La virulence de Bordetella pertussis, agent de la coqueluche, est liée à un arsenal de facteurs de virulence dont l’expression est régulée par le système à deux composants BvgAS. BvgA est le régulateur de réponse et BvgS le capteur du système, qui possède 3 domaines putatifs de perception de signaux. Il s’agit de 2 domaines périplasmiques « Venus FlyTrap » (VFT), reliés par un segment transmembranaire à un domaine PAS (Per-ARNT-Sim) cytoplasmique qui fait la jonction avec l’histidine-kinase. Les signaux perçus par ces domaines capteurs sont inconnus, mais une température de 37°C est suffisante pour maintenir le système actif en laboratoire. Cette activité peut être modulée négativement par des composés chimiques, comme le MgSO4 ou le nicotinate, qui à concentrations suffisantes entraînent le passage de la bactérie en phase avirulente.Nous nous sommes intéressés aux domaines VFT de BvgS. Ces domaines, ubiquitaires, sont composés de 2 lobes reliés par une charnière délimitant une cavité qui permet la fixation d’un ligand spécifique stabilisant le VFT sous une forme fermée.Les domaines VFT de BvgS ont pu être cristallisés et s’organisent en un dimère entrelacé définissant de larges interfaces entre les 4 VFTs. Les VFT2 sont fermés sans ligand et les VFT1 ouverts, et la fermeture artificielle de ces domaines par des ponts disulfure a montré qu’il s’agit de la conformation active de BvgS. L’importance des interfaces entre les domaines VFT pour la fonction de BvgS a été démontrée par mutagenèse dirigée. Un signal positif proviendrait du périplasme pour être transmis à travers la membrane par les interfaces entre les VFT et intégré via un couplage fonctionnel en trans entre ces domaines et les hélices pré-membranaires, dites H19.Ces hélices se prolongeraient à travers la membrane et dans le cytoplasme jusqu’au domaine PAS. Les domaines PAS sont ubiquitaires, avec une structure fortement conservée en feuillet  à 5 brins recouvert d’hélices  délimitant une cavité. Ils sont impliqués dans diverses fonctions biologiques, selon leur capacité de liaison d’un ligand. Certains domaines PAS fonctionneraient sans ligand et pourraient servir d’adaptateurs ou d’amplificateurs de signal.Nous avons pu mettre en évidence la capacité de dimérisation de PASBvg, confirmant la nature dimérique du capteur BvgS. Des substitutions de résidus de la cavité de PASBvg indiqueraient que l’intégrité de la cavité de PASBvg est nécessaire au passage de signaux positifs et négatifs provenant du périplasme. La fixation de ligand dans la cavité n’a pu être démontrée mais n’est pas exclue. D’autre part, certains résidus sont nécessaires au couplage du domaine PAS avec ses hélices flanquantes pour la transmission de signal. La perte de ces interactions déstabilise significativement PASBvg et rend BvgS inactif.Un message positif proviendrait du périplasme et serait maintenu par le domaine PAS, dans une conformation rigide, permettant aussi la transmission des signaux modulateurs. / Virulence of Bordetella pertussis, the whooping cough agent, is due to a plethora of virulence factors which expression is regulated by the two-component system BvgAS. BvgA is a classical response regulator and BvgS the sensor. BvgS contains 3 putative sensor domains, 2 periplasmic Venus FlyTrap (VFT), linked through a transmembrane segment to a cytoplasmic PAS domain preceding the histidine-kinase. Signals perceived by those sensor domains are still unknown, but a 37°C temperature is sufficient to maintain the system active under laboratory conditions. This activity can be down-modulated by chemical compounds, such as MgSO4 or nicotinate, which at sufficient concentration allows the bacteria to switch to avirulent phase.We investigated the role of BvgS VFT domains. VFTs are ubiquitous domains composed of 2 lobes linked by a hinge hence forming a cleft where a specific ligand can bind and stabilize the VFT in its closed conformation.BvgS VFT domains were crystalized and form an intricate dimer defining large interfaces between the 4 VFTs. VFT2s are closed without a ligand and VFT1s are opened, artificial closure of these domains via a disulfide bond indicates that this is the active conformation of BvgS. The role of the interfaces was probed by site-directed mutagenesis. A positive signal might originate from the periplasm to be transmitted through the membrane by the interfaces and integrated by a functional coupling between the VFT2s and the helices preceding the membrane, H19.These helices should be continued through the membrane and the cytoplasm to the PAS domain. Pas domains are ubiquitous with a highly conserved structure, a 5 stranded sheet surrounded by  helices defining a cavity. Pas domains are involved in a wide variety of physiological processes, depending on their ability to bind a ligand. Some PAS might function without a ligand and could then be signal adaptors or amplifiers.We demonstrated PASBvg was dimeric, confirming the dimeric nature of BvgS. Cavity residues were substituted indicating that integrity of the cavity is necessary to maintain activity and modulation capacity coming from the periplasmic moiety. Ligand binding wasn’t demonstrated but couldn’t be excluded. Some residues are needed for the correct coupling of the PAS domain to its flanking helices and hence signal transmission. Loss of these connections generates a strong destabilization of PASBvg and turns BvgS inactive.A positive signal might come from the periplasmic moiety and shoul be maintaines by the PAS domain, which is in a rigid conformation also allowing the transmission of negative signals.

Chan Ming Lok. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-108). / Abstracts in English and Chinese. / Ackowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Chapter Chapter 1 --- Introduction and Objectives / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Geographical Distribution --- p.1 / Chapter 1.1.3 --- Sex and Age --- p.1 / Chapter 1.1.4 --- Etiology --- p.2 / Chapter 1.1.5 --- Molecular Basis of HCC --- p.3 / Chapter 1.1.6 --- Situation in China and Hong Kong --- p.4 / Chapter 1.2 --- The Hepatitis B Virus --- p.5 / Chapter 1.2.1 --- Morphology --- p.5 / Chapter 1.2.2 --- Structure of the HBV Genome --- p.6 / Chapter 1.2.3 --- Functional Domains of the HBV Genome --- p.9 / Chapter 1.2.4 --- Pathogenesis of HBV Infection --- p.11 / Chapter 1.3 --- HBx --- p.12 / Chapter 1.3.1 --- The HBV x Gene --- p.12 / Chapter 1.3.2 --- The HBX Protein --- p.13 / Chapter 1.3.3 --- "Preferential HBX Expression in Sera, Hepatitis, Cirrhosis and HCC" --- p.13 / Chapter 1.3.4 --- Cellular Localization of HBX --- p.14 / Chapter 1.3.5 --- Animal Studies --- p.15 / Chapter 1.3.6 --- Functional Studies on HBX --- p.15 / Chapter 1.3.7 --- Variations in the HBx Gene --- p.21 / Chapter 1.4 --- Objectives of this Study --- p.24 / Chapter Chapter 2 --- Methods and Materials Methods / Chapter 2.1 --- Paraffin Embedding of Patient Tissue Samples --- p.26 / Chapter 2.1.1 --- Tissue Processing --- p.26 / Chapter 2.1.2 --- Paraffin Embedding of Tissue Samples --- p.26 / Chapter 2.2 --- Sectioning of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3 --- Immunohistochemical Staining of Paraffin Embedded Tissue Sections --- p.26 / Chapter 2.3.1 --- Dewaxing of Paraffin-Embedded Tissue Sections --- p.26 / Chapter 2.3.2 --- Rehydration of Tissue Sections --- p.27 / Chapter 2.3.3 --- Antigen Retrieval --- p.27 / Chapter 2.3.4 --- Quenching of Endogenous Hydrogen Peroxidase --- p.27 / Chapter 2.3.5 --- Blocking of Endogenous Biotin and Non-Specific Protein Binding --- p.27 / Chapter 2.3.6 --- Antibody Incubation and Color Development --- p.27 / Chapter 2.3.7 --- Counterstaining and Coverslip Mounting --- p.28 / Chapter 2.3.8 --- Interpretation of Immunostaining Results --- p.28 / Chapter 2.4 --- DNA Extraction from HCC Tissues --- p.28 / Chapter 2.4.1 --- Sectioning of Frozen HCC Specimens --- p.28 / Chapter 2.4.2 --- Proteinase K Digestion and Phenol Chloroform Extraction --- p.29 / Chapter 2.4.3 --- Ethanol Precipitation and Re-suspension in Tris-EDTA (TE) Buffer --- p.29 / Chapter 2.5 --- Quantitation and Purity Check of Extracted DNA --- p.29 / Chapter 2.6 --- Quality Check for Extracted Genomic DNA --- p.30 / Chapter 2.6.1 --- Agarose Gel Electrophoresis --- p.30 / Chapter 2.6.2 --- Polymerase Chain Reaction (PCR) of the β-globin Gene --- p.30 / Chapter 2.6.3 --- Analysis of PCR Fragments by Agarose Gel Electrophoresis --- p.30 / Chapter 2.7 --- Polymerase Chain Reaction Amplification of HBs and HBx Genes of the Hepatitis B Virus --- p.31 / Chapter 2.8 --- Southern Blot of HBx PCR Fragments --- p.31 / Chapter 2.8.1 --- Immobilization of DNA onto a Positively Charged Nylon Membrane and Pre-hybridization --- p.31 / Chapter 2.8.2 --- Radio-labeling of an HBV Probe --- p.32 / Chapter 2.8.3 --- Hybridization of a 32P-labeled HBV Probe and Film Exposure --- p.32 / Chapter 2.9 --- Cloning of PCR Fragments into pGEM®-T Vector for Sequencing --- p.33 / Chapter 2.9.1 --- Gel Extraction and Purification --- p.33 / Chapter 2.9.2 --- Ligation --- p.33 / Chapter 2.10 --- Transformation of Competent DH5a cells --- p.34 / Chapter 2.10.1 --- Preparation of Competent DH5α Using Calcium Chloride --- p.34 / Chapter 2.10.2 --- Heat Shock of Competent DH5α Cells --- p.34 / Chapter 2.10.3 --- Plating of Transformed Cells onto LB Agar Plates --- p.34 / Chapter 2.10.4 --- Screening of Transformants for Inserts --- p.35 / Chapter 2.11 --- Miniprep of Plasmid DNA --- p.35 / Chapter 2.11.1 --- Inoculation of Bacterial Clones --- p.35 / Chapter 2.11.2 --- DNA Extraction by Alkaline Lysis and Phenol/Chloroform --- p.35 / Chapter 2.11.3 --- Ethanol Precipitation and Re-suspension in TE Buffer --- p.35 / Chapter 2.11.4 --- Confirmation of Positive Clones --- p.36 / Chapter 2.12 --- Sequencing of pGEM®-T Cloned HBx PCR Fragments --- p.36 / Chapter 2.13 --- Construction of the HBx-GFP Plasmid --- p.36 / Chapter 2.13.1 --- PCR Amplification of HBx Gene Inserts --- p.36 / Chapter 2.13.2 --- Confirmation of HBx Insert Sequence by DNA Sequencing --- p.37 / Chapter 2.13.3 --- Restriction Digest of HBx-pGEM®-T Plasmids to Obtain HBx Inserts --- p.37 / Chapter 2.13.4 --- Restriction Digest of pEGFP-Nl Cloning Vector for Cloning --- p.37 / Chapter 2.13.5 --- Ligation of HBx Inserts into the pEGFP Cloning Vector --- p.37 / Chapter 2.14 --- Large Scale Plasmid DNA Preparation --- p.38 / Chapter 2.15 --- Cell Culture --- p.39 / Chapter 2.16 --- Transfection using LipofectAminéёØ --- p.39 / Chapter 2.16.1 --- Seeding of Cells for Coverslip Growth --- p.39 / Chapter 2.16.2 --- Transfection using LipofecAminéёØ --- p.39 / Chapter 2.17 --- Cell Fixation and DAPI Staining Materials --- p.40 / Chapter 2.18 --- Chemicals --- p.41 / Chapter 2.19 --- Antibodies --- p.41 / Chapter 2.20 --- "Formalin-fixed, Paraffin Embedded Tissues of HCC Tissues from Xiamen" --- p.41 / Chapter 2.21 --- Frozen Liver Tissues --- p.41 / Chapter 2.22 --- PCR Reagents --- p.43 / Chapter 2.23 --- Primers --- p.43 / Chapter 2.24 --- Plasmid --- p.43 / Chapter 2.25 --- Enzymes --- p.43 / Chapter 2.26 --- Ligation Reagents --- p.43 / Chapter 2.27 --- Cloning Vectors --- p.45 / Chapter 2.28 --- Competent Cell --- p.45 / Chapter 2.29 --- Hela and HepG2 Cell Line --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Hepatitis B Virus Status of HCC Patients from Hong Kong and Xiamen --- p.46 / Chapter 3.2 --- Immunohistochemical Studies of the HBx Protein in Hong Kong and Xiamen HCC --- p.46 / Chapter 3.2.1 --- Cross Reaction of Anti-99 with Cytokeratin 18 (CK18) --- p.46 / Chapter 3.2.2 --- HBx Expression in HCC Patient Tissue Samples from Hong Kong --- p.50 / Chapter 3.2.3 --- HBxAg Staining in HCC Tissue Samples from Xiamen --- p.50 / Chapter 3.3 --- Agarose Gel Electrophoresis of DNA Extracted from Frozen Liver Tissues --- p.50 / Chapter 3.4 --- PCR Amplification of the β-globin Gene --- p.55 / Chapter 3.5 --- PCR Amplification of the HBs Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.6 --- PCR Amplification of the HBx Gene from Liver Samples of HCC Patients from Hong Kong --- p.55 / Chapter 3.7 --- Amplification of the HBx Gene from Serum Samples of Chronic Hepatitis B Virus from Hong Kong Using Nested PCR --- p.61 / Chapter 3.8 --- Southern Blot of HBx PCR Fragments --- p.61 / Chapter 3.9 --- Cloning and Sequencing of the HBx Gene in HCC and Chronic Hepatitis B Patient Samples from Hong Kong --- p.61 / Chapter 3.10 --- Expression Pattern of Wild-type HBx-GFP Fusion Protein in Transiently Transfected HeLa and HepG2 Cells --- p.73 / Chapter 3.11 --- Expression Patterns of HBx-GFP with and without Mutations at Codons 130 and 131 in HeLa and HepG2 Cell Line --- p.78 / Chapter 3.12 --- Growth Kinetics of HeLa Cells Transfected with GFP and Wild-type HBx-GFP with and without Mutations in Codons 130 and131 --- p.81 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- HBxAg Expression in Tumorous and Surrounding Non-tumorous Tissues --- p.83 / Chapter 4.2 --- "Detection of the HBx Gene in Sera, Non-tumorous and Tumorous Tissues" --- p.84 / Chapter 4.3 --- HBx Gene Mutations in Chronic Hepatitis and HCC --- p.85 / Chapter 4.3.1 --- Codon 127 (HBV nt 1752-1754) --- p.85 / Chapter 4.3.2 --- Codons 130 and 131 (HBV nt 1761-1766) --- p.86 / Chapter 4.3.3 --- Lack of Correlation between HBx Gene Mutations and Lack of HBxAg Expression --- p.87 / Chapter 4.4 --- Cellular Localization of HBxAg in Transiently Transfected Cells Lines --- p.88 / Chapter 4.5 --- Functional Difference Between Wild-type and Mutant HBX Protein --- p.89 / Chapter Chapter 5 --- Conclusions and Directions for Further Studies / Chapter 5.1 --- Conclusions --- p.91 / Chapter 5.2 --- Directions for Further Studies --- p.92 / References --- p.93 / Appendix / Chapter A1 --- Recipes of Reagents Used in this Study --- p.109 / Chapter A2 --- Schematic Setup of Downward Capillary Transfer of DNA --- p.112 / Chapter A3 --- Circle Map of the pGEM®-T Cloning Vector and Construct of the HBx-pGEM®-T Plasmid --- p.113 / Chapter A4 --- Circle Map of the pEGFP-Nl Cloning Vector and Construct of the HBx-GFP Plasmid --- p.114

by Woo Yiu Ho, Anthony. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 162-171). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of Contents --- p.iv / List of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Bacterial Adhesion and Aggregation --- p.1 / Chapter 1.1.1 --- Significance of Bacterial Adhesion Studies --- p.1 / Chapter 1.1.2 --- Definitions --- p.4 / Chapter 1.1.3 --- Colonization Process --- p.7 / Chapter 1.1.4 --- Specific and Nonspecific Interactions --- p.8 / Chapter 1.1.5 --- Models of Bacterial Adhesion and Aggregation Processes --- p.14 / Chapter 1.1.6 --- Experimental Systems in Adhesion Research --- p.16 / Chapter 1.1.7 --- Experimental Systems in Aggregation Research --- p.19 / Chapter 1.2 --- Pseudomonas aeruginosa --- p.21 / Chapter 1.2.1 --- General Description and Clinical Significance --- p.21 / Chapter 1.2.2 --- Adhesins of Pseudomonas aeruginosa --- p.22 / Chapter 1.2.3 --- "Alginate, Mucoidity, Biofilm Formation and Cystic Fibrosis" --- p.23 / Chapter 1.2.4 --- Lipopolysaccharides --- p.26 / Chapter 1.2.5 --- Pili --- p.29 / Chapter 1.2.6 --- Flagella --- p.30 / Chapter 1.2.7 --- Lectins --- p.31 / Chapter 1.2.8 --- Other Adhesins --- p.31 / Chapter 1.2.9 --- Rhamnolipids --- p.32 / Chapter 1.3 --- Current Study --- p.33 / Chapter 2 --- MATERIALS AND EQUIPMENT --- p.35 / Chapter 2.1 --- Bacterial Strain --- p.35 / Chapter 2.2 --- Solid Surfaces --- p.35 / Chapter 2.3 --- Chemicals --- p.36 / Chapter 2.4 --- Recipes --- p.38 / Chapter 2.5 --- Equipment --- p.38 / Chapter 3 --- METHODS --- p.40 / Chapter 3.1 --- Maintenance and Culturation --- p.40 / Chapter 3.1.1 --- Maintenance of Bacterial Strains --- p.40 / Chapter 3.1.2 --- Seed Culture Preparation --- p.40 / Chapter 3.1.3 --- Culturation in Defined Growth Media --- p.40 / Chapter 3.2 --- Bacterial Adhesion and Aggregation Assay Methods --- p.41 / Chapter 3.2.1 --- Bacterial Adhesion on Glass Assay --- p.41 / Chapter 3.2.2 --- Bacterial Adhesion on Plastic Assay --- p.44 / Chapter 3.2.3 --- Bacterial Adhesion under Shear Assay --- p.44 / Chapter 3.2.4 --- Bacterial Aggregation Examination by Adhesion on Glass Assay --- p.45 / Chapter 3.2.5 --- Bacterial Aggregation Examination by Top-agar Assay --- p.45 / Chapter 3.2.6 --- Bacterial Aggregation Examination by Epi-fluorescence Microscopy --- p.46 / Chapter 3.2.7 --- Bacterial Aggregation Screening Test --- p.46 / Chapter 3.3 --- Determination of the Effects of Various Factors on Adhesion and Aggregation --- p.47 / Chapter 3.3.1 --- Culturation Period --- p.47 / Chapter 3.3.2 --- Osmotic Shock during the Washing Procedure --- p.47 / Chapter 3.3.3 --- Growth Media --- p.48 / Chapter 3.3.4 --- Assay Conditions --- p.48 / Chapter 3.3.5 --- Cell Pretreatments --- p.48 / Chapter 3.4 --- Isolation of Aggregation-deficient Mutants --- p.49 / Chapter 3.5 --- Outer Membrane Protein Profiles --- p.50 / Chapter 3.5.1 --- Isolation of Outer Membrane Fraction --- p.50 / Chapter 3.5.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 3.6 --- Determination of the Mobility of the Bacteria on Surfaces --- p.52 / Chapter 3.6.1 --- Subsurface Twitching Assay --- p.52 / Chapter 3.6.2 --- Soft-agar Swarm Assay --- p.53 / Chapter 3.7 --- Detection of Alginate Production --- p.53 / Chapter 3.7.1 --- Extraction of Alginate from Spent Growth Medium --- p.53 / Chapter 3.7.2 --- Releasing Cell Surface-associated Alginate --- p.54 / Chapter 3.8 --- Other Assay Methods --- p.55 / Chapter 3.8.1 --- Protein Assay --- p.55 / Chapter 3.8.2 --- Carbohydrate Determination --- p.55 / Chapter 3.8.3 --- Alginate Determination --- p.55 / Chapter 4 --- RESULTS --- p.57 / Chapter 4.1 --- Standardization of the Assays for Bacterial Adhesion and Aggregation --- p.57 / Chapter 4.1.1 --- Effects of Cell Density and Exposure Time on the Number of Adhered Bacteria Detected in Bacterial Adhesion on Glass Assay --- p.57 / Chapter 4.1.2 --- Characterization of Bacterial Aggregation by Different Examination Methods --- p.62 / Chapter 4.1.3 --- Effects of Culturation Period on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.67 / Chapter 4.1.4 --- Effects of Osmotic Shock during Washing on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.70 / Chapter 4.1.5 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 as a Function of Time under the Standard Assay Condition --- p.71 / Chapter 4.1.6 --- Consistency of Bacterial Adhesion on Glass Assay --- p.74 / Chapter 4.2 --- Effects of Growth Media on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.77 / Chapter 4.3 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 in Different Assay Media --- p.77 / Chapter 4.3.1 --- Effects of Various Buffers on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.77 / Chapter 4.3.2 --- Effects of pH on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.78 / Chapter 4.3.3 --- Effects of Various Electrolytes on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.81 / Chapter 4.3.4 --- Concentration Effects of Monovalent and Divalent Cations on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.88 / Chapter 4.3.5 --- Concentration Effects of Phosphate Buffers on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.93 / Chapter 4.3.6 --- Concentration Effects of Ammonium Sulfate and Cyclohexylammonium Sulfate on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.96 / Chapter 4.3.7 --- Effects of Cation Chelation on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.99 / Chapter 4.3.8 --- Effects of Sugars on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.100 / Chapter 4.3.9 --- Effects of Amino Acids on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.101 / Chapter 4.4 --- Adhesion and Aggregation after Pretreatments of the Cells --- p.103 / Chapter 4.4.1 --- Effects of Protease Treatments on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.103 / Chapter 4.4.2 --- Effects of Externally Added Proteins on Adhesion and Aggregation of Pronase-treated Cells --- p.107 / Chapter 4.4.3 --- Effects of Acid or Base Treatments on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.108 / Chapter 4.4.4 --- Effects of Heat Treatment on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.108 / Chapter 4.4.5 --- Effects of Extensive Washing on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.110 / Chapter 4.5 --- Isolation and Growth Characteristics of Aggregation-deficient Mutants --- p.111 / Chapter 4.6 --- Comparisons of the Adhesion and Aggregation Characters of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.115 / Chapter 4.6.1 --- Under Standard Condition --- p.115 / Chapter 4.6.2 --- On Different Surfaces and in Different Electrolytes --- p.115 / Chapter 4.6.3 --- Under Shear --- p.118 / Chapter 4.6.4 --- Adhesion and Aggregation of Combined Suspensions of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.122 / Chapter 4.7 --- Characterization of the Cell Surface Properties of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.125 / Chapter 4.7.1 --- Outer Membrane Protein Profiles --- p.125 / Chapter 4.7.2 --- Pili-elicited Twitching Mobility --- p.125 / Chapter 4.7.3 --- Mobility Due to Flagella --- p.128 / Chapter 4.7.4 --- Production of Alginate --- p.128 / Chapter 5 --- DISCUSSIONS --- p.130 / Chapter 5.1 --- Choice of the Materials --- p.130 / Chapter 5.2 --- Development of the Assay Methods --- p.130 / Chapter 5.2.1 --- Development of the Procedures for Bacterial Adhesion Assays --- p.130 / Chapter 5.2.2 --- Development of the Assay Methods for Bacterial Aggregation --- p.132 / Chapter 5.2.3 --- Standardization of the Assays --- p.133 / Chapter 5.2.4 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 as a Function of Time under the Standard Assay Condition --- p.134 / Chapter 5.2.5 --- Consistency of Bacterial Adhesion on Glass Assay --- p.135 / Chapter 5.2.6 --- Limits of Bacterial Adhesion on Glass Assay --- p.135 / Chapter 5.3 --- Effects of Growth Media on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.135 / Chapter 5.4 --- Effects of Various Chemicals in the Assay Media on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.136 / Chapter 5.4.1 --- Effects of Electrolytes on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.137 / Chapter 5.4.2 --- Effects of Aggregation on Adhesion --- p.140 / Chapter 5.4.3 --- Effects of Cyclohexylammonium Sulfate and Ammonium Sulfate on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.141 / Chapter 5.4.4 --- Effects of Sugars and Amino Acids on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.143 / Chapter 5.5 --- Effects of Various Cell-surface Modifications on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.144 / Chapter 5.6 --- Isolation and Growth Characteristics of Aggregation-deficient Mutants --- p.146 / Chapter 5.7 --- Comparisons of the Adhesion and Aggregation Characters of Pseudonomas aeruginosa ATCC 10145 and Mutant 9 --- p.147 / Chapter 5.7.1 --- Adhesion and Aggregation of Pseudonomas aeruginosa ATCC 10145 and Mutant 9 on Different Surfaces In Different Electrolytes --- p.147 / Chapter 5.7.2 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 Under Shear --- p.147 / Chapter 5.7.3 --- Adhesion and Aggregation of Combined Suspensions of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.148 / Chapter 5.8 --- Characterization of the Cell Surface Properties of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.148 / Chapter 5.9 --- General Discussions --- p.151 / Chapter 6 --- APPENDIX --- p.154 / Chapter 6.1 --- Visual Examination of Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 on Glass --- p.154 / Chapter 6.2 --- Fractal Analysis of Bacterial Aggregates --- p.154 / Chapter 7 --- REFERENCES --- p.162

Influenza A virus (IAV) circulates in waterfowl, causing mostly asymptomatic infections. IAV can undergo host adaptation and evolve to cause significant disease and mortality in domestic poultry and mammals, applying an enormous socio-economic burden on society. Sporadically, IAV causes global pandemics in man due to its zoonotic nature, and this can result in millions of deaths worldwide during a single outbreak. Host adaptation of IAV is an incompletely understood phenomenon, but is known to involve both host and viral determinants. It is essential to improve the understanding of the factors governing host range and pathogenicity of avian IAV, especially given the absence of a universal influenza vaccine and a limited weaponry of effective antiviral compounds. This study set out to improve the understanding of host adaptation of avian IAV, focussing on segment 8 (NS segment) of the virus genome. The NS segment of non-chiropteran IAV circulates as two phylogenetically distinct clades – the ‘A-’ and ‘B-alleles’. The A-allele is found in avian and mammalian viruses, but the B-allele is considered to be almost exclusively avian. This might result from one or both of the major NS gene products (NS1 and NEP) being non-functional in mammalian host cells, or from an inability of segment 8 RNA to package into mammalian-adapted strains. To investigate this, the NS segments from a panel of avian A- and B-allele strains were introduced into human H1N1 and H3N2 viruses by reverse genetics. A- and B-allele reassortant viruses replicated equally well in a variety of mammalian cell types in vitro. Surprisingly, the consensus B-allele segment 8 out-competed an A-allele counterpart when reassortant H1N1 viruses were co-infected, with the parental WT segment 8 being most fit in this system. A- and B-allele NS1 proteins were equally efficient at blocking the mammalian IFN response both in the context of viral infection and in transfection-based reporter assays. Consensus A- and B-allele H1N1 viruses also caused disease in mice and replicated to high virus titre in the lung. Interestingly, the B-allele virus induced more weight-loss than the A-allele, although the parental WT virus was most pathogenic in vivo. To re-address the hypothesis that B-allele NS genes really are avian-restricted, the relative rates of independent Aves to Mammalia incursion events of A- and B-allele lineage IAV strains was estimated and compared using phylogenetic analyses of all publically available segment 8 sequences. 32 A-allele introduction events were estimated compared to 6 B-allele incursions, however the total number of avian Aallele sequences outnumbered B-allele sequences by over 3.5 to 1, and the relative rates of introduction were not significantly different across the two lineages suggesting no bias against avian B-allele NS segments entering mammalian hosts in nature. Therefore, this study provides evidence that avian B-allele NS genes are not attenuating in mammalian hosts and are able to cause severe disease. Thus, this lineage of IAV genes, previously assumed to be avian-restricted, should be considered when assessing zoonotic potential and pandemic risk of circulating avian IAVs.

by Wu Che-yuen Justin. / "September 2000 (amendment)." / Thesis (M.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 237-267). / 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.

by Zhong Sheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 186-203). / 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.

Bibliography: leaves 159-192. / 11, 192, [58] leaves, [16] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Discusses the evidence implicating papillomaviruses in the development of cervical intraepithelial neoplasia (CIN) and carcinomas and documents derivation of clones and validation of experimental procedures, epidemiological studies of ano-genital neoplasia, HPV transcription mapping in genital neoplastic lesions and cell lines, and mechanisms of tumor development. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1992

<p><italic>Cryptococcus neoformans</italic> pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of <italic>C. neoformans</italic> yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.</p> / Dissertation

Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.

Equine influenza virus causes a mild, self-limiting upper respiratory disease in its natural host. In stark contrast, equine influenza viruses of the H7N7 subtype produce lethal infection in BALB/c mice. This dissertation explored the mechanism underlying the differential pathogenicity of the equine H7N7 influenza virus observed in horses and BALB/c mice. Initially, a comparative study of the pathogenesis was conducted in BALB/c mice inoculated intranasally with a representative isolate of either H7N7 or H3N8 subtype equine influenza virus. All H3N8 virus-infected mice survived the infection whereas 100% mortality was documented for the mice receiving the H7N7 virus by day 8 post infection. Both viruses replicated to a similar degree in the lungs at the early stages of infection. However, after day 2 post infection until the death of the mice, the pulmonary viral loads of the H7N7 group were significantly higher than those of the control, whereas the H3N8 virus was eventually eradicated from the mice at day 7 p.i. Correspondingly, a vigorous pro-inflammatory cytokine response in the lung was induced by the H7N7 virus but not the H3N8 virus, which reflected a desperate attempt by the host immune responses to restrain the overwhelming infection. The H7N7 virus was poorly sensitive to the innate immune containment, resulting in a significant higher cumulative mortality rate than that of the control virus in chicken embryos aged 9 days and older. On the contrary, in horses, replication of the paired viruses was completely cleared by the host immune responses at day 7 p.i. and the infections produced an acute yet non-lethal illness, albeit the H3N8 virus induced generally more pronounced clinical manifestations than the H7N7 virus. The clinical severity correlated to the difference in cytokine-inducing capacity between the two viruses in horses, as evidenced by the finding that the H3N8 virus triggered significantly higher levels of gene transcription of multiple key inflammatory cytokines in the circulation than those seen for the H7N7 virus. In addition, equine peripheral monocyte-derived macrophages were found to be a target of equine influenza virus and can support the productive replication of the virus in vitro.