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Adaptation of Plasmodium Falciparum in Human RBC Supplemented NSG Mice This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Adaptation of Plasmodium falciparum in human RBC supplemented NSG mice Chew, Marvin 2019 Chew, M. (2019). Adaptation of Plasmodium falciparum in human RBC supplemented NSG mice. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/142942 https://doi.org/10.32657/10356/142942 This work is licensed under a Creative Commons Attribution‑NonCommercial 4.0 International License (CC BY‑NC 4.0). Downloaded on 07 Oct 2021 21:19:51 SGT Adaptation of Plasmodium falciparum in human RBC supplemented NSG mice Marvin Chew SCHOOL OF BIOLOGICAL SCIENCES 2019 1 Adaptation of Plasmodium falciparum in human RBC supplemented NSG mice Marvin Chew SCHOOL OF BIOLOGICAL SCIENCES A thesis submitted to the Nanyang Technological University in partial fulfilment of the requirement for the degree of Doctor of Philosophy 2019 2 Statement of Originality I hereby certify that the work embodied in this thesis is the result of original research done by me except where otherwise stated in this thesis. The thesis work has not been submitted for a degree or professional qualification to any other university or institution. I declare that this thesis is written by myself and is free of plagiarism and of sufficient grammatical clarity to be examined. I confirm that the investigations were conducted in accord with the ethics policies and integrity standards of Nanyang Technological University and that the research data are presented honestly and without prejudice. Date Marvin Chew 3 Supervisor Declaration Statement I have reviewed the content and presentation style of this thesis and declare it of sufficient grammatical clarity to be examined. To the best of my knowledge, the thesis is free of plagiarism and the research and writing are those of the candidate’s except as acknowledged in the Author Attribution Statement. I confirm that the investigations were conducted in accord with the ethics policies and integrity standards of Nanyang Technological University and that the research data are presented honestly and without prejudice. Date Professor Peter Preiser 4 Authorship Attribution Statement This thesis does not contain any materials from papers published in peer- reviewed journals or from papers accepted at conferences in which I am listed as an author. Date Marvin Chew 5 Acknowledgements I have been blessed by many who have helped me along my PhD endeavor. Firstly, I thank God for His goodness, love and mercy in my life and His providence throughout my PhD journey. My sincerest gratitude to my wonderful parents, Mark and Wendy, and my family who have supported me wholeheartedly all my life and to my lovely wife, Jasmine, for her unwavering support and love. I am grateful to my PhD supervisors, Prof. Peter Preiser and Prof. Jianzhu Chen, who gave me the opportunity to pursue a PhD and to work with them under the SMART PhD Fellowship. Thank you for teaching me to become a critical thinker and guiding me to be a good scientist. It has been my privilege to be your student. I also would like to thank all my colleagues and friends at SMART and NTU, especially Dr Ye Weijian, for guiding me in the lab since the first day I joined. I am thankful to you for our joint publication which is my first co-first authored paper. My thanks to Dr Regina Hoo for her immense help with the microarray, bioinformatics and analysis as well as teaching and training me. I am especially grateful to Ms Lan Hiong Wong for her help with many of the experiments which involves both animal and lab work and Ms Hooi Linn Loo for her help and expertise in flow cytometry. My thanks to my fellow PhD 6 students in Preiser lab, Ameya and Radek, that we went supported one another and journeyed through our PhDs together. There are so many to thank through this whole PhD journey and my apologies if I have not acknowledged you by name. I offer you my thanks and gratitude and wish you success in all that you do. 7 Table of Contents Table of Contents ................................................................................................. 8 List of Figures .................................................................................................... 13 List of Tables ..................................................................................................... 17 Abbreviations ..................................................................................................... 18 Abstract .............................................................................................................. 19 Chapter 1. Introduction .................................................................................. 21 Overview of Malaria ........................................................................... 21 Global burden of Malaria ............................................................. 21 Malaria Lifecycle ......................................................................... 22 Variant Surface Antigens (VSAs) ................................................ 24 P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) ........ 26 Repetitive interspersed family (RIFINs) ...................................... 29 Sub-telomeric variable open reading frame (STEVOR) .............. 30 Naturally acquired immunity to malaria ............................................. 30 Innate immunity and malaria .............................................................. 32 Macrophages ................................................................................ 34 Natural Killer (NK) cells ............................................................. 37 NK cell heterogeneity .................................................................. 39 8 Humanized mice .................................................................................. 41 Project Objectives ............................................................................... 43 Chapter 2. Materials and Methods ................................................................. 44 Adaptation of P. falciparum in huRBC-NSG mice ............................ 44 P. falciparum culture and infection ..................................................... 45 P. falciparum microarray and analysis ................................................ 48 Cloning of pSN054 plasmid ................................................................ 51 Peripheral blood mononuclear cell (PBMC) purification. .................. 55 Monocyte-derived Macrophage and phagocytosis assay .................... 56 Primary NK cell parasitemia reduction co-culture .............................. 57 Immunofluorescence assay (IFA) ....................................................... 58 qPCR ................................................................................................... 60 Chapter 3. Characterization of adapted P.falciparum in huRBC-NSG mice 61 Adaptation of P. falciparum in huRBC-NSG mice ............................ 61 Generating huRBC-NSG mice and P. falciparum infection ........ 61 Adapted P. falciparum maintains adaptation after long term in vitro culture ................................................................................................ 64 Adaptation of P. falciparum to huRBC-NSG mice not due to genetic heterogeneity of parasite cultures .................................................. 66 9 Adapted parasites are genetically identical to parental non-adapted parasites .......................................................................................................... 67 Gene expression differences between non-adapted and adapted parasites enrich for variant surface antigens .................................................. 70 Adapted parasites maintain adaptation after plasmid transfection ...... 78 PfEMP1 surface expression affects adapted parasite growth in huRBC-NSG mice .......................................................................................... 86 Plasmid construction of pSN054_PTEF, MAHRP1 and SBP1 ... 88 MAHRP1 knockdown in no aTc condition affects surface expression of PfEMP1 ................................................................................ 93 MAHRP1 conditional knockdown adapted parasites affects growth in huRBC-NSG mice ...................................................................... 96 Discussion ........................................................................................... 98 Adaptation of P. falciparum in huRBC-NSG not a result of genetic changes ........................................................................................... 98 Adaptation of P. falciparum in huRBC-NSG due to change in adapted parasite gene expression ................................................................ 98 Adapted parasites can be genetically modified without affecting competency in huRBC-NSG mice ............................................................ 100 Surface PfEMP1 expression needed for adapted P. falciparum to thrive in huRBC-NSG mice ...................................................................... 101 10 Chapter 4. Macrophage phagocytosis of adapted P. falciparum ................. 103 Adapted parasites do not accumulate in the spleen and lungs in huRBC-NSG mice ........................................................................................ 103 Need for parasite adaptation abolished in clodronate-treated huRBC- NSG mice ....................................................................................................
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