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Function of ALS genes of Candida albicans in catheter adhesion.

by Chan Ping Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 108-118). / Abstracts in English and Chinese. / Abstract (in Chinese) --- p.ii / Abstract (in English) --- p.iv / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Appendices --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of catheter associated infections --- p.1 / Chapter 1.1.1 --- Catheter associated infections --- p.1 / Chapter 1.1.2 --- Risk and mortality of CAI --- p.2 / Chapter 1.1.3 --- Etiology of CAI --- p.3 / Chapter 1.1.3.1 --- Venous catheters --- p.4 / Chapter 1.1.3.2 --- Urinary catheters --- p.4 / Chapter 1.2 --- Pathogenesis of CAI --- p.5 / Chapter 1.2.1 --- Central venous catheters (CVC) --- p.6 / Chapter 1.2.2 --- Urinary catheters --- p.7 / Chapter 1.3 --- Adhesion mechanisms --- p.7 / Chapter 1.3.1 --- Definition of adhesion --- p.7 / Chapter 1.3.2 --- Adhesion mechanism --- p.8 / Chapter 1.3.2.1 --- The phase one --- p.8 / Chapter 1.3.2.2 --- The phase two --- p.10 / Chapter 1.4 --- Catheters --- p.10 / Chapter 1.5 --- Biology of Candida albicans --- p.11 / Chapter 1.5.1 --- Taxonomy of Candida albicans --- p.11 / Chapter 1.5.2 --- Morphology --- p.12 / Chapter 1.5.3 --- Genome --- p.13 / Chapter 1.5.4 --- Biology of Candida albicans cell wall --- p.14 / Chapter 1.5.4.1 --- Constituting molecules of Candida albicans cell wall --- p.14 / Chapter 1.5.4.2 --- Organization of Candida albicans cell wall --- p.15 / Chapter 1.6 --- Agglutinin like sequence gene family --- p.16 / Chapter 1.6.1 --- Gene structure of agglutinin like sequence genes --- p.16 / Chapter 1.6.2 --- Sequence similarity --- p.17 / Chapter 1.6.3 --- Sequence variability --- p.18 / Chapter 1.6.4 --- Expression of ALS genes --- p.19 / Chapter 1.6.5 --- The Als proteins --- p.20 / Chapter 1.6.6 --- Functions of Als proteins --- p.21 / Chapter 1.7 --- Adhesion assay --- p.23 / Chapter 1.7.1 --- Adhesion model --- p.24 / Chapter 1.7.2 --- Factors affecting static adhesion model --- p.25 / Chapter 1.7.3 --- Quantitation methods of adherent cells --- p.27 / Chapter 1.7.3.1 --- Sonication --- p.27 / Chapter 1.7.3.2 --- Staining methods --- p.28 / Chapter 1.7.3.3 --- ATP bioluminescence --- p.28 / Chapter 1.8 --- Research model --- p.29 / Chapter Chapter 2 --- Aim of study --- p.31 / Chapter Chapter 3 --- Materials and Methods --- p.32 / Chapter 3.1 --- Preparation of bacteriological reagents --- p.32 / Chapter 3.2 --- Confirmation of identity of Candida albicans and of Saccharomyces cerevisiae --- p.33 / Chapter 3.3 --- Cell culture of fibroblasts --- p.36 / Chapter 3.3.1 --- Preparation of cell culture reagents --- p.36 / Chapter 3.3.2 --- Recovery of freezing fibroblasts --- p.37 / Chapter 3.3.3 --- Establishment of cell line --- p.37 / Chapter 3.4 --- Preliminary study of adherence of Candida albicans to fibroblasts and to catheters --- p.38 / Chapter 3.4.1 --- Adherence to fibroblasts --- p.38 / Chapter 3.4.1.1 --- Preparation of fibroblasts --- p.38 / Chapter 3.4.1.2 --- Preparation of culture of Candida albicans and of Saccharomyces cerevisiae --- p.39 / Chapter 3.4.1.3 --- Adhesion assay --- p.41 / Chapter 3.4.2 --- Adherence to catheters --- p.42 / Chapter 3.4.2.1 --- Preparation of catheters --- p.42 / Chapter 3.4.2.2 --- Adhesion assay --- p.42 / Chapter 3.5 --- "Confirmation of expression of ALS1, ALS5 smaller allele, and ALS6 of Candida albicans in YPD broth" --- p.44 / Chapter 3.5.1 --- RNA extraction of Candida albicans --- p.45 / Chapter 3.5.2 --- "RT-PCR of ALS1, ALS5 smaller allele, and ALS6" --- p.46 / Chapter 3.5.2.1 --- Primers --- p.46 / Chapter 3.5.2.2 --- RT-PCR --- p.47 / Chapter 3.6 --- Establishment of quantitation system of adhesion assay --- p.49 / Chapter 3.6.1 --- Absorbance measurement of Candida albicans stained with safranin --- p.49 / Chapter 3.6.1.1 --- Preparation of Candida albicans culture --- p.49 / Chapter 3.6.1.2 --- Staining of Candida albicans --- p.50 / Chapter 3.6.1.3 --- Viable count of Candida albicans adhered on the 6-well plate --- p.51 / Chapter 3.6.2 --- ATP bioluminescence --- p.52 / Chapter 3.7 --- Effect of inoculum size on adhesion to catheters --- p.53 / Chapter 3.7.1 --- Preparation of adhesion chambers --- p.53 / Chapter 3.7.2 --- Preparation of catheters --- p.54 / Chapter 3.7.3 --- Preparation of Candida albicans culture --- p.54 / Chapter 3.7.4 --- Adhesion assay --- p.55 / Chapter 3.8 --- "Transformation of Saccharomyces cerevisiae with ALS1, ALS5 smaller allele, and ALS6" --- p.57 / Chapter 3.8.1 --- DNA extraction of Candida albicans --- p.58 / Chapter 3.8.2 --- "PCR of ALS1, ALS5 smaller allele, and ALS6" --- p.59 / Chapter 3.8.3 --- Gel extraction --- p.60 / Chapter 3.8.4 --- Restriction digestion of PCR products of ALS genes and cloning plasmids --- p.61 / Chapter 3.8.5 --- "Ligation of ALS1, ALS5 smaller allele, ALS6 with pYES6CT cloning plasmids" --- p.62 / Chapter 3.8.6 --- Transformation of ligated plasmid into Escherichia coli --- p.63 / Chapter 3.8.7 --- Miniprep of plasmids --- p.64 / Chapter 3.8.8 --- DNA sequencing --- p.65 / Chapter 3.8.9 --- Transformation of Saccharomyces cerevisiae --- p.66 / Chapter 3.8.10 --- "Detection of Alsl,Als5, and Als6 protiens expression" --- p.68 / Chapter 3.8.10.1 --- Preparation of cultures in SC synthetic medium --- p.68 / Chapter 3.8.10.2 --- Protein extraction --- p.69 / Chapter 3.8.10.3 --- Dot blot of cell wall lysates --- p.69 / Chapter 3.9 --- Adhesion of transformed Saccharomyces cerevisiae to fibroblasts --- p.71 / Chapter 3.9.1 --- Preparation of fibroblasts and of Saccharomyces cerevisiae cultures --- p.71 / Chapter 3.9.2 --- Adhesion assay --- p.72 / Chapter 3.10 --- "Adhesion of transformed Saccharomyces cerevisiae to FEP, polyurethane, and silicone catheters" --- p.72 / Chapter 3.10.1 --- "Preparation of catheters, adhesion chambers and transformed Saccharomyces cerevisiae cultures" --- p.73 / Chapter 3.10.2 --- Adhesion to catheter fragments --- p.73 / Chapter 3.11 --- Statistical analysis --- p.74 / Chapter Chapter 4 --- Results --- p.75 / Chapter 4.1 --- Confirmation of identity of Candida albicans and of Saccharomyces cerevisiae --- p.75 / Chapter 4.1.1 --- Candida albicans --- p.75 / Chapter 4.1.2 --- Saccharomyces cerevisiae --- p.75 / Chapter 4.2 --- Cell culture of fibroblasts --- p.76 / Chapter 4.3 --- "Preliminary studies of adherence of Candida albicans to fibroblasts and to FEP, polyurethane, and silicone catheters" --- p.76 / Chapter 4.3.1 --- Adherence to fibroblasts --- p.76 / Chapter 4.3.2 --- Adherence to catheters --- p.77 / Chapter 4.4 --- "Confirmation of expression of ALSl, ALS5 smaller allele, and ALS6 of Candida albicans in YPD broth" --- p.78 / Chapter 4.5 --- Establishment of quantitation system of adhesion assay --- p.79 / Chapter 4.5.1 --- Absorbance measurement of Candida albicans stained with safranin --- p.79 / Chapter 4.5.2 --- ATP bioluminescence --- p.79 / Chapter 4.6 --- Effect of inoculum size on adhesion to catheters --- p.80 / Chapter 4.7 --- "Transformation of Saccharomyces cerevisiae with ALS1, ALS5 smaller allele, and ALS6" --- p.81 / Chapter 4.7.1 --- "PCR of ALSl, ALS5 smaller allele, and ALS6" --- p.81 / Chapter 4.7.2 --- Ligation of PCR products with pYES6CT plasmids --- p.82 / Chapter 4.7.3 --- "DNA sequencing results of ALS1, ALS5 smaller allele, and ALS6 ligated plasmids" --- p.83 / Chapter 4.7.4 --- "Detection of Alsl, Als5, and Als6 proteins expression" --- p.84 / Chapter 4.8 --- Adhesion of transformed Saccharomyces cerevisiae to fibroblasts --- p.84 / Chapter 4.9 --- "Adhesion of transformed Saccharomyces cerevisiae to FEP, polyurethane and silicone catheters" --- p.85 / Chapter Chapter 5 --- Discussion --- p.89 / Chapter 5.1 --- Limitations of static adhesion assay model --- p.89 / Chapter 5.2 --- Quantitation System --- p.90 / Chapter 5.2.1 --- Staining method --- p.90 / Chapter 5.2.2 --- ATP bioluminescence assay --- p.91 / Chapter 5.3 --- "Preliminary studies of adherence of Candida albicans to fibroblasts and to FEP, polyurethane, and silicone catheters" --- p.93 / Chapter 5.4 --- Effect of inoculum size on adhesion to catheters --- p.94 / Chapter 5.5 --- Selection of ALS genes --- p.96 / Chapter 5.6 --- Adhesion assay of transformed Saccharomyces cerevisiae to fibroblasts --- p.97 / Chapter 5.7 --- Adhesion assay of transformed Saccharomyces cerevisiae to catheters --- p.99 / Chapter 5.8 --- Alternative research model --- p.101 / Chapter 5.9 --- Implications and future work --- p.102 / Chapter Chapter 6 --- Conclusion --- p.107 / References --- p.108 / Tables --- p.119 / Figures --- p.123 / Appendices --- p.136

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325626
Date January 2006
ContributorsChan, Ping Lung., Chinese University of Hong Kong Graduate School. Division of Microbiology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 150 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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