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Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Unidad Zacatenco T E S

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Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Unidad Zacatenco T E S CENTRO DE INVESTIGACIÓN Y DE ESTUDIOS AVANZADOS DEL INSTITUTO POLITÉCNICO NACIONAL UNIDAD ZACATENCO DEPARTAMENTO DE GENÉTICA Y BIOLOGÍA MOLECULAR “Seguimiento de la Variabilidad de la Microbiota Intestinal durante un Tratamiento con Dieta Cetogénica para Control de Peso en Pacientes con Obesidad y Diabetes tipo 2” T E S I S Que presenta L. N. CYNTHIA ARACELY LÓPEZ TORRES Para obtener el grado de MAESTRA EN CIENCIAS EN LA ESPECIALIDAD DE GENÉTICA Y BIOLOGÍA MOLECULAR Director de la Tesis: Dr. en C. Jaime García Mena. Ciudad de México Agosto 2016 El presente trabajo se realizó en el Laboratorio de Referencia y Apoyo para la Caracterización de Genomas, Transcriptomas y Microbiomas del Departamento de Genética y Biología Molecular, bajo la dirección del Dr. en C. Jaime García Mena en el Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Zacatenco y en colaboración con el Dr. Víctor Armando Martinez Silva, Dra. Virginia Samaniego Méndez y la Dra Arlen Martínez Alvarado. Se extiende un agradecimiento al CINVESTAV, al Biól. Alberto Piña Escobedo, por su apoyo técnico, a Rodrigo García Gutiérrez por su asistencia en el material de trabajo, al Dr. Selvasankar Muruesan y M.C. Otoniel Maya Lucas por sus contribuciones a lo largo del proyecto. Para la realización de este trabajo se recibió apoyo del Cinvestav, CONACyT 163235 INFR-2011-01 y FONSEC SS/IMSS/ISSSTE- CONACYT-233361. L.N. Cynthia Aracely López Torres recibió el apoyo de la beca CONACYT 339410. ii AGRADECIMIENTOS Quiero agradecer al Dr. en C. Jaime García Mena por haberme aceptado en su laboratorio y por su valiosa contribución en el desarrollo del proyecto. A todos mis compañeros de laboratorio, sobre todo al Dr. Selvasankar Murugesan y al M.C Otoniel Maya Lucas por su entera disposición y apoyo en el desarrollo del proyecto, así como a mis asesores Dr. Efraín Garrido Guerrero y Dr. Roberto Ruíz Medrano siempre atentos a mis necesidades. A mis compañeros y amigos de generación que en conjunto nos apoyamos para cumplir esta meta. A mis compañeras y amigas: Dra. Rosa Ma. Pineda, IBT Valeria Díaz Sabillón, IBT Daniela Ramírez Sánchez, Med. Cir. Janet Pineda por su apoyo y sobre todo su amistad. Al personal administrativo del departamento de Genética y Biología Molecular, especialmente a Gabriela García Mora por su importante labor administrativa de la coordinación académica y su siempre amable atención. Un especial agradecimiento a la Dra. Virginia Samaniego Méndez, Dr. Armando Martinez Silva y Dra. Arlen Martínez Alvarado por su contribución al desarrollo del proyecto, sin ellos esto no habría sido posible. Al M.C. Francisco Armendáriz Toledano por todas sus asesorias. A Dios, mi familia, mis padres, pareja y amigos por su apoyo siempre incondicional. ¡A todos muchas gracias! iii ÍNDICE ÍNDICE ........................................................................................................... iv ÍNDICE DE FIGURAS .................................................................................... vi ÍNDICE DE TABLAS ...........................................¡Error! Marcador no definido. NOTACIÓN .................................................................................................... ix RESUMEN ...................................................................................................... x ABSTRACT ................................................................................................... xi I. INTRODUCCIÓN ......................................................................................... 1 Obesidad ........................................................................................................................................ 1 Epidemiología. ....................................................................................................................................... 1 Definición y clasificación. .................................................................................................................. 4 Consecuencias de la obesidad en la salud. ........................................................................... 6 Diabetes tipo 2. ...................................................................................................................................... 8 Factores que contribuyen al desarrollo de obesidad. ..................................................... 10 Microbiota intestinal .......................................................................................................................... 12 Alternativas terapéuticas en obesidad. .................................................................................. 14 Dieta cetogénica. ............................................................................................................................... 16 II. JUSTIFICACIÓN ...................................................................................... 18 III. ALCANCE ............................................................................................... 18 IV. HIPÓTESIS ............................................................................................. 19 V. OBJETIVO GENERAL ............................................................................. 19 VI. OBJETIVOS PARTICULARES ............................................................... 19 VII. METODOLOGÍA .................................................................................... 20 VII. 1 PLAN DE TRABAJO .................................................................................................... 20 VII. 2 DISEÑO EXPERIMENTAL Y MÉTODOS .............................................................. 21 Selección de pacientes ......................................................................................................... 22 Instauración aleatoria de un tipo de régimen: cetogénico o mediterráneo .......... 24 Obtención y análisis de variables antropométricas y bioquímicas .................. 28 Recolección de muestras de copro ................................................................................ 30 Caracterización de la microbiota bacteriana intestinal a partir de las muestras de copro .................................................................................................................. 31 Secuenciación masiva semiconductora de iones. ............................................................ 34 Análisis de datos de secuenciación y diversidad microbiana. .................................... 35 Relación entre Variables Antropométricas y Bioquímicas vs. Diversidad ............ 37 VIII. RESULTADOS ...................................................................................... 38 Perfil Clínico de Pacientes Sometidos a Dieta Cetogénica .................................. 38 Perfil Clínico de Pacientes Sometidos a Dieta Mediterránea ............................... 42 Análisis Multivariado de las variables Antropométricas y Bioquímicas ........ 45 Caracterización de la Microbiota Intestinal Bacteriana a partir de las Muestras de Copro ................................................................................................................. 50 Generación de genotecas V3 del 16S rDNA. ...................................................................... 51 iv Estructura y Diversidad de la Comunidad Bacteriana ............................................ 52 Estructura de la comunidad (alfa diversidad) ...................................................................... 53 Estructura de la Comunidad (beta diversidad) ................................................................... 59 Relación entre Variables Antropométricas y Bioquímicas vs. Diversidad Microbiana .................................................................................................................................. 65 IX. DISCUSIÓN ............................................................................................. 70 X. CONCLUSIONES ..................................................................................... 77 XI.PERSPECTIVAS ...................................................................................... 78 XII.BIBLIOGRAFÍA ....................................................................................... 79 XIII.ANEXOS ................................................................................................ 83 ANEXO A. Cronograma de actividades del proyecto de investigación ......... 83 ANEXO B. Instrucciones para toma de muestra de copro .................................... 84 ANEXO C. Consentimiento informado para los pacientes .................................... 86 ANEXO D. Menús del régimen tipo cetogénico ......................................................... 91 ANEXO E. Ejemplos de menús hipocalóricos de 7 días para pacientes ...... 105 ANEXO F. Listado de alimentos para elaboración de menús para pacientes asignados a dieta control .................................................................................................. 107 ANEXO G. Hoja de Diario de alimentos para pacientes ....................................... 109 ANEXO H. Concentraciones de DNA genómico de muestras de pacientes de dieta mediterránea ................................................................................................................ 110 ANEXO I. Concentraciones de DNA genómico de muestras de pacientes de dieta cetogénica ..................................................................................................................... 112 ANEXO J. Lista de primers con barcodes
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