published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy

Jin, Dawei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / ABSTRACT (IN CHINESE) --- p.ii / ABSTRACT (IN ENGLISH) --- p.iv / ACKNOWLEDGEMENTS --- p.vii / CONTENTS --- p.ix / LIST OF TABLES --- p.vxiii / LIST OF FIGURES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- Biology of C. albicans --- p.2 / Chapter 1.1.1 --- Taxonomy --- p.2 / Chapter 1.1.2 --- Basic cell biology --- p.2 / Chapter 1.1.2.1 --- Cell cycle and phenotypic switch --- p.2 / Chapter 1.1.2.2 --- Cell wall --- p.3 / Chapter 1.1.3 --- "Morphological, culture and biochemical characteristics" --- p.4 / Chapter 1.1.4 --- Genomics --- p.5 / Chapter 1.1.5 --- Pathogenecity --- p.6 / Chapter 1.2 --- Catheter-related bloodstream infections (CRBSI) caused by C. albicans --- p.7 / Chapter 1.2.1 --- Intravenous catheter type --- p.7 / Chapter 1.2.2 --- Epidemiology of CRBSI caused by C. albicans --- p.8 / Chapter 1.2.3 --- Pathogenesis of intravascular catheter-related infections --- p.9 / Chapter 1.2.4 --- Diagnosis of catheter-related infections --- p.10 / Chapter 1.2.5 --- Prevention and control --- p.11 / Chapter 1.3 --- Mechanism of C. albicans adhesion to catheters --- p.12 / Chapter 1.3.1 --- The definition of microbial adhesion --- p.12 / Chapter 1.3.2 --- Relationship between microbial adhesion and biofilm formation --- p.12 / Chapter 1.4 --- Agglutinin-like sequence (ALS) gene family of C. albicans --- p.14 / Chapter 1.4.1 --- Members of ALS gene family --- p.14 / Chapter 1.4.2 --- Chromosomal location of ALS genes --- p.14 / Chapter 1.4.3 --- ALS gene organization --- p.14 / Chapter 1.4.3.1 --- Three-domain structure of ALS genes --- p.15 / Chapter 1.4.3.2 --- Characterization of ALS genes. --- p.15 / Chapter 1.4.4 --- ALS gene allelic variation --- p.17 / Chapter 1.5 --- Experimental models for catheter adhesion study of C. albicans --- p.17 / Chapter 1.5.1 --- "Static adhesion model for C, albicans" --- p.18 / Chapter 1.5.1.1 --- Advantage of static adhesion model --- p.19 / Chapter 1.5.1.2 --- Limitation of static adhesion model --- p.19 / Chapter 1.5.2 --- Dynamic adhesion model for C. albicans --- p.19 / Chapter 1.5.2.1 --- Advantage of dynamic adhesion model --- p.20 / Chapter 1.5.2.2 --- Limitation of dynamic adhesion model --- p.20 / Chapter 1.5.3 --- Quantification methods of adherent cells --- p.21 / Chapter 1.5.4 --- ALS gene expression study in the in vitro model --- p.22 / Chapter 1.6 --- Aim of study --- p.22 / Chapter CHAPTER II --- MATERIALS & METHODS --- p.24 / Chapter 2.1 --- Strains used in this study --- p.25 / Chapter 2.2 --- Design of an in vitro dynamic adhesion model for C. albicans --- p.26 / Chapter 2.2.1 --- Flask --- p.26 / Chapter 2.2.2 --- Peristaltic pump --- p.26 / Chapter 2.2.3 --- Glass tube and vascular catheters. --- p.27 / Chapter 2.2.4 --- Sterility check of in vitro dynamic adhesion model --- p.27 / Chapter 2.3 --- Construction of C. albicans growth curve --- p.27 / Chapter 2.4 --- Measurement of C. albicans adhesion to catheters --- p.29 / Chapter 2.5 --- Detection of C. albicans ALS genes --- p.30 / Chapter 2.5.1 --- DNA extraction of C. albicans --- p.30 / Chapter 2.5.2 --- ALS primers design --- p.31 / Chapter 2.5.3 --- PCR reaction --- p.32 / Chapter 2.5.4 --- Gel electrophoresis --- p.32 / Chapter 2.5.5 --- Purification of PCR products --- p.33 / Chapter 2.6 --- Construction of E. coli plasmid containing gene --- p.34 / Chapter 2.6.1 --- Ligation using the pGEM®-T Easy Vector --- p.34 / Chapter 2.6.2 --- Preparation of E. coli DH5a electro-competent cells --- p.35 / Chapter 2.6.3 --- Clean up of DNA ligation reaction for electro-transformation --- p.36 / Chapter 2.6.4 --- Electro-transformation of E. coli DH5a electro-competent cells --- p.37 / Chapter 2.6.5 --- Blue / white screening for positive transformation of E. coli DH5a. --- p.37 / Chapter 2.6.6 --- Extraction of plasmid containing ALS1 gene --- p.39 / Chapter 2.6.7 --- Plasmid validation by PCR and gel electrophoresis --- p.39 / Chapter 2.6.8 --- Serial dilution of plasmid solutions for ALS1 standard curve construction --- p.40 / Chapter 2.7 --- C. albicans ALS1 gene expression in dynamic adhesion model --- p.41 / Chapter 2.7.1 --- Design of real-time PCR primers specific for C. albicans ALS1 --- p.41 / Chapter 2.7.2 --- Validation of primers specificity --- p.42 / Chapter 2.7.3 --- RNA extraction of C. albicans cells adhered on catheters --- p.43 / Chapter 2.7.4 --- Complementary DNA (cDNA) synthesis --- p.45 / Chapter 2.7.5 --- Quantitative real-time RT-PCR --- p.46 / Chapter 2.8 --- Statistical analyses --- p.48 / Chapter CHAPTER III --- RESULTS --- p.49 / Chapter 3.1. --- Validation of the in vitro dynamic adhesion model for C. albicans --- p.50 / Chapter 3.2. --- C. albicans growth curve construction --- p.50 / Chapter 3.3. --- Measurement of C. albicans adhesion on catheters --- p.50 / Chapter 3.4. --- Detection of C. albicans SC5314 ALS genes --- p.52 / Chapter 3.5. --- Validation of E. coli plasmid containing ALS1 gene --- p.54 / Chapter 3.6. --- C. albicans ALS 1 gene expression in dynamic adhesion model --- p.54 / Chapter 3.6.1. --- Specificity validation of ALS1 real-time primers --- p.55 / Chapter 3.6.2. --- Quantitative real-time RT-PCR --- p.55 / Chapter CHAPTER IV --- DISCUSSION --- p.57 / Chapter 4.1 --- Experimental design of the in vitro dynamic adhesion model --- p.58 / Chapter 4.1.1 --- Advantages of this in vitro dynamic adhesion model --- p.58 / Chapter 4.1.2 --- Limitation of this in vitro dynamic adhesion model --- p.58 / Chapter 4.1.3 --- Catheter arrangement inside the glass tube --- p.60 / Chapter 4.1.4 --- Reproducibility of experiments in the model --- p.62 / Chapter 4.1.5 --- Identification of potential contamination in the model --- p.63 / Chapter 4.1.6 --- Advantages of removing method for C. albicans adherent cells --- p.64 / Chapter 4.1.7 --- Limitation of removing method for C. albicans adherent cells --- p.64 / Chapter 4.1.8 --- Limitation of statistical analysis --- p.66 / Chapter 4.1.9 --- Primers design --- p.67 / Chapter 4.1.9.1 --- Primers of C. albicans ALS gene detection --- p.67 / Chapter 4.1.9.2 --- Validation of ALS 1 real-time primers specificity --- p.69 / Chapter 4.2 --- C. albicans adhesion to catheters --- p.70 / Chapter 4.2.1 --- Theoretical explanation of C. albicans adhesion to different catheters --- p.71 / Chapter 4.3 --- C. albicans ALS gene expression --- p.74 / Chapter 4.3.1 --- Functions of Als proteins --- p.75 / Chapter 4.3.1.1 --- Adhesive functions --- p.75 / Chapter 4.3.1.2 --- Other functions in C. albicans pathogenesis --- p.75 / Chapter 4.3.2 --- Analysis of ALS1 gene expression pattern in the in vitro model --- p.76 / Chapter 4.4 --- Clinical application of our study --- p.78 / Chapter 4.5 --- Future study --- p.80 / Chapter 4.6 --- Conclusion --- p.81 / REFERENCES --- p.83