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Gerson Ney Hernandez Acevedo UNIVERSIDAD AUTÓNOMA DEL ESTADO DE MORELOS INSTITUTO DE INVESTIGACIÓN EN CIENCIAS BÁSICAS Y APLICADAS CENTRO DE INVESTIGACIÓN EN DINÁMICA CELULAR “Identificación de los complejos proteicos asociados a beta- catenina en linfocitos T CD8 de adultos y neonatos humanos” TESIS QUE PARA OBTENER EL GRADO DE DOCTOR EN CIENCIAS PRESENTA GERSON NEY HERNANDEZ ACEVEDO DIRECTOR DE TESIS Dra. MARIA ANGELICA SANTANA CALDERON CUERNAVACA, MORELOS JUNIO, 2019 Índice 1. INTRODUCCIÓN ........................................................................................................ 7 1.1 El Sistema Inmune .......................................................................................................................................7 1.1.1 Linfocitos T CD8+ o CTL citotóxicos ......................................................................................................................... 9 1.2 Sistema inmune neonatal ....................................................................................................................... 11 1.3 Vía de WNT ................................................................................................................................................. 12 1.3.1 Vía canónica de Wnt ..................................................................................................................................................... 14 1.3.2 Fosforilación de β-catenina/ Degradación por el complejo de degradación .................................... 15 1.3.3 Receptores de Wnt: Frizzled y LRP5/6 ............................................................................................................... 16 1.3.4 Dishevelled ........................................................................................................................................................................ 17 1.3.5 APC ........................................................................................................................................................................................ 17 1.3.6 Axina..................................................................................................................................................................................... 18 1.3.7 Casein kinase 1a (CK1a) ............................................................................................................................................. 19 1.3.8 Glycogen synthase kinase (GSK3) .......................................................................................................................... 19 1.3.9 Antagonistas y agonistas de Wnt............................................................................................................................ 20 1.3.10 Translocación al núcleo de β-catenina.............................................................................................................. 21 1.3.11 LEF/TCF ........................................................................................................................................................................... 22 1.3.12 Coactivadores asociados a β-catenina .............................................................................................................. 22 1.3.13 Estudios fosfoproteomicos de la asociación de CBP y P300 con β-catenina .................................. 24 2. ANTECEDENTES ...................................................................................................... 26 2.1 Señalización de Wnt en la auto-renovación de células madre hematopoyéticas ................... 27 2.2 La vía de señalización de Wnt en la proliferación y diferenciación celular: Papel de dos coactivadores ................................................................................................................................................... 27 2.2.1 Participación de Wnt en HSCs y en el desarrollo de timocitos ................................................................ 31 2.3 Influencia de Wnt en linfocitos T maduros ....................................................................................... 32 2.4 MPEC y SLEC ............................................................................................................................................... 34 3. HIPÓTESIS ............................................................................................................... 36 4. OBJETIVO GENERAL ................................................................................................ 37 4.1 Objetivos específicos ............................................................................................................................... 37 5. MATERIALES Y MÉTODOS ....................................................................................... 38 5.1 Desarrollo experimental: ....................................................................................................................... 38 5.1.1 Colecta de las muestras ............................................................................................................................................... 38 5.1.2 Purificación de linfocitos T CD8+ ............................................................................................................................ 38 5.1.3 Evaluación de pureza por tinción de marcadores de superficie celular ............................................. 40 5.1.4 Estimulación de linfocitos con Wnt3a ................................................................................................................. 40 5.1.5 Evaluación de las proteínas β-catenina, CBP y P300 por tinción intracelular con metanol ...... 40 5.1.6 Citometría de flujo ......................................................................................................................................................... 41 5.1.7 Lisis celular ....................................................................................................................................................................... 41 5.1.8 Inmunoprecipitación de β-catenina, CBP y P300 ........................................................................................... 42 5.1.9 Electroforesis y transferencia de proteínas para inmunoblots ............................................................... 42 5.1.10 Inmunodetección......................................................................................................................................................... 42 5.1.11 Espectrometría de masas ........................................................................................................................................ 43 5.1.12 Diseño de oligos ........................................................................................................................................................... 44 5.1.13 Inmunoprecipitación de cromatina .................................................................................................................... 45 5.1.14 PCR en tiempo real ..................................................................................................................................................... 47 5.1.15 Análisis estadístico ..................................................................................................................................................... 48 5.2 Equipos, marca y modelo ....................................................................................................................... 48 Agitador Enviro-Genie®; model S1-1200 ......................................................................................................................... 48 Balanza analítica Denver Instrument Company; model TR-104 .............................................................................. 48 Rotador IKA® Loopster digital ............................................................................................................................................. 49 5.3 Reactivos con especificaciones (marca, modelo y lote) ................................................................. 49 I 5.4 Disoluciones y composición .................................................................................................................. 50 5.5 Programas .................................................................................................................................................. 51 6. RESULTADOS .......................................................................................................... 53 6.1 Purificación de Linfocitos T CD8+ vírgenes ....................................................................................... 53 6.1.1 Purificación de linfocitos T CD8+ de adultos y neonatos ............................................................................ 53 6.2 Evaluación de β-catenina, CBP y P300 en linfocitos T CD8+ de adultos y neonatos ............... 53 6.3 Inmunoprecipitación de β-catenina en linfocitos T CD8+ de adultos y neonatos ................... 56 6.4 Proteómica ................................................................................................................................................. 59 6.4.1 Identificación de cambios en las vías de Wnt ................................................................................................... 62 6.4.2 Análisis del interactoma ............................................................................................................................................. 63 6.4.3 Expresión
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