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A Small Molecule Modulator of Prion Protein Increases Human Mesenchymal Stem Cell Lifespan, Ex Vivo Expansion and Engraftment to Bone Marrow in Nod/Scid Mice

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A Small Molecule Modulator of Prion Protein Increases Human Mesenchymal Stem Cell Lifespan, Ex Vivo Expansion and Engraftment to Bone Marrow in Nod/Scid Mice A SMALL MOLECULE MODULATOR OF PRION PROTEIN INCREASES HUMAN MESENCHYMAL STEM CELL LIFESPAN, EX VIVO EXPANSION AND ENGRAFTMENT TO BONE MARROW IN NOD/SCID MICE THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY SINDHU TANAYA MOHANTY DEPARTMENT OF HUMAN METABOLISM MEDICAL SCHOOL UNIVERSITY OF SHEFFIELD Registration No: 070236424 SUBMITTED June 2014 I want to acknowledge my teacher and supervisor Professor. Ilaria Bellantuono, for her excellent guidance, co-ordinated supervision and being a constant moral support in guiding me to complete my project while at Sheffield as well as far away from lab, in India. I fondly remember the days in lab when you have acknowledged me by saying - , eautiful- . I miss working with you. You are and will forever remain a I would like to dedicate this piece of hard work to my parents (Dr. Binoy Kumar Mohanty and Mrs. Supriti Mohanty) and late grandparents (Kishore Charan Mohanty, Chayamani Devi, Nrusinha Charan Mohanty and Satyabhama Devi) from whom I have learnt the art of sincerity, perseverance and patience which has helped me throughout the work period. The mere assurance all the way from India, that attending my phone calls at awry hours of night to listen to my plight when I was down and most importantly believing in me has been the greatest support from my parents. I hanking you for everything is just not enough. Your love and blessings were and will always remain my constant guide. My late Badabapa (Ajaya Kumar Mohanty) and his entire family will be always remembered fondly for their timely help in need. I would also like to thank each and every member of my family for their never ending moral support and blessings from time immemorial. A special mention about my youngest cousin sister Jigeesha Das, who has been unknowingly a constant source of positive encouragement. You all inspire me positively in many ways that I cannot describe. d to do is just love them back. u - Shyamal Misra. I remember the long nights you were awake just to give me company while I was writing my thesis, to bear my mood- i | P a g e finish my write- in every aspect of life. My in-laws (Dr. Naresh Chandra Misra and Mrs. Ranjita Misra) without whose support and relentless encouragement, I would not have made this far. My maternal -laws (Dr. Ram Shankar Rath and Mrs. Suprava Rath) need a special mention as they have never quit to constantly motivate me to wrap up my PhD work even at their age. I am simply blessed with my new family and counting on all their blessings for years to come. I would like to express my special thanks to Manikandan Thangavel, a silent friend in my life who was by my side from the very first day of my PhD work to cheer and motivate me, especially after having those days of roller-coaster rides at lab. How can I forget my little sister (Himanandini Mohanty) and brother-in-law (Satwik Patnaik) (Kamal Misra Jiksha Kamal Misra) for their timely reminders to support me in submitting my write-up. After parents, you are the only people I count on and I am blessed to have you in my life. And it would be very unfair not to have mentioned Juhi Misra - whose company as lab mate has evolved into an undefinable and beautiful bond of lifelong friendship. I fondly remember those days of working late in laminar air-flow cabinets and jumping into a piece of dance for a short break, long discussions at Royal Hallamshire cafeteria about work and life in general, the train journeys which comprised only of loud laughter, escape to Meadow hall shopping and movies. I am glad that this has extended to our long lasting skype chats. It was so much fun teaching you and at the same time learning with and from you. Indeed, I have made a beautiful friend for life and cheers to our friendship. Thank you so much for being there for me always. I would Subhas Chandra Pattanayak and Dr. Niranjan Rout for their motivation to pursue a career in research and to go abroad to broaden my horizon in every aspect of life including professional and personal. ii | P a g e I would like to thank my friends Lucksy Kottam, Alessandra Gambardella, Brindha and Sravanth, Mark Baduain, Chandan Kadur, Satyabrata Acharya, Soumya Sudhal and my school friends, whose timely help and moral support has been enormous. I would like to say a special thank you to my Uncle and Aunty (Manoranjan Das and Sabitha Sutrave) and cousins Sarthak and Mahan from London, who have been very warm and welcoming to accommodate me whenever I wanted few days of break from lab. I would like to thank all my colleagues from DU04, DU10 and DU16 for their tremendous moral support and helping me out with my experiments whenever needed. Lastly I want to express my sincere gratitude and thanks to all those people who have helped me during my work but whose names I am unable to mention here. It goes without saying about my gratitude to the -Maa Kundes whose bountiful iii | P a g e This study was supported by Biotechnology and Biological Sciences Research Council (BBSRC). The human bone marrow samples were kindly provided by Dr. Andrew D Chantry1, Dr. Sanjeev Madan2 and Dr. James Fernandes2. Flow cytometry and cell sorting facility of the Medical School is run by Susan Newton1 and Kay Hopkinson1. Steven J Howe3 and Adrian Thrasher3 assisted in setting up the lentiviral transduction in the laboratory. The gene expression profiling was funded by Cancer Research UK and was assisted by W Nicol Keith4 and Claire J Cairney4. Karyotype analyses were performed by Duncan Baker5. We are grateful to Orla Gallagher1, Daren Lough1 and Julia Hough1 for their expert technical assistance. We are thankful to Beining Chen6 and Mark J Thompson6 for providing the small molecule 3/689. We thank Peter Andrews7 and Harry Moore7 for helpful discussion. 1Mellanby Centre for Bone Research, Department of Human Metabolism, University of Sheffield, UK; 2Department of Paediatric Orthopaedic, and Trauma Surgery, Sheffield , UK; 3Molecular Immunology Unit, Department of Infection and Immunity, University College London, UK; 4Institute of Cancer Sciences, University of Glasgow, UK; 5 , UK; 6Department of Chemistry, University of Sheffield, UK; 7Centre for Stem Cell Biology, University of Sheffield, UK. iv | P a g e Publications Sindhu T Mohanty -femoral injection of human Stem cells, Methods and protocols-Methods in Molecular Biology series, 2012) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, A Small Molecule Modulator of Prion Protein Increases Human Mesenchymal Stem Cell Lifespan, Ex Vivo Expansion and Engraftment to Bone Marrow in NOD/SCID Mice . (Stem cells, 2012) Oral presentations Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. increases self-renewal of mesenchymal stem cells and delays their ageing in th UKMSCN Meeting, Birmingham, UK. (July 2011) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. increases self-renewal of mesenchymal stem cells and delays their ageing in . (June 2010) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. A small molecule binding to prion protein increases the number of mesenchymal progenitor cells Mellanby Centre Research Day, Sheffield, UK. (June 2009) v | P a g e Poster presentations Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. enhances mesenchymal stem cells expansion with age and their transplantation st European Conference on Mesenchymal Stem Cells, Toulouse, France. (November, 2010) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. increases self-renewal of mesenchymal stem cells and delays their ageing in th UKMSCN Meeting, Leeds, UK and was awarded the best poster prize award. (March 2010) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. mesenchymal stem cell rd UK Mesenchymal Stem Cell meeting, Octagon Centre, University of Sheffield, UK. (January 2009) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. increased self-renewal in IL-1ra-/- mouse model of rheumatoid arthritis in the Edinburgh Conference Centre at Heriot-Watt University, Edinburgh, UK. (November 2008) Sindhu T Mohanty, Claire Cairney, Andy Chantry, Sanjeev Madan, James Fernandes, Mark J Thompson, Steven J Howe, Adrian Thrasher, Beining Chen, W N Keith and Ilaria Bellantuono. nd SCIBS Initiative Grant . (November 2008) vi | P a g e Table of Contents Acknowledgements _____________________________________________________ i Declaration ___________________________________________________________ iv List of publications, presentations and posters ______________________________ v Table of Contents ____________________________________________________ vii List of figures ________________________________________________________
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