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Análisis Bioquímico Y Transcriptómico De Eritrocitos Expuestos in Vitro a Metilmercurio En La Tortuga Marina Caretta Caretta

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Análisis Bioquímico Y Transcriptómico De Eritrocitos Expuestos in Vitro a Metilmercurio En La Tortuga Marina Caretta Caretta Análisis bioquímico y transcriptómico de eritrocitos expuestos in vitro a metilmercurio en la tortuga marina Caretta caretta Javier Adolfo Hernández Fernández Pontificia Universidad Javeriana Facultad de Ciencias Doctorado en Ciencias Bogotá DC, Colombia, Julio de 2021 i Análisis bioquímico y transcriptómico de eritrocitos expuestos in vitro a metilmercurio en la tortuga marina Caretta caretta Javier Adolfo Hernández Fernández Tesis presentada como requisito para optar al título de: DOCTOR EN CIENCIAS BIOLÓGICAS DIRECTORA: Ellie Anne López Barrera, Ph.D. CODIRECTOR: Andrés Mauricio Pinzón Velasco, Ph.D. CODIRECTOR: Leonardo Mariño Ramirez, Ph.D. Pontificia Universidad Javeriana Facultad de Ciencias Doctorado en Ciencias Bogotá D.C., Colombia, Julio de 2021 ii Análisis bioquímico y transcriptómico de eritrocitos expuestos in vitro a metilmercurio en la tortuga marina Caretta caretta Javier Adolfo Hernández Fernández Aprobado ___________________________ __________________________ Ellie Anne López Barreta, Ph.D. Andrés Mauricio Pinzón, Ph.D. Tutora Co-tutor ______________________________ __________________________ Leornardo Mariño Ramirez, Ph.D. Ángela Moncaleano, Ph.D. Co-tutor Jurado _____________________________ __________________________ King Jordan, Ph.D. Carlos Carreras Huergo, Ph.D. Jurado Jurado _____________________________ __________________________ Diego Romero García, Ph.D. Miguel Rodríguez Rojas, Ph.D. Jurado Jurado iii Análisis bioquímico y transcriptómico de eritrocitos expuestos in vitro a metilmercurio en la tortuga marina Caretta caretta Javier Adolfo Hernández Fernández Alba Alicia Trespalacios, Ph.D. Jhon Jairo Sutachan Rubio, Ph.D. Decana Facultad de Ciencias Director de Posgrados Ciencias Bogotá, julio de 2021 iv NOTA DE ADVERTENCIA "La universidad no se hace responsable por los conceptos emitidos por sus alumnos en sus trabajos de tesis. Sólo velará porque no se publique nada contrario al dogma y a la moral católica y porque las tesis no contengan ataques personales contra persona alguna, antes bien se vea en ellas el anhelo de buscar la verdad y la justicia" Artículo 23 de la Resolución No. 13 de julio de 1946 v Dedicatoria La energía, el tiempo y la pasión dedicada a este estudio la dedico muy especialmente a mis hijos Pablo Miguel y Gabriela que guían mi camino y son los generadores de mis mayores alegrías y felicidad. vi Agradecimientos Especial agradecimiento a mi directora Ellie Anne López por aceptar dirigir mi tesis, más que una revisora, un apoyo y comprensión, con su visión el trabajo final se consolido. A mi codirector Andrés Pinzón, mis mayores agradecimientos que con sus sugerencias y recomendaciones guio el trabajo. A Leonardo Mariño, un amigo especial, que impulso la idea de hacer el doctorado y claro, discutió minucias de los diferentes capítulos y artículos publicados, gracias totales. A Pilar Rodríguez, asistente de investigación, por su dedicación, trabajo incansable y compromiso inconmensurable, mis más sinceros agradecimientos. A Carlos Zuluaga por su apoyo en la consecución de muestras de sangre de tortugas cabezonas, mi gratitud y aprecio. Al Acuario El Rodadero en Santa Marta, Colombia, gracias por el apoyo técnico. A Alejandro Arias director del Programa de Ciencias Naturales y Ambientales de la UJTL, por su apoyo y el ánimo que me ofreció en cada momento que necesite. A Alejandro López, Ignacio Briceño y Carlos Yesid Soto por las recomendaciones hechas al manuscrito final, mis más sinceros agradecimientos. A la dirección de Investigaciones de la Universidad Jorge Tadeo Lozano, pero muy especialmente a Marcela Lozano por su apoyo y dedicación mis mayores agradecimientos. Al Ministerio del Medio Ambiente y Desarrollo Territorial por los permisos de investigación concedidos para el desarrollo de este proyecto, agradecimientos especiales. Quiero agradecer muy especialmente a la Universidad Jorge Tadeo Lozano por apoyar mi postulación y el desarrollo de mi tesis. vii viii Tabla de contenido Lista de Tablas ....................................................................................................................................... xiii Lista de Figuras ...................................................................................................................................... xiv Lista de figuras ........................................................................................................................................ xv Lista de Anexos ...................................................................................................................................... xvi Resumen .................................................................................................................................................... 1 Abstract ..................................................................................................................................................... 3 Capítulo 1 .................................................................................................................................................. 5 Generalidades de la tortuga cabezona, Caretta caretta y metil-mercurio ................................................ 5 1.1 Caretta caretta, tortuga cabezona ....................................................................................................... 6 1.2 El metilmercurio (MeHg) .................................................................................................................... 8 1.2.1 Mercurio en tortugas marinas .......................................................................................................... 9 1.2.2 Daños producidos por metilmercurio en órganos .......................................................................... 12 1.2.3 Daños producidos por metilmercurio en células ............................................................................ 13 1.2.4 Daños producidos por el metilmercurio a nivel molecular ............................................................ 13 1.2.5 Metilmercurio y transcriptómica .................................................................................................... 14 1.2.6 Interacciones del mercurio ............................................................................................................. 16 1.3 Eritrocitos nucleados ......................................................................................................................... 19 1.4 Referencias bibliográficas ................................................................................................................. 21 Capítulo 2 ................................................................................................................................................ 28 2.1 Resumen del capítulo ........................................................................................................................ 29 2.2 Introducción ...................................................................................................................................... 29 2.3 Materiales y métodos ........................................................................................................................ 31 2.3.1 Área de estudio, recolección y análisis de muestras ...................................................................... 31 2.3.2 Diseño del experimento ................................................................................................................. 33 2.3.3 Procedimientos experimentales ...................................................................................................... 34 2.3.3.1 Aislamiento y pre-adaptación de eritrocitos de tortuga cabezona ............................................... 34 2.3.3.2 Determinación de CL50 de metilmercurio en eritrocitos de tortuga cabezona ............................ 35 2.3.3.3 Determinación de la viabilidad celular de los eritrocitos de las tortugas cabezonas .................. 35 2.3.3.4 Estrés oxidativo de los eritrocitos de la tortuga cabezona .......................................................... 36 2.3.3.4 Análisis de los datos .................................................................................................................... 38 2.4 Resultados ......................................................................................................................................... 39 2.4.1 Conteo sanguíneo y estado de salud de las tortugas cabezonas ..................................................... 39 ix 2.4.2. Cuantificación de mercurio en eritrocitos de tortugas cabezonas ................................................. 39 2.4.3 Concentración letal (CL50) y viabilidad celular de eritrocitos de tortugas cabezonas ................... 40 2.4.4 Bioensayo y estrés oxidativo.......................................................... ¡Error! Marcador no definido. 2.4.4 Bioensayo y estrés oxidativo.......................................................................................................... 42 2.5 Discusión ........................................................................................................................................... 44 2.6 Conclusiones ..................................................................................................................................... 51 2.7 Referencias bibliográficas ................................................................................................................. 52 Capítulo 3 ...............................................................................................................................................
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