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The Biochemical Analysis of Mucus and Mucins in Respiratory Diseases with a Focus on Tuberculosis

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The Biochemical Analysis of Mucus and Mucins in Respiratory Diseases with a Focus on Tuberculosis THE BIOCHEMICAL ANALYSIS OF MUCUS AND MUCINS IN RESPIRATORY DISEASES WITH A FOCUS ON TUBERCULOSIS Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY University Department of Capeof Surgery Town UNIVERSITY OF CAPE TOWN February 2016 Supervisor: Prof. Anwar Suleman Mall The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Declaration I, Henrietta Refiloe Mofokeng, hereby declare that the work on which this dissertation/thesis is based is my original work (except where acknowledgements indicate otherwise) and that neither the whole work nor any part of it has been, is being, or is to be submitted for another degree in this or any other university. I hereby grant the University of Cape Town free licence to reproduce this thesis in whole or in part, for the purpose of research. Signature: Signature removed Date: 15 February 2016 ii Dedication I dedicate this thesis to my mom (Domina), my dad (Anthony), my sister (Antoinette) and my brother (Bashi). With much love! iii Acknowledgements First and foremost, I would like to thank my heavenly Father for the strength He has given me to complete the project. It really has been a long journey. I would like to express my sincere gratitude to the following people for their contribution towards the success of the project. I am grateful mainly to the National Research Foundation for financial assistance they provided for the project and the Health and Welfare SETA (HWSETA) for financial assistance to complete the project. I express my gratitude to my supervisor Prof. Anwar Suleman Mall, for accepting my request to do the research. I thank him for his constant guidance, wisdom, support and encouragement. I extend my gratitude to Prof. Delawir Kahn, for his support, encouragement and giving ideas around the project. According to his classification, we have passed gold stage to platinum. To my fellow lab family, I have no words. Thanks to Mrs. Zoe Lotz, Mrs. Marilyn Tyler and Mrs. Lizette Fick for the assistance with techniques around the lab. I am a better person because of you. Not only did I learn lab techniques but I discovered by passion for cooking and baking, and picked up some life lessons. To my fellow lab mates, Julia, Yolanda, Sam, Rufaro and Baxolele, you made each day in the lab worthwhile. One thing I do miss about the lab is the company and Friday tea sessions. I thank Groote Schuur hospital, DP Marais hospital, Gugulethu clinic, Seeisoville clinic and Thusanong clinic for granting access to their facilities for the collection of sample. To Prof. Keertan Dheda, for assistance with sample collection and advice. To the staff at respective facilities, thank you. A big thank you to the department of anaesthesia, especially Dr. Marcus Rademeyer, for the collection of tracheal aspirates from patients who underwent surgery. iv To Prof. Bongani Ndimba from the University of the Western Cape, thank you for the permission to use your lab for the proteomics part of project. To Dr. Salome Smit from the Central Analytical Facility at the Stellenbosch University, for assisting me in obtaining the identities of the proteins and assisting with the troubleshooting of O-glycan release. A very big thank you to Dr. Jeronimo (Jerry) Rodriguez for assisting with the amino acid analysis; and Prof. Dhiren Govender for assisting with the analysis of the work on immunohistochemistry. His expertise advice as a pathologist was high appreciated. A heartfelt thank you to Dr. Niclas Karlsson and Dr. Chunsheng Jin at Gothenburg University in Sweden, for conducting the O-glycan analysis. Without your assistance the O-glycan analysis would be at the optimising stages. To Dr. Jackson Marakalala and Dr. Claudius Marondedze, for proofreading the work. Last but not least, I’m grateful for the support I have received from my family throughout this long journey. To my friends for their continued support and encouragement. The time we lost shall be regained. v Table of Contents Declaration ............................................................................................................................. ii Dedication ............................................................................................................................. iii Acknowledgements ............................................................................................................... iv List of Figures......................................................................................................................... xi List of Tables ........................................................................................................................ xiii List of abbreviations ............................................................................................................ xiv Abstract .............................................................................................................................. xvii Chapter 1 : Introduction and literature review ......................................................................... 1 1.1 Background .................................................................................................................. 1 1.2 Lower respiratory tract mucus .................................................................................... 2 1.3 Mucus as a barrier ....................................................................................................... 3 1.4 Mucins ......................................................................................................................... 3 1.4.1 Mucin definition and nomenclature .................................................................... 3 1.4.2 Membrane bound mucins.................................................................................... 5 1.4.3 Secreted mucins ................................................................................................... 5 1.4.4 Mucin structure ................................................................................................... 5 1.4.5 The structure of mucin monomers ...................................................................... 6 1.4.6 Glycosylation of mucin subunits .......................................................................... 7 1.5 Mucin production in the respiratory tract .................................................................. 8 1.6 Respiratory mucus and mucins in disease ................................................................ 10 1.6.1 Asthma ............................................................................................................... 12 1.6.2 COPD .................................................................................................................. 13 1.6.3 Cystic Fibrosis ..................................................................................................... 14 1.6.4 Tuberculosis (TB) ................................................................................................ 15 1.7 Interaction of TB and the respiratory mucosa .......................................................... 17 1.8 Previous studies in our laboratory ............................................................................ 18 1.9 Rationale of the study ............................................................................................... 19 1.10 Aims and objectives .................................................................................................. 20 Chapter 2 : Materials and Methods ......................................................................................... 21 2.1 Materials and suppliers ............................................................................................. 21 vi 2.2 Research ethics and permissions .............................................................................. 23 2.3 Sample collection ...................................................................................................... 24 2.3.1 Healthy or uninfected sample collection (n=17) ............................................... 24 2.3.2 Bronchioalveolar lavage (BAL) collection (n=95) ............................................... 24 2.3.3 Sputum collection (n=56) ................................................................................... 24 2.4 Sample preparation ................................................................................................... 25 2.4.1 Mucus sample preparation ................................................................................ 25 2.4.2 Preparation of crude mucus .............................................................................. 25 2.4.3 Purification of mucins with caesium chloride .................................................... 25 2.4.4 Reduction and alkylation of samples ................................................................. 26 2.4.5 Dialysis of samples ............................................................................................. 26 2.4.6 Freeze drying ...................................................................................................... 26 2.5 Amino acid analysis ..................................................................................................
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