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Caracterización De Complejos CDK-Ciclina Atípicos Humanos Eva Quandt Herrera

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Caracterización De Complejos CDK-Ciclina Atípicos Humanos Eva Quandt Herrera Caracterización de complejos CDK-Ciclina atípicos humanos Eva Quandt Herrera ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tesisenxarxa.net) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. 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On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tesisenxarxa.net) service has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized neither its spreading and availability from a site foreign to the TDX service. Introducing its content in a window or frame foreign to the TDX service is not authorized (framing). This rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. Caracterización de complejos CDK-Ciclina atípicos humanos Eva Quandt Herrera Tesis Doctoral 2017 Universitat Internacional de Catalunya Facultat de Medicina i Ciències de la Salut UNIVERSITAT INTERNACIONAL DE CATALUNYA FACULTAD DE MEDICINA Y CIENCIAS DE LA SALUD CARACTERIZACIÓN DE COMPLEJOS CDK-CICLINA ATÍPICOS HUMANOS Eva Quandt Herrera 2017 CARACTERIZACIÓN DE COMPLEJOS CDK-CICLINA ATÍPICOS HUMANOS Eva Quandt Herrera Memoria presentada para optar al título de doctor por la Universitat Internacional de Catalunya, Barcelona 2017 Trabajo dirigido por el Dr. Josep Clotet Erra del Departamento de Ciencias Básicas de la Universitat Internacional de Catalunya Programa de Doctorado en Ciencias de la Salud de la Universitat Internacional de Catalunya Dr. Josep Clotet Erra Eva Quandt Herrera Director de tesis Doctoranda ÍNDICE ÍNDICE ÍNDICE ÍNDICE ÍNDICE ........................................................................................................................................................... 1 INTRODUCCIÓN ......................................................................................................................................... 1 1. EL CICLO CELULAR ............................................................................................................................................. 3 1.1 VISIÓN GENERAL DEL CICLO CELULAR .................................................................................................. 3 1.2 QUINASAS DEPENDIENTES DE CICLINA ................................................................................................ 4 1.3 COMPLEJOS CDK-CICLINA DE CICLO CELULAR ................................................................................... 6 2. CICLINAS Y CDKS DE TRANSCRIPCIÓN ...................................................................................................... 9 3. CICLINAS Y CDKS ATÍPICAS .......................................................................................................................... 11 3.1 NUEVAS CDKs ................................................................................................................................................... 12 3.2 CICLINAS ATÍPICAS ....................................................................................................................................... 14 3.2.1 SPY1 ................................................................................................................................................................................................ 15 3.2.2 CCNI, CCNG1 y CCNG2 ............................................................................................................................................................ 16 3.2.3 CNTD2 ........................................................................................................................................................................................... 19 3.2.4 CCNY ............................................................................................................................................................................................... 19 OBJETIVOS ................................................................................................................................................ 23 MATERIALES Y MÉTODOS ................................................................................................................... 27 1. CONSTRUCCIÓN DE DENDROGRAMAS Y DIAGRAMAS DE ESTRUCTURAS PROTEÍNICAS ........................................................................................................................................................................................ 29 2. CONSTRUCCIONES PLASMÍDICAS ............................................................................................................. 29 3. ENSAYO DE DOBLE HÍBIDO EN LEVADURA ......................................................................................... 30 3.1 Transformación de levaduras ................................................................................................................................................. 30 3.2 Mating ............................................................................................................................................................................................... 31 3.3 Evaluación de la capacidad de crecimiento de los clones positivos ....................................................................... 31 3.4 Preparación de extractos proteínicos de levadura para WB ..................................................................................... 31 4. TÉCNICAS DE BIOLOGÍA CELULAR ........................................................................................................... 32 4.1 Cultivo celular y transfección .................................................................................................................................................. 32 4.2 Preparación de extractos celulares ...................................................................................................................................... 32 4.3 Preparación de extractos de cerebro de rata ................................................................................................................... 33 5. TÉCNICAS BIOQUÍMICAS ............................................................................................................................... 33 5.1 Expresión y purificación de proteínas fusionadas a GST ............................................................................................ 33 5.2 Unión covalente (crosslinking) de anticuerpos a esferas magnéticas ................................................................... 35 5.3 Ensayo de traPPing ...................................................................................................................................................................... 35 5.4 Co-Inmunoprecipitación ........................................................................................................................................................... 35 5.5 Tiofosforilación y captura de sustratos de CDK16 ........................................................................................................ 36 5.6 Ensayo de fosforilación in vitro .............................................................................................................................................. 37 5.7 Análisis de proteínas mediante Western Blot ................................................................................................................. 38 6. Análisis MS ........................................................................................................................................................... 39 RESULTADOS ........................................................................................................................................... 41 PARTE 1 ..................................................................................................................................................................... 43 IDENTIFICACIÓN DE NUEVOS COMPLEJOS CDK-CICLINA .................................................................
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