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Integrierte Omics-Analysen Zur Charakterisierung Physiologischer Effekte Von Schilddrüsenhormonen Und Von Spezifischen Schilddrüsenhormon-Metaboliten

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Integrierte Omics-Analysen Zur Charakterisierung Physiologischer Effekte Von Schilddrüsenhormonen Und Von Spezifischen Schilddrüsenhormon-Metaboliten Integrierte Omics-Analysen zur Charakterisierung physiologischer Effekte von Schilddrüsenhormonen und von spezifischen Schilddrüsenhormon-Metaboliten INAUGURALDISSERTATION zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Janine Golchert geboren am 08.02.1991 Greifswald, 09.03.2020 Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Uwe Völker 2. Gutachter: Prof. Dr. Klaudia Brix Tag der Promotion: 10.09.2020 Inhaltsverzeichnis Inhaltsverzeichnis Abkürzungsverzeichnis ..................................................................................................... V Zusammenfassung ........................................................................................................... VII Summary ............................................................................................................................ IX 1 Einleitung ...................................................................................................................... 1 1.1 Biosynthese der klassischen Schilddrüsenhormone T3 und T4 in der Schilddrüse . 1 1.2 Transport und Metabolismus der klassischen Schilddrüsenhormone T3 und T4 ..... 3 1.3 Regulation der Schilddrüsenhormone .................................................................... 4 1.4 Molekulare Wirkmechanismen von Schilddrüsenhormonen ................................... 5 1.5 Physiologische Wirkungen von Schilddrüsenhormonen ......................................... 7 1.6 Fehlfunktionen im Schilddrüsenhormonhaushalt ................................................... 8 1.7 Schilddrüsenhormon-Metabolite ............................................................................ 9 1.7.1 3-Iodthyronamin (3-T1AM) .......................................................................... 9 1.7.2 3,5-Diiodthyronin (3,5-T2) ......................................................................... 12 1.8 Die Leber als zentrales Stoffwechselorgan .......................................................... 13 1.8.1 Die Fettleber (Steatosis hepatis) .............................................................. 14 1.9 Wirkungen von Schilddrüsenhormonen auf die Leber.......................................... 14 1.9.1 Wirkungen von T3 auf die Leber ............................................................... 14 1.9.2 Wirkungen von 3,5-T2 auf die Leber ......................................................... 15 1.10 Zielstellungen dieser Arbeit ................................................................................. 17 2 Material ........................................................................................................................ 19 2.1 Biologische Materialien ........................................................................................ 19 2.2 Laborgeräte und Hilfsmittel .................................................................................. 19 2.3 Verbrauchsmaterialien ......................................................................................... 21 2.4 Chemikalien ........................................................................................................ 21 2.5 Enzyme und Antikörper ....................................................................................... 22 2.6 Kits ...................................................................................................................... 23 2.7 Puffer und Lösungen ........................................................................................... 23 2.8 Software und Datenbanken ................................................................................. 26 I Inhaltsverzeichnis 3 Methoden .................................................................................................................... 27 3.1 Arbeiten mit biologischem Material ...................................................................... 27 3.1.1 Tierhaltung und –behandlung .................................................................. 27 3.1.2 Kultivierung von PCCL3-Zellen ................................................................ 28 3.2 Arbeiten mit RNA................................................................................................. 29 ® 3.2.1 RNA-Präparation (TRIzol -Methode) ....................................................... 29 3.2.2 Konzentrationsbestimmung von Nukleinsäuren ....................................... 30 3.2.3 Qualitätskontrolle von RNA ...................................................................... 30 3.2.4 DNase-Verdau und RNA-Aufreinigung ..................................................... 31 3.2.5 Transkriptomanalyse ............................................................................... 31 3.2.6 Auswertung der Transkriptomdaten ......................................................... 34 3.3 Arbeiten mit Proteinen ......................................................................................... 35 ® 3.3.1 Protein-Präparation (TRIzol -Methode) .................................................... 35 3.3.2 Bestimmung der Proteinkonzentration nach Bradford .............................. 36 3.3.3 Trypsinverdau mit vorangehendem Reduzieren und Alkylieren ................ 37 3.3.4 Entsalzung von Peptidlösungen an C18-Material ..................................... 37 3.3.5 Massenspektrometrische Analyse ........................................................... 38 3.3.6 Auswertung der Proteomdaten ................................................................ 40 3.3.7 Eindimensionale SDS-Polyacrylamid-Gelelektrophorese (1D-SDS-PAGE) 42 3.3.8 Western Blot-Analyse .............................................................................. 43 3.4 Arbeiten mit Metaboliten ...................................................................................... 44 3.4.1 Analyse von Sexualhormon-Konzentrationen .......................................... 44 4 Ergebnisse .................................................................................................................. 47 4.1 Wirkungen von 3-T1AM auf das Transkriptom von PCCL3-Zellen........................ 47 4.2 Wirkungen von 3,5-T2 auf das hepatische Transkriptom von Mäusen unter Hochfettdiät ......................................................................................................... 49 4.2.1 Vergleich der Wirkungen von 3,5-T2 auf das hepatische Transkriptom von Mäusen unter Hochfett- und Standarddiät ......................................... 53 4.3 Wirkungen von 3,5-T2 auf das hepatische Proteom von Mäusen unter Standard- und Hochfettdiät .................................................................................................. 55 4.3.1 Pathway-Analysen von 3,5-T2 induzierten Veränderungen des Leberproteoms von Mäusen unter Standard- und Hochfettdiät ................ 60 4.3.2 Ergebnisse der Western Blot-Analysen .................................................... 64 II Inhaltsverzeichnis 4.4 Vergleich der Wirkungen von 3,5-T2 auf das hepatische Transkriptom und Proteom von Mäusen unter Standard- und Hochfettdiät ...................................... 65 4.5 Wirkungen von 3,5-T2 auf Sexualhormon-Spiegel in verschiedenen Geweben .... 72 4.6 Wirkungen von Hypo- und Hyperthyreose auf das hepatische Proteom von Mäusen ............................................................................................................... 74 4.6.1 Pathway-Analysen von Hypo- und Hyperthyreose-induzierten Veränderungen des Leberproteoms von Mäusen .................................... 79 4.6.2 Vergleich der Wirkungen von Hypo- und Hyperthyreose auf das hepatische Proteom und Transkriptom von Mäusen ................................ 83 5 Diskussion .................................................................................................................. 85 5.1 Wirkungen von 3-T1AM auf das Transkriptom von PCCL3-Zellen ........................ 86 5.2 Wirkungen von 3,5-T2 auf das hepatische Transkriptom und Proteom von Mäusen unter Standard- und Hochfettdiät ........................................................... 89 5.2.1 Wirkungen von 3,5-T2 auf das hepatische Transkriptom von Mäusen unter Hochfettdiät .................................................................................... 91 5.2.2 Wirkungen von 3,5-T2 auf das hepatische Proteom von Mäusen unter Standard- und Hochfettdiät ...................................................................... 97 5.2.3 Vergleich der Wirkungen von 3,5-T2 auf das hepatische Transkriptom und Proteom von Mäusen unter Standard- und Hochfettdiät .................. 121 5.3 Wirkungen von T4- und Methimazol-induzierter Hyper- und Hypothyreose auf das hepatische Proteom von Mäusen ................................................................ 123 5.4 Schlussfolgerung und Ausblick .......................................................................... 129 Literaturverzeichnis ........................................................................................................ 131 Abbildungsverzeichnis ................................................................................................... 145 Tabellenverzeichnis ........................................................................................................ 149 Anhang ............................................................................................................................. 151 Eigenständigkeitserklärung ...........................................................................................
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