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Drug Targets of the Heartworm, Dirofilaria Immitis

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Drug Targets of the Heartworm, Dirofilaria Immitis Drug Targets of the Heartworm, Dirofilaria immitis Inauguraldissertation zur Erlangung des Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Christelle Godel aus La Sagne (NE) und Domdidier (FR) Schweiz Avenches, 2012 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Jürg Utzinger Prof. Dr. Pascal Mäser P.D. Dr. Ronald Kaminsky Prof. Dr. Georg von Samson-Himmelstjerna Basel, den 26th of June 2012 Prof. Dr. M. Spiess Dekan To my husband and my daughter With all my love. Table of Content P a g e | 2 Table of Content Table of Content P a g e | 3 Acknowledgements ............................................................................................................... 5 Summary ............................................................................................................................... 8 Introduction ..........................................................................................................................11 Dirofilaria immitis ..............................................................................................................12 Phylogeny and morphology ...........................................................................................12 Repartition and ecology .................................................................................................14 Life cycle .......................................................................................................................16 Diagnosis, treatment and resistance ..............................................................................18 Old and new sequencing strategies ..................................................................................21 DNA sequencing ...........................................................................................................21 Premises - Sanger, Maxam and Gilbert .....................................................................21 Dye-labeling terminator sequencing ...........................................................................23 Automated sequencing and high-throughput sequencing development .....................23 454-Pyrosequencing ..................................................................................................24 Illumina sequencing ...................................................................................................25 Third generation of high-throughput sequencing ........................................................27 Summary and further analysis ...................................................................................27 Assembly methods ........................................................................................................29 Example of de novo genome sequencing ......................................................................30 Anthelmintics and targets ..............................................................................................31 Nicotinic acetylcholine receptors and cys-loop ligand-gated ion channels .........................33 Aims and objectives .............................................................................................................36 Chapter 1. Understanding the magic bullet: molecular opportunities for antiparasitic drug selectivity ..............................................................................................................................38 Chapter 2. Efficacy testing of AADs against D. immitis (unpublished) ...................................61 Chapter 3. The genome of the heartworm, Dirofilaria immitis ...............................................66 Genome-wide survey for ligand gated ion channels in D. immitis .................................... 104 Chapter 4. Loss of DEG-3-subfamily acetylcholine receptors and lack of stereoselective monepantel sensitivity in Dirofilaria immitis ......................................................................... 106 Table of Content P a g e | 4 Deeper investigations through the DEG-3-subfamily ....................................................... 117 In silico novel potential drug targets of D. immitis ............................................................ 118 Chapter 5. Drug sensitivity tests (unpublished) ................................................................... 121 RNA-dependant RNA polymerase .................................................................................. 122 Chitin synthase ............................................................................................................... 123 Potential novel receptors of Wolbachia, endosymbiont of D. immitis ............................... 124 Discussion .......................................................................................................................... 126 1. Nucleic acid synthesis and repair receptors ............................................................. 127 RNA-dependant RNA polymerase ............................................................................... 127 2. Glycosylation and sugar metabolism ........................................................................ 128 Beta-1,4-mannosyltransferase ..................................................................................... 128 UDP-galactopyranose mutase ..................................................................................... 130 Chitin synthase ............................................................................................................ 131 3. Parallel study performed on Wolbachia of D. immitis ............................................... 132 Conclusions ........................................................................................................................ 134 References ......................................................................................................................... 136 Appendices ........................................................................................................................ 146 List of abbreviations ........................................................................................................ 147 List of figures .................................................................................................................. 152 List of tables ................................................................................................................... 153 Curriculum vitae ................................................................................................................. 155 Acknowledgements P a g e | 5 Acknowledgements Acknowledgements P a g e | 6 I would like to thank all the people who contributed to the success of this doctoral thesis: Pascal Mäser for his precious support and supervision throughout my PhD duration; for his help in innovation and for bringing relevant new input. And I would like to specially thank him for being present to answer important questions and to bring judicious advices. Christian Epe for the internal support and supervision within Novartis Animal Health and good advices, trust and friendship. The CAP, companion animal parasiticides, team for their help in laboratory work and parasites maintenance. Ronald Kaminsky to lead the project in house and help me with the internal questions, to guide me through the meanders of Novartis guidelines; to believe in me through my entire PhD and in my results. Georg von Samson-Himmelstjerna for his invaluable advice and comments as my co-referee. Jacques Bouvier for his support and help concerning screening tests and precious advices and information about chemical compounds as well as for his valuable new scientific input and ideas; for our open-minded interesting discussions. Lucien Rufener for his precious advices in molecular biology; especially to help to come across difficulties in the laboratory work; for brainstorming situations and interesting scientific discussions as well as for friendship and sharing the same office. Sandra Schorderet-Weber for her help and precious advices concerning the in vivo tests with the rodents. The Rodents Model team for the laboratory work and the parasites maintenance. Daniel Nilsson from the Karolinska Institutet in Sweden for his great help and knowledge in informatics, computer science and bioinformatic studies. Acknowledgements P a g e | 7 Sujai Kumar, Georgios Koutsovoulos and Mark Blaxter from the University of Edinburgh for their help to share their knowledge and success in bioinformatics as well as their huge education concerning nematode parasites to set a good publication. Frederik Bringaud to share his educative information concerning transposons and apply it to the heartworm. Philipp Ludin and Christoph Schmid from the Swiss Tropical and Public Health Institute for their collaboration and work in the genome publication. Novartis Animal Health St-Aubin FR and the Swiss Tropical and Public Health Institute to allow me to perform my PhD and share the results and interesting data with relevant persons and for the fellowship grant and the internal support. Special thanks go to Timothy Geary and Roger Prichard for their adequate and quick advices as well as for their interesting scientific opinions and for their support to my studies. Finally, I would like to thank my husband, Sylvain, and my daughter, Mélie, who supported me during the whole PhD and were always behind me as well as my family and my friends. S u m m a r
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