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Thesis Harshal Patil.Pdf DISSERTATION Submitted to The combined faculties for Natural Sciences and Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Harshal Patil born in Savda Tal Raver, India Date of oral examination: ___________________ Studies on zygote morphogenesis in rodent malaria parasite, Plasmodium berghei Referees: Prof. Andrew P. Waters Prof. Dr. Freidrich Frischknecht Prof. Dr. Michael Lanzer Eidesstattliche Erklärung Hiermit erkläre ich, dass alle Ergebnisse in dieser Doktorarbeit auf meiner eigenen Arbeit beruhen, mit Ausnahme der folgenden Daten, Datenanalysen und Plasmid-Konstruktion: die Umwandlung der primären Datensets in Excel Datensets der RNA Sequenzierungs Daten wurde von Dr Nicholas Dickinson, Genome and Data Analyst and Glasgow Polyomics Team, University of Glasgow, durchgeführt; elektonmikroskopische Bilder und deren Analyse wurde mit Hilfe von Frau Margaret Mullin, EM technische Angestellte, University of Glasgow, durchgeführt; die Bedienung des FACS Gerätes sowie die FACS Datenanalyse wurde mit Hilfe von Dr Katie Hughes, Postdoctoral Researcher, Professor Andrew Waters Laboratory, durchgeführt; die Ookineten Mobilität wurde mit Hilfe von Dr Nishia Philip, Research Associate, Professor Andrew Waters Laboratory, berechnet; das ISP::mCherry Plasmid wurde von Ms Christina Manakanata, einer ERASMUS Studentin, die unter meiner Anleitung gearbeitet hat, generiert. Alle Beiträge anderer sind an den entsprechenden Stellen der Doktorarbeit erwähnt und anerkannt. Diese Doktorarbeit ist nicht für einen akademischen Grad (Dr. rer. nat.) an einer anderen Institution eingereicht worden. Die hier präsentierte Doktorarbeit wurde von dem Autoren der Arbeit in der Zeit von Oktober 2011 bis May 2015 im Rahmen des EU FP7 Progamms „EVIMalaR“ unter der gemeinsamen Anleitung von Professor Andrew Paul Waters, Wellcome Trust Centre for Molecular Parasitology, University of Glasgow und Professor Dr. Friedrich Frischknecht, Zentrum für Infektiologie - Parasitologie, Ruprecht Karls Universität, Heidelberg, durchgeführt. ……… …………………… Datum Harshal Patil Acknowledgements Acknowledgements This PhD project is funded by the European Union’s Seventh Framework Programme for Research (EU FP7) under European Virtual Institute for Malaria Research (EVIMalaR). This project has immensely benefited from the wisdom and experience of Prof. A. P. Waters, University of Glasgow. I thank him for his patience, trust and open-mindness throughout the period of PhD and giving me an opportunity to work under for EVIMalaR PhD programme and critically reviewing my project time to time and for guidance and inspiration. I also thank Prof. Dr. Friedrich Frischknecht and Prof. Dr. Michael Lanzer, University of Heidelberg for acting as my supervisors their comments, suggestions and guidance. I am grateful to Dr. Katie Hughes, University of Glasgow who has been very kind to supervise me and to teach me the fundamentals at the bench. I also thank following people at the University of Glasgow, University of Heidelberg and European Molecular Biology Laboratory including group members of Prof. Waters, Prof.Dr. Frischknecht and Prof. Dr. Lanzer lab for their kind co-operation during my PhD. Mrs. Rachael Cameron for parasite transfections Mrs. Angela Mcbridge for mosquito maintenance Mrs. Margaret Mullin and Dr. Sonya Taylor for their introduction to and help during electron microscopy Dr. Mhairi Stewart during RNA extraction Dr. Nisha Philip for motility assays and discussion Dr. Anubhav Srivastava for statistical analysis and IT help Dr. Abhinav Sinha for regular discussions and suggestions Ms. Anne Graham for Lab support Dr. Volodymyr Nechyporuk-Zloy for imaging Glasgow Polyomics Facility for sequencing RNA Dr Nickolas Dickens for processing raw RNA-Sequencig data Dr. Scott Johnston & Dr. Ian Salt for introducing and allowing me to use their Odyssey® Sa Infrared Imaging System Ms. Sandra Niebel, Ms. Alessia Valdarno, Ms. Elisa Kless, Ms. Yvonne Maier for EVIMalaR related administrative support in Heidelberg I Acknowledgements Ms. Miriam Griesheimer, Secretory to Prof. Dr. Michael Lanzer for admistration related support Mr. Kartik Bane, Ms. Sonia Moliner Cubel and Ms. Priyanka Fernandes for helping with regular discussions Ms. Maria Mayorga, Ms. Pauline Conlon for coordinating stipend Ms. Diah Yulianti and Mr. Tim Nuernberger during EVIMalaR|BioMalPar conferences Ms. Jill Moore and Ms. Lisa Milne for all the visa applications All EVMalaR mates for making my PhD a wonderful time Ms. Gillian Murray, Ms. Amanda Baird, Ms. Hansa Pertab for EVIMalaR related administrative support in Glasgow Ms. Martina Galvan and The Hartmut Hoffmann-Berling International Graduate School, University of Heidelberg for administrative support The College of Medical, Veterinary and Life Sciences-Graduate School, University of Glasgow for administrative support I am also thankful to my students- Ms. Christina Manakanata, EARASMUS student and Mr. Tim Hannay, Medical Student for providing me an opportunity to supervise them which helped enhance my leadership abilities. Further, I am grateful to Prof. Sylke Muller for translating the summary and declaration of this thesis in German, Prof. Markus Meissner, Dr. Lisa Ranford Cartwright – University of Glasgow for assessing my work every year and providing various ideas and research directions. I am also thankful to our collaborators Dr. Oliver Bilker, Wellcome Trust Sanger Institute, UK; Dr. Marc-Jan Gubbels, Boston College, USA; Prof. Judith Green, London School of Hygiene & Tropical Medicine, UK; Dr. Jacquin C. Niles, Massachusetts Institute of Technology, USA and Dr. Inga Siden-Kiamos, Institute of Molecular Biology and Biotechnology, Greece for providing project related materials and protocols. Finally, I would like to appreciate the efforts of my entire family, especially my parents- Mrs. Prabhavati Patil and Mr. Prakash Patil, my wife- Rucha and my sister- Mrs. Priti Gavade without whom I would not have reached this far.------------------------------------------------------- II Abbreviations Abbreviations > haploid more than haploid µm micrometer 2DG 2-Deoxy-D-glucose 2N diploid 4N tetraploid 5FC 5-fluorocytosine 8N octaploid or octoploid ACT Artemisinin Based Combination Therapy AMA-1 apical merozoite antigen 1 aPKC atypical Protein Kinase C AUFG Activated- unfertilized female gamete bp base pairs cdc24 cell division cycle 24 cdc42 cell division cycle 42 CDPK1 calcium-dependent protein kinase CelTOS cell traversal protein of Plasmodium ookinetes and sporozoites CITH homolog of worm CAR-I and fly Trailer Hitch) CO2 carbon di-oxide CRISPER/Cas9 clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing CRMPs cysteine repeat modular proteins CSP circumsporozoite protein CTRP circumsporozoite- and thrombospondin-related adhesive protein DAPI 4', 6-diamidino-2-phenylindole DDT Dichlorodiphenyltrichloroethane DG dense granules DOZI development of zygote inhibited EBA erythrocyte binding antigen ECP-1 egress cysteine protease 1 EGF epidermal growth factor ELISA Enzyme-Linked Immunosorbent Assay ER endoplasmic reticulum FIP family of interacting proteins FPKM fragments per Kilobase of open reading frame per million reads G6PD Glucose-6-phosphate dehydrogenase GAK cyclin G-associated kinase GAP GTPase-activating protein GAP40 glideosome associated protein 40 GAP45 glideosome associated protein 45 GAP50 glideosome associated protein 50 GAP70 glideosome associated protein 70 GAPM3 glideosome associated protein with multiple membrane spans 3 III Abbreviations GDI Guanine nucleotide dissociation inhibitor gDNA genomic DNA GEF GDP/GTP exchange factor OR Guanine nucleotide exchange factor GFP Green Fluorescent Protein GPI glycosylphosphatidylinositol GRA granule protein h hour hpa hours post-activation hpi hours post-infection i.p. intraperitoneal i.v. intravenously IMC Inner Membrane Complex IPTP intermittent preventive treatment of pregnant women IPTP-SP intermittent preventive treatment of pregnant women using sulfadoxine-pyrimethamine iRBC infected red blood cell IRS Indoor Residual Spraying ISP1 IMC sub-compartment protein 1 ISP3 IMC sub-compartment protein 3 ITN Insecticide-treated nets kd Kilobase pairs LCCL Limulus clotting factor C, Coch-5b2, Lgl1 LLIN Long-Lasting Insecticidal Nets MACPF membrane-attack complex/perforin MAEBL membrane antigen/erythrocyte binding-like MAEBL membrane antigen/erythrocyte binding-like MAOP membrane-attack ookinete protein MAP Kinase Mitogen-activated Protein Kinase MDCK Madin-Darby Canine Kidney MDV/PEG3 male development-1/protein of early gametocyte 3 min minutes mRNP messenger ribonucleoprotein particles MSP1 Merozoite Surface Protein 1 MTOC microtubule-organizing center N haploid NEK 2/4 NIMA (never in mitosis/Aspergillus) related Kinases NIMA never in mitosis/Aspergillus nm nano meter NSF N-ethylmaleimide-sensitive factor O2 oxygen ODS oocyst derived sporozoites Pb Plasmodium berghei PbG2 Plasmodium berghei glycine at position 2 IV Abbreviations PbRab11A Plasmodium berghei Rab11A PC12 cells cell line derived from a Pheochromocytoma of the rat adrenal medulla. PCR Polymerase Chain Reaction PDEδ Phosphodiesterases δ Pf Plasmodium falciparum PfEMP1 P. falciparum-infected erythrocyte membrane protein 1 PI4K phosphatidylinositol-4 kinases PK7 protein kinase 7 PPLP3/4/5 perforin-like proteins PPLPs perforin-like proteins PPM metallo-dependent protein phosphatases 2 and 5 PV parasitophorous vacuole Pv Plasmodium
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