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Comparative Surface and Molecular Investigations of the Sandfish’S Epidermis (Squamata: Scincidae: Scincus Scincus )“

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Comparative Surface and Molecular Investigations of the Sandfish’S Epidermis (Squamata: Scincidae: Scincus Scincus )“ “Comparative surface and molecular investigations of the sandfish’s epidermis (Squamata: Scincidae: Scincus scincus )“ Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologe Konrad Staudt aus Erlangen Berichter: Prof. Dr. techn. Werner Baumgartner Prof. Dr. rer. nat. Wolfgang Böhme Tag der mündlichen Prüfung: 09.07.2012 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. „Var försiktig med vad du önskar, för det kan hända att du får det “ Content Content Abstract ............................................................................................................................... 7 1. Introduction ............................................................................................................ 9 1.1 Comparative taxonomy of Scincus scincus ...................................................................................... 9 1.2 Skin properties of Scincus scincus ................................................................................................... 10 1.4 The epidermis of Scincus scincus .................................................................................................... 12 1.3 Biomimetic aim ................................................................................................................................ 15 1.5 Aims of the thesis ............................................................................................................................. 16 2. Materials and Methods ........................................................................................... 17 2.1 Materials .......................................................................................................................................... 17 2.1.1 Chemicals and enzymes ................................................................................................................... 17 2.1.2 Devices ............................................................................................................................................. 20 2.1.3 Kits ................................................................................................................................................... 21 2.1.4 Buffers and solutions ....................................................................................................................... 21 2.1.5 Primers ............................................................................................................................................. 25 2.1.6 Animals and skin samples ................................................................................................................ 26 2.2 Surface investigations ...................................................................................................................... 26 2.2.1 Comparative friction angle measurements ....................................................................................... 26 2.2.2 Scanning electron microscopy ......................................................................................................... 27 2.2.3 Atomic force microscopy ................................................................................................................. 28 2.2.4 Skin replicas ..................................................................................................................................... 28 2.2.5 Abrasion resistance .......................................................................................................................... 28 2.2.6 Other possible functions of dorsal serrations ................................................................................... 29 2.3 DNA analysis ................................................................................................................................... 29 2.3.1 General methods .............................................................................................................................. 30 2.3.2 Rapid amplification of cDNA ends (RACE) .................................................................................... 31 2.3.3 RNA hybridization ........................................................................................................................... 34 2.3.4 T4 RNA ligation .............................................................................................................................. 37 2.3.5 Amplification with semi-specific primers ........................................................................................ 39 2.3.6 Sequencing of a β-keratin coding gene fragment of Meroles anchietae .......................................... 43 2.4 Proteomics........................................................................................................................................ 43 2.4.1 General methods .............................................................................................................................. 43 2.4.2 Visualization of β-keratin proteins through specific antibodies ....................................................... 45 2.4.3 Visualization of glycoproteins ......................................................................................................... 45 2.4.4 Comparative glycosylation intensities ............................................................................................. 45 2.4.5 Enzymatical deglycosylation ........................................................................................................... 46 2.4.6 Chemical deglycosylation through β-elimination ............................................................................ 47 2.5 Glycomics ........................................................................................................................................ 49 2.5.1 Prediction of glycosylation sites ...................................................................................................... 49 2.5.2 Silanisation ...................................................................................................................................... 49 III Content 2.5.3 Surface characteristics of immobilised glycans ............................................................................... 50 2.5.4 Quantification of linked glycans ...................................................................................................... 51 2.5.5 Glycan analysis ................................................................................................................................ 51 2.5.6 Monosaccharide classification ......................................................................................................... 53 2.5.7 Glycopeptide analysis ...................................................................................................................... 53 3. Results ..................................................................................................................... 54 3.1 Surface investigations ...................................................................................................................... 54 3.1.1 Comparative friction angle measurements ....................................................................................... 54 3.1.2 Topography investigation by scanning electron microscopy ........................................................... 55 3.1.3 Surface properties of resin replicas of Scincus scincus .................................................................... 55 3.1.4 Other possible functions of dorsal serrations ................................................................................... 56 3.2 DNA analysis ................................................................................................................................... 58 3.2.1 Rapid amplification of cDNA ends (RACE) .................................................................................... 58 3.2.2 RNA hybridization ........................................................................................................................... 58 3.2.3 T4 RNA ligation .............................................................................................................................. 59 3.2.4 Amplification with semi-specific primers ........................................................................................ 59 3.2.5 Sequencing of a β-keratin coding gene fragment of Meroles anchietae ......................................... 60 3.3 Proteomics........................................................................................................................................ 61 3.3.1 In-silico translation of β-keratin coding DNA sequences ................................................................ 61 3.3.2 Visualization of β-keratin proteins through specific antibodies ....................................................... 63 3.3.3 Comparative glycosylation intensities ............................................................................................. 63 3.3.4 Enzymatical deglycosylation ........................................................................................................... 66 3.3.5 Chemical deglycosylation through β-elimination ............................................................................ 67 3.4 Glycomics .......................................................................................................................................
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