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Investigating the Role of Amino Acid Transporter SLC6A14 As a Modifier of Pseudomonas Aeruginosa Lung Infection in Cystic Fibrosis

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Investigating the Role of Amino Acid Transporter SLC6A14 As a Modifier of Pseudomonas Aeruginosa Lung Infection in Cystic Fibrosis Investigating the Role of Amino Acid Transporter SLC6A14 as a Modifier of Pseudomonas aeruginosa Lung Infection in Cystic Fibrosis by Michelle Di Paola A thesis submitted in conformity with the requirements for the degree of Master of Science Biochemistry University of Toronto © Copyright by Michelle Di Paola (2016) Investigating the Role of Amino Acid Transporter SLC6A14 as a Modifier of Pseudomonas aeruginosa Lung Infection in Cystic Fibrosis Michelle Di Paola Master of Science Biochemistry University of Toronto 2016 Abstract The most common disease causing mutation in CF is F508del, however there is considerable variability in the clinical phenotype of patients homozygous for this mutation. To address this variation, genetic modifiers are studied. Li et al found that SNPs in the putative promoter region of SLC6A14 were significantly associated with severity and age of first Pseudomonas aeruginosa lung infection in CF. Experiments were aimed at studying if SLC6A14-mediated amino acid uptake is regulated by bacterial pathogens and in turn regulates bacterial growth. Arginine uptake from the ASL of non-CF and CF primary bronchial cultures was measured, after treatment with purified flagellin from Pseudomonas. This treatment resulted in a 26.4% (n=5; p<0.01) and 16.6% (n=6; p<0.0204) increase in arginine uptake, in non-CF and CF cultures respectively. Currently, the impact of SLC6A14 function on Pseudomonas growth in co-culture is being investigated, to better understand the role which SLC6A14 plays in modulating infection. ii Acknowledgments I first need to extend my deepest gratitude to my supervisor, Dr. Christine Bear, for providing me with endless support and guidance. Your scientific inputs and patience has truly been instrumental to this work, and I could not imagine a supervisor who is more kind, helpful and understanding to their students. It has been a true joy to work in your lab. Thank you to my supervisory committee members, Dr. Patricia Brubaker and Dr. Igor Stagljar, for taking the time to provide me with helpful inputs and questions, which have pushed me to think more critically about this project. Next, I need to thank my family, especially my mother Antonietta and my father Antonio, for their unwavering support and belief in me and my abilities, in addition to my beautiful baby sister Jessica and grandmother Gerarda, for being amazing and understanding always. No words can express how lucky I am to have all of your love and support. I also need to thank many researchers at SickKids, specifically members of the Bear Lab (past and present), which is filled with many of my friends and mentors. Firstly, thank you to Dr. Saumel Ahmadi, for being not only a great friend, but someone who is always willing to help with any problem, providing endless amounts of ideas and energy, no matter what the situation. Thank you Sunny Xia, for being an amazing friend, someone who I can laugh with in times of stress, and who has always been helpful and kind. Thank you to all the students of the Bear Lab, Dr. Steven Molinski, Stephanie Chin, Maurita Hung, Dr. Stan Pasyk, Andrew Lloyd-Kuzik, Onofrio Laselva, Donghe Yang, Wilson Wu, Ida Szárics, Randolph Kissoon, and Alec Popa, for making each day in the lab enjoyable and full of energy. I know you all will have bright and successful futures. Also, thank you to Ling Jun Huan, for being a great lab technician and friend, in addition to all remaining members of the Bear Lab and the SickKids community, Dr. Mohabir Ramjeesingh, Dr. Danny Li, Dr. Paul Eckford, Catherine Luk, Dr. Leigh Wellhauser, Angela Skoutakis, Dr. Kai Du, Dr. Zoltán Bozóky and Natalie Workewych, for helping to make the lab like a second home. Thank you to Dr. Cezar Khursigara, Dr. Amber Park and the rest of their group at Guelph University, for their great work and enthusiasm on this project. Lastly, I need to thank my friends outside the lab, Jonathan Desmond, John Kedzierski, Josie Vu and Harishni Ramesha, for their continuing friendship and support over these past years. iii Table of Contents Abstract .......................................................................................................................................... ii Acknowledgments ........................................................................................................................ iii Table of Contents ......................................................................................................................... iv List of Tables ................................................................................................................................ vi List of Figures .............................................................................................................................. vii List of Appendices ........................................................................................................................ ix List of Abbreviations .................................................................................................................... x Chapter 1: Introduction ............................................................................................................... 1 1.1 General Airway Physiology ............................................................................................ 1 1.1.1 Airway Surface Fluid .................................................................................................. 1 1.1.2 Importance of CFTR in Regulating Airway Surface Fluid ......................................... 2 1.2 Cystic Fibrosis ................................................................................................................ 5 1.2.1 Cystic Fibrosis Transmembrane Conductance Regulator ........................................... 5 1.2.2 CFTR Genotypes ........................................................................................................ 7 1.2.3 Lung Infection in CF ................................................................................................... 7 1.2.4 Linking Infection to Inflammation .............................................................................. 8 1.2.5 Heterogeneity in CF .................................................................................................... 9 1.2.6 Genetic Modifiers ..................................................................................................... 12 1.3 Solute Carrier Family 6 (Amino Acid Transporter) Member 14 .................................. 13 1.4 Alternative Arginine Transporters ................................................................................ 15 1.5 Project Rationale ........................................................................................................... 18 1.6 Hypothesis and Specific Aims ...................................................................................... 19 Chapter 2: Methods .................................................................................................................... 21 2.1 Cell Culture ................................................................................................................... 21 2.2 Lentiviral Infection ....................................................................................................... 23 iv 2.3 Quantitative Real-Time Polymerase Chain Reaction ................................................... 23 2.4 Western Blotting ........................................................................................................... 25 2.5 Amino Acid Uptake Studies ......................................................................................... 25 2.5.1 L-[2,3-3H]-arginine Uptake ....................................................................................... 25 2.5.2 Treatment and Collection of Airway Surface Fluid .................................................. 27 2.6 Reagents ........................................................................................................................ 28 2.7 Statistical Analysis ........................................................................................................ 28 Chapter 3: Modulation of SLC6A14 messenger RNA expression in lung epithelium ......... 29 3.1 SLC6A14 expression is enhanced by an immune activating component from Pseudomonas aeruginosa ......................................................................................................... 29 3.2 SLC6A14 expression can be modified by shRNA-mediated knock-down ................... 33 Chapter 4: SLC6A14 plays an important role in regulating amino acid uptake in lung epithelium .................................................................................................................................... 34 4.1 Inhibition of SLC6A14 causes a significant reduction in L-[2,3-3H]-arginine uptake..34 4.2 Inhibition or up-regulation of SLC6A14 significantly modifies uptake of arginine in non-CF and CF primary airway cells ........................................................................................ 36 4.3 Changes in arginine uptake positively correlates with up-regulation of SLC6A14 expression following treatment with purified flagellin ............................................................. 43 Chapter 5: Discussion ................................................................................................................. 44 References ...................................................................................................................................
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