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Binding Protein in Monitoring the Formation of Mrnp Granules in Toxoplasma Gondii

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Binding Protein in Monitoring the Formation of Mrnp Granules in Toxoplasma Gondii University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 10-19-2015 Utilization of Poly(A)-Binding Protein in Monitoring the Formation of mRNP Granules in Toxoplasma gondii Emad Manni University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Manni, Emad, "Utilization of Poly(A)-Binding Protein in Monitoring the Formation of mRNP Granules in Toxoplasma gondii" (2015). Electronic Theses and Dissertations. 5443. https://scholar.uwindsor.ca/etd/5443 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). Under this license, works must always be attributed to the copyright holder (original author), cannot be used for any commercial purposes, and may not be altered. Any other use would require the permission of the copyright holder. Students may inquire about withdrawing their dissertation and/or thesis from this database. For additional inquiries, please contact the repository administrator via email ([email protected]) or by telephone at 519-253-3000ext. 3208. Utilization of Poly(A)-Binding Protein in Monitoring the Formation of mRNP Granules in Toxoplasma gondii By Emad Manni A Thesis Submitted to the Faculty of Graduate Studies through the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Master of Science at the University of Windsor Windsor, Ontario, Canada 2015 © 2015 Emad Manni Utilization of Poly(A)-Binding Protein in Monitoring the Formation of mRNP Granules in Toxoplasma gondii By Emad Manni APPROVED BY: ______________________________________________ A. Swan Department of Biological Sciences ______________________________________________ M. Boffa Department of Chemistry & Biochemistry ______________________________________________ S. Ananvoranich, Advisor Department of Chemistry & Biochemistry June 26, 2015 ii DECLARATION OF ORIGINALITY I hereby certify that I am the sole author of this thesis and that no part of this thesis has been published or submitted for publication. I certify that, to the best of my knowledge, my thesis does not infringe upon anyone’s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my thesis, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. Furthermore, to the extent that I have included copyrighted material that surpasses the bounds of fair dealing within the meaning of the Canada Copyright Act, I certify that I have obtained a written permission from the copyright owner(s) to include such material(s) in my thesis and have included copies of such copyright clearances to my appendix. I declare that this is a true copy of my thesis, including any final revisions, as approved by my thesis committee and the Graduate Studies office, and that this thesis has not been submitted for a higher degree to any other University or Institution. iii ABSTRACT Toxoplasma regulates its gene expression throughout its complex life cycle to survive and confront different kind of stresses. Here I aimed to gain a better understanding of how this regulation is maintained in Toxoplasma at the post-transcriptional level and in the aspect of the formation of messenger ribonucleoprotein complex (mRNPs). I chose to study the poly(A) binding protein (PABP). Metazoans usually encode multiple isoforms of PABP to function in nucleus and/or cytoplasm but in Toxoplasma there is only one predicted PABP. In this thesis, I studied the localization of the only predicted PABP in Toxoplasma (TgPABP). It was detected that TgPABP is predominantly localized in the cytoplasm under treatment with actinomycin D. Second, we also found that TgPABP aggregates into microscopically visible granules, as an indicative of mRNPs. Upon salubrinal treatment, mRNPs were formed in both extracellular and in intracellular parasites whereas cycloheximide can diffuse these granules in less than one hour. iv Dedication I dedicate this work to my family, friends, and my wife, Alanoud, who help and encourage me to success v Acknowledgments I would like firstly to express my heartiest gratitude to the Almighty Allah who has created me in a good shape and has given me the patient, ability, and strength to carry out towards success and finish the writing of this thesis. My sincere gratitude to my supervisor Dr. Sirinart Ananvoranich whose sincerity, patience and encouragement I will never forget. I thank her first for accepting me to join her research team and support throughout the learning process of this master thesis. I also have to express my sincere gratitude to my master’s committee members Dr. Michael Boffa and Dr. Andrew Swan for their constructive comments and inputs. During my master’s research projects, many people support me in both theory and practice. Dr. Michael Holmes, thank you for spending a great deal of time giving me advice and support on troubleshooting and a big thank you for proofreading the introduction chapter of this thesis. Anna Crater-Poter, your help and guidance on experimental aspects was highly appreciated. I would like to thank also the master’s students, who recently joined our lab, Farzana Afrin and Md Amran Hossain for assistance. A special thank you to Natalie Gabriel for taking care of the cell culture while I was away. I would like to extend Marlene Bezaire for everything she has done through the past two years. I thank Elizabeth Fidalgo for her training and guidance for using the Flourescence Microscopy. My M.Sc study would have been impossible without the financial support from the King Abdullah program. May Allah rest his soul in peace and grant him the best place in paradise, jannatul-firdaus. Employees in the Saudi Cultural Bureau in Canada, thank you for always being available to all Saudi students, and thank you to Dr. Hamid Al-Zaidi. Last but not least, there is no enough words to describe my greatest gratitude to my parents Mohammed Manni and Fatema Mohammed, THANK YOU from my bottom of my heart. vi Table of Contents Declaration of Originality .............................................................................................................. iii Abstract .......................................................................................................................................... iv Dedication ........................................................................................................................................v Acknowledgments.......................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures ..................................................................................................................................x List of Appendices ......................................................................................................................... xi List of Abbreviation ...................................................................................................................... xii Chapter I Literature Review 1.1 The discovery of Toxoplasma gondii .....................................................................................2 1.2 History ....................................................................................................................................3 1.3 Life cycle of Toxoplasma gondii ............................................................................................3 1.3.1 Sexual stage ...............................................................................................................4 1.3.2 Asexual stage .............................................................................................................4 1.4 Toxoplasma genotypes ...........................................................................................................7 1.5 Toxoplasmosis symptoms ......................................................................................................7 1.6 The regulation of gene expression .........................................................................................8 1.6.1 Transcriptional and Post-transcriptional regulations ..................................................9 1.7 Structure and functions of mRNPs .......................................................................................14 1.7.1 Neuronal transport RNP granules .............................................................................14 1.7.2 Sponge bodies ...........................................................................................................15 1.7.3 U bodies ....................................................................................................................15 1.7.4 RNP-rich cytoplasmic germline granules .................................................................15 1.7.5 Processing bodies ......................................................................................................16 1.7.6 Stress
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