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2.4. Microarray Experiments https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] University of Glasgow Identification of gene markers for in vitro toxicity control testing of pertussis vaccines Noela Moncassin BSc Hons, MSc A thesis submitted to the University of Glasgow for the degree of Master of Science Division of Bacteriology and Immunology National Institute for Biological Standards and Control Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow May 2005 __ ProQuest Number: 10390729 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10390729 Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 ^GLASGOW UNIVERSITY LIBRARY: J l mon grand-père, iCdevert 'Moncassin (1924-2004), qui a gratifié fa ^cherche de ses dernières vobntés. Table of content Acknowledgments............................................................................................. 6 List of figures............................................................................................................................. 7 List of tables...................... .....9 List of abbreviations............................................................................................................... 10 Abstract........................................................................................ 13 1. INTRODUCTION......... ................................................................................ 14 1.1. Pathogenesis of whooping cough.......................................................................... 14 1.2. Epidemiology and burden of Pertussis disease....................................................15 1.3. Bordetella pertussis virulence factors ............................................... .16 1.3.1. Pertussis toxin (PT).............................................................................. 18 1.3.1.1. Structure........................................................ 18 1.3.1.2. Gene transcription, protein synthesis and excretion ..... 18 1.3.1.3. Mechanism of action......................................................... 18 1.3.1.4. Pathophysiological effects of PT............................... 19 1.3.1.5. Cell-binding properties of PT and its consequences............................. 19 1.3.2. Detoxified P T ........................................................................................... ..........21 1.3.3. Adenylate cyclase toxin (A CT).............................................. 23 1.3.4. Dermonecrotic toxin (D N T )..................... 24 1.3.5. Lipopolysaccharide (LP S )................... ..24 1.3.6. Tracheal cytotoxin (TCT)........................................................................ ...26 1.3.7. Adhesins............................................................... 26 1.3.7.1. Filamentous haemagglutinin (FH A )....................................... 26 1.3.7.2. Fimbriae (Fim ).................................... 27 1.3.7.3. Pertactin (PRN) and tracheal colonization factor (TCF)....................... 28 1.3.7.4. Other 6vg-regulated virulence determinants.................. 28 1.4. Immunology................................................................... .29 1.4.1. Innate immunity........................................................................................... 30 1.4.1.1. The surface barrier.............................................. 30 1.4.1.2. Inflammation................. ...30 1.4.1.3. Polymorphonuclear neutrophils ........................ 30 1.4.1.4. Macrophages ........................... 31 1.4.1.5. Natural killer cells.......................................................... 31 1.4.1.6. Complement.................................................. 31 1.4.1.7. Dendritic cells........................................................................ ...31 1.4.2. Adaptive or acquired immunity................. 32 1.4.2.1. Cell-mediated immunity..............................................................................32 1.4.2.2, Humoral immunity........................ 33 1.4.3. Immunity to B. pertussis............................................. 33 1.5. Vaccines ................................... 35 1.5.1. Whole-cell vaccines (W CV)...................................................................... 36 1.5.2. Acellular vaccines (ACV).................................................................. 37 1.5.3. Vaccine-induced immunities......................................................... ..38 1.5.4. Comparison of whole-cell and acellular vaccines.............. 39 1.5.5 The re-emergence of Pertussis.................................... 40 1.5.6. Potency tests.......................................................................................................41 1.5.7. Toxicity tests ........................ 42 1.5.7.1. Whole-cell vaccines... ........ 42 1.5.7.2. Pertussis toxin................. 43 1.5.7.2.1. Histannine sensitisation test (HIST).................. 43 1.5.7.2.2. Role of PT in the histamine sensitisation test................... .44 1.5.7.2.3. CHO cell assay........................... 44 1.5.7.3. LPS..................... 45 1.6. Cell lines for in vitro studies of the molecular mechanisms of whooping cough ........................ 46 1.6.1. Bronchial epithelial cell lines BEAS-2B and NL20........................................ 46 1.6.2. Human umbilical endothelial cell (HUVEC) ........................................47 1.7. Microarrays...................................................... 48 1.7.1. Advantages and limitations of microarrays .............. 48 1.7.2. Applications.......................................................................................................... 50 1.7.2.1. Study of host-pathogen interactions........................................................ 50 1.7.2.2. Pharmacogenetics and toxicogenomics ............................................. 52 1.8. Aims of the study ................. 54 2. MATERIALS AND M ETHO DS............................. 55 2.1. Biological reagents.............................................................. 55 2.2. Cell lines and culture conditions.................................................. 56 2.3. Cytotoxicity te s t.............................. 57 2.3.1. Alamarblue assay....................................................................... 57 2.3.2. MTT assay.................................................................. 59 2.4. Microarray experiments...................................................................................... 60 2.4.1. Cell treatment ............. 60 2.4.2. Total RNA extraction.............. 62 2.4.3. Microarray template ....................................... 62 2.4.4. Preparation of cDNA...................................................... 63 2.4.4.1. Annealing primers ......................... 63 2.4.4.2 Reverse transcription..................................................... ...63 2.4.4.3 Hybridization..................................................................................................63 2.4.5. Microarray analysis.................................................................................. 64 2.4.5.1. Template alignment ................................................... 64 2.4.5.2. Normalisation and calculation of statistical significance of internal control........................ 65 2.4.5.3. Filtering of the data..................................... ..65 2.4.5.4. Reproducibility between microarray experiments.................................. 66 2.4.5.5. Selection of candidate genes.....................................................................67 2.5. Semi-quantitative R T-PC R ............................................................................67 2.5.1. Cell treatments.................. 67 2.5.1.1. PT and detoxified PT treatments on HUVEC and NL20 cells..............67 2.5.1.2. Pertussis toxin and interleukin-1 beta (IL-1 beta) on HUVEC cells. ...67 2.5.1.3. Pertussis toxin and tumor necrosis factor alpha (TNF alpha) on HUVEC cells ..................... 68 2.5.2. Total RNA extraction..................................................................... 68 2.5.3. Semi-quantitative RT-PCR .....................................................................68 2.6. Cell migration assay .........
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