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Identification of Deubiquitinating Enzyme Genes Relevant for the Regulation of Retina-Specific Genes

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Identification of Deubiquitinating Enzyme Genes Relevant for the Regulation of Retina-Specific Genes Identification of deubiquitinating enzyme genes relevant for the regulation of retina-specific genes Mariona Esquerdo Barragan Aquesta tesi doctoral està subjecta a la llicència Reconeixement 3.0. Espanya de Creative Commons. Esta tesis doctoral está sujeta a la licencia Reconocimiento 3.0. España de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution 3.0. Spain License. Table of content List of abbreviations ................................................................................................................. 9 Introduction ........................................................................................................................... 13 1. The Retina ...................................................................................................................... 15 1.1. Structure and Function ........................................................................................... 15 1.2. Formation and development .................................................................................. 17 1.3. Photoreceptor Cells ................................................................................................. 20 1.3.1. Morphology and function .............................................................................. 20 1.3.2. Development ................................................................................................. 23 2. Ubiquitin and Ubiquitination ......................................................................................... 27 2.1. Ubiquitin chains structure and function ................................................................. 30 2.2. Deubiquitinating enzymes ...................................................................................... 32 2.2.1. DUB Families .................................................................................................. 33 2.2.2. Physiological relevance of DUBs .................................................................... 40 2.3. Ubiquitin-like molecules: SUMO ............................................................................. 42 3. Ubiquitin and SUMO in photoreceptor development.................................................... 47 Objectives............................................................................................................................... 51 Materials and methods .......................................................................................................... 55 Bacterial transformation and culture ................................................................................. 57 GENE EXPRESSION Constructs............................................................................................ 57 Mammalian Cell culture ..................................................................................................... 58 Transfection IN CELL CultureS ............................................................................................ 58 shRNA silencing .................................................................................................................. 59 siRNA silencing ................................................................................................................... 60 Luciferase assay ................................................................................................................. 61 5 Ethics statement for Animal procedures ........................................................................... 62 Animal handling, tissue dissection and preparation of samples ........................................ 62 RNA extraction and cDNA synthesis ................................................................................... 62 Real Time qPCR .................................................................................................................. 63 In situ hybridization ............................................................................................................ 64 Fluorescent immunohistochemistry .................................................................................. 70 Protein immunodetection by western blot ........................................................................ 72 Immunoprecipitation ......................................................................................................... 73 Mass Spectrometry ............................................................................................................ 74 Phylogenetic analyses ........................................................................................................ 75 Transcriptomics database search ....................................................................................... 76 In vivo DNA electroporation in mouse retinaS ................................................................... 76 Results .................................................................................................................................... 79 Chapter 1. Deubiquitinating enzymes in the mouse retina ............................................... 81 1. Expression pattern of DUBs in the mouse retina ....................................................... 82 1.1. mRNA expression levels .................................................................................... 82 1.2. mRNA localization ............................................................................................. 86 1.3. Protein localization ........................................................................................... 92 2. Sequence and functional conservation of DUBs through phylogeny ......................... 95 2.1. Phylogenetic analysis ........................................................................................ 95 2.2. Phenotypic analysis of DUBs through species ................................................ 101 3. Transcriptomic analysis of DUBs during retinal development ................................. 102 3.1. RNA-sequencing analysis ................................................................................ 103 3.2. Immunohistochemistry assays for RNA-seq data validation .......................... 112 3.3. In vivo DNA electroporation in the mouse retina ........................................... 114 3.4. AON design for intravitreal injection in the mouse retina .............................. 117 Chapter 2. Establishment of a cell system for assaying the activity of retinal transcription factors. ............................................................................................................................. 120 1. Design of the assay ................................................................................................... 120 6 1.1. Rationale ......................................................................................................... 120 1.2. Selection of the cell line and lipofection system ............................................ 123 1.3. Lipofection conditions .................................................................................... 127 1.4. Proof of principle ............................................................................................ 128 2. assessment of DUB silencing in our cellular system ................................................ 130 2.1. shRNA-MEDIATED silencing ............................................................................ 131 2.2. siRNA-MEDIATED silencing ............................................................................. 139 2.3. GapmeR®-MEdiated SILENCING ..................................................................... 148 Chapter 3. Unveiling CRX post-translational modifications. ............................................ 153 1. Evidences in favour of post-translational modifications of CRX .............................. 153 2. Determination of SUMO and/or ubiquitin modifications of CRX ............................. 155 3. CRX Mass-spectometry ............................................................................................ 158 Discussion............................................................................................................................ 161 1. Approaching the function of DUBs in the mouse retina physiology and development ......................................................................................................................................... 163 1.1. Defining a landscape for DUB expression in the mouse retina ............................. 164 1.1.1. Low expression levels in genes involved in the control of cell replication and division ................................................................................................................... 165 1.1.2. Variable expression levels in genes involved in neuronal physiology ......... 166 1.1.3. Layer-specific expression and retinal function ............................................ 167 1.2. Unveiling possible roles for DUBs in retinal development .................................... 168 1.2.1. Transcriptomic analysis showED a wide range of DUB expression patterns during retinal developement ................................................................................. 169 1.2.2. Validating transcriptomic analysis data performing in vivo experiments .... 171 2. Generating a cell culture system to simulate retinal physiological conditions ............ 173 2.1. Establishment of A RETINAL-LIKE cell system ........................................................... 174
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