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Universidad Nacional De La Plata Facultad De Ciencias Exactas Departamento Biología UNIVERSIDAD NACIONAL DE LA PLATA FACULTAD DE CIENCIAS EXACTAS DEPARTAMENTO BIOLOGÍA Trabajo de Tesis Doctoral Estudio de las melaninas de Pseudocercospora griseola, agente causante de la mancha angular del poroto y rol biológico. Tesista Lic. Alejandra Bárcena Director Dr. Mario, Carlos Nazareno Saparrat, Codirector Dr. Pedro Alberto Balatti Asesora académica Dra. Mariela, Bruno Año 2016 AGRADECIMIENTOS A la Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICBA) y a la Comisión Nacional de Investigaciones Científicas y Tecnológicas (CONICET) por las becas otorgadas. A la Universidad Nacional de La Plata, Facultad de Ciencias Exactas por permitirme realizar esta investigación. Al Dr. Mario Saparrat y el Dr. Pedro Balatti, por guiarme, enseñarme, entusiasmarme cuando los resultados eran como esperábamos, y por estimularme cuando así no era. Por cada una de nuestras discusiones para enriquecer el trabajo. Por enseñarme que la paciencia es la madre de la ciencia y el esfuerzo su padre. A la Dra. Mariela Bruno, por ser mi Asesora Académica, por escucharme, por sus palabras de aliento en los momentos más difíciles con esa ternura que la caracteriza. A la Dra. Laura López por asesorarme cuando comencé la tesis. A la Dra. Fernanda Rosas, Dra. María Mirifico, Dra Gabriela Petroselli, Dra Rosa Erra- Balsells, Dra. Ana Gennaro, Dr. José Estévez, por su colaboración con los espectros FTIR, UV MALDI-TOF, EPR, microscopía confocal, y sobre todo por la buena predisposición que siempre demostraron. A los chicos del laboratorio Carla, Darío, Ernest, Grace, Inés, Pepe, Silvina, Rocío, por estar siempre dispuestos a dar una mano en cuestiones de estadística, biología molecular, y también por los lindos momentos extra laborales. Al INFIVE y a cada uno de sus integrantes por hacer que cada día fuera un placer ir a trabajar. A Agus, Ali, Ana, Lucía, Iné, Fer, Pepe, Diego, Emi, Anita, Mari, Andre, Facu, Nico, Canari, Cristian, Diana, Laurita, Mari, Nadia, Guille, Santi, Taty, por las charlas y discusiones laborales, por compartir los almuerzos al sol (en invierno) o a la sobra (en verano en el Botánico) y cenas por fuera del trabajo con alguna que otra cervecita artesanal. A los amigos que conocí en este lugar, Agus, Emi, Santi, Pepe que sé que aunque nuestras vidas tomen rumbos distintos ellos siempre van a estar para compartir una charla, una cerveza o un momento. i A los amigos de la facultad, Ana, Cacho, Canari, Caro, Cristian, Diego, Lara, María, Pau, Tincho, de los cuales algunos también fueron compañeros de trabajo. A Ceci, Mari, Pau y Romi, por nuestras salidas y charlas. A las chicas de Maca, Carli, Dani, Pao, Tati y Vale, por sus palabras de aliento con cada uno de los capítulos que terminaba, por estar siempre por más que el tiempo y la distancia nos separan desde hace más de 10 años. A Sebastián, por ser mi compañero durante una gran parte de este camino. A Ángel, por la colaboración con las imágenes, y a él y Grace por bancarme en especial en el último tiempo. A mi familia, Papi, Mami, Dai y Nahuel por bancarse mis estados anímicos en la etapa de escritura, por interesarse y preguntar por mi trabajo. GRACIAS por acompañarme y entender mis elecciones. ii ÍNDICE AGRADECIMIENTOS .......................................................................................... i ÍNDICE ............................................................................................................... iii ÍNDICE DE TABLAS ......................................................................................... vii ÍNDICE DE FIGURAS ........................................................................................ ix ABREVIATURAS .............................................................................................. xiv RESUMEN ....................................................................................................... xvi ABSTRACT .................................................................................................... xviii CAPÍTULO I: INTRODUCCIÓN GENERAL Introducción general ..................................................................................................... 1 1.1. Pigmentos .............................................................................................................. 4 1.2. ¿Qué son las melaninas? ...................................................................................... 6 1.3. Diversidad de melaninas ........................................................................................ 7 1.4. Características y propiedades de las melaninas .................................................. 12 1.5. Roles de las melaninas ........................................................................................ 15 1.5.1. Resistencia a estreses ambientales ............................................................. 15 1.5.2. Patogénesis y virulencia ............................................................................... 17 1.6. Sitios de deposición ............................................................................................. 19 1.7. Aplicaciones ......................................................................................................... 20 1.8. Pseudocercospora griseola: un hongo bajo estudio ............................................. 21 Hipótesis ..................................................................................................................... 24 Objetivos ..................................................................................................................... 24 CAPÍTULO II: VARIABILIDAD MORFOLÓGICA Y DEPOSICIÓN DE MELANINAS EN REPRESENTANTES DE LOS GRUPOS GÉNICOS DE PSEUDOCERCOSPORA GRISEOLA. 2.1. Introducción ......................................................................................................... 26 Objetivos ..................................................................................................................... 31 Hipótesis ..................................................................................................................... 31 2.2. Materiales y métodos ........................................................................................... 32 2.2.1. Aislamientos de Pseudocercospora griseola. ............................................... 32 2.2.2. Conservación de los aislamientos. ............................................................... 32 2.2.3. Inoculación y condiciones de cultivo de los hongos ...................................... 33 2.2.4. Estimación del crecimiento miceliar. ............................................................. 33 iii 2.2.5. Análisis de la pigmentación de las colonias. ................................................. 34 2.2.6. Análisis microscópicos de P. griseola en cultivos sólidos y en la presencia de Triciclazole. ................................................................................................................. 34 2.2.7. Análisis estadístico ....................................................................................... 36 2.3. Resultados ........................................................................................................... 37 2.3.1. Crecimiento y pigmentación de las colonias ................................................. 37 2.3.2. Análisis microscópicos de P. griseola en cultivos sólidos y en la presencia de triciclazole. ............................................................................................................. 42 2.4. Discusión ............................................................................................................. 47 CAPÍTULO III: CARACTERIZACIÓN DE LAS MELANINAS DE PSEUDOCERCOSPORA GRISEOLA 3.1. Introducción ......................................................................................................... 53 Objetivos ................................................................................................................ 55 Hipótesis ................................................................................................................ 55 3.2. Materiales y métodos ........................................................................................... 56 3.2.1. Cultivos de Pseudocercospora griseola. ....................................................... 56 3.2.2. Extracción de los pigmentos. ........................................................................ 56 3.2.3. Espectroscopía UV-visible. ........................................................................... 57 3.2.4. Espectroscopía de fluorescencia. ................................................................. 57 3.2.5. Espectroscopía de resonancia paramagnética electrónica. .......................... 58 3.2.6. Espectroscopía infrarroja transformada de Fourier. ...................................... 58 3.2.7. Determinación de grupos fenólicos. .............................................................. 59 3.2.8. Identificación de intermediarios de la síntesis de melanina por UV-MALDI MS.60 3.2.9. Actividad antioxidante de melaninas. ............................................................ 61 3.2.10. Actividad quelante de melaninas. ............................................................... 62 3.2.11. Análisis estadístico ..................................................................................... 63 3.3. Resultados ........................................................................................................... 64 3.3.1. Espectroscopía UV-visible ...........................................................................
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