Method Development for Valid High-Resolution Profiling Of

Method Development for Valid High-Resolution Profiling Of

Method development for valid high‐resolution profiling of mitochondria and Omics investigation of mitochondrial adaptions to excess energy intake and physical exercise Dissertation der Mathematisch‐Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Lisa Claudia Charlotte Kappler aus Weingarten Tübingen 2018 Gedruckt mit Genehmigung der Mathematisch‐Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 27.03.2018 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatterin: Prof. Dr. Carolin Huhn 2. Berichterstatter: Prof. Dr. Rainer Lehmann Over the long term, symbiosis is more useful than parasitism. More fun, too. Ask any mitochondria. ‐Larry Wall‐ Scopes and aims of the thesis Index 1 SCOPES AND AIMS OF THE THESIS .............................................................................. 1 2 INTRODUCTION ................................................................................................................. 2 2.1 Insulin resistance and type 2 diabetes ................................................................................................. 2 2.1.1 Diet and physical activity ...................................................................................................................... 3 2.2 Mitochondria, structure and function ................................................................................................. 3 2.2.1 Function of mitochondria ..................................................................................................................... 3 2.2.1.1 Tissue‐specificity of mitochondria ............................................................................................... 4 2.2.1.2 Oxidative stress ............................................................................................................................ 5 2.2.2 Mitochondrial lipids ............................................................................................................................. 6 2.2.2.1 (Patho)physiological role of mitochondrial lipids ........................................................................ 6 2.2.2.2 Oxysterols, a lipid class of oxidised sterols .................................................................................. 6 2.2.3 Dysfunction of mitochondria ................................................................................................................ 7 2.2.3.1 Dysfunction of mitochondria and insulin resistance .................................................................... 7 2.3 Investigation of mitochondrial composition and function .................................................................... 8 2.3.1 Mitochondrial purification ................................................................................................................... 8 2.3.2 Mitochondrial respiration: Investigation of OXPHOS capacity ............................................................. 9 2.3.2.1 Complex 1 linked substrates‐ NADH (Pyruvate/Glutamate and Malate) ..................................... 9 2.3.2.2 Complex 2 associated substrate‐ Succinate ............................................................................... 10 2.3.2.3 Complex 1 and 2 associated substrates‐ Octanoyl‐/Palmitoylcarnitine and Malate ................. 10 2.3.3 Lipidome analyses of mitochondria ................................................................................................... 11 2.3.3.1 Reversed‐phase (RP) chromatography ...................................................................................... 11 2.3.3.2 Electrospray ionisation (ESI) ...................................................................................................... 12 2.3.3.3 Mass spectrometry (MS) ............................................................................................................ 12 2.3.3.4 Tandem mass spectrometry (MS/MS) ....................................................................................... 13 2.3.3.5 “Top down” and “bottom up” lipidomics .................................................................................. 13 3 MATERIAL AND METHODS ........................................................................................... 15 3.1 Material ........................................................................................................................................... 15 3.1.1 Chemicals ........................................................................................................................................... 15 3.1.2 Buffers and solutions .......................................................................................................................... 18 3.1.2.1 General buffers .......................................................................................................................... 18 3.1.2.2 Respiratory measurement buffers for high resolution respirometry (HRR) and Seahorse XF96 measurements ........................................................................................................................... 20 3.1.2.3 Citrate synthase (CS) activity assay buffers ............................................................................... 23 3.1.3 Cells, animals and human biopsies ..................................................................................................... 23 3.1.3.1 Cells ............................................................................................................................................ 23 3.1.3.2 Animals ...................................................................................................................................... 23 Scopes and aims of the thesis 3.1.3.2.1 Mouse diets ................................................................................................................... 24 3.1.3.3 Human biopsies and tissues ...................................................................................................... 24 3.1.4 Gels .................................................................................................................................................... 25 3.1.5 Culture media and supplements ....................................................................................................... 26 3.1.6 Kits ..................................................................................................................................................... 27 3.1.7 Internal standards for lipidomics analysis ......................................................................................... 27 3.1.8 Enzymes and inhibitors ...................................................................................................................... 28 3.1.9 Molecular markers ............................................................................................................................ 29 3.1.10 Consumables ..................................................................................................................................... 29 3.1.11 Laboratory equipment ....................................................................................................................... 30 3.1.12 Software ............................................................................................................................................ 31 3.1.13 Primers .............................................................................................................................................. 32 3.1.13.1 Primers for real time PCR .......................................................................................................... 32 3.1.14 Antibodies.......................................................................................................................................... 32 3.1.14.1 Primary antibodies .................................................................................................................... 32 3.1.14.2 Secondary antibodies ................................................................................................................ 33 3.2 Methods ........................................................................................................................................... 34 3.2.1 Cell culture ......................................................................................................................................... 34 3.2.1.1 Cultivation, passaging and seeding for experiments ................................................................ 34 3.2.1.1.1 HEPG2 hepatoma cells ................................................................................................... 34 3.2.1.1.2 C2C12 mouse skeletal muscle cells ................................................................................ 35 3.2.1.2 Cell lysis ..................................................................................................................................... 35 3.2.1.3 Cryopreservation of cell lines .................................................................................................... 35 3.2.2 Animal studies ................................................................................................................................... 36 3.2.2.1 Tissue lysis ................................................................................................................................

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