / Chapter 2.2.5 --- Trypsin digestion assay for the design of stable fragment --- p.64 / Chapter 2.2.6 --- Partial purification of the N protein amino acid residue 214-422 fragment --- p.65 / Chapter 2.2.7 --- Sumoylation of the SARS-CoV N protein --- p.67 / Chapter 2.2.7.1 --- In vitro sumoylation assay --- p.67 / Chapter 2.2.7.2 --- Sample preparation for mass spectrometric analysis --- p.68 / Chapter 2.3 --- RESULTS --- p.70 / Chapter 2.3.1 --- Construction of the vector pMAL-c2P --- p.70 / Chapter 2.3.2 --- "Construction of recombinant N protein-pAC28m, N-protein- pGEX-6P-l,N protein-pMAL-c2E and N protein-pMAL-c2P plasmids" --- p.72 / Chapter 2.3.3 --- Optimization of expression conditions --- p.79 / Chapter 2.3.4 --- Screening of purification strategies --- p.82 / Chapter 2.3.4.1 --- Purification of His6-N protein --- p.82 / Chapter 2.3.4.2 --- Purification of MBP-N protein --- p.84 / Chapter 2.3.4.3 --- Purification of GST-N protein --- p.85 / Chapter 2.3.4.4 --- Purification of untagged N protein --- p.87 / Chapter 2.3.5 --- Limited trypsinolysis for the determination of discrete structural unit --- p.91 / Chapter 2.3.6 --- Partial purification of the N protein 214-422 fragment --- p.94 / Chapter 2.3.7 --- Sumoylation of N protein --- p.97 / Chapter 2.2.7.1 --- Sumoylation site prediction --- p.97 / Chapter 2.2.7.2 --- In vitro sumoylation assay --- p.99 / Chapter 2.2.7.3 --- Mass spectrometric identification of sumoylated SARS-CoV N protein --- p.103 / Chapter 2.4 --- DISCUSSION --- p.109 / Chapter Chapter III --- Characterization of the Nucleic Acid Binding Ability of N protein --- p.119 / Chapter 3.1 --- INTRODUCTION --- p.119 / Chapter 3.2 --- METHODOLOGY --- p.120 / Materials --- p.120 / Methods --- p.124 / Chapter 3.2.1 --- Spectrophotometric Measurement of ratio OD260/ OD280 --- p.124 / Chapter 3.2.2 --- Native gel electrophoresis --- p.124 / Chapter 3.2.3 --- Quantitative determination of nucleic acids content --- p.125 / Chapter 3.2.3.1 --- Dische assay - quantitative determination of DNA content --- p.125 / Chapter 3.2.3.2 --- Orcinol assay - quantitative determination of RNA content --- p.126 / Chapter 3.2.4 --- RNase digestion of the N protein-bound RNA --- p.128 / Chapter 3.2.5 --- Isolation of RNA from purified GST-N proteins --- p.128 / Chapter 3.2.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.129 / Chapter 3.2.7 --- Vero E6 cell line maintenance and total RNA extraction --- p.131 / Chapter 3.2.8 --- Electrophoretic mobility shift assay (EMSA) --- p.131 / Chapter 3.3 --- RESULTS --- p.133 / Chapter 3.3.1 --- Detection of nucleic acids in the purified N proteins byspectrophotometric Measurement of ratio OD260/ OD280 --- p.133 / Chapter 3.3.2 --- Native gel electrophoresis --- p.135 / Chapter 3.3.3 --- Quantitative determination of nucleic acids content in purified GST-N proteins --- p.136 / Chapter 3.3.3.1 --- Dische assay for the determination of DNA --- p.136 / Chapter 3.3.3.2 --- Orcinol assay for the determination of RNA --- p.138 / Chapter 3.3.4 --- RNase digestion treatment --- p.139 / Chapter 3.3.5 --- Extraction of RNA from GST-N proteins --- p.140 / Chapter 3.3.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.142 / Chapter 3.3.7 --- Electrophoretic mobility shift assay (EMSA) --- p.144 / Chapter 3.4 --- DISCUSSION --- p.147 / Chapter Chapter IV --- Discussion --- p.154 / Chapter 4.1 --- "Purity, Aggregation and RNA Binding Property of the SARS-CoV Nucleocapsid Protein" --- p.154 / Chapter 4.2 --- Future perspectives --- p.156 / Chapter 4.2.1 --- Structural study of the SARS-CoV N protein through x-ray crystallography --- p.156 / Chapter 4.2.2 --- Mapping the RNA binding domain in the SARS-CoV N protein --- p.156 / Chapter 4.2.3 --- Determination of aggregation state by lateral turbidimetry analysis --- p.156 / Chapter 4.2.4 --- Exploring protein interacting partners that enhance RNA binding specificity --- p.157 / Appendix --- p.159 / Chapter I. --- Sequence of the SARS-CoV N protein --- p.159 / Chapter II. --- Sequence of the SARS-CoV genome fragment used for RNA binding assay in section 3.37.1 --- p.161 / Chapter III. --- Vector maps --- p.161 / Chapter a) --- Vector map of pACYC177 --- p.161 / Chapter b) --- Vector map and MCS of pET28a --- p.163 / Chapter c) --- Vector map and MCS of pAC28 --- p.164 / Chapter d) --- Vector map and MCS of pGEX-6P-1 / Chapter e) --- Vector map of pMAL-c2X and MCS of pMAL-c2E / Chapter IV. --- Electrophoresis markers --- p.166 / Chapter V. --- SDS-PAGE gel parathion protocol --- p.169 / References --- p.170

by Luk Choi Wan. / Thesis submitted in: November 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 125-132). / Abstracts in English and Chinese. / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.viii / Tabel of contents --- p.x / List of Figures --- p.xii / List of Tables --- p.xiv / List of Abbreviations --- p.xv / List of Abbreviations --- p.xv / Chapter Chapter One --- Literature review --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- General aspects of plant lectins --- p.4 / Chapter 1.3 --- Monocot mannose-binding lectins --- p.5 / Chapter 1.3.1 --- General introduction --- p.5 / Chapter 1.3.2 --- Sugar specificity --- p.6 / Chapter 1.3.3 --- Isolation and purification --- p.7 / Chapter 1.3.4 --- Molecular cloning --- p.9 / Chapter 1.3.5 --- Molecular structure and modifications --- p.10 / Chapter 1.3.6 --- Molecular evolution --- p.12 / Chapter 1.3.7 --- Transformation --- p.12 / Chapter 1.3.8 --- Physiological roles --- p.14 / Chapter 1.3.9 --- Application --- p.19 / Chapter 1.4 --- Dendrobium nobile --- p.31 / Chapter 1.4.1 --- Background --- p.31 / Chapter 1.4.2 --- Chemical analysis --- p.32 / Chapter Chapter Two --- Biological activities of a mannose-binding lectin isolated from Dendrobium mobile --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- Mannose-binding lectin from D. nobile --- p.35 / Chapter 2.3 --- Biological activities of mannose-binding lectin from D. nobile --- p.36 / Chapter 2.3.1 --- Hemagglutinating activity --- p.36 / Chapter 2.3.2 --- In vitro anti-proliferative assay --- p.37 / Chapter 2.3.3 --- In vitro antiviral assay --- p.40 / Chapter 2.3.4 --- Statistical Analysis --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Physiochemical properties of D. nobile lectins --- p.43 / Chapter 2.4.2 --- Cytotoxicity to cancer cell lines --- p.53 / Chapter 2.4.3 --- Antiviral activity --- p.59 / Chapter 2.5 --- Discussion --- p.60 / Chapter Chapter Three --- Molecular cloning of lectin gene of Dendrobium nobile --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Methods --- p.67 / Chapter 3.2.1 --- RNA extraction --- p.67 / Chapter 3.2.2 --- RT-PCR synthesis of D. nobile lectin cDNA --- p.68 / Chapter 3.2.3 --- RACE of D. nobile agglutinin gene --- p.69 / Chapter 3.2.4 --- Generation of DNL full-length lectin gene sequence --- p.70 / Chapter 3.2.5 --- Cloning and sequencing of PCR product --- p.71 / Chapter 3.2.6 --- Data analyses --- p.72 / Chapter 3.2.7 --- Synthesis of single-stranded DIG-labeled DNA probe --- p.74 / Chapter 3.2.8 --- Northern blot analysis --- p.74 / Chapter 3.2.9 --- Genomic DNA extraction --- p.75 / Chapter 3.2.10 --- Southern blot analysis --- p.76 / Chapter 3.2.11 --- Expression of DNL in E. coli --- p.76 / Chapter 3.2.12 --- Western blot analysis --- p.78 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Isolation and characterization of DNL gene --- p.80 / Chapter 3.3.2 --- Sequence analysis of DNL --- p.89 / Chapter 3.2.3 --- Secondary and tertiary structure --- p.96 / Chapter 3.2.4 --- Southern blot analysis --- p.99 / Chapter 3.2.5 --- Northern blot analysis --- p.99 / Chapter 3.2.6 --- Expression of fusion protein in E.coli --- p.102 / Chapter 3.4 --- Discussion --- p.104 / Chapter Chapter Four --- General Discussion --- p.110 / Chapter 4.1 --- General discussion --- p.110 / Chapter 4.2 --- Isolation and Characterization of monocot mannose-binding lectin of D. nobile --- p.111 / Chapter 4.3 --- Molecular cloning of monocot mannose-binding lectin of D. nobile --- p.115 / Chapter 4.4 --- Further investigations --- p.122 / Chapter Chapter Five --- Conclusion --- p.124 / References: --- p.125 / Appendix --- p.133

by Chu Ka-yi. / Thesis submitted in: October 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 160-174). / Abstracts in English and Chinese. / ABSTRACT (English) --- p.i / ABSTRACT (Chinese) --- p.iii / ACKNOWLEDGMENT --- p.v / LIST OF CONTENTS --- p.vi / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xii / ABBREVIATIONS --- p.xiv / TERMS --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Taxonomy of Acinetobacter - historical and current --- p.1 / Chapter 1.2 --- Ecology and clinical significance of Acinetobacter --- p.5 / Chapter 1.3 --- General identification and typing methods for Acinetobacter species / Chapter 1.3.1 --- Identification at species level --- p.9 / Chapter 1.3.2 --- Identification at strain level --- p.11 / Chapter 1.4 --- Methods used in this study for characterization of Acinetobacter species / Chapter 1.4.1 --- Amplified ribosomal DNA restriction analysis (ARDRA) --- p.14 / Chapter 1.4.2 --- tDNA spacer fingerprinting (tDNA) --- p.15 / Chapter 1.4.3 --- Fluorescent amplified fragment length polymorphism (FAFLP) --- p.16 / Chapter 1.4.4 --- Phenotypic methods including carbon utilization tests --- p.20 / Chapter 1.5 --- Objectives --- p.25 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.27 / Chapter 2.1 --- Bacterial strains and isolates --- p.27 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Antimicrobial agents and chemicals --- p.30 / Chapter 2.2.2 --- "Carbohydrates, enzymes and other materials" --- p.32 / Chapter 2.2.3 --- Commercial media and media prepared manually --- p.33 / Chapter 2.2.4 --- "Buffers, solutions and list of instruments" --- p.35 / Chapter 2.3 --- General Bacteriological Techniques / Chapter 2.3.1 --- Isolation of acinetobacters --- p.38 / Chapter 2.3.2 --- Routine bacteriological identification --- p.39 / Chapter 2.4 --- General Molecular Biology Techniques / Chapter 2.4.1 --- DNA isolation --- p.40 / Chapter 2.4.2 --- Transformation --- p.41 / Chapter 2.4.3 --- Agarose gel electrophoresis --- p.43 / Chapter 2.5 --- Genospeciation of acinetobacters by Amplified Ribosomal Restriction DNA Analysis (ARDRA) --- p.44 / Chapter 2.6 --- Characterization of ARDRA unclassifiable acinetobacters (AUA) by Phenotypic methods / Chapter 2.6.1 --- Temperature tolerance tests --- p.47 / Chapter 2.6.2 --- Carbon utilization tests --- p.47 / Chapter 2.6.3 --- Gelatin and hemolysis tests --- p.48 / Chapter 2.6.4 --- Minimum Inhibitory Concentration (MIC) --- p.49 / Chapter 2.7 --- Characterization of AUA by tDNA spacer fingerprinting (tDNA) method --- p.51 / Chapter 2.8 --- Characterization of AUA by Fluorescent Amplified Fragment Length Polymorphism analysis (FAFLP) --- p.55 / Chapter 2.9 --- Relatedness study of isolates within the same AUA group by Enterobacterial Repetitive Intergenic Consensus (ERIC) typing method --- p.58 / Chapter CHAPTER 3 --- COLLECTION OF UNCLASSIFIABLE ACINETOBACTERS by ARDRA (AUA) METHOD --- p.59 / Chapter 3.1 --- Results / Chapter 3.1.1 --- Isolation and genospeciation of acinetobacters from hospital environments and raw food --- p.59 / Chapter 3.1.2 --- Collection of ARDRA unclassifiable acinetobacters (AUA) --- p.63 / Chapter 3.2 --- Discussion / Chapter 3.2.1 --- Limitations and merits of ARDRA method --- p.68 / Chapter 3.2.2 --- Potential significance of the representative AUA groups --- p.71 / Chapter CHAPTER 4 --- CHARACTERIZATION OF ARDRA UNCLASSIFIABLE ACINETOBACTERS (AUA) BY tDNA SPACER (tDNA) FINGERPRINTING METHOD --- p.72 / Chapter 4.1 --- Results / Chapter 4.1.1 --- Assessment of reproducibility --- p.72 / Chapter 4.1.2 --- Construction of the database with the reference strains --- p.75 / Chapter 4.1.3 --- Characterization of the representative AUA groups --- p.78 / Chapter 4.2 --- Discussion / Chapter 4.2.1 --- Evaluation of the reproducibility and discriminatory power --- p.89 / Chapter 4.2.2 --- Possible genospeciation of the representative AUA groups --- p.92 / Chapter 4.2.3 --- Limitations and merits --- p.96 / Chapter CHAPTER 5 --- CHARACTERIZATION OF ARDRA UNCLASSIFIABLE ACINETOBACTERS (AUA) BY FLUORESCENT AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (FAFLP) METHOD --- p.98 / Chapter 5.1 --- Results / Chapter 5.1.1 --- Assessment of robustness and reproducibility --- p.98 / Chapter 5.1.2 --- Construction of the database with the reference strains --- p.104 / Chapter 5.1.2 --- Characterization of the representative AUA groups --- p.108 / Chapter 5.2 --- Discussion / Chapter 5.2.1 --- "Evaluation of robustness, reproducibility and discriminatory power" --- p.116 / Chapter 5.2.2 --- Possible genospeciation of the representative AUA groups --- p.120 / Chapter 5.2.3 --- Merits and limitations --- p.122 / Chapter CHAPTER 6 --- CHARACTERIZATION OF ARDRA UNCLASSIFABLE ACINETOBACTERS (AUA) BY PHENOTYPIC METHODS --- p.125 / Chapter 6.1 --- Results Characterization of the representative AUA groups --- p.125 / Chapter 6.2 --- Discussion / Chapter 6.2.1 --- Possible genospeciation of the representative AUA groups --- p.134 / Chapter 6.2.2 --- Limitations in classification of Acinetobacter species at genomic species level --- p.135 / Chapter CHAPTER 7 --- RELATEDNESS OF ISOLATES WITHIN THE SAME AUA GROUPS --- p.139 / Chapter 7.1 --- Results Typing results of the studied AUA groups by ERIC method --- p.139 / Chapter 7.2 --- Discussion Relatedness of the isolates within the same AUA group --- p.146 / Chapter CHAPTER 8 --- GENERAL DISCUSSION --- p.148 / Chapter 8.1 --- Possible genospeciation of the representative AUA groups --- p.150 / Chapter 8.2 --- "Comparison of ARDRA, tDNA fingerprinting, FAFLP and phenotypic methods" --- p.154 / Chapter 8.3 --- Conclusion and significance of the AUA groups studied --- p.158 / Chapter 8.4 --- Future work --- p.159 / REFERENCES --- p.160 / APPENDIX --- p.176

Cheung, Hang Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 138-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction of Lectins --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Definition and History of Lectins --- p.1 / Chapter 1.1.2 --- More than Just Carbohydrate Binding --- p.2 / Chapter 1.1.3 --- Classification of Lectins --- p.3 / Chapter 1.2 --- Plant Lectins --- p.4 / Chapter 1.2.1 --- History of Plant Lectins --- p.4 / Chapter 1.2.2 --- Occurrence of Plant Lectins --- p.5 / Chapter 1.3 --- Physiological Roles of Plant Lectins --- p.6 / Chapter 1.3.1 --- Lectins as Storage Proteins --- p.6 / Chapter 1.3.2 --- Lectins as Defense Proteins --- p.7 / Chapter 1.3.3 --- Lectins as mediator in symbiosis with bacteria --- p.8 / Chapter 1.4 --- Biological Activities of Plant Lectins --- p.9 / Chapter 1.4.1 --- Immunomodulatory Activity --- p.9 / Chapter 1.4.2 --- Lectins and Cancer --- p.10 / Chapter 1.4.3 --- A ntiviral A ctivity --- p.12 / Chapter 1.5 --- Lectins in Glycomic Study --- p.14 / Chapter 1.5.1 --- Background --- p.14 / Chapter 1.5.2 --- Glyco-catch method --- p.15 / Chapter 1.5.3 --- Lectin Blot Analysis --- p.16 / Chapter 1.6 --- Aim of current study --- p.17 / Chapter Chapter 2: --- Purification and Characterization of a Lectin from Musa acuminata --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Purification Scheme --- p.20 / Chapter 2.2.2 --- Assay of Hemagglutinating A ctivity --- p.21 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.22 / Chapter 2.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.22 / Chapter 2.2.5 --- Protein Concentration Determination --- p.22 / Chapter 2.2.6 --- N-terminal amino acid sequence analysis --- p.22 / Chapter 2.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.23 / Chapter 2.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.23 / Chapter 2.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.24 / Chapter 2.2.10 --- Assay of Nitric Oxide Production by Murine Peritoneal Macrophages --- p.25 / Chapter 2.2.11 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.25 / Chapter 2.2.12 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.26 / Chapter 2.2.13 --- RNA Extraction --- p.27 / Chapter 2.2.14 --- Reverse Transcription: First Strand cDNA Synthesis --- p.28 / Chapter 2.2.15 --- Polymerasae Chain Reaction (PCR) --- p.28 / Chapter 2.3 --- Results --- p.32 / Chapter 2.4 --- Discussion --- p.46 / Chapter Chapter 3: --- Purification and Characterization of a Lectin from Gymnocladus chinensis Baill. --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Material and Methods --- p.50 / Chapter 3.2.1 --- Purification Scheme --- p.50 / Chapter 3.2.2 --- Assay of Hemaggl utinating Activity --- p.51 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 3.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.51 / Chapter 3.2.5 --- Protein Concentration Determination --- p.51 / Chapter 3.2.6 --- N-terminal amino acid sequence analysis --- p.52 / Chapter 3.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.52 / Chapter 3.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.52 / Chapter 3.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.52 / Chapter 3.2.10 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.52 / Chapter 3.2.11 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.53 / Chapter 3.2.12 --- Assay of Anti-fungal Activity --- p.53 / Chapter 3.3 --- Results --- p.56 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4: --- Introduction to Protease Inhibitors --- p.70 / Chapter 4.1 --- General Introduction --- p.70 / Chapter 4.2 --- Serine Protease Inhibitors --- p.71 / Chapter 4.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.73 / Chapter 4.2.2 --- Bowman-Birk Type Serine Protease Inhibitors (BBI) --- p.74 / Chapter 4.2.3 --- Squash Type Serine Protease Inhibitors --- p.75 / Chapter 4.3 --- Roles of Pis in Plants --- p.76 / Chapter 4.3.1 --- Pis as a defense protein --- p.76 / Chapter 4.3.2 --- Pis in seed germination --- p.78 / Chapter 4.4 --- Applications of Protease Inhibitors --- p.79 / Chapter 4.4.1 --- Pis in Cancer Prevention --- p.79 / Chapter 4.4.2 --- Pis in Crop Protection --- p.81 / Chapter 4.5 --- Aim of Current Study --- p.83 / Chapter Chapter 5: --- Isolation and Characterization of a Trypsin Inhibitor from the seeds of Lens culinaris --- p.84 / Chapter 5.1 --- Introduction --- p.84 / Chapter 5.2 --- Materials and Methods --- p.86 / Chapter 5.2.1 --- Purification Scheme --- p.86 / Chapter 5.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.87 / Chapter 5.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.88 / Chapter 5.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.88 / Chapter 5.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.88 / Chapter 5.2.6 --- Protein Concentration Determination --- p.89 / Chapter 5.2.7 --- N-terminal amino acid sequence analysis --- p.89 / Chapter 5.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.89 / Chapter 5.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.90 / Chapter 5.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.90 / Chapter 5.2.11 --- Assay of Anti-fungal Activity --- p.90 / Chapter 5.3 --- Results --- p.93 / Chapter 5.4 --- Discussion --- p.103 / Chapter Chapter 6: --- Isolation and Characterization of trypsin inhibitors trom the seeds of Vigna mungo (L.) Hepper --- p.106 / Chapter 6.1 --- Introduction --- p.106 / Chapter 6.2 --- Materials and Methods --- p.107 / Chapter 6.2.1 --- Purification Scheme --- p.107 / Chapter 6.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.109 / Chapter 6.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.109 / Chapter 6.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.109 / Chapter 6.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.109 / Chapter 6.2.6 --- Protein Concentration Determination --- p.109 / Chapter 6.2.7 --- N-terminal amino acid sequence analysis --- p.110 / Chapter 6.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.110 / Chapter 6.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.110 / Chapter 6.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.110 / Chapter 6.2.11 --- Assay of Anti-fungal Activity --- p.110 / Chapter 6.3 --- Results --- p.113 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7: --- General Discussion --- p.135 / References --- p.138

submitted by Liu Dan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 66-80). / Abstract also in Chinese. / CONTENTS --- p.i / LIST OF TABLES --- p.iii / LIST OF FIGURES --- p.iv / LIST OF ABBREVIATIONS --- p.v / ACKNOWLEDGEMENTS --- p.vi / ABSTRACT --- p.vii / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- History --- p.1 / Chapter 1.2 --- Immunoglobulins --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Properties of immunoglobulins --- p.7 / Chapter 1.3 --- Monoclonal proteins and monoclonal gammopathies --- p.12 / Chapter 1.3.1 --- Monoclonal proteins --- p.12 / Chapter 1.3.2 --- Monoclonal gammopathies --- p.14 / Chapter 1.4 --- Laboratory investigation of monoclonal immunoglobulin --- p.17 / Chapter 1.4.1 --- The current procedure of investigation in laboratory --- p.17 / Chapter 1.4.2 --- Problems in identifying monoclonal immunolgobuin --- p.19 / Chapter 1.5 --- Comparison of different techniques --- p.20 / Chapter 1.5.1 --- Immunoelectrophoresis --- p.20 / Chapter 1.5.2 --- Immunofixation electrophoresis --- p.22 / Chapter 1.5.3 --- Isoelectric focusing and immunoisoelectric focusing --- p.24 / Chapter 1.6 --- Aim of the present study --- p.27 / Chapter 1.7 --- Design of experiment --- p.27 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- Study subjects --- p.30 / Chapter 2.2 --- Apparatus --- p.30 / Chapter 2.2 --- Apparatus --- p.30 / Chapter 2.3 --- Reagents and materials --- p.32 / Chapter 2.4 --- Preparation of gels --- p.35 / Chapter 2.5 --- Isoelectric focusing procedure --- p.36 / Chapter 2.6 --- Acid fixation and staining --- p.37 / Chapter 2.7 --- Technical factors affecting results --- p.38 / Chapter Chapter 3 --- RESULTS --- p.40 / Chapter 3.1 --- Interpretation of results in isoelectric focusing --- p.40 / Chapter 3.2 --- Affecting factors --- p.47 / Chapter 3.3 --- Comparison of the results between IEF and IFE --- p.53 / Chapter Chapter 4 --- DISCUSSION --- p.59 / Chapter Chapter 5 --- CONCLUSION --- p.65 / References --- p.66

Background: Accurate diagnostic methods for Clostridium difficile infection (CDI) are required for optimal patient management, appropriate implementation of infection control measures and surveillance. An assay that also provides rapid results, is easy to implement in a routine diagnostic lab and is cost-effective would be ideal. Laboratory testing for Clostridium difficile infection is rapidly evolving. Recently published literature has shown that immunoassays for toxin detection, whilst being cheap and easy to implement, lack sensitivity. Molecular diagnostics that are sensitive and provide rapid results are now available. However, the high cost of these assays is of concern. As reflected in the literature the optimal test or testing algorithm for Clostridium difficile infection diagnosis is not clear. Objectives: This study aimed to compare the performance of a real-time PCR assay and two immunoassays, and to establish the optimal testing strategy for Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). Methods: Using toxigenic culture as the gold standard, the Roche PCR assay for the detection of the tcdC gene, the Immuno Card Toxins A & B immunoassay and the C. Diff Quik Chek Complete immunoassay were evaluated as stand alone assays and as part of testing algorithms. Results: The sensitivity, specificity, positive predictive value and negative predictive value of the various assays and algorithms ranged from 38% to 81%, 98% to 100%, 92% to 100% and 85% to 95%, respectively. The charge per sample tested varied widely depending on the assay and algorithm used. The maximum turnaround time ranged between four and twenty four hours. Conclusion: The algorithm combining glutamate dehydrogenase and toxin immunoassay testing of all samples followed by PCR testing of only a subset of samples, performed the best, providing accurate results rapidly and cost-effectively.

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