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Dissertation.Pdf Genomweite Sequenzierung des Fledermaus- Malariaparasiten Polychromophilus murinus und bioinformatische Analyse von Parasit-Wirt- Interaktions-Proteinen DISSERTATION zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.) am Fachbereichs Biologie, der Fakultät für Mathematik, Informatik und Naturwissenschaften der Universität Hamburg vorgelegt von ELSA AUL (geb. Ziegler) aus Hamburg Hamburg im März 2020 “According to Darwin’s Origin of Species, it is not the most intellectual of the species that survives; it is not the strongest that survives; but the species that survives is the one that is able best to adapt and adjust to the changing environment in which it finds itself” Leon C. Megginson, 1963 Tag der Disputation: 03. Juli 2020 Gutachter der Dissertation: 1. Gutachterin: Prof. Dr. rer. nat. Iris Bruchhaus Bernhard-Nocht-Institut für Tropenmedizin Abteilung Zelluläre Parasitologie Bernhard-Nocht-Straße 74, 20359 Hamburg 2. Gutachterin: Prof. Dr. rer. nat. Susanne Dobler Universität Hamburg, Institut für Zoologie Abteilung Molekulare Evolutionsbiologie Martin-Luther-King-Platz 3, 20146 Hamburg 2 Inhalt Inhalt Abbildungsverzeichnis ......................................................................................................... 7 Tabellenverzeichnis .............................................................................................................. 9 Zusammenfassung .............................................................................................................. 11 1 Einleitung ..................................................................................................................... 12 1.1 Das Phylum Apikomplexa ....................................................................................................... 12 1.1.1 Malaria und malariaähnliche Parasiten ........................................................................... 14 1.1.2 Phylogenetische Beziehungen der Haemosporida ....................................................... 15 1.2 Polychromophilus als Modelorganismus ..................................................................................... 17 1.2.1 Biologie und Ökologie der Wirte .................................................................................... 19 1.2.2 Epidemiologische Aspekte .............................................................................................. 22 1.2.3 Morphologie und Lebenszyklus ...................................................................................... 22 1.3 Parasit-Wirt-Interaktionen auf Proteinebene ........................................................................ 27 1.3.1 Export putativ wirtszell-interagierender Proteine ........................................................ 27 1.3.2 Genomanalyse zur Identifizierung relevanter Proteine und damit verbundene Hürden ................................................................................................................................ 28 1.4 Zielsetzung ................................................................................................................................. 30 2 Material ......................................................................................................................... 31 2.1 Arbeitsgeräte .............................................................................................................................. 31 2.2 Gebrauchsmaterialien ............................................................................................................... 32 2.3 Chemikalien ................................................................................................................................ 32 2.4 Lösungen und Puffer ................................................................................................................ 33 2.5 Molekularbiologische Kits ....................................................................................................... 34 2.6 Enzyme und andere biologische Substanzen ........................................................................ 34 2.7 Oligonukleotide ......................................................................................................................... 35 2.8 Datenbanken und Programme ................................................................................................ 37 3 Methoden..................................................................................................................... 43 3.1 Gewinnung von Untersuchungsmaterial ............................................................................... 43 3.2 Anfertigung von Blutausstrichen und Bestimmung der Parasitämie ................................. 43 3.3 Anreicherung von Oozysten, Mikrogameten und Gametozyten ....................................... 44 3.3.1 Mitteldarmpräparation der Fledermausfliegen ............................................................. 44 3.3.2 In vitro Exflagellation ......................................................................................................... 44 3.3.3 Dichtegradientenzentrifugation in Ficoll ....................................................................... 44 3.4 Isolierung genomischer DNA ................................................................................................. 45 3.4.1 Isolierung genomischer DNA aus Fledermausfliegen ................................................. 45 3 Inhalt 3.4.2 Isolierung genomischer DNA aus Oozysten und Mikrogameten ............................. 45 3.4.3 Isolierung genomischer DNA aus Blut.......................................................................... 45 3.5 Amplifizierung der genomischen DNA ................................................................................. 46 3.6 Selektive Amplifizierung der genomischen DNA ................................................................ 47 3.6.1 Komplementärstrangsynthese ......................................................................................... 47 3.7 PCR-basierte Methoden ........................................................................................................... 48 3.7.1 Nested-PCR ....................................................................................................................... 49 3.7.2 Stepdown-PCR .................................................................................................................. 49 3.7.3 Banden-Reamplifizierung ................................................................................................ 49 3.7.4 Kolonie-PCR ..................................................................................................................... 49 3.7.5 Quantitative Real-Time-PCR .......................................................................................... 50 3.8 Analyse und Aufreinigung von Nukleinsäuren ..................................................................... 52 3.8.1 Konzentrationsbestimmung von Nukleinsäuren ......................................................... 52 3.8.2 Elektrophoretische Auftrennung von Nukleinsäuren ................................................. 52 3.8.3 Extraktion von DNA aus Agarosegelen ........................................................................ 53 3.8.4 Enzymatische Aufreinigung der PCR-Produkte .......................................................... 53 3.8.5 Phenol/Chloroform-Extraktion ..................................................................................... 53 3.8.6 Ethanol- und Isopropanol-Präzipitation ....................................................................... 54 3.9 Klonierungsmethoden .............................................................................................................. 54 3.9.1 Ligation ............................................................................................................................... 54 3.9.2 Transformation und Selektion ........................................................................................ 55 3.9.3 Plasmidpräparation ........................................................................................................... 55 3.10 Sequenzierung ............................................................................................................................ 55 3.10.1 Sanger-Sequenzierung ...................................................................................................... 55 3.10.2 Paired-End-Sequenzierung .............................................................................................. 56 3.10.3 Mate-Pair-Sequenzierung ................................................................................................. 56 3.11 Allgemeine bioinformatische Methoden ................................................................................ 56 3.11.1 DNA-Sequenzanalyse ....................................................................................................... 56 3.11.2 BLAST ................................................................................................................................ 57 3.11.3 Primerdesign ...................................................................................................................... 57 3.11.4 Herstellung der SWGA-Primer ....................................................................................... 58 3.12 Allgemeine Methoden mit NGS-Daten ................................................................................. 58 3.12.1 Bearbeitung
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