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Aspectos Biológicos Y Químicos Del Alga Roja Gymnogongrus Torulosus (Hooker Et Harvey) Schmitz (Phyllophoraceae, Rhodophyta) Estevez, José Manuel 2003

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Aspectos Biológicos Y Químicos Del Alga Roja Gymnogongrus Torulosus (Hooker Et Harvey) Schmitz (Phyllophoraceae, Rhodophyta) Estevez, José Manuel 2003 Tesis de Posgrado Aspectos biológicos y químicos del alga roja gymnogongrus torulosus (Hooker et Harvey) Schmitz (Phyllophoraceae, Rhodophyta) Estevez, José Manuel 2003 Tesis presentada para obtener el grado de Doctor en Ciencias Biológicas de la Universidad de Buenos Aires Este documento forma parte de la colección de tesis doctorales y de maestría de la Biblioteca Central Dr. Luis Federico Leloir, disponible en digital.bl.fcen.uba.ar. Su utilización debe ser acompañada por la cita bibliográfica con reconocimiento de la fuente. This document is part of the doctoral theses collection of the Central Library Dr. Luis Federico Leloir, available in digital.bl.fcen.uba.ar. It should be used accompanied by the corresponding citation acknowledging the source. Cita tipo APA: Estevez, José Manuel. (2003). Aspectos biológicos y químicos del alga roja gymnogongrus torulosus (Hooker et Harvey) Schmitz (Phyllophoraceae, Rhodophyta). Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. http://digital.bl.fcen.uba.ar/Download/Tesis/Tesis_3677_Estevez.pdf Cita tipo Chicago: Estevez, José Manuel. "Aspectos biológicos y químicos del alga roja gymnogongrus torulosus (Hooker et Harvey) Schmitz (Phyllophoraceae, Rhodophyta)". Tesis de Doctor. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. 2003. http://digital.bl.fcen.uba.ar/Download/Tesis/Tesis_3677_Estevez.pdf Dirección: Biblioteca Central Dr. Luis F. Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Contacto: [email protected] Intendente Güiraldes 2160 - C1428EGA - Tel. (++54 +11) 4789-9293 UNIVERSIDAD DE BUENOS AIRES ASPECTOS BIOLOGICOS Y QUIMICOS DEL ALGA ROJA GYMNOGONGRUS TORULOSUS (HOOKER ET HARVEY) SCHMITZ (PHYLLOPHORACEAE, RHODOPHYTA) José Manuel Estevez DIRECTOR:Dr. Alberto S. Cerezo CO-DIRECTOR:Dr. Eduardo J. Cáceres *-—-c «w-N Tesis presentada para optar al Titqu de DOCTOR DE LA UNIVERSIDAD DE BUENOS AIRES Departamento de Biodiversidad y Biología Experimental 2003 5.0.0.0....OOOOOOOOOOOOOQOOOOOOOOOOOOOOÓOOOOOOO... AGRADECIMJENTOS Al Dr. Alberto S. Cerezo, quien dirigió este trabajo de Tesis, por haberme transmitido su amplia experiencia en el estudio de polisacáridos, además del constante estímulo, apoyo y afecto. Al Dr. Eduardo J. Cáceres, por haberme recibido en su laboratorio, además de su dirección en los aspectos relacionados con la Microscopía Electrónica de Transmisión. A la Dra. Marina Ciancia, por su valiosa guía y comprensión durante toda la realización de esta Tesis. Además, por su calidad humana, rectitud y tenacidad para superar los problemas. Le agradezco también, la lectura critica del manuscrito de la Tesis y los innumerables consejos recibidos. A mi familia, justamente, por ser mi familia. A mis padres, por el amor incondicional que siempre me brindaron y por enseñarme que las cosas importantes sólo se consiguen con esfuerzo. A mis hermanos Fran, Maria y Male, por saber que siempre estánjunto a mí. A mis tlos y primos, por el cariño recibido desde siempre. A Josefina Pons, por ser mi “hermana” del alma. A mis cinco sobrinos, Josefina, Lunita, Manolo, Morita y Rocío, que me quieren y los quiero mucho. A Guille, por ser mi mejor amigo y tan buena persona. A Marco y Mario, por la amistad de tantos años. A Andrea Lavigne y Josefina Alberghina, por ser ambas excelentes personas a las que aprecio mucho y por su aliento constante, para que nunca bajara los brazos en este camino recorrido. A Josefina A. además, por ayudarme con los cultivos de carposporas. Al Dr. Carlos A. Stortz. por su honestidad y sentido del bien. A la Dra. Cristina Matulewicz, por su humildad y buena predisposición para ayudar. A la Dra. Elsa Damonte, Dr. Carlos Pujol y Dra. Josefina Carlucci, por la determinación de la actividad antiviral de los polisacáridos. A Carlos Pujol además, por la lectura critica del Capítulo de Actividad Antiviral. A la Dra. Carmen Pujals, por permitinne consultar su bibliografia A la Dra. Suzanne Fredericq, por la secuenciación del material de Gymnogongrus Iorulosus. A la Lic. Maria Luz Piriz, por la ayuda en la recolección de Gymnogongrus de Santa Isabel, Chubut. A Andrea Ponce, Diego, Cecilia y Gustavo, por compartir buenos momentos en el laboratorio, tenerme paciencia y'ser buenos compañeros. A Andrea y Diego, también, por danne una mano en los momentos críticos. A Adriana, María lnés y Carlos Lima, por su buena onda. A Adriana, además por los CGL-EM. A Carlos Lima, además, por sus buenos consejos en los momentos justos. A Susana Vigna, Memo V., Teresita W., Alicia B., Nora M., Susana P., por las horas de docencia compartidas. A Roxi, Coti y Mariana B., por la buena onda que siempre me transmitieron. A la Coneja y Amelia, por su calidez humana. A Patricia Leonardi, por su ejemplo y humildad. A toda la gente del laboratorio de Micología y Ficologia de Bahía Blanca, por su ayuda cuando estuve trabajando con ellos. A los Ficólogos M. Adela C., Eugenia S., lnés S., Taty C., Jose Man'a G., Marta F., Ricardo E., Silvia S., Dr. Guarrera, y Micólogos del Museo de la Plata como la Vasca Arambarri, Claudia, Cecilia C., Vilma R. por los primeros pasos recorridos juntos tanto en la docencia como en la investigación. A los operadores del RMN del Departamento de Quimica Orgánica, por los numerosos espectros nocturnos. A Gcmot y a José, por su excelente disposición. A Jorge Aznares, por estar siempre a dispuesto a dar una mano. Al Centro de Estudios en Hidratos de Carbono (ClHlDECAR-CONlCET) por brindanne un lugar de trabajo y ofrecerme la infraestructura para llevar a cabo el desarrollo experimental de algunos aspectos de esta Tesis. A los Departamentos de Química Orgánica y Biodiversidad-Biología Experimental de la Facultad de Ciencias Exactas y Naturales de la Universidad de Buenos Aires, por brindanne la posibilidad de hacer esta Tesis. A Mabel, Maripi, Olga, Rosa y Sergio, del Departamento de Química Orgánica, y a Ana, Claudio y Silvina, del Departamento de Biodiversidad y Biologia Experimental, por su ayuda constante y buena voluntad. Al Centro de lnvestigaciones Básica y Aplicadas de Bahía Blanca (CRIBBAB), por permitirme utilimr el Microscopio Electrónico de Transmisión. Al Consejo Nacional de Investigaciones Cientificas y Tecnológicas (CONICET) y a la Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC), por las becas de Postgrado que me otorgaron para realizar esta Tesis. A la Phycological Society of America (PSA) por los subsidios otorgados como alumno de doctorado, durante los años 2002-2004, para la realización de algunos aspectos de esta investigación. A todas aquellas personas que de una u otra manera me ayudaron a cumplir este objetivo y no las nombré antes. Graciasll. INDICE RESUMEN .­ ABSTRACT PUBLICACIONES ABREVIATURAS mua“ PARTE A. INTRODUCCION GENERAL CAPITULO l. ASPECTOS BlOLOGlCOS Y QUIMICOS DE LAS ALGAS ROJAS l.l. ASPECTOS BlOLOGlCOS DE LAS ALGAS ROJAS |.| .I. Algas rojas. Características principales l.I.2. Algas rojas. Reproducción y ciclos de vida 1.1.3. Diversidad de algas rojas I.l .4. Familia Pliyllophoraceae ¡.2. PARED CELULAR DE LAS ALGAS ROJAS 1.2.I. Carragcnanos ¡2.2. Agaranos |.2.3. Galactanos híbridos DL |.2.4. Significado quimiotaxonómico de los galactanos en el orden Gigartinales 1.2.5. Determinación estructural de polisacáridos de algas rojas por melilación y espectroscopía de RMN '3C PARTE B. SECCION EXPERIMENTAL 59 CAPITULO 2. METODOS EXPERIMENTALES 6| 2. l. CONSIDERACIONES GENERALES 62 2.2. ASPECTOS BlOLOGlCOS: CULTIVOS DE CARPOSPORAS 63 2.2.I. Material alga! 63 2.2.2. Medio y condiciones de cultivo 63 2.2.3. Liberación de las carposporas 64 2.2.4. Desarrollo de las carposporas 64 2.3. ANALISIS POR MICROSCOPIA OPTICA (MO): LOCALIZACION IN SITU DE POLISACARIDOS 64 2.3.]. Lugo! 64 2.3.2. Rojo de rutenio 65 2.3.3. Rojo de mtenio/lugol 65 2.3.4. Azul de toluidina () 65 2.3.5. Azul de metileno/fucsina básica 65 2.3.6. Coomasie brillant blue 65 2.4. ANALlSlS POR MlCROSCOPlA ELECTRONICA DE TRANSMISION (MET) 65 2.4. l. Ultraestructura celular 65 2.4.1.]. Material algal 65 2.4. l .2. Fijación 66 2.4. l .3. Deshidratación 66 2.4.1.4. lnclusión 66 2.4. l .5. Ultramicrotomia 67 2.4.1.6. Contrastado 67 2.4.2. Localización in situ de polisacáridos 68 2.4.2.]. Material algal 68 2.4.2.2. Fijación 68 2.5. ANALISIS POR METODOS QUIMICOS Y ESPECTROSCOPICOS: ESTRUCTURA QUlMlCA DE POLISACARIDOS 2.5.1. Material algal 2.5.1.]. Limpieza del material algal 2.5. l.2. Aislamiento de las estructuras reproductivas 2.5.2. Extracción de los polisacáridos con agua a temperatura ambiente 2.5.2.]. Plantas cistocárpicas de Gymnogongrus Iorulosus 2.5.2.2. Plantas cistocárpicas de Gigurlinu skoll.s'bergii 2.5.2.3. Estructuras reproductivas de Gigarlina skollsbergii 2.5.3. Extracción de los polísacáridos con agua a 90 °C 2.5.3.1. Plantas cistocárpicas de Gymnogongru.s-tom/05m" 2.5.3.2. Cistocarpios de Gymnogongrus tom/asus 2.5.3.2. Plantas tetraspóricas de (iigurlinu .s-koltsbergii 2.5.2.3. Estructuras reproductivas de Gigurlinu slmllxbergii 2.5.4. Fraccionamiento de los carragenanos con cloruro de potasio 2.5.4.]. Precipitación analítica 2.5.4.2. Fraccionamiento preparativo 2.5.5. Tratamiento alcalino 2.5.6. Tratamiento con a-amilasa 2.5.7. Determinación del contenido total de hidratos dc carbono 2.5.8. Determinación del contenido de 3,6-anhidrogalactosa 2.5.9. Determinación del contenido dc sulfato 2.5.10. Detenninación del contenido en proteínas totales 73 2.5.] I. Determinación del peso molecular 74 2.5.1 l .l . Por dosaje de extremos reductores 74 2.5.! l.2. Por cromatografía de pcrmeacíón en gel 75 2.5. ¡2. Hidrólisis de los polisacáridos 75 2.5. l2. l. l-lidrólisis ácida total 75 2.5.122.
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