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