¡BIENVENIDOS al CONGRESO ANUAL DE LA SOCIEDAD DE FARMACOLOGÍA DE CHILE! Concepción 2019 El evento científico contará con la participación de 5 conferencistas internacionales, 70 expositores en simposios y mini simposios, junto a cerca de 200 jóvenes científicos que exhibirán los avances obtenidos en sus diferentes líneas de investigación.

1 2 ¡BIENVENIDOS al CONGRESO ANUAL La Revista de Farmacología de Chile tiene un panel de editores conformado DE LA SOCIEDAD DE por connotados farmacólogos nacionales que son miembros de la Sociedad de FARMACOLOGÍA DE CHILE! Farmacología de Chile y académicos de las principales universidades chilenas. Concepción 2019 El evento científico contará con la participación de 6 conferencistas internacionales, 70 expositores en simposios y mini Comité Editorial simposios, junto a cerca de 200 jóvenes científicos que exhibirán los avances obtenidos en sus diferentes líneas de investigación. Dr. Ramón Sotomayor Z., Editor en Jefe (Universidad de Valparaíso, Chile)

Dr. Mario Rivera M., Psicofarmacología (Universidad de Chile, Chile)

1 Dr. Jorge Fuentealba A., Neurofarmacología Directiva (Universidad de Concepción, Chile)

Presidente: Dra. Viviana Noriega S., Farmacología Clínica Dr. Jorge Fuentealba A. (Universidad de Chile, Chile) Universidad de Concepción [email protected] Dr. Miguel Reyes P., Química Médica (Universidad de Santiago de Chile, Chile)

Vicepresidente: Dra. María Angélica Rivarola, Neuroendocrinología Dr. Javier Bravo V. (Universidad Nacional de Córdova, Argentina) Pontificia Universidad Católica de Valparaíso [email protected] Dr. Juan Pablo García Huidobro T., Farmacodinamia (Universidad de Santiago de Chile, Chile)

Secretario General: Dra. Marcela Julio-Pieper, Farmacología Gastrointestinal Dr. Claudio Coddou A. (P. Universidad Católica de Valparaíso, Chile) Universidad Católica del Norte [email protected] Dra. Georgina Renard C., Co-Editor, Psicofarmacología (Universidad de Valparaíso, Chile)

Tesorero: Dra. Gonzalo Cruz N., Farmacología Endocrina Dr. Ramón Sotomayor Z. (Universidad de Valparaíso, Chile) Universidad de Valparaíso [email protected] Dra. Carolina Gómez G., Ciencias Farmacéuticas (Universidad de Concepción, Chile)

Past-presidente: Dr. Edgar Pastene N., Fitofarmacología Dr. Edgar Pastene N. (Universidad de Concepción, Chile) Universidad de Concepción [email protected] Dr. Rodrigo Castillo P., Farmacología Cardiovascular (Universidad de Chile, Chile)

Directores: Dr. Patricio Iturriaga V., Química Médica Dra. Georgina Renard D. (Universidad de La Frontera, Chile) Universidad de Santiago [email protected] Dr. Mauricio Dorfman P., Metabolismo y Diabetes (University of Washington, Seattle-USA) Dr. Gustavo Moraga C. Universidad de Concepción Dr. Javier Bravo V., Neurofarmacología [email protected] (P. Universidad Católica de Valparaíso, Chile)

Dra. Viviana Noriega S. Universidad de Chile Revista de Farmacología de Chile es publicada por la Sociedad de Farmacología de [email protected] Chile Derechos Reservados a la Sociedad de Farmacología de Chile. ISSN 0718-8811 versión impresa ISSN 0718-882X versión digital Todos los derechos reservados. Nin- guna parte de esta publicación puede ser reproducida, almacenada en sistema alguno Dr. Gonzalo Cruz N. de tarjetas perforadas o transmitida por otro medio -electrónico, mecánico, fotocopia- Universidad Valparaíso dor, registrador, etcétera- sin permiso previo por escrito del comité editorial. [email protected] IMPRESO EN TRAMA IMPRESORES.

3 4 Indice

Volumen 13, número 2, AÑO 2019

Página 22: Magíster en Neurobiología: Formando especialistas en investigación experimental

Página 50: Especial XLI Congreso Anual de la Sociedad de Farmacología de Chile

Página 52: Bienvenida

Página 53: Programa

Página 54: Conferencistas Internacionales

Página 60: Abstracts for XLI of the chilean Society of Pharmacology Conferences

Página 62: Symposia

Página 76: Minisymposia

Página 78: Dr Jorge Mardones Restat Award

Página 80: Incorporations

Página 83: Oral Communications

Página 88: Posters

5 a Revista de Farmacología de Chile es una revista de Linformación científica vigente desde el año 2008. Su objetivo inicial ha sido ser un órgano de difusión de la farmacología en Chile para luego ser una plataforma de difusión para el resto de Latinoamérica. Desde sus comienzos, la recepción de artículos científicos fue aumentando año a año, llegando a un máximo de artículos publicados el año 2014. Además, hemos logrado contar con varios artículos internacionales. Sin embargo, desde esa fecha en adelante se produjo una disminución en los artículos recibidos que coincidió con nuevas revistas de la disciplina de libre acceso y una gran competencia con revistas del área que ya se encuentran indexadas.

6 Editorial

En este sentido, se nos ha planteado resúmenes correspondientes a las un gran desafío para los próximos conferencias, simposios y paneles años en pro de revertir esta tendencia. que serán presentados del 4 al 8 de Por un lado, debemos aumentar la noviembre en la sede del congreso, “Por un lado, difusión y distribución de la revista Universidad de Concepción. debemos a nivel latinoamericano y, asimismo, Nuevamente invitamos a todos a ser aumentar la estamos evaluando la posibilidad parte del crecimiento de la Revista difusión y de asociarnos a una casa editorial de Farmacología de Chile y lograr distribución de internacional y cambiar el idioma de difundir información científica del la revista a nivel publicación de nuestra Revista. área a nivel internacional. Latinoamericano En el volumen de este año, hemos y, asimismo, logrado volver a aumentar el número estamos de Revistas publicadas con respecto evaluando la al año anterior. En este número de posibilidad de la Revista se publican dos artículos asociarnos a una originales internacionales (Cuba y casa editorial España) y dos artículos nacionales. Dra Georgina Renard internacional Además, teniendo en cuenta que Co - Editora y cambiar este número corresponde al XLI el idioma de Congreso Anual de la Sociedad Directora por Santiago Sociedad de publicación de de Farmacología, se publican los Farmacología de Chile nuestra Revista”.

7 La sociedad y Sofarchi: un vínculo que se expande

Estimadas socias y socios:

stimadas socias y socios, junto de integración. Además, como actividad con extenderles un cordial sa- de divulgación a la sociedad se realizó “En esta ludo desde nuestra directiva, les una charla científica a alumnos del Cole- invitamos a leer nuestro nuevo gio Alemán de La Serena. conversación, Enúmero de la revista, Editado con el in- A nivel internacional, 4 miembros de le expresamos cansable trabajo del Dr. Ramón Sotoma- nuestra sociedad nos representaron en al Ministro yor como editor en jefe, Dra. Georgina la Reunión Anual de la Sociedad Espa- Renard como co-Editora, Fabiola Valde- ñola de Farmacología, realizada en el pa- la total benito, nuestra periodista, y todos quie- sado mes de Julio en La Palma de Gran disposición nes de una u otra forma colaboran para Canaria, España. En este congreso, don- de la Sofarchi que la revista siga activamente siendo de además participaron representantes un canal de comunicación con ustedes de las sociedades de farmacología de para ser un y la sociedad. En este número podrán Alemania y Holanda, pudimos compar- modulador encontrar el informe de algunas de las tir e intercambiar experiencias y generar positivo en más recientes actividades donde nuestra colaboraciones entre nuestras socieda- sociedad ha estado presente, actualidad des que esperamos den sus frutos en el esta tarea, y científica, artículos científicos originales mediano plazo. pusimos a su y revisiones, entre otros. Por último, estamos trabajando sin pau- disposición Respecto a la actualidad científica nacio- sa, y con nuestros mejores esfuerzos, nal, durante el primer semestre, hemos para organizar nuestra próxima reu- los diferentes participado en una interesante reunión nión en Noviembre, la que contará con canales de con el Ministro de Ciencia Dr. Andres importantes expositores nacionales e comunicación Couve, quien nos contó los avances que internacionales en 7 conferencias ple- su ministerio ha experimentado, y las narias, 10 simposios, comunicaciones de nuestra metas de mediano y largo plazo que se orales, comunicaciones en póster y ac- sociedad para han impuesto, como el acercar la acti- tividades satélites que complementaran ayudar en ese vidad científica al centro de atención una intensa jornada científica de 5 días. de la sociedad. En esta conversación, le A nivel personal, tengo el secreto anhelo objetivo”. expresamos al Ministro la total disposi- que puedan re-descubrir la belleza del ción de la SOFARCHI para ser un modu- Campus Concepción, en el centenario de lador positivo en esta tarea, y pusimos a la Universidad de Concepción, y sus al- su disposición los diferentes canales de rededores, y les reitero mi más ferviente comunicación de nuestra sociedad para invitación para que nos acompañen en ayudar en ese objetivo. esta jornada científica que nos prepara En este plan de acercar la ciencia a la so- varias sorpresas. ciedad, la SOFARCHI realizó la pasada Un atento saludo a todos Uds. y disfru- reunión de la Directiva en la ciudad de ten de este nuevo número de nuestra Coquimbo. Esta reunión fue realizada en nueva revista la Facultad de Medicina de la Universi- dad Católica del Norte, reuniéndose con Dr. Jorge Fuentealba Arcos nosotros el decano de la facultad con Presidente SOFARCHI quien discutimos nuevas oportunidades

8

“El encuentro, realizado en el Palacio de La Moneda, contó con la participación de cerca de 25 organizaciones dedicadas a la investigación y promoción de diversas áreas del saber, entre ellas, la Sociedad de Farmacología de Chile”.

10 Sociedades científicas se reúnen con Ministro Andrés Couve para conocer lineamientos para promover investigación

“La principal preocupación on el objetivo de entregar ción del estado que va a potenciar la in- que manifesté al detalles sobre el estado de vestigación científica”. ministro dice en avance de la instalación del Uno de los ejes de la reunión giró en relación a cómo las Ministerio de Ciencias, Tec- torno a las estrategias que las organiza- 20 o 25 sociedades ciones podrían desarrollar en conjunto nología e Innovación, además que estábamos en de exponer sobre las directrices que se para contribuir a potenciar el desarrollo C la mesa podíamos van a implementar para el desarrollo de de la ciencia en Chile. En este sentido, investigación, se realizó una reunión en- Fuentealba agregó que “la principal pre- contribuir a tre el jefe de la cartera, Andrés Couve, y ocupación que manifesté al ministro dice potenciar el los representantes de las principales so- en relación a cómo las 20 o 25 socieda- desarrollo de la des que estábamos en la mesa podíamos ciedades científicas del país. ciencia en términos El encuentro, realizado en el Palacio de contribuir a potenciar el desarrollo de la La Moneda, contó con la participación de ciencia en términos de darle difusión, de de darle difusión, cerca de 25 organizaciones dedicadas a demostrar la calidad de la investigación de demostrar la investigación y promoción de diversas que se hace en los distintos ámbitos, des- la calidad de la áreas del saber, entre ellas la Sociedad de de las ciencias sociales hasta las ciencias investigación naturales y exactas”, expuso. Farmacología de Chile Sofarchi. Su pre- que se hace en los sidente, Jorge Fuentealba, indicó que “la El líder de la Sofarchi añadió que “el mi- actividad ha sido muy productiva, en el nistro dijo que confía plenamente en que distintos ámbitos, sentido de poder avizorar cuáles son las vamos a ser actores activos y relevantes desde las ciencias etapas que ha cumplido el Ministerio y en la difusión y fortalecimiento de los sociales hasta las cuáles son las que quedan dentro de la conceptos de ciencia y de generación de ciencias naturales conocimientos”, puntualizó. instalación de esta importante reparti- y exactas”, Jorge Fuentealba A.

11 asta La Serena, en la región de Coquim- Hbo, se trasladó la directiva de la Sociedad de Farmacología de Chile, con el objeto de realizar diversas actividades orientadas a for- talecer el desarrollo de este organismo cientí- fico en regiones y a motivar la incorporación de nuevos socios. La visita estuvo encabezada por su presidente, Jorge Fuentealba, acompañado por el Vice- presidente de la entidad, Ramón Sotomayor, junto al académico de la UCN y Secretario General de Sofarchi, Claudio Coddou y las directoras del organismo, Georgina Renard y Viviana Noriega. El equipo sostuvo un encuentro con el Decano de la Facultad de Medicina de la Universidad Católica del Norte, Osvaldo Iribarren, quien pudo conocer detalles sobre las principales líneas de trabajo que está desarrollando la or- ganización durante este año, lo que permitió sentar las bases para próximas acciones con- juntas. “La idea de reunirnos en Coquimbo es poten- ciar las actividades en regiones y fortalecer el interés de los científicos de esta zona del país para integrarse a Sofarchi y colaborar, de esta forma, al desarrollo de la disciplina”, señaló Jorge Fuentealba. Agregó que “actividades como estas refuerzan nuestro rol social, dan- do a conocer la ciencia y la farmacología. Por eso, estamos muy contentos con la recepción que hemos tenido de parte del Decano, ya que es importante contar con ese entusiasmo y creo firmemente que será un motor de empu- je para esta iniciativa en la región”. En este sentido, el decano de la Facultad de Medicina de la Universidad Católica del Nor- te, Osvaldo Iribarren, comentó que “la UCN sustenta su funcionamiento y su desarrollo en tres pilares: la docencia, la investigación y

12 Directiva de Sofarchi visita Coquimbo para promover la participación de socios

la vinculación. Para nosotros, como Facultad de Medicina, tenemos que ir empujando la frontera del conocimiento cada vez mas y una sociedad científica como Sofarchi representa exactamente está construcción de vínculos” En este sentido, agregó que esta asociación “nos permite ir abriendo otros lazos sutiles, El presidente de Sofarchi, Jorge Fuentealba, se pero muy firmes, con el resto de las universida- reunió con el Decano de la Universidad Católica des, porque la Sofarchi está en la Universidad de Concepción, en la de Valparaíso, en la de del Norte, Doctor Osvaldo Iribarren; para exponer Santiago, etcétera; así es que para nosotros no sobre los principales lineamientos de trabajo de puede ser mejor y más bienvenida”, manifestó. la organización farmacológica. Con ello, se busca Respecto a las actividades a realizar en con- junto, Irribarren indicó que “me invitaron a la sentar las bases de futuras acciones conjuntas. inauguración del Congreso de Sofarchi que se realizará en noviembre próximo en Concep- ción, lo cual me parece muy bien porque está en perfecta sintonía con la vinculación cada vez más fuerte que pretendo hacer con el am- biente académico”, expuso.

Ciencia más cerca

Otra de las actividades en las que participó la directiva se desarrolló en el Colegio Alemán de La Serena, donde cerca de 70 escolares de octavo básico a segundo medio asistieron a la charla “Mirando la naturaleza, el laboratorio maestro”. “Tuvimos una grata jornada con niños y jóve- nes que nos dieron una muy buena recepción, fueron muy interactivos, hicieron muchas preguntas. Fue muy grato sentir que en Co- quimbo hay un sustrato para poder desarro- llar y potenciar esta área, que permitiría fa- vorecer el desarrollo de la biomedicina en la región”, explicó Fuentealba. A través de la exposición, los escolares apren- dieron sobre el valor de la observación como mecanismo para entender diversos procesos de la naturaleza, desde donde la humanidad ha extraído fármacos desde tiempos inmemo- riales. El investigador agregó que “hoy en día esos principios farmacológicos son estudia- dos científicamente y aplicados a la biomedi- cina, para transformarlos en medicamentos que permiten mejorar la calidad de vida de millones de personas en todo el mundo y que tienen su origen en este laboratorio maestro que es la naturaleza”. Otro de los presentes en la jornada fue el secre- tario de la organización científica, Claudio Co- ddou, quien comentó que “es muy importante tener estos espacios, porque gracias al link con la Sociedad de Farmacología estamos promo- viendo la Universidad y generando interés de los estudiantes hacia este mundo de la ciencia”.

13 Delegación de la Sofarchi participó en el Congreso de la Sociedad Española de Farmacología

on la colaboración internacional grande como la española nos reconozca de las asociaciones farmacológi- y nos trate como pares, invitándonos a cas alemana, holandesa, irlan- participar en su Congreso”. El directivo desa y chilena, se efectuó el 39° agregó que “fue una actividad muy in- CCongreso Anual de la Sociedad Española teresante, científicamente de alto nivel de Farmacología (Socesfa), realizado por y con bastantes oportunidades para el primera vez en Las Palmas de Gran Ca- intercambio, para la vinculación y para naria. Más de 60 expositores de diversas el desarrollo de articulaciones de estu- partes de globo divididos en 13 sesiones diantes y de trabajos de investigación”, de simposios, compartieron sus conoci- expuso. mientos con farmacólogos, científicos, Los representantes nacionales realiza- médicos, profesionales de área de la sa- ron cuatro exposiciones plenarias dentro lud, pacientes y representantes de la in- de los simposios, la primera a cargo del dustria farmacéutica, convirtiendo a este doctor Guillermo Díaz, quien se refirió al encuentro en uno de los más relevantes rol del fibroblasto cardíaco en procesos del planeta en su tipo. inflamatorios y la interacción con células La delegación de la Sofarchi estuvo inte- inmunes. grada por los doctores Jorge Fuentealba En tanto, el doctor Ramón Sotomayor y Leonardo Guzmán, de la Universidad abordó la programación de neuronas do- de Concepción; junto a Ramón Sotoma- paminérgicas por exposición temprana a yor, de la Universidad Católica de Valpa- hormonas sexuales y su posible efecto en raíso y Guillermo Díaz, de la Universidad la vulnerabilidad ante la drogadicción. de Chile. Además, valoró la posibilidad que otor- Respecto a esta participación, el presi- gan estos eventos internacionales para dente de la Sofarchi, Jorge Fuentealba, potenciar el intercambio de experien- indicó que “como directiva nos pare- cias en nuestro país, “nos contactamos ce muy relevante que una Sociedad tan con Michael Spedding, secretario gene-

14 ral de la International Union of Basic & nuevos avances en neurofarmacología. Cuatro Clinical Pharmacology para invitar a la En el desarrollo de este último tema, representantes de representante de la UPHAR el próximo abordado en un simposio en el que expu- año a Chile. Además, logramos confir- sieron otros tres investigadores; participó la organización mar la participación de la Doctora Con- el Doctor Leonardo Guzmán con su char- científica chilena cha Peiró, de la Universidad Autónoma la titulada “efectos de los dendrímeros fueron invitados de Madrid y miembro de la directiva de PAMAM en la funcionalidad neuronal In por la Socesfar para la Socesfar a nuestro próximo Congre- Vitro”. El investigador detalló que en la exponer parte de so Anual de Sofarchi que se realizará en ocasión “se mostraron avances en neuro- Concepción durante este año”, detalló el fármacos que se están desarrollando para sus investigaciones. doctor Sotomayor. ayudar en procesos de adicciones; tam- En la actividad Aporte científico bién se mostraron investigaciones sobre también La farmacología es entendida como una intervenciones para la liberación de neu- participaron ciencia necesariamente interdisciplina- rotransmisiones y, por mi parte, mostré sociedades ria, por lo que en el Congreso se trataron cómo los nanotransportadores podrían temáticas tan diversas como cáncer, re- ayudar a la entrega de fármacos en sitios farmacológicas de ceptores, diseño de drogas, farmacoge- sinápticos”, detalló. Holanda, Irlanda y nética, farmacología clínica en áreas de En tanto, Jorge Fuentealba presentó Alemania. endocrinología, gastroenterología y res- “Sobreexpresión de P2xr contribuye a piratorios; además de farmacología del la toxicidad por Beta Amiloides: nuevo dolor y la inflamación, entre otros. blanco farmacológico para la AD”, en El equipo de académicos de la Universi- la que expone parte de su investigación dad de Concepción presente en este 39° orientada al análisis de los mecanismos Congreso de la Socesfar, participó tanto celulares y moleculares que inducen el en el área de envejecimiento, conside- desarrollo de enfermedades neurodege- rando desde los mecanismos hasta las nerativas, especialmente la Enfermedad perspectivas farmacológicas; como en de Alzheimer.

15 Cien años de Química y Farmacia UdeC

“Hace exactamente 100 años tenía lugar la primera clase del curso de Farmacia, dictada por don Salvador Gálvez Rojas a 28 estudiantes, todas ellas mujeres, constituyéndose en una de las primeras carreras universitarias en el sur de Chile que ofrecía una opción de educación superior para las mujeres del país”.

a conmemoración de los 100 años de la Carrera de Química Ly Farmacia, de la Facultad de Farmacia de la Universidad de Con- cepción en este año 2019, constituye uno de los más importantes hitos en la historia de la también centenaria Uni- versidad de Concepción y es especial- mente significativa para todos quienes hemos tenido el privilegio de realizar nuestros estudios universitarios en esta Alma Mater. Hace exactamente 100 años tenía lu- gar la primera clase del curso de Far- macia, dictada por don Salvador Gál- vez Rojas a 28 estudiantes, todas ellas mujeres, constituyéndose en una de las primeras carreras universitarias en el sur de Chile que ofrecía una opción de educación superior para las muje- res del país. Cien años desde aquel sencillo pero visionario comienzo en que nacía la carrera de Química y Farmacia y al mismo tiempo, la entonces, Escuela de Farmacia, cuando la creación de la ca- rrera - la segunda históricamente en el país - fue promovida con decisión por los fundadores desde el momento en que el Comité Pro Universidad y Hos- pital Clínico creado en 1917 empieza a definir la forma en que la novel Casa de Estudios iniciaría sus actividades.

16 Cien años de Química y Farmacia UdeC

Respondiendo a una sentida necesi- el recientemente creado programa de dad, no sólo de la región sino del país, Especialización en Farmacia Clínica. pues con su creación se buscaba el Sumando también Cursos, Diplomas mejoramiento sanitario de la pobla- y Diplomados. ción. La investigación como pilar para la Cien años después, la Facultad de generación de nuevo conocimiento Farmacia ha formado más de 2.800 también ha sido un área destacada en profesionales Químicos Farmacéuti- el quehacer de la Facultad de Farma- cos, que se desempeñan en los distin- cia, reflejado en publicaciones en re- tos ámbitos del ejercicio profesional, vistas de corriente principal y adjudi- representando un importante aporte cación de proyectos de investigación “Cien años después, al desarrollo del país, particularmen- con financiamiento externo (Fonde- te en el sector sanitario, el sector pro- cyt, Fondef, entre otros). Desde 2015 la Facultad de ductivo, la academia y también en la a la fecha, la Facultad ha adjudicado Farmacia ha política universitaria y ciudadana. 4 iniciativas Fondequip para la adqui- formado más de En este contexto es relevante destacar sición de equipamiento mediano que 2.800 profesionales la figura de Ligia Gargallo González, ha significado para la Universidad de Químicos químico farmacéutica, académica e Concepción la atracción de más de Farmacéuticos, investigadora, Premio Nacional de 1.000 millones de pesos para inves- que se desempeñan Ciencias Naturales 2014 y Premio tigación, permitiendo el desarrollo y en los distintos L’Oreal UNESCO 2007 para Muje- crecimiento de la Facultad y la insti- ámbitos del ejercicio res en Ciencias. Del mismo modo, tución, además de mayores posibili- reconocer el esfuerzo, compromiso, dades de formación terminal para es- profesional, dedicación y liderazgo de recordados tudiantes de pre y postgrado. representando un maestros y profesores. Muchos desafíos y tareas se proyec- importante aporte al Durante estos 100 años son muchísi- tan para la Facultad, sin embargo, es desarrollo del país, mos los hitos que marcan la historia de necesario destacar nuevamente el pe- particularmente en la Carrera de Química y Farmacia: la renne sello UdeC del profesional quí- el sector sanitario, el Farmacia Modelo en 1920, un centro mico farmacéutico, esa característica sector productivo, la de práctica, único e innovador para tan propia que permite reconocernos academia y también la época, con un fuerte sentido social; con nuestros pares cualquiera que la estructura y organización definitiva sea el área laboral y lugar en que nos en la política como Facultad en 1927; el surgimien- encontremos, haciendo que nuestros universitaria y to de nuevas iniciativas de formación profesionales sean preferidos y respe- ciudadana”. profesional como la creación de las ca- tados. rreras de Bioquímica en 1957 y de Nu- La invitación es a seguir construyen- trición y Dietética en 1975; las fiestas do y nutriendo con orgullo, respeto, universitarias, juegos florales y carna- empatía, comportamiento ético y de- vales de antaño; la difícil y anhelada dicación esta hermosa profesión far- Autorización Ministerial para que la macéutica, para proyectarla exitosa Universidad de Concepción pudiera hacia el futuro. otorgar los títulos de Químico Farma- céutico y de Bioquímico hacia fines de 1974; la ampliación del actual edificio Dr. Ricardo Godoy Ramos y el surgimiento del Postgrado. En Decano Facultad de Farmacia este ámbito, la Facultad de Farmacia Universidad de Concepción ofrece actualmente, un programa de doctorado, 3 programas de magíster y

17 18 Formando profesionales en las grandes líneas de desarrollo científico básico y clínico de la farmacología

a necesidad de contar con es- Neurociencias de la Universidad de pecialistas que comprendie- Valparaíso. Además, hay estudiantes ran en profundidad las bases profesionales, incluso, en esta opor- modernas de la farmacología tunidad contamos con un estudiante Ldel Sistema Nervioso y que, además, polaco que es médico”. adoptaran conceptos biomédicos re- El Doctor Sotomayor destacó tam- lacionados con la farmacología mole- bién la amplia presencia de la Socie- cular y clínica, motivó en 2012 a un dad Chilena de Farmacología en esta equipo de académicos de la Facultad instancia, “este curso ha sido patro- de Ciencias de la Universidad de Val- cinado desde su origen por la Sofar- paraíso a generar nuevas instancias chi, lo que nos ha permitido contar de formación continua. con la participación de profesores de Desde hace Fue así como el Doctor Ramón So- alto nivel provenientes de destacadas siete años, tomayor, académico del Instituto de Universidades del país”. este programa Fisiología de la Facultad de Ciencias En este sentido, indicó que “gracias prepara con de esta casa de estudios, encabezó la a esta alianza hoy contamos con una idea de promover el desarrollo de un decena de académicos que son socios conocimiento programa de postgrado en Neurofar- titulares de la Sofarchi, quienes inte- teórico y macología. El investigador, quien ac- gran del staff de docentes de este cur- habilidades tualmente es el coordinador de esta so”. Entre ellos, se cuentan los docto- instancia, comentó que “este curso se res Jorge Fuentealba (Universidad de experimentales realiza todos los años en la Universi- Concepción), Javier Bravo (Pontificia tanto a dad de Valparaíso y ha sido muy bien Universidad Católica de Valparaíso), profesionales evaluado en cuanto a los contenidos Claudio Coddou (Universidad Católi- del área que se imparten, así como también ca del Norte) y Georgina Renard (Uni- por las habilidades experimentales versidad de Santiago), entre otros. clínica, como a que adquieren los estudiantes duran- especialistas en te el transcurso del programa” Alumnos exitosos neurociencia, Agregó que el curso está orientado farmacología y tanto a profesionales del área clíni- Diversas son las habilidades y cono- ca, como neurólogos, neurociruja- cimientos que los alumnos de este biomedicina. nos, psiquiatras y psicólogos, entre curso de Neurofarmacología han ob- otros; como a especialistas en neuro- tenido generación tras generación. A ciencia, farmacología y biomedicina. través de una metodología que inclu- Según indicó el Doctor Sotomayor, ye clases magistrales y seminarios de esta séptima versión “cuenta con es- discusión, se han formado profesio- tudiantes del Doctorado en Ciencias, nales con pensamiento crítico, capa- mención Neurociencias y del Magís- ces de analizar las grandes líneas de ter en Ciencias Biológicas, mención desarrollo científico básico y clínico

19 que han permitido tanto la genera- determinado que “aquellas ratas que ción de los tratamientos farmacoló- ingirieron dietas altas en grasa du- gicos actuales, como su evolución y rante gran parte de su vida, a diferen- proyecciones futuras. cia de las control, siguen prefiriendo Junto a ello, la propuesta académi- esta dieta en grandes proporciones. ca de este programa permite a los Hay una diferencia bien importante” alumnos desarrollar trabajos experi- Respecto al aporte que ha sido el pro- mentales originales que contribuyen grama de Neurofarmacología en el de- a la generación y desarrollo de trata- sarrollo de su investigación, sostuvo mientos farmacológicos del sistema que “el curso entrega una base neuro- nervioso. biológica en todos los ámbitos, que van Estos y otros conocimientos teóricos desde las adicciones, el movimiento, y prácticos entregados en el progra- la fisiología, lo molecular, etcétera. El ma de Neurofarmacología, le han en- ramo de Estructura y Función del Sis- tregado herramientas para avanzar tema Nervioso, por ejemplo, es súper en sus investigaciones a la estudiante importante y exigente”. de segundo año del Magíster en Cien- La séptima versión del curso de Neu- cias Biológicas, mención Neurocien- rofarmacología tiene una duración cias de la Universidad de Valparaíso, de 18 semanas y considera un total de Victoria Collio, 162 horas semestrales. “Estoy haciendo mi tesis sobre el efec- to del consumo de dieta alta en grasa en algunos núcleos, como el septum Mayor información: lateral cerebral, en ratas, para eva- Sra. Francisca Ramírez Coordi- luar el impacto en el control de la co- nación de Postgrado, Facultad de mida, del hambre y de la saciedad”, Ciencias, Universidad de Valparaí- indicó la pedagoga en biología de la so Avda. Gran Bretaña #1111, Pla- Pontificia Universidad Católica de ya Ancha, Valparaíso. Fono +56 32 Valparaíso. Hasta ahora, los resulta- 2508000 [email protected] ; dos obtenidos por Victoria Collio han [email protected]

20 21 Magíster en Neurobiología: Formando especialistas en investigación experimental

El programa de la Facultad de Ciencias Biológicas de la UdeC proporciona un contenido académico orientado a la formación de especialistas en esta área, poniendo énfasis en el aprendizaje práctico de técnicas, protocolos, procedimientos de experimentación en neurobiología celular y molecular, además de estudio de comportamiento animal.

22 Magíster en Neurobiología: Formando especialistas en investigación experimental

as bondades del ajo han sido am- en el marco del Magíster en Neurobiolo- pliamente valoradas desde tiem- gía impartido por la Facultad de Ciencias pos remotos. Es considerado uno Biológicas de la UdeC. de los productos alimenticios Ella es parte de la primera generación de Lmás completos en la naturaleza gracias estudiantes que ingresó a este programa a la larga lista de vitaminas y minerales de postgrado, el cual también ha impul- que posee, que le otorgan comprobadas sado líneas de investigación en torno a propiedades cardioprotectoras, hipogli- enfermedades neurodegenerativas como cemiantes y antioxidantes. Parkinson y Creutzfeld Jacob, además A este listado de beneficios se agrega una de estudios sobre receptores de glicina y nueva cualidad descubierta recientemen- niveles fisiológicos de nutrientes, entre Gracias a este te por un equipo de investigadores de la otros. enfoque, el Universidad de Concepción, quienes ana- “Cuando este magíster se abrió fue como lizaron las capacidades neuroprotectoras una luz, fue súper bueno porque era es- programa ha de una variedad fermentada del Allium pecíficamente lo que yo quería aprender desarrollado líneas ampeloprasum o ajo chilote, una espe- y lo que me hacía falta también en mi ca- de investigación cie endémica de la Isla Grande. Según la rrera como bióloga”, señaló Gavilán. en enfermedades bióloga que encabeza el estudio, Javiera Este es, precisamente, el principal enfo- neurodegenerativas Gavilán, este alimento cambia tras de ser que del Magíster en Neurobiología que sometido a condiciones específicas de ca- imparte la Facultad de Ciencias Biológi- como Alzheimer, lor y humedad, lo que le otorga su carac- cas de la UdeC, programa que apunta a Parkinson y terístico color oscuro y potencia sus pro- formar científicos especialistas en neuro- Creutzfeld Jacob; piedades. “Nos dimos cuenta de que su biología, con una base teórica y práctica compuestos composición química es distinta al ajo que sólida en estudios relacionados al siste- naturales, además comemos normalmente, porque adquiere ma nervioso y con una fuerte orientación nuevos compuestos sulfurados. Desde ahí hacia lo experimental, donde se encuen- de estudios sobre partió el interés de probar su efecto neu- tra el sello distintivo de este programa. receptores de glicina roprotector en modelos de Alzheimer”. Este se fundamenta en la calidad de los y niveles fisiológicos Los datos que obtuvo en modelos de ra- grupos de investigación vinculados al de nutrientes, entre tón comprobaron que el ajo contiene una claustro académico del programa, en la otros. serie de compuestos bioactivos que pro- disponibilidad de equipamiento de pri- porcionan un blindaje a las neuronas, mera línea para la investigación de fron- haciéndolas más resistentes ante agen- tera en neurobiología y en la vigencia de tes tóxicos que están presentes en pato- proyectos con financiamiento externo. logías neurodegenerativas. Es por ello que, a largo plazo, este estudio “apunta a desarrollar un nutracéutico, porque he- Enfoque hacia lo experimental mos visto que este extracto tiene efectos preventivos en la Enfermedad de Alzhei- El Magister en Neurobiología de la FCB mer”, señaló la bióloga UdeC. busca formar profesionales especiali- Los resultados de esta investigación, que zados en la comprensión científica pro- por su impacto ya fue destacada en varios funda de problemáticas neurobiológicas medios de comunicación; están conteni- relevantes en nuestra sociedad actual, dos en la tesis titulada “Efectos neuro- como por ejemplo, enfermedades neu- protectores del extracto de ajo negro chi- rodegenerativas, adicción, dolor crónico, lote sobre la toxicidad del péptido beta desórdenes alimenticios, y depresión, amiloide”, que Javiera Gavilán desarrolla entre otros.

23 Uno de los creadores del programa fue el A través de su programa académico, que doctor Leonardo Guzmán, quien comen- considera asignaturas básicas, de espe- tó que este “pretende ser un magíster cialidad, complementarias y seminarios; con parámetros modernos, con mucho el alumno egresado será especialista en énfasis en generar nuevo conocimiento neurobiología experimental y contará a través de los proyectos de tesis, por lo con competencias científicas que permi- que está pensado para quienes estén mo- tan el inicio de una carrera asociada a tivados en aprender técnicas, protocolos, la investigación y a la divulgación de la procedimientos de experimentación en disciplina. Además, tendrá la capacidad neurobiología celular y molecular y com- de comunicar, integrar y analizar crítica- portamiento animal” mente y generar nuevos conocimientos El académico detalló que este programa en el área de la neurobiología en un con- está orientado a “estudiantes que hayan texto bioético. tenido una base de fisiología y bioquími- ca más o menos importante, por lo tanto, Mayor información: alumnos de Bioquímica, de Bioingenie- Paula Veloso Aguilera ría, Química y Farmacia, Biología, Bio- Secretaría de Magíster, Facultad de logía Marina; también para las carreras Ciencias Biológicas, Universidad de del área de la salud, como Kinesiología, Concepción. Medicina, Tecnología Médica, Obstetri- Fono: (56-41) 2204159 cia, Odontología”, entre otras. E-mail: [email protected]

24

26 26 La formación de doctores: Nuevas tendencias y oportunidades de internacionalización

Concepción Peiró Vallejo Profesora Titular, Departamento de Farmacología, Facultad de Medicina Coordinadora del Programa de Doctorado en Farmacología y Fisiología Subdirectora de la Escuela de Doctorado (EDUAM) Universidad Autónoma de Madrid, España Email: [email protected]

Significado de los estudios de de escritura de trabajos científicos o de pro- Doctorado yectos de investigación, o de pensamiento crí- tico, entre otros. El título de Doctor es el máximo grado acadé- Pero más allá de los aspectos específicos re- mico que puede otorgar la institución univer- lacionados con la actividad puramente inves- sitaria. En Europa, tras el proceso de armo- tigadora, se busca también hoy en día pro- nización del Espacio Europeo de Educación veer a los estudiantes de doctorado con una Superior (EEES), los estudios universitarios serie de capacidades de carácter transversal se ordenan en los niveles sucesivos de Grado, e intersectorial, que les puedan ser útiles en áreas que no sean las puramente académicas Máster y Doctorado, coincidiendo con la ma- “La educación yoría de las instituciones académicas a nivel o investigadoras. Hablamos, por ejemplo, de global. La finalidad primordial de los estudios habilidades de comunicación oral y escrita, doctoral hoy en de Doctorado es la formación de profesiona- de habilidades interpersonales, de manejo día tiene como de conflictos y negociación, de desarrollo de les de la investigación, que representan pie- meta, junto con zas clave de una sociedad basada en el cono- carrera profesional (Figura 1). En efecto, hay cimiento, como es la actual. Por otra parte, no que tener en cuenta que cada vez más institu- la realización de hay que olvidar que el término “doctor” deri- ciones no académicas y empresas demandan la tesis doctoral, doctores, que puedan ejercer de líderes de su va del verbo latín “docere”, es decir, enseñar. ayudar a que el No en vano el título de Doctor es un requisito sector. Por otra parte, es una realidad que en imprescindible para el desarrollo completo de la actualidad un gran número de los doctores futuro Doctor una carrera académica universitaria. egresados ya no permanecen en la academia, adquiera una serie por lo que conviene dotarles de este tipo de herramientas profesionales que puedan ser- de conocimientos les de utilidad tanto dentro como fuera de la y habilidades, Educación doctoral: más allá de institución universitaria. la tesis doctoral relacionadas o no con la Durante muchos años, el eje central de los es- Internacionalización y investigación, que tudios de Doctorado ha sido la realización de movilidad en el Doctorado, la tesis doctoral, consistente en un trabajo de le proporcionen una investigación original desarrollado por el can- ¿cuáles son sus ventajas? preparación más didato a recibir el título de Doctor. Hoy en día, completa e integral la tesis doctoral sigue siendo sin duda el ele- Uno de los principales aspectos transversales mento principal de los estudios de Doctorado, que se busca desarrollar hoy en día durante los de cara al mundo si bien se hace cada vez más hincapié en una estudios de Doctorado es la internacionaliza- profesional”. formación más amplia e integral del doctoran- ción y la movilidad de estudiantes. Pero, ¿qué do que le permita adquirir una serie de com- ventajas puede ofrecer al candidato a Doctor el petencias y habilidades, relacionadas con la realizar una estancia de investigación en el ex- investigación, pero también con otros aspectos tranjero que puedan compensarle del esfuerzo más transversales y de utilidad más allá de la personal, logístico y económico que supone carrera investigadora (Figura 1). ese desplazamiento? Si el grupo receptor y el Efectivamente, uno de los objetivos actua- momento de realización de la estancia se eli- les de los programas de doctorado es poner gen bien, sin duda las ventajas son múltiples y a disposición de sus estudiantes actividades abarcan diferentes planos de interés. formativas que les permitan manejar mejor En primer lugar, la movilidad puede, y de- aspectos generales directamente asociados bería, suponer una expansión del horizonte con la labor investigadora, como cursos de técnico y científico del estudiante, con opor- conducta responsable en investigación, de tunidades para intercambiar conceptos, ideas análisis de datos, de manejo de bibliografía, y experiencias en entornos diferentes del ha-

27 bitual. Esto además favorece la creación de te a su capacidad de adaptación y crecimiento redes de contactos, lo que en inglés se conoce personal. De hecho, la superación de los retos como “networking”, que pueden ser de gran encontrados durante la estancia de movilidad utilidad para la empleabilidad y el desarrollo puede también fomentar y afianzar la confianza de la carrera profesional futura del candidato en sí mismo del candidato a Doctor. a Doctor. Por último, no olvidemos que, más allá del Por otra parte, la movilidad puede suponer beneficio para los propios estudiantes de Doc- un claro beneficio en cuanto a competencias torado, la movilidad puede resultar altamente idiomáticas y de comunicación, especialmen- provechosa para los propios grupos de investi- te si la estancia se realiza en países que no gación e instituciones implicados, que pueden comparten la lengua materna del estudiante beneficiarse mutuamente de su respectiva vi- de Doctorado. En cualquier caso, e indepen- sión investigadora y educativa. De hecho, no dientemente del idioma hablado, el estudian- cabe duda de que la movilidad de los estudian- te tendrá que desarrollar más que nunca su tes de Doctorado se verá claramente favoreci- capacidad organizativa y sus habilidades de da si se fomentan acuerdos y alianzas formales comunicación y de relaciones interpersona- entre instituciones académicas investigadoras les, al salir de su zona de confort en el centro de diferentes países, ya sea a nivel global de la de origen. propia institución, ó de manera más particu- Por otra parte y más allá del beneficio profe- lar entre los programas de Doctorado y grupos sional, durante la movilidad el estudiante toma de investigación implicados. En este sentido, consciencia de otros usos y costumbres, de otros no sólo la Universidad como institución, sino ámbitos culturales y amplía su círculo relacio- también otras agencias de investigación y fi- nal, lo que puede contribuir muy positivamen- nanciación, adquieren un rol de responsabili-

28 dad en la facilitación de la movilidad y en la Referencias supresión de barreras económicas y logísticas que permitan a los doctorandos desarrollar The European Council of Doctoral Candidates una experiencia internacional beneficiosa para and Junior Researchers. “Identifying trans- su desarrollo profesional y personal. ferable skills and competences to enchance early-career researchers employability and competitiveness” (2018). http://eurodoc.net/ Conclusión y perspectivas skills-report-2018.pfd (consultado el 20 de marzo de 2019). En resumen, la educación doctoral hoy en día Siemers, O. “Skills for here or to take away? tiene como meta, junto con la realización de Outcomes of academic mobility for expatriate la tesis doctoral, ayudar a que el futuro Doc- researchers”. (2016). Journal of International tor adquiera una serie de conocimientos y Mobility: 4, 149-170. habilidades, relacionadas o no con la investi- Universities UK International focus group on gación, que le proporcionen una preparación PhD mobility. “PhD student outward mobility: más completa e integral de cara al mundo perceived barriers and benefits”. (2016). https:// profesional y que fomenten su empleabilidad www.universitiesuk.ac.uk/policy-and-analysis/ en un mercado cada vez más competitivo. Es reports/Documents/International/Phd-stu- por tanto una obligación y un reto actual de dent-outward-mobility-December-2016.pdf los Programas de Doctorado y de los agentes (consultado el 1 de junio de 2019). responsables de la educación doctoral velar por facilitar, en la medida de posible, la mejor formación investigadora y transversal, para que los egresados puedan constituirse en pro- fesionales de referencia que contribuyan al avance de sus respectivos sectores.

29 30 Efectos del ozono médico sobre la Peroxidación Lipídica y la actividad de la Aldehído Deshidrogenasa 2 cerebralen ratas alcohólicas en abstinencia

María Teresa Díaz-Soto1* (Dra. Farmacéuticas, Profesora Auxiliar), Ángela Fraga Pérez (MC Toxicología), Jacqueline Dranguet Vaillant1 (MC Farmacología), Erik Jerez (Lic. Cs. Farmacéuticas), María de los Angeles Bécquer (MC Farmacología), Mayté Casanova (MC Toxicología), Maikel Arteaga Cruz (MC Farmacología), Olga Sonia León Fernández (Dra. Ciencias Farmacológicas, Profesora Consultante),

Instituto de Farmacia y Alimentos, Universidad de la Habana .San Lázaro y L .Habana 10 400, Cuba

. Tel.: +53 54978238 E-mail: [email protected] [email protected] (M .T Díaz).

Resumen: en soluciones de 10, 20, 30, y 40 % (56 días). El consumo crónico de etanol provoca aumen- Sesenta ml de agua o solución de etanol fue- to en la acumulación de acetaldehído y forma- ron colocados en cada caja diariamente por ción de Especies Reactivas de Oxígeno, lo que 8 semanas. Cada 24 horas fueron medidos provoca daño a nivel cerebral, esto se prolonga los volúmenes de etanol o agua consumidos. durante el estado de abstinencia. Actualmente Fue evaluada la tolerancia farmacológica. Se el tratamiento utilizado para contrarrestar las determinó las concentraciones en suero de afectaciones provocadas por el alcoholismo y MDA antes y después de la aplicación de ozo- que se manifiestan durante la abstinencia al- no Finalmente los cerebros fueron removidos cohólica es extremadamente limitado. para estudiar el estado redox mediante Alde- Objetivos: Este estudio se propuso determi- hído deshidrogenasa 2 mitocondrial y malo- nar los efectos protectores del Post-condicio- nildialdehído. namiento Oxidativo con Ozono sobre la acti- Resultados: Fueron significativos los efec- vidad de la enzima Aldehído Deshidrogenasa tos del ozono sobre los niveles de MDA. La 2 cerebral y las concentraciones de malonil- aplicación de Ozono restableció los niveles dialdehído sistémica y cerebral después de 2 de MDA y la actividad dela ALDH2 mitocon- semanas de abstinencia alcohólica en ratas drial, enzima que transforma el Acetaldehído Lewis machos en acetato inactivo Método: La administración de etanol por Conclusiones: El tratamiento con Ozono vía oral fue “ad libitum” .Las ratas se distri- disminuyó el estrés oxidativo sistémico du- buyeron en cuatro grupos: (I) Control, re- rante la abstinencia alcohólica a nivel de Sis- cibió agua durante todo el experimento, (II) tema Nervioso Central. Estos resultados de- Etanol (ET-OH), (III) Etanol + Ozono, como muestran el papel que juega el Ozono como el grupo II pero después de la abstinencia al- protector del daño a este nivel en la abstinen- cohólica fueron sometidas a postcondiciona- cia alcohólica. Estos resultados demostra- miento oxidativo con ozono a concentración ron que el ozono ejerció un efecto protector de 20 μg/mL, dosis de 1 mg/kg, 15 trata- contra el daño oxidativo en el cerebro, pre- mientos por l insuflación rectal y (IV) Etanol servando funciones importantes del Sistema + Oxígeno, como el grupo III pero tratado Nervioso Central(SNC) con oxígeno (26 mg/kg). . Se les fue introdu- Abstract: Ethanol withdrawal (EW) increa- ciendo gradualmente el consumo de etanol ses acetaldehyde accumulation and reactive

31 oxygen species formation that promote da- ET-OH solutions from 10, 20, 30, and 40% “El consumo crónico mage to the brain. It is necessary to emphasi- (56 days). The determination systemic con- de etanol puede ze that while EtOH abuse and dependence are centrations of MDA was before and after the widespread, treatment options are extremely application provocar daño limited. of ozone. Afterwards, brains were prompt- mitocondrial lo que Purpose: This study aimed at investiga- ly removed for oxidative stress studies. conlleva a disfunción ting the protective effects of Ozone Oxida- (Mitochondrial aldehyde dehydrogenase 2 tive Postconditioning (Ozone OxPost) on (ALDH2) and MDA concentrations) de la ALDH y por mitochondrial aldehyde dehydrogenase 2 Results: Of special note were ozone´s effects tanto aumento de las (ALDH2) and MDA concentrations induced on MDA levels. Ozone therapy re-established concentraciones de by oxidative stress after 2 weeks of EW in rats. MDA and mitochondrial aldehyde dehydro- Method: Oral “ad libitum” administration genase 2 (ALDH2), the same enzyme that acetaldehído al cual of ethanol was used. Rats were divided into 4 transforms acetaldehyde into inactive aceta- se le ha adjudicado groups. The groups were (I) Control, received te. un papel importante tap water during the entire duration of the Conclusions: Ozone treatment improved experiment, (II) Ethanol (ET-OH), (III) Etha- the systemic oxidative stress during EW. The- en el daño cerebral nol + Ozone, as group II, but at the beginning se results demonstrated that Ozone protected característico of EW, rats were submitted to Ozone OxPost the brain against oxidative injury, improving del alcoholismo at a concentration of 20 μg/mL, dose 1 mg/ important functions of the Central Nervous kg, 15 treatments by rectal insufflation was Systems (CNS). y del Síndrome used and (IV) Ethanol + Oxygen, as group III de Abstinencia but rather than ozone, rats were treated with Keywords Ozone, Ethanol Withdrawal, Oxi- Alcohólica” oxygen (26 mg/kg).The rats were gradually dative Stress, Central Nervous System introduced to ethanol consumption through

32 Introducción

El alcoholismo es una enfermedad crónica caracterizada por la ingesta compulsiva y ex- cesiva de bebidas alcohólicas a un nivel que interfiere con la salud física y mental, al igual Síndrome de Abstinencia Alcohólica (6, 7, 8). que con las responsabilidades sociales, fami- Por otra parte, las vías no enzimáticas por las liares, económicos o laborales. El alcoholismo que se metaboliza el etanol provocan la for- es un tipo de drogadicción, en la cual hay tan- mación de Especies Reactivas de Oxigeno fa- to dependencia física como mental (1, 2). Pro- voreciéndose la Peroxidación Lipídica (POL) duce una fuerte dependencia al consumo de generándose malonildialdehído (MDA) (cuyo alcohol etílico, manifestándose el Síndrome metabolismo también ocurre por la ALDH). “A los grupos que de Abstinencia Alcohólica cuando no es posi- Se ha evidenciado la formación de aductos fueron tratados ble su ingesta (3). entre el acetaldehído y el MDA. Ambos pue- con ozono se le El alcoholismo es incompatible con la vida den provocar afectaciones en el Sistema Ner- (4), existen dos factores que afectan de forma vioso tanto Autónomo como Central (9) administró por significativa la calidad de vida del paciente al- Numerosos estudios han demostrado que la insuflación rectal cohólico: Por una parte, los efectos directos aplicación del Ozono médico constituye una 1mg/kg, es decir, 5 del etanol sobre diferentes sistemas de neu- conducta beneficiosa para muchas enferme- rotransmisión a nivel de Sistema Nervioso dades degenerativas y afecciones orgánicas cc durante 14 días Central (SNC) como son la transmisión GA- (Diabetes Mellitus, Artritis Reumatoide, her- de abstinencia. Los BAérgica, dopaminérgica y la glutamatérgica, nia discal, presencia de convulsiones). Traba- grupos que fueron al provocar desequilibrio neuronal y con ello jos experimentales y clínicos confirman sus alteraciones conductuales que se prolongan acciones terapéuticas. En estudios realizados tratados con oxígeno durante el Síndrome de Abstinencia Alcohó- en modelos de hepatotoxicidad por CCl4 y en se le administraron lica (SAA). Otro factor que afecta la calidad modelos de isquemia/reperfusión hepática, 5 cc por insuflación de vida del paciente alcohólico está relaciona- se han evidenciado los efectos pleiotrópicos do con el metabolismo del etanol, que ocurre del ozono, el cual ha restablecido el balance rectal durante por vías enzimáticas y vías no enzimáticas. redox y preservado la integridad mitocondrial los 14 días de la Las enzimas que metabolizan al etanol son lo que se relaciona con la protección de la ac- abstinencia” la Citocromo P4502E1 que se localiza en los tividad de la enzima Superóxido Dismutasa a microsomas, la Catalasa localizada en peroxi- nivel hepático. soma y la Alcohol Deshidrogenasa citosólica, A partir de los objetivos de este trabajo se el metabolito formado es el acetaldehído que realizó un estudio después de dos semanas es degradado por la enzima Aldehído Des- de abstinencia alcohólica sobre la influencia hidrogenasa mitocondrial (ALDH2) (5). El del ozono sobre el metabolismo del etanol y consumo crónico de etanol puede provocar la ocurrencia de peroxidación lipídica a nivel daño mitocondrial lo que conlleva a disfun- cerebral mediante la determinación de la ac- ción de la ALDH y por tanto aumento de las tividad cerebral de la enzima Aldehído deshi- concentraciones de acetaldehído al cual se le drogenasa mitocondrial y las concentraciones ha adjudicado un papel importante en el daño de MDA respectivamente. cerebral característico del alcoholismo y del

33 Materiales y Métodos Determinación del consumo de líquido Se utilizaron 20 ratas Lewis (250-300g) pro- venientes del Centro Nacional de Producción Se estableció dos grupos iniciales: Grupo de Animales de Laboratorio de (CENPALAB; Control de 5 ratas y Grupo tratado con etanol Mayabeque, Cuba). Las ratas fueron alimen- de 15 ratas. El Grupo tratado con alcohol se le tadas con alimento proveniente del Centro administró dosis ascendentes cada 14 días de Nacional para la Producción de Animales de experimentación (5% día 1, 10% día 15, 20% Laboratorio (CENPALAB; Mayabeque, Cuba) día 29, 30% día 43 y 40% día 57). Los anima- y agua/etanol “ad libitum” (45 g y 60 mL) en les tenían acceso solamente a la botella con cajas hogar bajo ciclos de luz y oscuridad de solución de etanol. A partir del día 70 hasta 12 horas a una temperatura de 20+2°C. Para el día 84 el grupo tratado con etanol se some- medir el peso y el consumo de alimentos se tió a una etapa de Tolerancia Farmacológica empleó una balanza analítica (Denver Instru- suministrándole “ad libitum” etanol a las dos ment XP-3000). Todos los procedimientos concentraciones (10% y 40%) para evaluar el con los animales se realizaron según lo esta- biomodelo de alcoholismo desarrollado. Se blecido por la unidad de gestión de la calidad realizó control del peso corporal, consumo de (UGC) del Centro de Investigaciones y En- comida y líquido durante las distintas etapas sayos Biológicos del Instituto de Farmacia y de inducción y tolerancia al consumo crónico Alimentos de la Universidad de La Habana en del etanol (84 días) y al final de los 14 días concordancia con la regulación de la Unión siguientes de Abstinencia Alcohólica. Europea para el tratamiento de animales de experimentación (Bethesda, MD, USA)(10) El estudio fue dividido en diferentes etapas Distribución de los grupos las cuales se muestran a continuación: experimentales

A- Etapa administración de etanol (In- Al terminar el día 84 del experimento el gru- ducción del alcoholismo) po tratado con etanol fue dividido en 3 grupos Etapa cuyo objetivo es inducir y estimular el de 5 ratas formando un total de 4 grupos de desarrollo de conductas reforzadoras a tra- ratas con 5 ratas cada uno: vés del consumo exploratorio, recompensa y Grupo 1: AGUA adicción al etanol. Se desarrolla con una es- Grupo 2: ETANOL calada de consumo in crescendo para generar Grupo 3: ETANOL+OXÍGENO dependencia física al etanol (11). Grupo 4: ETANOL+OZONO B- Etapa Tolerancia Farmacológica Etapa cuyo objetivo es la determinación de Aplicación de la Ozonoterapia y conductas compulsivas al consumo del etanol Oxígeno. propias de los alcohólicos. La preferencia de mayores concentraciones del etanol (entre El Ozono (O3/O2) se generó mediante el 10% y 40% durante 2 semanas) permite esta- OZOMED (equipo producido por el Centro blecer el grado de tolerabilidad y adaptación Nacional de Investigaciones Clínicas de Cuba, en el SNC. CNIC) a partir del oxígeno clínico, el cual constituyó alrededor del 3% de la mezcla O3/ C- Etapa de Abstinencia O2 + O2. A los grupos que fueron tratados Etapa cuyo objetivo es analizar los resultados con ozono se le administró por insuflación de la deprivación del consumo de etanol ca- rectal 1mg/kg, es decir, 5 cc durante 14 días racterísticos de los alcohólicos en Abstinencia de abstinencia. Los grupos que fueron trata- Alcohólica. Al grupo tratado con etanol se le dos con oxígeno se le administraron 5 cc por retiró abruptamente el suministro de etanol insuflación rectal durante los 14 días de la durante 2 semanas. El comportamiento de abstinencia. los grupos experimentales en esta etapa de- muestra la necesidad o no que presenten de consumir etanol lo cual es evaluado mediante Obtención del Suero test conductuales. Las ratas de estudio fueron sedadas en una campana de cristal con éter dietílico. Después de haber corroborado el efecto, se procedió a la extracción de 5ml de sangre por el plexo ocular. La sangre extraída se depositó en via-

34 les para centrifugar, debidamente rotulados y Determinación de la actividad de se mantuvo en reposo a temperatura ambien- ALDH te durante 10 min para la retracción del coá- gulo. Posteriormente el coágulo fue separado Fraccionamiento celular. Obtención cuidadosamente de las paredes del vial con de mitocondria. un capilar y se centrifugó a 3 600 rmin-1 du- Para la obtención de la fracción mitocondrial rante 15 min para la obtención del suero. El de células cerebrales en la medición de la acti- suero obtenido se congeló a -20˚C para luego vidad de ALDH, el homogenado se centrifugó ser empleado en una serie de determinacio- a 900 g por 15 minutos obteniéndose el pellet. nes bioquímicas. La extracción de sangre se El pellet obtenido de este paso se hizo resus- “Los resultados realizó en 3 etapas: al inicio (día 0), después pender con 10 ml de buffer sucrosa-tris-HCl de la etapa de consumir el 20% de alcohol (día 29) y al pH=7,4 0,2M. Posteriormente se rehomoge- de tolerancia finalizar la abstinencia (día 98) (12). nizó a 340 rpm y se centrifugó a 9000g por 10 minutos. El sobrenadante obtenido se vol- farmacológica en vió a centrifugar a 104.000 g por 60 min y el la que se expone Preparación de Homogenado de precipitado mitocondrial se lavó dos veces y al animal a dos cerebro. se re suspendió en 10 ml de la solución tam- pón, que se utilizó para la determinación de la concentraciones Los homogenados se obtuvieron en el día ALDH mitocondrial de cerebro. Se comprobó de etanol, una 99 del experimento empleando el homoge- la pureza de la fracción mitocondrial aislada baja 10% y otra nizador de cuchillas (Edmund Bühler HO4, mediante la determinación de la enzima Alco- Alemania) para lo cual se utilizó 2 g de tejido hol Deshidrogenasa. La enzima alcohol des- alta 40%, para en 20 ml de buffer sucrosa-tris-HCl pH=7,4 hidrogenasa fue utilizada, como enzima mar- que consuma la 0,2M según se describe en el PNO/TEC/0303. cadora de la fracción microsomal. Menos del que prefiera se Posteriormente se centrifugó 10 min, 3000 5% de la actividad alcohol deshidrogenasa se rpm a 4oC (Sigma Centrifuge 2K15, Reino encontró en la fracción microsomal, indican- observó que hubo Unido). El sobrenadante se guardó para la ob- do una contaminación mínima de enzimas un incremento tención de las fracciones subcelulares y para citosólicas (15). la determinación de los marcadores de estrés significativo oxidativo (13) (p < 0.05) en el Determinación de proteínas totales consumo de la solución etílica Determinación de La concentración de proteínas totales en el Malonildialdehído (MDA) homogenado de cerebro fue determinada por más concentrada el método descrito por Bradford (1976). Este lo cual indica la El MDA se midió como indicador de la POL método se basa en la coloración de las proteí- preferencia de las utilizando el método colorimétrico que em- nas presentes en el medio con el reactivo azul plea el 1-metil-2-fenil indol como cromógeno de Coomassie (Sigma), detectable a 595 nm. ratas en estudio reportado por Esterbauer, H en 1990. La con- La cuantificación se llevó a cabo mediante por el etanol”. densación de una molécula de MDA con dos una curva de calibración con albúmina sérica moléculas de 1-metil-2-fenil indol, bajo con- bovina (BSA) (Sigma) como patrón de refe- diciones de acidez da como resultado la for- rencia (16). mación de un cromóforo con una absorbancia máxima de 586 nm. Una solución de 7,6 mM de 1-metil-2-fenil indol en metanol al 33% en Determinación de aldehído deshidro- acetonitrilo se preparó inmediatamente antes genasa cerebral. de ser utilizada. Una alícuota de 325 μL de este reactivo se hizo reaccionar con 200 μL de La actividad de la ALDH2 se midió por el au- muestra se agitó y se añadió 75 μL de ácido mento de la producción de NADH a 340 nm. clorhídrico 37 %. Tras una nueva agitación de La mezcla de reacción contenía: 60 mM de microtubos de ensayo se incubaron 40 min a tampón de fosfato de sodio (pH 8,5), 1 mM de 45 ºC. Posteriormente, cuando alcanzaron la NAD+ + EDTA 1 mM y las proteínas mitocon- temperatura ambiente, se centrifugó a 3000 driales (0,5 mg / ensayo). La reacción se man- rpm durante 15 min y se leyó la absorbancia a tuvo durante 2 min a temperatura ambiente, 586nm contra blanco reactivo. Se utilizó una la reacción enzimática se inició mediante la curva patrón de 1,1,3,3-tetraetoxipropano adición del sustrato (10 mM propionil alde- para calcular las concentraciones finales (14). hído). El cambio de absorbancia se controló durante 30 segundos, 1, 2, 3 y 4 minutos para calcular la tasa de producción de NADH. La actividad específica de ALDH2 se calculó uti-

35 Figura 1

Figura 1. Esquema gráfico de habili- dades y competencias transferibles de interés para la mejor emplea- bilidad y competitividad de slos estudiantes de Doctorado. Fuente: “The European Council for Doctoral “La Candidates and Junior Researchers (EURODOC)”. http://eurodoc.net/ implementación skills-report-2018.pdf de un modelo de alcoholismo, que mimetice la sintomatología lizando el coeficiente de extinción molar de Resultados del humano la reducción de NAD+ de 6,22 × 106 cm2 a alcohólico, es 340 nm (Merck Index) y 1 unidad representa La Figura 1 muestra el consumo de líquido en una reducción de NAD+ (+ 1 nmol/min/mg los animales objeto de estudio durante las di- un requisito de proteína) a temperatura ambiente (17,18). ferentes etapas del desarrollo del modelo, el indispensable Procesamiento estadístico grupo control que consumió agua (Control) y para el estudio el grupo que ingirió etanol a diferentes con- Los datos experimentales obtenidos fueron centraciones. A todas las concentraciones que de los efectos sometidos a un análisis exploratorio donde se les fue suministrado el etanol los animales terapéuticos detectaron puntos aberrantes (outiers). Ade- en estudio consumieron la solución alcohó- de fármacos más se estimaron parámetros descriptivos lica, no existieron diferencias significativas como media y desviación estándar. (p<0.05) en el volumen de solución etanólica candidatos a ser Para determinar diferencias entre grupos en (10%-40%) consumido durante la inducción utilizados en el una variable en cuestión se utilizó un ANOVA del alcoholismo (56 días). Los resultados de tratamiento del de clasificación simple, seguida de un ensayo la etapa de tolerancia farmacológica en la que de comparación de medias (Student-New- se expone al animal a dos concentraciones de alcoholismo, man-Keuls) y el test de Student. El nivel de etanol, una baja 10% y otra alta 40%, para que principalmente significación estadístico empleado en todos consuma la que prefiera se observó que hubo en la etapa de los casos fue como mínimo de p< 0.05. Para un incremento significativo (p < 0.05) en el las correlaciones lineales se empleó el coefi- consumo de la solución etílica más concentra- la “sufrida” ciente de correlación de Pearson. Los datos da lo cual indica la preferencia de las ratas en abstinencia”. fueron procesados utilizando en paquete es- estudio por el etanol. En la etapa de Abstinen- tadístico: STATISTICA versión 6.0 para WIN- cia a las ratas alcoholizadas se les retiró el eta- DOWS. nol y durante 2 semanas se registró la ingesta de agua. Como puede apreciarse los animales alcoholizados consumieron más agua (p < 0.05) que los controles indicando las afecta-

Tabla 1 Grupos Actividad de Aldehído Deshidrogenasa experimentales mitocondrial en cerebro (mM/mL/min de NADH) Control (Agua) 6 .33 ± 0.021(a) Etanol 1 .23 ± 0.050(b) Etanol + Ozono 4 .85 ± 0.430(c) Etanol + Oxígeno 0 .97 ± 0.031(b)

Tabla I: Actividad de la Aldehído Deshidrogenasa 2 en homogenado de cerebro de las ratas estudia- das.

36 ciones metabólicas, entre otras manifestacio- un modelo de alcoholismo, que mimetice la nes, asociadas a la avidez por el agua durante sintomatología del humano alcohólico, es un la abstinencia. requisito indispensable para el estudio de los La Figura 2 representa las concentraciones efectos terapéuticos de fármacos candidatos a de malonildialdehído (MDA) a nivel sistémi- ser utilizados en el tratamiento del alcoholis- co en los diferentes grupos objeto de estudio, mo, principalmente en la etapa de la “sufrida” antes y después de la aplicación del Ozono. El abstinencia. El modelo seleccionado en nues- grupo que consumió etanol mostró elevadas tro trabajo tuvo en cuenta su acercamiento concentraciones de MDA respecto a los res- a las prácticas del alcohólico de ahí que se tantes grupos, mientras que el tratado con empleó el consumo de alcohol “ad libitum” ozono manifestó disminución de las concen- ya que es el más semejante al alcoholismo en traciones de MDA con respecto a la etapa pre- humanos (20). El consumo de etanol a las di- via a la administración de Ozono. ferentes concentraciones (Figura 1) demostró En la Tabla I se presenta la actividad de la la tendencia de los grupos experimentales a ALDH2 cerebral en los grupos estudiados, adquirir la conducta adictiva, lo cual se co- manifestándose aumento de la actividad de la rroboró con los resultados de la Prueba de enzima en los grupos ozonizados con respecto Tolerancia Farmacológica, al manifestarse la a los no ozonizados que consumieron etanol. preferencia por la solución etanólica de ma- La Figura 3 representa las concentraciones de yor concentración. Estos resultados se corres- MDA a nivel cerebral, en el grupo que fue ozo- ponden con lo planteado por otros autores al nizado existió un comportamiento en las con- afirmar que “El consumo crónico genera un centraciones del producto de la peroxidación progresivo requerimiento de dosis mayores lipídica similar al grupo control mientras que de sustancia para conseguir un efecto similar. en los restantes grupos las concentraciones de La tolerancia puede considerarse un intento MDA fueron significativamente mayores del organismo para retornar a la homeostasis, esto es, a las condiciones anteriores al consu- mo” (21, 22). Este modelo experimental per- Discusión mite no solo profundizar en los mecanismos de acción terapéutica, del agente en cuestión, El alcoholismo es una de las toxicomanías sino que hace posible ampliar los conocimien- que se manifiesta con más frecuencia a nivel tos relacionados con la etiopatogenia alcohó- mundial. El paciente alcohólico pasa por di- lica. Por otra parte, los hallazgos científicos ferentes etapas desde la adaptación al consu- que fundamentan los efectos terapéuticos del mo del alcohol hasta llegar al llamado punto ozono, por un mecanismo denominado Pre/ de “no retorno” (19). La implementación de Post condicionamiento oxidativo, constituye-

Figura 2

Figura 2. Concentración de MDA séricos en los grupos experimentales estudiados. Antes del PostOx Ozono corresponde al final de la etapa de Tolerancia, antes de la abstinencia y antes del PostOx. Des- pués del PostOx Ozono representa el final del tratamiento con ozono, el final de la Abstinencia y el pun- to final del estudio experimental. Se representan la media ± EEM. Letras diferentes indican diferencias significativas (* p<0.05), para cada grupo experimental, antes y después del PostOx Ozono

37 Figura 3

Figura 3. Concentraciones de MDA, en homogenado de cerebro, en las diferentes condiciones experi- mentales. Las concentraciones de MDA en cerebro de rata, corresponde con el final de la Abstinencia, del PostOx y del experimento (105 días) Se representan la Media ± EEM. Letras diferentes indican diferencias significativas (* p<0.05).

ron la base científica que justifica abordar el lesiones, inducidas por el consumo crónico estudio de los efectos del ozono, sobre la so- de etanol incrementan la producción de su- breproducción de ERO y las alteraciones de peróxido y peróxido de hidrógeno dentro de algunas de las funciones básicas del SNC que la mitocondria afectando a la integridad mi- se modifican o alteran en el sujeto alcohólico. tocondrial y con ello la función de la enzima Los cambios fisiopatológicos en la función ce- ALDH2 (25). lular, inducidos por la exposición crónica de El consumo de etanol es un detonante muy alcohol, se derivan del propio metabolismo importante para la generación de estrés oxi- del etanol que incluye la generación de acetal- dativo- nitrosativo en el hepatocito y ya sea a dehído, radicales libres (como el hidroxietil) corto o a largo plazo, puede desencadenar la y NADH, entre otros causando Estrés Oxi- muerte de la célula, el tejido y el organismo dativo. Es así como mucha de la sintomato- en general. Algunos factores que están invo- logía asociada a las disfunciones orgánicas y lucrados en la generación de estrés oxidati- conductuales del alcohólico son el resultado vo-nitrosativo por etanol son: cambios en el del daño que promueven estas moléculas oxi- estado redox intracelular como resultado del dadas, sobre el hígado y el Sistema Nervioso metabolismo oxidativo del etanol, hipoxia ce- Central, tanto durante la tolerancia como en lular, porque las alteraciones en el metabolis- la abstinencia. (23) mo redox afectan el consumo de oxígeno y la El acetaldehído, derivado del metabolismo producción de ATP en la mitocondria. del etanol y de reconocida toxicidad, es trans- El metabolismo del etanol y el estrés oxida- formado a ácido acético por la enzima Aldehí- tivo asociado, producen una serie de com- do Deshidrogenasa mitocondrial, isoforma 2 puestos de alta reactividad, capaces de unirse (ALDH2). Esta enzima metaboliza eficiente- a un gran número de residuos proteicos. El mente el acetaldehído, producido durante el resultado de estas uniones es la generación de metabolismo del etanol, pero también meta- nuevas entidades bioquímicas que, de forma boliza otros aldehídos lipídicos tóxicos como genérica, reciben el nombre de aductos. el MDA y 4-hidroxi-2-nonenal, entre otros Estos aductos, según su composición, pueden (24). El consumo crónico de etanol induce clasificarse en cuatro grandes grupos: aductos lesiones en los complejos I y III de la cadena derivados del acetaldehído, que provienen de de transporte electrónico mitocondrial, con la formación de enlaces entre el acetaldehído afectaciones en la transferencia de electrones y residuos lisina o grupos amino de distintas desde el flavín-mononucleótido del complejo proteínas. Estos enlaces pueden ser covalen- I al complejo III, se produce acumulación del tes o no covalentes, lo que determina la esta- complejo flavín-mononucleótido-semiquino- bilidad del aducto formado, algunos produc- na y una generación incrementada de radi- tos aldehídicos derivados de la peroxidación cales superóxido dentro del complejo I. Estas lipídica, como el MDA o el 4-hidroxinonenal,

38 también son capaces de formar aductos con provoca efectos antioxidantes se resumen en proteínas, también se ha documentado la for- dos moléculas intermediarias: Peróxido de mación de aductos mixtos o híbridos, que son Hidrógeno y los productos de POL (MDA). aquellos formados por la unión de acetaldehí- El O3/O2 induce la formación de H2O2 e do y MDA a un mismo sustrato, finalmente, hidroperóxidos, en cantidades pequeñas, los en presencia de átomos de hierro, los radica- cuales son capaces de actuar sobre los eritro- les hidroxietilos formados en la oxidación del citos, plaquetas, monocitos y células endote- etanol, pueden formar aductos con proteínas. liales estimulando la acción de la Glutatión La unión de grupos aldehído a una biomolé- Peroxidasa (GSH-Px) para formar H2O. Esta cula resulta en una alteración estructural y actividad prooxidante estimula no sólo la consecuentemente, funcional de dichos sus- GSH-Px sino también la Glutatión Reductasa tratos. Así, se ha descrito que tras su unión (GSH-Rx) y aumenta los niveles de Glucosa con moléculas de acetaldehído (formación 6-Fosfato (G-6P). Como consecuencia se pro- “Estos resultados se de aductos), la actividad de algunas enzimas duce un aumento del intercambio iónico, de corresponden con se reduce o incluso se suprime. El metabo- los niveles de 2,3 difosfoglicerato (2,3-DPG), estudios anteriores lismo del etanol y la consecuente formación de los niveles de ATP y, por ende, de la glicó- de aductos en el SNC, están implicados en los lisis. El O3/O2 también estimula un aumento (27) donde se efectos tóxicos del etanol en otros órganos y de la relación NAD+/NADH activando la vía evidencia, tanto tejidos y en la generación de alteraciones en del monofosfato de hexosa dependiente indu- preclínica como el cerebro. ciendo la liberación de ATP de los eritrocitos Los aductos de acetaldehído pueden formar y vasodilatación (28). clínicamente, el complejos con serotonina llamados tetrahi- Se ha demostrado que el ozono es capaz de efecto antioxidante drocarbolinas, ambos complejos aumentan la proteger a las células hepáticas frente al daño del ozono y sus preferencia por etanol y pueden inhibir com- producido por los radicales libres del oxígeno; petitivamente la MAO y la COMT. así como la preservación de las concentracio- consecuencias En el presente estudio existió un incremento nes de glucógeno hepático, evitando la sobre- favorables en las concentraciones de MDA en los grupos producción de lactato, que en el homogenado sobre trastornos no tratados con ozono lo cual indica peroxida- de hígado está asociado al daño hepatotóxico ción lipídica. Este aldehído tiene una especial (29). El restablecimiento en las concentra- conductuales importancia en el alcoholismo y en la absti- ciones de MDA, sugiere la preservación de la durante la nencia alcohólica ya que el MDA es un sustra- enzima ALDH2, evitando la acumulación de abstinencia to de la Aldehído Deshidrogenasa (ALDH2). acetaldehído, a nivel sistémico y del SNC, con Esta enzima es responsable del metabolismo la consiguiente disminución en la formación alcohólica”. del acetaldehído (inductor del daño cerebral de aductos. por consumo crónico de etanol) y otros alde- La disminución de las concentraciones de hídos lipídicos producidos por el metabolis- MDA y el aumento de la actividad de la enzi- mo del etanol. Por lo tanto el incremento del ma ALDH2 sugieren que el tratamiento con MDA sugiere la inhibición de ALDH2, la cual ozono protegió la integridad mitocondrial puede ser consecuencia de las especies reacti- manifestada con la disminución de la peroxi- vas de oxígeno y nitrógeno (26). La adminis- dación lipídica lo cual puede ser con secuen- tración de ozono preservó la actividad de la cia del incremento en el metabolismo tanto enzima ALDH2 cerebral y disminuyó las con- del acetaldehído como del malonildialdehido centraciones de MDA, séricas y cerebrales. sustratos de la enzima y relacionados con el Estos resultados indican el efecto protector daño cerebral presente en la Abstinencia Al- del ozono sobre el daño que provocó el eta- cohólica. nol y por tanto la capacidad de estimular la La aplicación de nuevos procedimientos ex- recuperación funcional de una de las enzimas perimentales, así como la identificación de que de forma más significativa participa en su posibles biomarcadores que sean confiables y metabolismo. Al tener en cuenta que la pro- sensibles, es un gran avance que puede con- pia ALDH2 participa en el metabolismo del tribuir a implantar un adecuado sistema de MDA, la disminución de las concentraciones prevención y diagnóstico para detectar, en lo del mismo indican que también el ozono fue posible, el daño ocasionado por la ingestión capaz de disminuir el proceso de POL. Estos de etanol. resultados se corresponden con estudios an- teriores (27) donde se evidencia, tanto preclí- Agradecimientos: nica como clínicamente, el efecto antioxidan- Los autores queremos expresar nuestra grati- te del ozono y sus consecuencias favorables tud al Instituto de Farmacia y Alimento de la sobre trastornos conductuales durante la abs- Universidad de la Habana por todo el soporte tinencia alcohólica. profesional en el desarrollo de este estudio El mecanismo por el cual el ozono médico

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40 41 42 Función fisiológica y fisiopatológica de las membranas asociadas a mitocondrias (MAMs): Posible rol en el procesamiento de APP y en la disfunción mitocondrial en la enfermedad de Alzheimer.

Panes-Fernandez J 1, Flores-Nuñez O 1, Fuentealba J1* 1 Laboratory of Screening of Neuroactive Compound, Physiology Department, Faculty of Biological Sciences, Universidad de Concepción, Concepción, Chile

Resumen desconoce su causa, histológicamente se han identificado dos marcadores; las placas seni- La Enfermedad de Alzheimer (EA) es la princi- les y los ovillos neurofibrilares (Ballard et al., pal forma de demencia que afecta a los adultos 2011). El péptido Aβ se postula como princi- mayores. Se caracteriza por una muerte neu- pal agente responsable de la EA (Hardy and ronal progresiva, especialmente en hipocam- Higgins, 1992), por su capacidad de generar po y corteza, y uno de los principales eventos dishomeostasis del Ca+2, ATP, y lípidos (Blass celulares con los que cursa esta patología es la et al., 2000; Lal et al., 2007)electrophysiolo- disfunción mitocondrial. Se postula que esta gical, and neuroimaging examinations. The- enfermedad neurodegenerativa se produce refore, impairment in energy metabolism in principalmente por el aumento en los nive- AD can not be attributed to loss of brain subs- les del péptido beta-amiloide (Aβ), que se tance or to electrophysiological abnormali- genera a partir del procesamiento proteolíti- ties. Among the characteristic abnormalities co de la proteína precursora amiloide (APP). in the AD brain are deficiencies in several Se ha descrito que tanto APP como la ma- enzyme complexes which participate in the quinaría enzimática de la vía amiloidogénica mitochondrial oxidation of substrates to yield y no amiloidogénica presentaría diferente energy. There include the pyruvate dehydro- localización espacial; mientras que los com- genase complex (PDHC. En este contexto, la ponentes de la via no amiloidogénica se en- mitocondria es un factor clave, ya que funcio- contrarían en la membrana plasmática y na como tampón de Ca+2, productor de ATP, y compartimentos secretores tardíos, los com- participa en el metabolismo de los lípidos, por partimentos endosómales, Golgi y estructuras ende, se ha asociado la disfunción mitocon- tipo balsas lipídicas denominadas MAMs (del drial como evento temprano de la pérdida de inglés Mitochondria-Associated RE Mem- la conexión de la red neuronal, falla sináptica, branes) serían los encargados de procesar y finalmente de la muerte neuronal (Onyango APP por la vía amiloidogénica, y producir Aβ. et al., 2016). Las MAMs son dominios especializados entre La producción del péptido Aβ comienza por Retículo endoplásmico (RE) y mitocondrial, el procesamiento proteolítico de la proteína donde existe una alta aposición entre ambos precursora amiloide (APP), proteína inte- organelos de entre 10-20 nm, que les per- gral de membrana que se puede procesar por mite comunicarse entre sí tanto física como dos vías. La vía no amiloidogénica involucra bioquímicamente, lo que facilita las funcio- la participación de α-secretasa, una enzima nes de esta región tales como la homeosta- que pertenece a la familia de metaloproteasas sis del calcio, colesterol, fosfolípidos, y el ADAM (del inglés A Disintegrin and Metallo- metabolismo mitocondrial. Existe evidencia protease), y γ-secretasa, complejo compuesto reciente que ha encontrado que las funcio- por 4 proteínas: Presenilina 1 o 2 (PS1 o PS2), nes localizadas en las MAMs estarían au- Nicastrina (Nct), Aph-1 (del iglés Anterior mentadas significativamente en los modelos Pharinx-Defective-1) y Pen-2 ( del inglés pre- animales y muestras de pacientes con la EA. senilin enhancer 2); de estas proteínas, PS1 En esta revisión bibliográfica estudiaremos y PS2 tienen la actividad catalítica y el resto los principales roles fisiológicos y fisiopatoló- tiene funciones regulatorias. Estas enzimas gicos de las MAMs, y su impacto en la funcio- forman los productos: APPsα (APP soluble nalidad mitocondrial y en el procesamiento α), C83(APP-C83), AICD(Dominio intrace- de APP. lular carboxi terminal de la proteína APP) , y p3. Por otra parte, en la vía amiloidogénica, actúan β y γ-secretasa, que forman APPsβ, Introducción C99 (APP-C83), AICD y el péptido Aβ (Muller et al., 2017; Suh and Checler, 2002)the cause La Enfermedad de Alzheimer (EA) es una and progression of both familial and sporadic enfermedad neurodegenerativa, caracteriza- AD have not been fully elucidated. The auto- da por la pérdida progresiva de la memoria y somal-dominant inherited forms of early-on- aprendizaje (Castellani et al., 2010). Si bien se set Alzheimer’s disease are caused by muta-

43 tions in the genes encoding APP, presenilin-1 meses). Además, en tejido cerebral de pacien- (chromosome 14. tes con EA (etapa Braak V-VI) los niveles de Se ha descrito que tanto APP como la maqui- VDAC1 aumentaron significativamente (55%) naría enzimática de ambas vías presentarían comparados con individuos control (Cuadra- diferente distribución subcelular; la vía no do-Tejedor et al., 2011). amiloidogénica se produciría principalmente En relación con el IP3R, existen trabajos que en la membrana plasmática y compartimentos muestran resultados distintos dependiendo secretorios tardíos, mientras que la vía ami- de los modelos de estudio. En células CHO- loidogénica se produciría principalmente en 7PA2 que expresan APPV717F, se encontró un compartimentos endosomales, balsas lipídicas incremento en los niveles del IP3R en com- (Zhang and Song, 2013) y estructuras denomi- paración a células control, mismo resultado nadas MAMs (Membranas de Retículo Endo- que se observó en modelos celulares de so- plásmico asociadas a mitocondrias) (Area-Go- breexpresión de APP swedish (APPswe); sin mez et al., 2012). Por lo tanto, sería en estos embargo, en muestras de corteza de ratones últimos compartimentos intracelulares donde transgénicos Tg2576 la expresión de IP3R no se produciría el péptido Aβ, y no en la super- se vio afectada significativamente (Oules et ficie de la membrana plasmática, como se ha al., 2012). sugerido clásicamente. Por otra parte, en ratones P301L tau, que fue- Las MAMs son dominios especializados entre ron inyectados en la amígdala con el péptido

retículo endoplásmico (RE) y mitocondrial, Aβ1-42, no se observaron cambios significati- de una naturaleza semejante a las balsas li- vos en muestras de corteza para la proteína pídicas ya que son microdominios ricos en GRP75 en comparación a muestras de corteza esfingomielina y colesterol resistentes a los control. Esto también fue observado por aná- detergentes (Aria Gómez et al., 2012), donde lisis de western blot de la corteza temporal existe una asociación estrecha entre ambos de individuos con EA, donde no se observa- organelos (10-20 nm), que les permite co- ron cambios significativos en los niveles de municarse entre sí física y bioquímicamente, GRP75 en comparación con individuos con- facilitando las funciones de esta región tales trol (David et al., 2006). como metabolismo del Ca+2, lípidos, y regu- Además, Dyzma describió una evidencia fun- lar funciones mitocondriales (Bernard-Ma- cional de que el incremento en las asociacio- rissal et al., 2018). Existe evidencia reciente nes MAMs estaría relacionada con una mayor de que las funciones asociadas a las MAMs transferencia de Ca+2 desde el retículo hacia la aumentan significativamente en los modelos mitocondria mediado por la vía IP3R-VDAC1 animales de la EA y en las células de pacien- en un modelo de la EA. En células SHSY5Y al tes con EA (Area-Gomez et al., 2012). Es por co-incubar con una agonista de la chaperona ello por lo que a continuación describiremos del IP3R Sigma R1 (Receptor Receptor intra- los principales roles que cumplen los MAMs celular no-opioide sigma 1), localizada en la y sus implicancias en la EA cuando estos se interfaz RE-mitocondria, y el péptido Aβ, se incrementan de forma patológica. producía una potenciación del flujo de Ca+2 desde el RE hacia la mitocondria (Dyzma et Rol de los MAMs en la transferencia al., 2012). de Ca+2 Rol de los MAMs en mitofagia Los MAMs tiene un rol en la transferencia de Ca+2 desde RE a la mitocondria, donde parti- El proceso de mitofagia es un proceso degra- ciparían los proteínas IP3R (receptor de IP3), dativo que regula el control de calidad mito- Sigma R1 (Receptor intracelular no opioide condrial, en el cual las mitocondrias depola- sigma 1) a nivel de RE, y VDAC1(canal selecti- rizadas asociadas un fenotipo hiperfisionado vo de aniones dependiente de voltaje 1) a nivel activan su reciclaje selectivo, y por lo tanto la de la membrana mitocondrial externa (MME). célula es capaz de adaptar la cantidad y cali- Esta comunicación inter-organelar coordina dad de las mitocondrias dependiendo de sus tanto el metabolismo de Ca+2 como preserva- necesidades energéticas. Este proceso estaría ción de la integridad de la MME (Vance, 2014). mediado por receptores Atg8 y Atg32 (Proteí- Los estudios de proteómica para evaluar los na relacionada con la autofagia 8 y 32), que niveles las proteínas del complejo IP3R- reclutan la maquinaria autofágica en la mito- VDAC1, mostraron un aumento dependiente condria; evento que promueve su envoltura de la edad de los niveles de proteína VDAC1 por un fagóforo que posteriormente se fu- en el hipocampo de ratones Tg2576, un mo- siona con un lisosoma (Bockler and Wester- delo de la EA (Hsiao et al., 1996), comparadas mann, 2014). con ratones control de la misma edad (6-12 De acuerdo al trabajo de Gelmetti (Gelmetti

44 et al., 2017), se logró describir que la forma- anterógrado, entre el soma neuronal y el axón ción de autofagosomas (mitofagosomas) ocu- (Bernard-Marissal et al., 2018). rriría en las MAMs; y que este proceso estaba Sin embargo, existe escasa información del mediado por la relocalización de las proteínas papel de las MAMs en el compartimento axo- mitofágicas PINK1, BECN1 en las MAMs. En nal, y si la disfunción de MAM puede afectar otro trabajo, Böckler en un modelo de leva- específicamente el transporte axonal, ya que duras realizó mutantes de proteínas MAMs la mayoría de los estudios están hechos en lí- Mmm1, Mdm10, Mdm34, Mdm12 encarga- neas celulares. En modelos de esclerosis late- das de estabilizar el acoplamiento RE-mito- ral amiotrófica (ELA) y de neuropatías here- condria, observado que estas mutantes eran ditarias motoras y sensoriales Villegas en un defectuosas específicamente para el proceso explante de nervio usando mutantes de pro- mitofágico. Además, encontró una asociación teínas MAMs SigmaR1 y Mfn2, logró visuali- entre atg8 y Mmm1, resultado que indicaría zar que había una degradación axonal asocia- que la asociación MAM es necesaria para ini- da a una falla en el transporte mitocondrial, ciar el proceso de la mitofagia de forma selec- lo que estaría sugiriendo que este transporte tiva (Bockler and Westermann, 2014). es dependiente de la formación de las MAMs (Villegas et al., 2014). Rol de los MAMs en transporte En motoneuronas, al suprimir la expresión de mitocondrial la proteína SigmaR1 disminuyó el transpor- te anterógrado mitocondrial, conduciendo La comunicación entre el cuerpo celular y a una acumulación mitocondrial en la zona los terminales sinápticos en las neuronas se distal del axón, interesantemente esta acu- basan en el transporte axonal, el cual es alta- mulación también condujo a un cambio en la mente eficiente para las mitocondrias, ya que morfología mitocondrial (Bernard-Marissal deben alcanzar sitios especializados con alta et al., 2018). energía de en un breve tiempo. Se ha descrito una correlación positiva entre el transporte Rol de los MAMs en dinámica de mitocondrias axonal con proteínas MAMs, mitocondrial en donde sería necesaria la interacción de Mfn2 (mitofusina 2), MIRO1/2 (Rho GTPa- La dinámica mitocondrial es el proceso que se 1 y 2 mitocondrial) y las proteínas moto- regula la fisión y fusión de mitocondrias, de- ras quinesinas y dineinas para promover el pendiendo de los requerimientos energéticos transporte de las mitocondrias retrogrado y de la célula, suceso donde participan pro-

45 teínas para fisión como DRP1 y Fis1, y para y observó que los MAMs principalmente pre- fusión, como Mfn1,2 y OPA1. Se ha descrito sentaban áreas de superficie de contacto que que estos complejos MAMs se encontrarían alcanzaban asociaciones cortas (menores a directamente asociados con un rol directo en los 50nm) (superiores al 50%), en tanto que la dinámica mitocondrial, ya que se ha encon- áreas de contacto larga (entre 50 a 200 nm) trado que tanto DRP1 como mitofusina 1 y 2 se encontraban en torno al 30%, y las áreas de se encontrarían reclutados en estos comple- contacto muy largas (mayores a los 200nm) jos, sin embargo, se le han adjudicado a los sólo alcanzaban entorno al 4%. En contrapar- MAMs un rol en la desarrollo un fenotipo más te, se observó que en fibroblastos de pacien- fisionado. De hecho, cuando hay una disrup- tes con FAD y SAD, tenían áreas de contacto ción de los complejos MAMs por la deleción principalmente largas y muy largas, alcanzan- de Sigmar1, se observó una alteración en la do porcentajes del 40% y del 35%, respectiva- dinámica mitocondrial, que condujo a que las mente (Area-Gomez et al., 2012). mitocondrias presentasen un fenotipo mito- Para lograr comprender por qué estas con- condrial más fusionado (Bernard-Marissal et formaciones altamente especializadas entre al., 2018). retículo y mitocondrias presentan una sobre- Otra proteína altamente estudiada que coor- expresión, el grupo de Aria Gómez comenzó dina la fusión mitocondrial es Mfn2, proteí- a estudiar si componentes de la vía amiloi- na que forma homodiméros entre RE y mi- dogénica estaban regulando este aumento. tocondria (ya que se encuentra expresada en Describió que las presinilinas en fibroblastos ambos organelos), o heterodímeros con Mfn1 humanos se encontraban enriquecidas en los (se encuentra sólo en mitocondria). En efecto, MAMs, utilizando marcadores contra proteí- en células MEFs ( del inglés Mouse Embryo- nas como es FACL-4, proteína descrita clási- nic Fibroblasts) de un ratón MFN2-KO, hubo camente como proteína MAM. Además, por una menor obtención de MAMs por fracciona- fraccionamiento subcelular (para separar los miento subcelular, lo que estaba directamen- MAMs del retículo y mitocondria), se observó te correlacionado con la evidencia funcional que las presenilinas 1 y 2 se encontraban al- que mostró que había una menor liberación tamente enriquecidas en los compartimentos de Ca+2 del RE a la mitocodnria, comparados MAM, respecto de la membrana plasmática, con MEFs WT (Filadi et al., 2018). Sin embar- la mitocondria y el retículo endoplasmático. go, en el año 2012 el grupo de Orci (Cosson Además, al analizar la actividad de esta pro- et al., 2012), en el mismo modelo, se observó teína por un ensayo enzimático se pudo con- un efecto opuesto; por microscopía de trans- cluir que son los compartimiento MAMs los misión electrónica e inmunofluorescenca en que tienen una mayor actividad de la γ-secre- células MEFs del ratón MFN2-KO hubo un tasa con respecto a los otros compartimien- incremento en la yuxtaposición de los MAMs tos subcelulares. En otro trabajo similar, el (~4.9%), comparadas con MEFs WT (~2,2%), grupo de Schreiner describió que además este y desde un punto de vista funcional hubo una compartimiento MAM era el que presentaba directa correlación con el aumento de MAMs, una mayor producción del péptido β-amiloi- ya que había una mayor liberación de Ca+2 del de, respecto de RE, mitocondria y membrana RE a la mitocondria en MEFs MFN2-KO que plasmática (Schreiner et al., 2015). en MEFs WT (Filadi et al., 2018). Interesan- Respecto de la localización de los otros com- temente, estos efectos estuvieron asociados ponentes de la vía amilodogénica, se analizó a una disminución de la expresión del MCU la localización de β-secretasa y APP en com- (uniportador de Ca2+ mitocondrial), el cual partimentos intracelulares. Primero, por frac- media el importe de Ca+2 por la membrana mi- cionamiento subcelular con separación de tocondrial interna. Por lo tanto, y a pesar de gradiente de sucrosa en fracciones mitocon- que aún es controversial el rol de Mfn2 en los driales y fracciones endosomales lisosomales, MAMs, si podemos establecer que participa se pudo observar con distintos marcadores activamente como espaciador de la conforma- para endosomas, Golgi, retículo y MAMs, que ción de las asociaciones RE-mitocondria, aun- APP y BACE1, el componente catalítico de la que serán necesarias más investigaciones para β-secretasa, cofraccionan parcialmente con abordar este problema. marcadores de endosomas tempranos y tar- díos, y con marcadores MAM. Sin embargo, Rol de los MAMs en la EA faltan evidencias funcionales de la actividad de β-secretasa en estos compartimentos (Pera En fibroblastos de pacientes humanos el gru- et al., 2017). Otra observación importante de po de Aria Gómez describió que existían dis- estos autores fue que en cerebros de ratones tintas conformaciones entre retículo y mito- WT no había un inmunomarcaje positivo condria en fibroblastos de pacientes control, para los fragmentos derivados de la escisión

46 en el fragmento C-terminal de APP por la ACAT1, enzima que transforma el colesterol al β-secretasa (APP-CTF), que corresponden al colesterol éster, se observó que en células WT fragmento C83 y al C99. Esto podría eviden- MEFs tratadas con un inhibidor de la γ-secre- ciar 2 fenómenos; el primero es que no esta- tasa (DAPT) había un aumento en la actividad rían presentes en estos complejos MAMs y la de ACAT1 en comparación al vehículo con otra posibilidad es que es la alta la actividad DMSO y este efecto se revirtió al añadir un in- de la γ-secretasa estaría rápidamente escindi- hibidor de la β-secretasa (BI). Cabe destacar do el fragmento APP-CTF de la vía amiloido- que este efecto no se vio revertido en este caso géncia (C99) para producir el péptido β-ami- por in inhibidor de la α-secretasa (TAPI); con- loide y el dominio intracelular AICD. firmando entonces que sería C99 y no C83 el Por ende, utilizando un doble KO para las pre- que estaría regulando las MAMs. sinilinas 1 y 2, observaron que los APP-CTF se Interesantemente, se observó que había una encuentran cofraccionando con endosomas y comunicación entre presinilinas y la proteína con los MAMs. En células COS-7, línea celu- Mfn2, ya que ambas participaban en la misma lar de riñón humano, tratadas con DAPT, un ruta de actividad de las MAMs (Area-Gomez inhibidor de la γ-secretasa, se observó una et al., 2012). Para ello, en células WT MEFs triple colocalización positiva entre C99, SE- comparadas con doble KO (DKO) de las presi- C61β, una proteína de retículo, y una sonda nilinas 1 y 2 se observó que el escisión de C99 mitocondrial, indicando la presencia de este se vio inhibida en células MEFs DKO. Por otra fragmento en los MAMs (Pera et al., 2017). parte, al utilizar un ratón KO para mitofusina Además, en células MEFs del doble KO de las 2 en células MEFs y al compararlos con rato- presenilinas utilizando anticuerpos APP-CTF, nes WT MEFs, se observó un efecto similar en se observó la presencia de C99 en los MAMs células MEFs DKO; ambas presentaban una (Pera et al., 2017). disminución de escisión de C99. Estos datos Sin embargo, falta evidencia que indique que establecen entonces que Mfn 2 regularía po- efectivamente sería C99 el fragmento que se sitivamente el clivaje de C99 y la actividad de encuentra en los MAMs, debido a que estos γ-secretasa, y, por ende, estaría potenciando autores utilizaron un anticuerpo que también la vía amiloidogenica. sería positivo para C83; por lo tanto, faltan más trabajos que indiquen efectivamente es Rol de los MAMs en la disfunción este fragmento de la vía amiloidogénica que mitocondrial estaría localizado en estos compartimientos. Se observó que los MAMs se encuentran regu- Ha sido ampliamente estudiado que en la lados negativamente por las presinilinas. En EA ocurre una disfunción mitocondrial, pero células MEFs de ratones DKO para las presi- aún se desconoce la razón de esta alteración. nilinas, se observó que había un incremento Una de las hipótesis que ha sido ampliamen- en la colocalización entre marcadores de RE y te estudiada por el grupo de Área Gómez mitocondria, respecto de células MEFs de ra- (Area-Gomez et al., 2009, 2012; Pera et al., tones WT. Además, al utilizar BI, un inhibidor 2017), es que el aumento de la comunicación de β-secretasa, se observó una disminución mitocondrial ER y la función MAM en la EA significativa de la colocalización entre ambas sería la responsable de esta alteración, y esta marcas. Por lo tanto, C99 estaría regulando di- alteración esta mediada por una potenciación rectamente la sobrerregulación MAM. Este re- de la vía amiloidogénica. En efecto, el rol que sultado también fue confirmado por ensayo de tendría C99 de la vía amiloidogénica tendría microscopía electrónica, en donde observó que un alto impacto en la funcionalidad mitocon- había un incremento en las áreas de contacto drial. Esta alteración en funcionalidad mito- largas y muy largas de los MAMs (>200nm) condrial fue observada mediante ensayos de en células DKO, que además presentaban una Seahorse de fracciones enriquecidas en mito- conformación punteada no superior al 20%, condrias, que permiten medir en tiempo real respecto de las células MEFs-WT, que princi- medir el consumo de oxígeno (ORC), fenóme- palmente tenían una conformación punteada no asociado directamente a la funcionalidad (sobre el 80%), en tanto que alcanzaban áreas de los complejos respiratorios de la cadena de contacto en bajo porcentaje (menores del transportadora de electrones, y por ende, a la 16%), y las muy largas no más de un 4%. Ade- capacidad de producir ATP. más, se observó un aumento de la funcionali- Se observó que células MEFs APP-DKO (do- dad de los MAMs en células MEFs de ratones ble KO de APP) presentaban un mayor consu- DKO, respecto de células MEFs de ratones WT mo de oxígeno en comparación a MEFs WT, (Area-Gomez et al., 2012). indicando que C99 estaría regulando negati- Desde un punto de vista funcional, por ensayos vamente la función mitocondrial. Este resul- de radioactividad donde se midió la activad de tado fue confirmado al utilizar un fragmento

47 En la siguiente esquema se encuentra un modelo propuesto que resume lo expuesto anteriormente en esta revisión:

Figura 1. Rol de las MAMs en la EA. Esquema representativo que muestra las vías que esta- rían involucradas en la falla sináptica. Se puede observar que el incremento en la asociación RE-mitocondria mediaría la disfunción mitocondrial mediante alteración de los componen- tes encargados de regular la homeostasis de calcio (I), mitofagia (II), transporte y dinámica mitocondria (III), y la producción del péptido Aβ (IV).

de C99 en el ratón APP-DKO donde hubo ble β que no se ha descrito hasta el momento, una disminución significativa de la función que y pudiesen también estar afectando la mitocondrial en ratones APP-DKO C99, com- funcionalidad mitocondrial. Por lo tanto, po- paradas con ratones APP-DKO. Es más, esta demos concluir que la alteración o mutación disminución se vio potenciada al utilizar este las presinilinas impiden el correcto corte de fragmento C99 en conjunto con un inhibidor C99, lo que estaría induciendo disfunción de la γ-secretasa (DAPT), indicando que C99 mitocondrial, evento que se ha asociado di- estaría promoviendo la pérdida de la correcta rectamente con la falla sináptica y la muerte función mitocondrial (Pera et al., 2017). neuronal en pacientes con EA. Por otra parte, en células MEFs DKO (para En suma, como hemos visto, es posible que la presinilina 1 y 2), hubo una disminución sig- intercomunicación entre RE y Mitocondrial va nificativa del consumo de oxígeno respecto más allá de los roles clásicamente atribuidos a células MEFs WT. Sin embargo, cuando se a estas dos importantes organelas; y permiten utilizó un inhibidor de la síntesis de Novo de pensar que frente a la carencia de selectivi- lípidos (Miriocina), y por ende, bloqueando dad o especificidad de los actuales fármacos el flujo de lípidos hacia la mitocondria, hubo indicados en las etapas leves a moderadas de una reversión de este fenómeno de disfunción la EA, la identificación de nuevas dianas tera- mitocondrial, reflejando en la disminución en péuticas, que permitan abrir nuevos espacios la capacidad respiratoria mitocondrial estaría de desarrollo de moléculas de síntesis, o mo- mediada por el aumento del metabolismo li- léculas biológicas que puedan modular la fun- ción de las MAMs, representa una interesante pídico en los MAMs (Pera et al., 2017). Cabe oportunidad para el desarrollo farmacológico destacar que falta evidencia de posibles efec- de medicamentos neuroprotectores. tos de AICD y de los fragmentos de APP solu-

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49 50 Especial Congreso

51 Estimadas socias y socios ean todas y todos muy bienvenidos a tra experiencia a disposición de la sociedad, Concepción, la ciudad Universitaria para convertirnos en referentes y líderes de de Chile, que les recibe en un año es- opinión en el área de nuestra experiencia. pecial donde la comunidad penquista Tenemos el deber ético de estar estrechamen- Scelebra a la Universidad de Concepción, su te vinculados a las necesidades de nuestros emblema más reconocido, en el aniversario ciudadanos. Ya hemos visibilizado este com- de sus primeros 100 años, periodo en el que promiso a través de actividades de extensión, ha aportado a la generación de conocimiento como lo hicimos con las charlas de difusión en las ciencias, las artes y las humanidades, realizadas en diversos colegios de la comuna a la política social del país y a su desarrollo. de Santa Cruz en el año 2018, las presenta- Hemos trabajado arduamente como directiva ciones en colegios de La Serena a comienzos para lograr que el presente Congreso cuente de este año y nuestra participación en el Bra- con toda la excelencia, experiencia y juven- in Awarennes Week Concepción 2019, donde tud que caracteriza a nuestra organización. ocho de nuestros socios realizaron exposicio- Recibiremos en Concepción a una veintena nes científicas para más de 2000 alumnos de de investigadores internacionales provenien- colegios municipales, un hecho inédito para tes desde México, Cuba, Venezuela, Uruguay, nuestros registros. Ecuador, Brasil y Argentina, quienes se han También hemos contribuido a cursos de ac- interesado en compartir con nosotros sus in- tualización en temas de Cáncer, Drogas de vestigaciones, exposiciones que se sumarán Abuso y Adicciones, Neurofarmacología y a las de destacados especialistas en farma- Fármaco-endocrinología, entre otros. Nues- cología del país y a las de nuestros invitados tra excelencia científica una vez más se ve especiales. reconocida por la revista Frontiers in Phar- Es así como hemos construido un programa macology, que nos ha dedicado una sección académico y científico de alto nivel que conta- especial (Research Topic) donde tenemos rá con 9 simposios, 2 mini simposios, más de cerca de 70 trabajos científicos comprometi- 70 expositores nacionales e internacionales, dos por los socios, de los cuales cerca de 40 entre quienes destacan 5 reconocidos confe- se encuentran ya evaluados o en proceso de rencistas de prestigio mundial. Las jóvenes revisión. huestes científicas también estarán presen- Finalmente quiero agradecer a los todos los tes, representados por cerca de 190 investi- miembros de la Directiva, al Editor de la Re- gadores que nos mostrarán sus avances en el vista de Farmacología de Chile, Dr. Ramón desarrollo de sus trabajos de investigación. Sotomayor, al Editor Asociado de Frontiers Especial mención y agradecimientos debe- in Pharmacology, Dr. Gonzalo Yévenes y a mos por la generosa participación del Ins- todos quienes han dedicado gran trabajo y tituto-Fundación Teófilo Hernando de la esfuerzo para lograr los hitos mencionados y Universidad Autónoma de Madrid, cuyos permitir el continuo crecimiento de nuestra representantes participarán impartiendo un organización. simposio de sus actividades de I+D del medi- Con todos estos elementos, esperamos que camento. También extendemos este recono- este XLI Congreso Anual de la Sociedad de cimiento a los representantes de La Sociedad Farmacología de Chile supere con creces sus Española de Farmacología (SEF), a quienes expectativas científicas y, además, nos permi- agradecemos también por haber acogido a ta compartir agradables jornadas de camara- una delegación de la Sofarchi en su reciente dería entre nosotros, disfrutando de las her- congreso anual en julio de 2019. mosas postales de nuestra histórica ciudad Queridas amigas y amigos, en sus 41 años de de Concepción. existencia la Sofarchi se encuentra en un mo- mento clave de su desarrollo. A consecuencia Un fraterno abrazo de las movilizaciones de millones de perso- nas y familias que han ocurrido a lo largo y Dr. Jorge Fuentealba Arcos, PhD. ancho de nuestro Chile, tenemos el enorme Presidente desafío y la responsabilidad de poner nues-

52 PROGRAMA XLI CONGRESO ANUAL SOCIEDAD DE FARMACOLOGIA DE CHILE 2019 UNIVERSIDAD DE CONCEPCION, CONCEPCION, CHILE LUNES 04 DE NOVIEMBRE

lunes 04 de noviembre 11:00 – 15:00 Inscripciones 15:00 – 17:00 Inauguración (Dr. J. Fuentealba) Conferencia 1: Dr. A. García, España 17:00 – 17:30 Coffee break 17:30 – 19:30 Simposio 1: “Purinergic signalling: from structure-activity to application in pathologies” 19:30 – 20:30 Conferencia 2: Dr. S. Moncada, UK 20:30 – 22:00 Cóctel bienvenida

MARTES 05 DE NOVIEMBRE 09:00 – 11:00 Simposio 2: “Toxicología como una ciencia multidisciplinaria” 11:00 – 11:30 Coffee break 11:30 – 12:30 Conferencia 3: Dr. S. Stojilkovic, USA 12:30 – 13:30 Premio Mardones (3) 13:30 – 15:00 Almuerzo libre 15:00 – 17:00 Simposio 3: “Translational options for the treatment of drug-abuse disorders: opportunities, successes and pitfalls” 17:00 – 17:30 Coffee break 17:30 – 19:30 Minisimposios: “Desregulación del metabolismo proteico en esquizofrenia”, “Envejecimiento cardiovascular” 19:30 – 22:00 Sesión de Posters

MIERCOLES 06 DE NOVIEMBRE 09:00 – 11:00 Simposio 4: “Estrategias para el diseño y desarrollo de nuevo fármacos” 11:00 – 11:30 Coffee break 11:30 – 12:30 Conferencia 4: Dr. T. Gallagher, UK 12:30 – 13:30 Incorporaciones (4) 13:30 – 15:00 Almuerzo libre 15:00 – 17:00 Simposio 5: “Enfermedades Neurodegenerativas y Neuroprotección” 17:00 – 17:30 Coffee break 17:30 – 19:30 Simposio 6: “Química Medicinal: Diseño Racional y Relaciones Estructura Actividad, la aproximación sintética de nuevas sustancias biológicamente activas” 19:30 – 22:00 Sesión de Posters

JUEVES 07 DE NOVIEMBRE 09:00 – 11:00 Simposio 7: “Antimicrobial activity of herbal extracts against clinically relevant pathogens” 11:00 – 11:30 Coffee break 11:30 – 12:30 Conferencia 5: Dr. C. Bauzat, Argentina 12:30 – 13:30 Incorporaciones (2), Comunicaciones orales (4) (2 Chile, 2 México) 13:30 – 15:00 Almuerzo libre 15:00 – 22:00 Tarde libre

VIERNES 08 DE NOVIEMBRE 09:00 – 11:00 Simposio 8: “Biotechonology Aspects of Asparaginase clinical and industrial development” 11:00 – 11:30 Coffee break 11:30 – 12:30 Conferencia 6: Dr. A. Bush, Australia 12:30 – 13:30 Comunicaciones orales (6) 13:30 – 15:00 Almuerzo libre 15:00 – 17:00 Simposio 9: “Neurobiological topics in chronic pain management: from molecules to behavior” 20:30 Cena clausura

53 Conferencistas internacionales

Dr. Salvador Moncada, School of Medical Sciences, Manchester Cancer Research Centre, University of Manchester, Inglaterra. Médico, cirujano y farmacólogo hondureño. Realizó labores docentes en las Universidades de El Salvador y Honduras y distintos estudios en los laboratorios Wellcome Research entre 1975 y 1985, cuando estuvo a cargo del equipo de investigación que trabajaba en las prostaglandinas. Su trabajo se ha centrado en efectos farmacológicos de las sustancias vasoactivas, inflamación e interacción entre plaquetas y pared vascular; además de realizar importantes trabajos sobre la trombosis y la arterioesclerosis. Respecto a este último punto, destacó en los años 70 por ser el descubridor de la prostaciclina, un vasodilatador muy potente que actúa como inhibidor de los trombos que obstruyen las arterias.

Fue profesor visitante en diferentes universidades de Europa, Estados Unidos, Hispanoamérica y Japón y desde 1972 ha sido asesor de la Organización Panamericana de la Salud. Sus muchos méritos profesionales le han valido el reconocimiento de todo el mundo; es miembro de la Royal Society, de la Sociedad Británica de Farmacología, de la Sociedad Colombiana de Medicina Interna, de la Sociedad Farmacológica Peruana y académico de Honor de la Real de Medicina de Valencia; es también doctor Honoris causa por las Universidades de Honduras, Cantabria y Complutense de Madrid. Está en posesión de cinco patentes correspondientes a distintos fármacos, y es autor, colaborador o director de unas cuatrocientas publicaciones científicas.

54 Conferencistas internacionales

Dr. Antonio G. García, Instituto Teófilo Hernando and Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid. MD, PhD Presidente de la Fundación Teófilo Hernando (FTH) y Profesor Emérito de Farmacología Universidad Autónoma de Madrid.

Desde 1975 sus estudios se han enfocado en la regulación farmacológica y neuroquímica de los canales iónicos, junto con la señalización del calcio; ambas líneas de investigación proyectadas hacia la comprensión de los principios básicos que gobiernan la liberación exocítica de catecolaminas, la supervivencia neuronal y la muerte neuronal. Más recientemente, se ha orientado al estudio de las enfermedades de Alzheimer (EA) y de Huntington (EH), además de la esclerosis lateral amiotrófica (ELA), a través de la búsqueda de un medicamento neuroprotector capaz de retrasar la progresión de la EA y / o la ELA y mejorar la calidad de vida de los pacientes afectados por estas patologías.

55 Dr. Ashely Bush, Melbourne Dementia Research Centre, Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Australia. Psiquiatra y neurocientífico translacional, Director del Centro de Investigación de la Demencia de Melbourne y Jefe de la Unidad de Biología de la Oxidación del Florey Institute of Neuroscience and Mental Health. Es considerado una de las mentes científicas más influentes del mundo, tras descubrir la interacción del beta- amiloide con el zinc como un factor importante en la patogénesis de la enfermedad de Alzheimer y centrarse en la neurobiología de los iones metálicos y del estrés oxidativo en los trastornos neurodegenerativos y psiquiátricos. Gracias a sus resultados, el estudio de la neurodegeneración se orientó hacia una apreciación de una perturbación subyacente en la homeostasis del metal del cerebro, proporcionando información sobre las enfermedades de Alzheimer, Parkinson y Esclerosis Lateral Amiotrófica. Además, ha desarrollado nuevas pruebas predictivas para la EA y estrategias innovadoras, potencialmente modificadoras de la enfermedad.

Su exitosa carrera ha sido merecedora de diversos galardones, entre ellos el Premio Beeson de la Federación Estadounidense de Investigación sobre el Envejecimiento, el Premio Senator Hatfield de la Asociación de Alzheimer de EE. UU., El Premio Potamkin para la investigación de la enfermedad de Alzheimer, el Premio de Investigación Superior Schering-Plough del Colegio Australiano de Psiquiatras. , una beca de la Federación ARC, el Premio Victoria de Ciencia e Innovación, y actualmente una beca principal de investigación del NHMRC; entre otros.

Es autor de más de 447 publicaciones (más de 41 mil citas), 28 patentes y fundador de 4 empresas de biotecnología.

56 Dra. Cecilia Bouzat, Instituto de Investigaciones Bioquímicas (INIBIBB), Consejo Nacional de InvestigacioneS Científicas y Técnicas (Conicet), Universidad Nacional del Sur, Argentina.

Doctora en Bioquímica de la Universidad Nacional del Sur (UNS), posteriormente realizó estudios postdoctorales en el Departamento de Fisiología y Biofísica, en la Clínica y Fundación Mayo (Rochester, USA). A partir de su experiencia, la Dra. Bouzat comenzó su carrera como investigadora independiente en el Instituto de Investigaciones Bioquímicas Bahía Blanca (INIBIBB), dependiente de la UNS, a fines de los años 90. En el área de la investigación ha contribuido significativamente al conocimiento a nivel funcional, farmacológico y bioquímico de los canales iónicos activados por acetilcolina y por serotonina del sistema nervioso central, a través de 87 publicaciones científicas, lo que incluye artículos publicados en Nature, Journal of Biological Chemistry y Molecular Pharmacology, entre otras, además de 30 proyectos de investigación financiados por entidades nacionales e internacionales. Producto de su trabajo y esfuerzo, la Dra. Bouzat ha sido distinguida con numerosos premios y distinciones, dentro cuales se destaca el premio L´Oréal-UNESCO for Women in Science, Latin America International Laureate, obtenido el año 2014. El liderazgo de la Dra. Bouzat también se ha reflejado en numerosas participaciones en comités nacionales e internacionales, entre las cuales se destacan la presidencia y vicepresidencia de la Sociedad Argentina de Investigación en Neurociencias (SAN), la membresía titular de la comisión de Ciencias Médicas del CONICET, la membresía del Comité Regional de América Latina de International Brain Research Organization (IBRO-LARC) y la membresía permanente del comité editorial de la revista Molecular Pharmacology. Actualmente, dirige el Laboratorio de Neurofisiología y Farmacología Molecular del INIBIBB y es la directora del INIBIBB, en Bahía Blanca. Además, preside el Comité Regional de América Latina de International Brain Research Organization (IBRO-LARC) y es la directora del programa de Doctorado en Ciencias Farmacéuticas de la UNS.

57 Dr. Tim Gallagher, PhD Universidad de Liverpool, profesor de Quimica Orgánica de la Escuela de Química de la Universidad de Bristol (Reino Unido ), Sus intereses de investigación abordan la química heterocíclica y abarcan una variedad de temas, los que incluyen el desarrollo de metodología sintética, el estudio del mecanismo de reacción, la síntesis de productos naturales y la aplicación de síntesis a áreas de interés biológico. Tiene intereses particulares en el diseño y síntesis de ligandos nicotínicos selectivos (basados ​​en la citisina) y en la comprensión del mecanismo de acción, así como la química de los sulfamidatos cíclicos como vehículos para la síntesis asimétrica. Tiene colaboraciones dentro del área de carbohidratos basadas en un interés en comprender y explotar las interacciones basadas en carbohidratos.

Su trabajo ha recibido amplios galardones, tales como el Premio Katritzky de la Sociedad Internacional de Química Heterocíclica (1999), el Tilden Lectureship de la Royal Society of Chemistry (RSC) (2004) y el Premio de Química Heterocíclica (2005). Además, es supervisor en el Centro EPSRC de Formación Doctoral en Síntesis Química.

Por otra parte, ha sido consultor y presentador de tres programas de televisión relacionados con la ciencia en el mundo antiguo, entre ellos “Antiguo Descubrimientos III “, serie encargada por History Channel.

58 59 Abstracts for XLI of the Chilean Society of Pharmacology

CONFERENCES importancia con relación a la salud y a la enfermedad. 1. THE EMOTION OF SCIENTIFIC DISCOVERY. 2. THE PLEASURE OF SCIENCE: MY Antonio G. García LIFE IN PHARMACOLOGY. Instituto Teófilo Hernando, Departamento Salvador Moncada de Farmacología y Terapéutica, Facultad de Manchester Cancer Research Centre, The Medicina, Universidad Autónoma de Madrid, University of Manchester, U.K. Spain I will describe the work that opened several Stephen Hawking said that “science is not fields of investigation. From the mechanism only a matter of reason; it is also a matter of action of non-steroidal anti-inflammatory of romance and passion”. This was already drugs, to the discovery of thromboxane written by Santiago Ramón y Cajal in his synthase and prostacyclin, to the identification famous book “Reglas y Consejos sobre of nitride oxide and its metabolic pathway of Investigación Científica”, a hundred years synthesis. I will finish with a reference of the ago. More recently, the National Academy role of mitochondria in oxidative stress. I of Sciences of the USA wrote a short will put all this work in a general context and guide entitled “ON being a scientist”, that its importance in health and disease will be summarizes the emotions that a scientist discussed. may feel along his carrier when pursuing a problem and finding the response 3. CELL TYPE- AND SEX-DEPENDENT sometimes after years of hard work. I will TRANSCRIPTOME PROFILES OF RAT illustrate the way science is practiced with ANTERIOR PITUITARY CELLS. some experimental findings on the topic of Stanko S. Stojilkovic calcium signaling and exocytosis in adrenal Section on Cellular Signaling, Eunice Kennedy chromaffin cells. In doing so, I will focus first Shriver National Institute of Child Health and on basic science, describing how we arrived Human Development, NIH, Bethesda, MD, to the concept of a functional triad that USA includes the voltage-gated calcium channels (VGCCs), the endoplasmic reticulum (ER) We are investigating receptors and channels and mitochondria (MIT). Such triad shapes expressed in neuronal and endocrine the cytosolic calcium signals that control both cells and their roles in signaling, gene pre-exocytotic and exocytotic responses, the transcription, and hormone secretion. To basis of the fight-or-flight stress response gain better understanding of the cell type- of W. Cannon. Then, I will focus on more specific expression and role of these and other recent translational research done in proteins in anterior pituitary cell functions chromaffin cells from mouse models of and related disorders, we performed single neurodegenerative diseases. I will comment cell RNA sequencing on freshly dispersed on dysfunctions of Ca2+ and exocytosis cells from adult male and female rats. Our occurring even at pre-symptomatic disease analysis based on over 7000 cells confirmed stages. I will next make some comments on the expression of six pituitary-specific the failure of clinical trials in AD, to end with cell: folliculostellate cells and hormone some hints on the pressure to “publish-or- producing corticotrophs, gonadotrophs, perish” and how science is becoming just a thyrotrophs, somatotrophs, and lactotrophs. mere business for editorials and else. Also identified were endothelial and blood cells from the pituitary capillary network. Recent and ongoing work from AGG’S The expression of numerous developmental laboratory is being founded by and neuroendocrine marker genes in both 1. European Union Horizon 2020 Research folliculostellate and hormone producing cells and Innovation Programme under the Marie supports that they have a common origin. For Skoldowska-Curie Grant Agreement 766124); several genes, the validity of transcriptome 2. Grant SAF-2016-48892-R, from Ministerio analysis was confirmed by qRT-PCR and single de Ciencia, Innovación y Universidades, cell immunocytochemistry. Folliculostellate Spain; and cells exhibit impressive transcriptome 3. Fundación Teófilo Hernando, Madrid, diversity, indicating their major roles in Spainen un contexto general y discutiré su production of endogenous ligands and

60 detoxification enzymes, and organization of 5. GENETIC, PROTEIN AND extracellular matrix. Transcriptome profiles PHARMACOLOGICAL MODULATION of hormone producing cells also indicate OF HUMAN α7 NICOTINIC contributions toward those functions, while RECEPTORS. also clearly demonstrating their endocrine Cecilia Bouzat function. This include the expression of Instituto de Investigaciones Bioquímicas de genes encoding numerous voltage-gated, Bahía Blanca, INIBIBB (CONICET-UNS), ligand-gated and other channels in hormone- Departamento de Biología, Bioquímica y producing but not follicolostellate cell. This Farmacia, Universidad Nacional del Sur, survey highlights many novel genetic markers Bahía Blanca, Argentina. contributing to pituitary cell type identity, sexual dimorphism, and function and points The α7 nicotinic acetylcholine receptor is to relationships between hormone producing a pentameric ligand-gated ion channel. It and folliculostellate cells. is widely expressed in the central nervous system where it is involved in cognition, 4. DIRECT C-H FUNCTIONALIZATION attention, and memory. It is also expressed OF CYTISINE. NICOTINIC RECEPTOR in many non-neuronal cells and its activation SELECTIVITY AND MECHANISM OF has anti-inflammatory and neuroprotective ACTIVATION. roles. Enhancement of α7 activity is emerging Tim Gallagher as a therapeutic strategy for cognitive, University of Bristol, UK neurodegenerative and inflammatory disorders. We have focused on understanding The talk will cover the application of α7 function and its different mechanisms subtype-selective nicotinic partial agonists of modulation associated to physiological, to manage nicotine addiction, with a focus pathological and therapeutic situations. By on the chemistry of cytisine. Already single-channel recordings we determined used for smoking cessation, cytisine (and that positive allosteric modulators (PAMs) varenicline) target subsets of nicotinic enhance α7 activation by increasing open- receptors, and the opportunity to generate channel lifetime and inducing prolonged novel structural variants of cytisine raises activation episodes, and we also identified the question of whether more subtype novel PAMs. Although α7 has been selective ligands are available and of value considered the homomeric member of the or indeed are even desirable. The talk will family, heteromeric α7β2 receptors have been cover recent work in this area, much of detected in human brain. We generated α7β2 which is underpinned by development of C-H receptors with different stoichiometries and functionalisation chemistry that provides determined how the β2 subunit modifies α7 very direct and efficient access to new C-10 kinetics and its allosteric modulation. This cytisine derivatives, which in turn, offer more information is required to decipher the role selective subtype profiles. Recent studies (in of α7β2 receptors in native cells. In humans, collaboration with Henry Lester and Dennis there is a truncated α7 subunit (dupα7) Dougherty) have probed the influence of that lacks part of the ACh-binding site and steric vs. electronic factors in determining results from partial duplication of the α7 the binding mode of cytisine. We have also gene. We demonstrated that dupα7 acts as a pursued extensive molecular dynamics negative modulator and can assemble with simulations to probe the mechanism (timing) α7 into functional heteromeric receptors. of signal propagation through the protein Deciphering the molecular basis underlying scaffold that occurs on ligand association α7 function has implications for the design of to the receptor, and addressed the question novel therapeutic compounds as well as for of the applicability and generality of that clarifying its pleiotropic actions. mechanism across other receptors.

61 6. IRON AND FERROPTOSIS SYMPOSIA IN AGING AND AGE-RELATED NEUROLOGICAL DISEASES. 1. PURINERGIC SIGNALLING: Ashley Bush FROM STRUCTURE-ACTIVITY TO The Melbourne Dementia Research Centre, APPLICATION IN PATHOLOGIES The Florey Institute of Neuroscience and Mental Health and the University of ALLOSTERIC REGULATION OF THE Melbourne, Australia. P2X4 RECEPTOR CHANNEL GATING. Stojilkovic, S.S. Recent research has implicated increased Section on Cellular Signaling, Eunice Kennedy brain iron as a trait that can propel various Shriver National Institute of Child Health and neurodegenerative diseases, including Human Development, NIH, Bethesda, MD, Alzheimer’s disease, Parkinson’s disease, USA. Motor Neuron disease and the complications of stroke. Ageing itself causes iron to increase In general, allosteric modulators of in the brain to a point where it is “too much of ligand-gated receptor-channels induce a good thing” and can set up conditions that conformational changes of the entire lead to neurodegeneration. During childhood protein that alter potencies and efficacies for and reproductive life, iron recruitment is orthosteric ligands, expressed as the EC50 and geared towards avoiding iron deficiency, maximum current amplitude, respectively. but there is no natural mechanism for off- Recently, we studied the influence of allostery loading excess iron. After reproductive life on channel gating using the rat P2X4 receptor the systems that harvest iron so efficiently expressed in HEK-293T cells and gated by do not turn off, and lead to accumulation ATP in the presence and absence of ivermectin in tissues that are not normally shed, like (IVM), an established positive allosteric brain. In the C. elegans model of ageing, we regulator of this channel. In the absence of find that such iron elevation limits lifespan. IVM, this channel activates and deactivates In Alzheimer’s disease brain iron elevation is rapidly, does not conduct NMDG+, a large associated with the rate of cognitive loss, lipid organic cation, desensitizes completely with a peroxidation products and features of the moderate rate, and recovers only fractionally regulated cell death mechanism, ferroptosis. during washout. IVM treatment increases the Anti-ferroptosis agents have been effective in efficacy of ATP to activate the channel, slows animal models of neurodegenerative disease, receptor desensitization during sustained and a recent phase 2 clinical trial of the anti- ATP application and receptor deactivation ferroptotic chelator deferiprone in Parkinson’s after ATP washout, and makes channel disease lowered nigral iron and improved permeable to NMDG+. Experiments with clinical readouts. We are currently testing vestibular and transmembrane domain this drug in a phase 2 RCT in Alzheimer’s receptor mutants further established that disease. CuATSM, has recently reported IVM has distinct effects on the channel benefits in phase 1 studies of Parkinson’s pore opening, the first accounting for disease and Motor Neuron Disease, and we increased peak current amplitude and the have identified that it possesses potent anti- latter correlating with changes in the EC50 ferroptotic properties. and kinetics of receptor deactivation. The corresponding kinetic (Markov state) models References: can reproduce many of the observed time Stockwell et al. Ferroptosis: A Regulated Cell series of evoked currents, as well as the Death Nexus Linking Metabolism, Redox transient changes in desensitization observed Biology, and Disease. Cell, 171, 273–285 upon IVM application, the significant increase (2017). in ATP-induced current amplitudes at low Ayton et al. Brain iron is associated with IVM concentrations, and the modest increase accelerated cognitive decline in people with in the unitary conductance. In summary, this Alzheimer pathology. Molecular Psychiatry, study provides a detailed analysis of P2X4R in press doi: 10.1038/s41380-019-0375-7. kinetics and elucidates the mechanism Southon et al. CuII(atsm) inhibits regulating its channel gating. ferroptosis: implications for treatment of neurodegenerative disease. British Journal of Pharmacology, in press.

62 CONTRIBUTIONS OF MOLECULAR P2X2 RECEPTOR: NEW DYNAMIC CALCULATIONS PHARMACOLOGICAL TARGET TO TO STRUCTURAL BIOLOGY AD. UNDERSTANDING, THE CASE OF Fuentealba, J. IVERMECTIN AND ALFAXOLONE AS Depatamento de Fisiología, F. de Ciencias P2X4R MODULATORS. Biológicas, Centro de Investigaciones Huidobro-Toro J.P.; Latapiat V.; Alveal N.; Avanzadas en Biomedicina (CIAB-UdeC), Montenegro F.; Barrera N. Universidad de Concepción, Chile. Laboratorio de Farmacología de Nucleótidos, Departamento de Biología, Facultad Soluble oligomers of amyloid beta peptide de Química y Biología, Universidad de (SOAβ) have been considered as central Santiago de Chile y Centro de Nanociencia y factors in Alzheimer’s disease (AD). Aβ Nanotecnología, CEDENNA. peptide is generated through the sequential cleavage of the amyloid precursor protein Multiple molecules modulate the (APP), a process that requires the previous electrophysiological activity of P2X receptors endocytosis of APP and that can be (P2XR). In particular, trace metals such modulated by the multidomain adaptor as Cu(II) or Zn(II), modulate P2X4R protein Fe65. This protein is able to regulate concentrated in neurons by selective and the transcription of key genes directly related specific cationic transporters; the putative site to AD pathogenesis, encoding proteins like of action of these trace metals is apparently APP and BACE 1. On the other hand, we restricted to the extracellular receptor domain. have described that chronic SOAβ treatment Drugs such as the antiparasitic antibiotic induces an increase in the expression of the ivermectin (IVN) or alfaxolone (A) a synthetic P2X2 purinergic receptor in PC12 cells and neuro-steroid, with hypnotic properties, also hippocampal neurons. Additionally, it has modulate the P2X4R but at intracellular sites, been described that the P2X2a isoform has near the transmembrane P2X4R domain. In an intracellular domain that can interact order to reveal and understand the site and with Fe65, a segment which is absent on the mechanism of the modulator effect, as well as P2X2b isoform. We found that SOAβ treated the allosteric agonism of A, we strategically cells displayed an increase in evoked ATP used molecular dynamic simulations/ currents (C: 100 ± 50%; SOAβ: 231 ± 70%; calculations to visualize the docking and n=9). Additionally, immunocytochemistry mode of action of these modulators. A (ICC) experiments demonstrated that these rP2X4R homology model was built; docking cells exhibited an increase in their P2X2R of either IVM or A revealed selective binding immunoreactivity (C: 100 ± 1 %; SOAβ: sites confined to the transmembrane domain 149 ± 15%; n=5). Moreover, cells treated as anticipated based on drug lipophilicity. In chronically with SOAβ showed a reduction addition, molecular dynamic simulations in in the Fe65 nuclear-cytoplasmic (N-C) ratio the APO and HOLO P2X4R states revealed (C: 100 ± 6%; SOAβ: 80 ± 4%; n=5). A allosteric-induced stability. Pore and lateral similar behavior was observed in PC12 cells P2X4R fenestrations measurements of the transfected to express the P2X2a isoform, but different receptor states, in the absence not in those transfected with P2X2b (C: 100 ± and next in the presence of either IVM or 5%; P2X2a: 70 ± 6%; P2X2b: 95 ± 6%; n=3). A, showed that both IVM and A can elicit a Colocalization analyses demonstrated that larger pore opening that proved larger in the SOAβ decreased the colocalization of Fe65 presence of ATP, as expected for allosteric with APP (C: 100 ± 17%; SOAβ: 47 ± 12%; modulators. Interestingly, in the case of A, n=5); results that correlate with the increase consonant and consistent with an allosteric observed in the colocalization of APP with agonist, the hole analysis demonstrated an clathrin (C: 100 ± 8%; SOAβ: 127 ± 8%; n=4) even larger opening. The present findings and Rab5 (C: 100 ± 6%; SOAβ: 132 ± 16%; reveal the power and strategy of using n=5). In conclusion, these results suggest simulation studies to understand molecular that chronic SOAβ treatment promotes aspects of the binding and intricate molecular the endocytosis of APP, potentiating its mechanism of allosteric agonist studies. amyloidogenic processing. Additionally, the Based on these findings, dynamic simulation calcium dyshomeostasis/overload induced calculations offer opportunities to design by P2X2R overexpression, alter the activation novel, P2X4R ligands not based on purines. and localization of CAMKIIα, in the context of AD. Using molecular biology techniques, we observed that after chronic SO-Aβ treatments, mice hippocampal neurons

63 showed an increase on the levels of P2X2R survival rates of gastric cancer patients. Our compared to the control cells (C: 100.0 ± latest experiments suggest that purinergic 6.4%; SOAβ: 130.1 ± 10.7%, n=5). This was signaling also can contributes to epithelial correlated with increased Ca2+ signal evoked to mesenchymal transition in CG-derived by ATP (C: 100.0 ± 12%, SOAβ: 194 ± 24%, cells and that transactivation of P2Y2/ n=4). Immunocytochemistry approaches on HER2 can also contribute to these effects. mice hippocampal neurons, showed that the Taken together, these results demonstrate overexpression of P2X2R induced changes on the involvement of purinergic signaling the immunorreactivity pattern of pCAMKIIα in GC, and that the changes in expression (in soma and neurites), which induced and nucleotides release observed in GC alterations on the cells morphology, and could direct nucleotide signaling from anti- electrophysiological recordings assessed by proliferative effects in healthy tissues to Sholl Analysis and Patch Clamp, respectively. proliferative effects in cancer. These results suggest that P2X2R overexpression can potentiate the toxicity of 2. TOXICOLOGY AS A SO-Aβ, due to the chronic Ca2+ overload and MULTIDISCIPLINARY SCIENCE inactivation of CAMKIIα, and thus, altering the mechanisms of neuronal plasticity, the TOXICOLOGY AS A basis of the pathophysiological mechanism of MULTIDISCIPLINARY SCIENCE. AD. Schulz B. Toxicología, Departamento de Farmacia, ROLE OF PURINERGIC SIGNALING Facultad de Farmacia, Universidad de AND IN GASTRIC CANCER. Concepción. Coddou C.1; Castro P.2; Cerda D.1; Reyna- Jeldes M.1; De la Fuente E.1. The development of the Toxicology is 1, Laboratorio de Señalización Purinérgica, strong related to the human history and its Departamento de Ciencias Biomédicas, relationship with the environment, as well as Facultad de Medicina, Universidad Católica geographic, social or politic environment. In del Norte, Coquimbo, Chile. 2, Departamento the primitive phase of the toxicology as health de Fisiología, Facultad de Ciencias Biológicas, topic, the principal focus of the knowledge Universidad de Concepción, Concepción, was the description of the toxic events on Chile. humans, and its accidental or intentional character. Many of the early register of Gastric cancer (GC) is the one of the most intoxications were based on the contact of prevalent cancers worldwide. Here, we studied plants, minerals or animals with the human in detail purinergic signaling in several gastric bodies and it deleterious results. This adenocarcinoma-derived cell lines: AGS, description of events summarized the botanic, MKN-45 and MKN-74, and compared them chemical, zoological and medical sciences. to a non-tumoral epithelial cell line: GES-1. In With the human enrichment on scientific GC-derived cells, we detected the expression information, the description of intoxications of several purinergic receptors, and found are more specific and included clinical important differences as compared to GES- symptoms, treatment of the intoxicated 1 cells. Functional pharmacological studies patients or forensic evidences in the body. with calcium imaging and proliferation For that are applied the physiopathology, assays revealed a strong contribution of forensic medicine, clinical medicine and P2YRs, especially P2Y2Rs to increases in cell pharmacology. The actual toxicology is proliferation and an antiproliferative effect related to many complex and heterogeneous induced by the activation of P2X4Rs. Also, knowledge, as well as analytical, molecular, we detected a tonic purinergic response that computational, clinical, legal, epidemiological is probably a reflect of the paracrine and and pharmacological methods. In a more autocrine nucleotide signaling, because to general aspect, toxicology is implicated in sole application of purinergic antagonists diverse applied areas, as clinical toxicology, changed the basal proliferation of GC-derived forensic and legal toxicology, occupational cells. In tumor-derived biopsies, we found toxicology, ecotoxicology and environmental an increase of P2Y2R and a decrease in toxicology. Therefore, toxicology is clearly a P2X4R expression; however, we found high multidisciplinary science, it may be used in variability between the biopsies and their other areas, and toxicology needs information respective adjacent healthy gastric mucosa. of other different disciplines as pharmacology. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and

64 STATE OF THE ART OF TOXICOLOGY regarding calls for toxicological emergencies IN CHILE. that entered CITUC, from the 15 regions, 54 Cavieres, F. provinces and 346 communes of Chile. During Toxicología, Facultad de Farmacia, the telephone call, the emergency center Universidad de Valparaíso, Chile. professionals collect all available information provided by the caller, required for the The first scientific meeting of Sociedad de evaluation of the case considering data of the Toxicología de Chile (SoTox) was held in agent, the circumstances of the exposure and november 2012. The event brought together the patient. After the background evaluation, toxicologists from academia, regulatory the technical recommendations for exposure agencies and industry that represented the management are communicated. The data practice of toxicology in Chile. Only a few is collected in the manual registration form months earlier, SoTox had acquired its legal and subsequently all the data is entered into personality becoming Chile´s first scientific the electronic Registration System called society in toxicology, so the meeting was an “Call Registry System CITUC SRL”. The unprecedented opportunity to talk about Central of toxicological emergencies provides the state of the art of basic and applied free telephone assistance with qualified toxicological sciences in Chile. In general professionals 365 days a year in continuous terms, it was acknowledged that toxicology hours (24/7), answering questions from played an important yet undefined role in health professionals, authorities, emergency the Chilean society, mostly due to the lack personnel and the general public. The of professionals who had received formal commitment and dedication of the academic training in toxicology. Seven years professionals, together with the excellence later, SoTox continues its efforts to position in the service, guarantee the 27 years of toxicology and toxicologists as an important existence of the center and the 35,000 calls discipline developed by professionals who can on average that CITUC receives annually. contribute to better decision making in the many different areas needed for the growth of CLINICAL MANAGEMENT OF the country. In this talk I will: i) introduce a POISONINGS: SPECIALISTS NEEDED. modern perspective of what toxicology should Müller C. be; ii) describe the state of development of the Toxicología, Departamento de Farmacia, science in the country and iii) communicate Facultad de Farmacia, Universidad de SoTox´s goals and achievements. Concepción.

EPIDEMIOLOGY OF INTOXICATIONS In Chile, the number of acutely poisoned IN CHILE. 2018 ANNUAL REPORT patients, admitted into emergency medicine OF THE TOXICOLOGICAL departments, is increasing every year. Under INFORMATION CENTER OF THE this circumstance, medical staff need to PONTIFICIA CATHOLIC UNIVERSITY be prepared, in terms of proving poisoned OF CHILE (CITUC). patients, with adequate clinical treatment. Silva, L. According to the National Poison Control Centro de Información Toxicológica, Facultad Center (CITUC), 57% of phone calls received de Medicina, Pontificia Universidad Católica at the center are from healthcare settings. de Chile (CITUC). This may be indicative of existing limitations related to the clinical management of The epidemiology of poisonings in countries poisoned patients. Thus, availability of is relevant when addressing public health basic up-to-date knowledge about clinical guidelines, evaluating exposure profiles management of poisoning events would and defining treatment and prevention optimize treatment regimes, economic strategies. This presentation describes the burden, and also improve patients’ prognosis. characterization of exposures to potentially A classic example of this is the use of gastric dangerous substances based on the cross- decontamination techniques, such as gastric sectional descriptive study of the universe lavage and activated charcoal, when patients of calls that entered the CITUC toxicological expose to chemicals through the oral route. emergency center during 2018. The variables However, there are some limitations when analyzed were: sex, age, circumstance of the employing these maneuvers. Specifically, the exposure, agent (s), interlocutors of the call, frame time elapsed between the exposure and location of the interlocutor and the incident, the use of the technique (up to 60 minutes). routes of exposure, symptomatology and After that time, there is a significant severity. This work collects information decrease of effectiveness, and the procedure

65 itself turns, not only very traumatic to the TRANSLATIONAL APPROACHES patient, but also in unnecessary expenses to TO THE TREATMENT OF COCAINE the healthcare setting. This inappropriate ADDICTION. practice has become common in several Gaetano Di Chiara health institutions, which are characterized Dept. of Biomedical Sciences, University of by a lack of specialized medical staff with Cagliari, Cagliari, Italia. knowledge in clinical toxicology. Cocaine addiction treatment is probably the 3. TRANSLATIONAL OPTIONS FOR most challenging and paradigmatic example of THE TREATMENT OF DRUG-ABUSE the difficulties in translating neurobiological DISORDERS: OPPORTUNITIES, knowledge into addiction therapy. Thus, to SUCCESSES AND PITFALLS date, in spite of the advances in the knowledge of the neural basis of cocaine addiction, INTRODUCTORY REMARKS. none, among the translationally-based Herrera-Marschitz, M. treatments proposed, has been approved by Programme of Mol. & Clin. Pharmacology, national or international agencies. Clearly, Medical Faculty, University of Chile. the difficulties with cocaine addiction might be a case of a general difficulty in Synthesized molecules and drugs can be used translating experimental results obtained in for rapidly achieving a high pleasant and/or animals into human therapy. Although drug euphoric mood, bypassing the homeostatic addiction is recognized as a brain disease, pathways for pleasure and reward. That can it is an exceedingly complex one and it is be escalated by repeating and increasing the unclear to which extent, as in the case of drug experience, abusing of the shortcutting schizophrenia and dementia, animal models pathway to pleasure, arriving to dependence are able to model the human condition. and addiction to the substances providing Therefore, critical analysis of the defaillances that shortcut, changing the physiological with cocaine can provide important clues substrate for perpetuating an addicting as to the models to utilize. As far as the behaviour, impacting on the social and main lines of research, simple approaches familiar environment for the compulsion of targeting DA transmission, the indirect site “experiencing a new trip”. Much has been of cocaine action, with DA receptor agonists investigated about the physiological and and antagonists or with low abuse liability neuronal framework sustaining that condition DAT blockers have been discouraging, in mammals, including humans, arriving although the development of allosteric DAT to the pivotal neurocircuitry of pleasure, ligands is currently a major line of research identifying a role for dopamine, glutamate at NIDA. Acknowledgment of the critical role and opioid neuropeptides. The obtained of neuroplastic changes at the level of the knowledge is enormous, but that has not led glutamatergic/dopaminergic cortico-ventral to a consensus for treating a medical issue, striatal circuit is the basis for pharmacological which is not only menacing the individual, and physical treatments (DBS and TMS), but is destroying the society. We have hereby aimed at reversing the neural changes induced sampled a group of international leaders by long lasting drug exposure. Preliminary, and experts, who have devoted a research small scale observations in humans indicate life to investigate the issue, both at basic that this might be the right way to go. and clinical levels, to discuss why preclinical results have not translated into the clinic. CORTICOTROPIN-RELEASING Thus, Gaetano Di Chiara (Cagliari), who first HORMONE RECEPTOR 1 (CRHR1) proposed a role for accumbens dopamine AND ADDICTION: WHY THE release as a common substrate for addictive PRECLINICAL RESULTS DID NOT substances will discuss about translational TRANSLATE INTO THE CLINIC?. approaches for treating cocaine addiction. Spanagel, R. Rainer Spanagel (Mannheim), a leader on Heidelberg-Mannheim, Germany neuropeptides and addiction will discuss on the role of corticotropin-releasing pathways ALCOHOL-INDUCED for sustaining drug abuse and addiction. Yedy NEUROINFLAMMATION-OXIDATIVE Israel (Santiago), an international referent STRESS CYCLE: INFLAMMATORY on alcohol and alcoholism will discuss on a AND ANTIOXIDANT DRUGS INHIBIT neuroinflammatory-oxidative stress cycle CHRONIC ALCOHOL INTAKE AND sustaining chronic alcohol intake. BLUNT RELAPSE BINGE-DRINKING. Israel Y. 1, Quintanilla ME.1, Ezquer F.3

66 , Morales P. 1,2 ,Ezquer M. 3, Herrera- Escuela de Química y Farmacia, Facultad Marschitz,M.1. de Medicina y ciencia, Universidad San 1 Pharmacology Program and 2 Dept Sebastian sede Patagonia Puerto Montt. Neuroscience, Fac.Medicine-ICBM, University of Chile, and 3 Centro de Med. Resistance to antibacterial agents is a growing Regenerativa, Universidad del Desarrollo, problem of global public health, which affects Santiago CHILE. the treatment of infectious diseases, reducing the effectiveness of available antibacterials, Brain of UChB rats chronically consuming causing an increase in mortality and over 10 g ethanol/kg/day show (i) a 200% morbidity of patients. If innovative initiatives increase in hippocampal oxidative stress, that seek to solve this problem are not determined as the ratio of oxidized/reduced generated, it is projected that, worldwide, in glutathione (GSSG/GSH), and (ii) marked the year 2050 there will be around 10 million neuroinflammation, shown as 60% increases deaths caused by resistant microorganisms, in astrocyte glial-fibrillary acidic protein with costs estimated at 100 trillion dollars. (GFAP) and increases in microglial density Unfortunately, the pharmaceutical industry (Iba-1). Noteworthy, these changes remain has been leaving research in this area which long after ethanol intake is discontinued; is reflected in the approval of only ten drugs in line with a proposed self-perpetuation by the FDA, during the last ten years, none (vicious cycle) of oxidative stress and of them with new mechanisms of action. neuroinflammation. Administration of Due to this is why it is necessary to promote a low dose of the antioxidant N-acetyl and encourage the development of a greater cysteine (40 mg/kg) reduces brain oxidative quantity of antibacterial compounds, stress and neuroinflammation and inhibits different from those already known. For this chronic alcohol intake by 50-60%. The co- reason, the discovery and development of administration of N-acetyl cysteine with new molecules is of vital importance. Thus, low doses of aspirin (ASA 15 mg/kg) inhibit different approaches have been used for the alcohol intake by 75%, showing a significant discovery of active compounds, such as the synergism of both drugs. Following chronic High-throughput screening, the extraction ethanol intake, co-administration of N-acetyl of compounds from natural products or the cysteine plus the anti-inflammatory drug design based on a biological target. But, what during a 2-week alcohol deprivation period can be done when an isolated molecule (or block neuroinflammation and oxidative series) is available and its pharmacological stress and inhibit by 85% the relapse binge- target is unknown? How to know which area like drinking (“ADE”) prompted by the of the molecule can be modified and what subsequent ethanol re-access. As will be type of modification can be made to obtain shown, studies tie neuroinflammation- compounds that are more active? How to oxidative-stress and hyper-glutamatergic know the relationship structure activity of this conditions as the likely mechanisms that family of molecules? In this work, we show the perpetuate chronic alcohol intake and experience of development of a new family of promote intoxicating relapse drinking, and antibacterial drugs using different strategies also indicate the pharmacological agents that of traditional medicinal chemistry. References block this condition. Studies suggest that W. H. Organization, Antimicrobial resistance: anti-oxidant and anti-inflammatory agents global report on surveillance. World Health may add significantly to interventions aimed Organization, 2014. J. Campanini-Salinas, at reducing alcohol-use disorders. J. Andrades-Lagos, J. Mella-Raipan, and D. Vasquez-Velasquez, “Novel classes of 4. STRATEGIES FOR THE DESIGN antibacterial drugs in clinical development, a AND DEVELOPMENT OF NEW hope in post antibiotic era.,” Curr. Top. Med. DRUGS Chem., 2018. J. Campanini-Salinas et al., “A New Kind of Quinonic-Antibiotic Useful DRUG DESIGN STRATEGIES BASED Against Multidrug-Resistant S. aureus and E. ON THE MOLECULE. faecium Infections,” Molecules, vol. 23, no. 7, Andrades-Lagos, J. 1,2; Vasquez-Velasquez, 2018. D, 2; Campanini-Salinas, J. 3. 1. Facultad de Medicina y ciencia, Universidad San Sebastian,Campus Los Leones. 2. Laboratorio de Desarrollo de Fármacos, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile. 3.

67 INTEGRATION OF STRUCTURAL structural relationship with the antibiotic BIOINFORMATICS AND CHEMICAL drugs predecessors. Under this look, the BIOLOGY FOR THE DISCOVERY OF present work addresses the development of NOVEL DRUGS. the latest antibacterial drugs in clinical phases Lagos C. F. II and III, analyzing the design strategies by Chemical Biology & Drug Discovery Lab, which these new molecules were obtained Facultad de Medicina y Ciencia, Universidad and the structure-activity relationship of San Sebastián. these new families of antibiotics, in order to define the state of the vanguard antibacterial The discovery and design of drugs is drugs in the post-antibiotic era. increasingly incorporating structural bioinformatics techniques to model and PRECLINICAL DEVELOPMENT OF A analyze proteins of biological or therapeutic NEW CLASS OF ANTIBACTERIALS, A interest, perform large-scale virtual screening NATIONAL EXPERIENCE. programs to identify lead compounds and Campanini-Salinas J. 1, Andrades-Lagos J. evaluate molecular interactions through 12, Vásquez D. 3. molecular dynamics simulations. These 1,Facultad de Medicina y Ciencia, Universidad techniques are fast, cost effective and San Sebastián, Lago, Panguipulli 1390, Puerto complementary to the existing experimental Montt 5501842, Chile.2. Facultad de Medicina techniques of chemical biology. In this y Ciencia, Universidad San Sebastián, presentation, we will discuss some examples Santiago,Lota 2465, Chile 3. Laboratorios de of strategies that combine different structural Desarrollo de Fármacos, Facultad de Ciencias bioinformatics approaches with chemical Químicas y Farmacéuticas. Universidad de biology tools to successfully discovery of Chile. Santiago, Chile. novel drugs, focusing on the analysis of the inherent strengths and limitations on the use The rapid emergence of resistant bacteria is of structural bioinformatics tools, as well as occurring worldwide, endangering the efficacy complementary biological assays. of antibiotics, reducing the therapeutics arsenal for treatment of infectious diseases. According DESIGN STRATEGIES FOR NEW this, it is a urgent need the development of CLASSES OF ANTIBACTERIAL new antibacterial drugs. In this study, we DRUGS, A HOPE IN A POST- develop a new class of compounds obtained ANTIBIOTIC ERA. with a simple synthesis in two single-step. Vásquez-Velásquez D. These compounds were screened for in vitro Drug Development Laboratory, Facultad antibacterial activity against ATCC strains and de Ciencias Químicas y Farmacéuticas, clinical isolates, using the broth microdilution Universidad de Chile, Sergio Livingstone method. In addition, a series of trials were 1007, 8380492, Santiago, Chile. conducted to gather information about the effectiveness and safety of derivatives, such as Bacterial resistance is a growing problem how; toxicity in mammalian cells and galleria worldwide and is estimated that deaths by mellonella, assays of potential for induction infectious diseases associated with resistant of mutations, among others. The compounds pathogens will generate 10 million deaths per exhibited MICs of 1-32 μg/ml against Gram- year in 2050. This problem becomes more positive ATCC strains. The MIC50 for serious due to the low level of research and compound 7 against the MRSA isolates tested development of new drugs, which has fallen were 2 mg/L, compound 16 exhibit 2 mg/L. drastically in the last 40 years. For example, For the VREF isolates the compound 7 showed in the last decade of a total of 293 new drugs MIC50 and MIC90 values of 2 and 4 mg/L, approved by FDA, only 9 corresponded to and the compounds 16 obtain values of 4 and antimicrobial drugs and none constituted 4 mg/L. The compounds were bactericidal a new structural class. The majority of the in all isolates tested. Both compounds were molecules in the clinical phase II or III, bactericidal in all clinical isolates tested. coming from modifications of drugs in clinical Neither compound affected cell viability in use, this strategy make easier the bacterial any of the mammalian cell lines and Galleria susceptibility to generate resistance through mellonella larvaes, at any of the concentrations the mechanisms expressed for their drug tested. These in vitro data indicate that predecessors. Under this scenario, is urgent compounds 7 and 16 can advance in assay on to generate the most novel strategies for the murine models of infection and determination development of antibacterial compounds with of pharmacokinetics parameters. new targets or mechanism of action, without

68 5. ALZHEIMER’S DISEASE: NEW de Madrid. 2.Instituto de Investigaciones TARGETS AND DRUGS. Biomédicas “Alberto Sols” Departamento de Bioquímica. Facultad de Medicina. INTRODUCTION. Universidad Autónoma de Madrid. España.3. García, A.G. Instituto de Química Médica. CSIC. Madrid. Instituto Teófilo Hernando, Departamento España. 4. Instituto de Investigación La de Farmacología y Terapéutica, Facultad de Princesa. Hospital Universitario La Princesa. Medicina, Universidad Autónoma de Madrid, Madrid, España. Spain. Alzheimer´s disease (AD) is the most common Alzheimer’s disease (AD) is becoming a form of dementia with still no effective devastating health, social, and economical treatment. From a histopathological point problem. Burden to society will increase of view, AD is characterized by extracellular as population ages. The search of disease- aggregates of betaamyloid and, intracellular modifying drugs has focused on over neurofibrillary tangles composed of 30 distinct targets, most of them linked hyperphosphorylated tau protein. During to amyloid beta (Aβ) aggregation or the last twenty years great effort has been hyperphosphorylated tau. Anti-oxidant, anti- made to develop therapeutic strategies, inflammatories, neurotransmitter receptors, mostly based on beta-amyloid pathology, or growth factors have been explored as but without success. On the other hand, AD targets to develop a medicine to delay disease shares with other neurodegenerative diseases progression. Ligands for those targets have pathological mechanisms like oxidative stress, been explored in cell and murine models of subchronic inflammation, mitochondrial AD. Although many of them have shown dysfunction and proteinopathy. Given this efficacy in this preclinical set-up, they have scenario, our group seeks to identify new failed in dozens of clinical trials performed therapeutic targets focused on the regulation during the last 20 years in AD patients. It is of oxidative stress and neuroinflammation, interesting that the Alzheimer’s Foundation processes that precede the accumulation of for Drug Discovery (AFDD) is not supporting aberrant proteins and cognitive impairment. any more clinical trials with compounds We have therefore centered out attention on targeted to Aβ or tau. So, new targets and ideas two targets: (1) the NADPH oxidases enzymes are urgently needed. In this symposium on (NOXs), which are the enzymes responsible new targets and drugs for AD, four scientists for the production of reactive oxygen species from the Institute “Teófilo Hernando for Drug such as superoxide and hydrogen peroxide, Discovery”, at the Universidad Autónoma and more specifically, in its NOX4 isoform de Madrid, Spain, will present their work and, (2) in the transcription factor NRF2 on new approaches to the search of new (Nuclear factor (erythroid-derived 2) -like 2), targets beyond conventional (Manuela García master regulator of the antioxidant response, López), multitarget compounds (Rafael León which also regulates the expression of genes Martínez), and Phosphatase PP2 (Raquel that participate in the anti-inflammatory López Arribas). A last communication focus response and autophagic processes. To on altered neurotransmission processes in validate NOX4 as a possible target, we have AD (Luis Gandía Juan). It is expected that used transgenic mice that do not express this only with these and other new strategies, we enzyme and a model of taupathy by injecting can find out the way to a medicine capable i.c.v. adeno viruses containing the human of slowing down the natural course of the tau protein mutated in P2301L, under the disease; and what it is even more challenging, promoter synapsin. In this model we have if administered at presymptomatic AD stages, been able to determine that animals that in patients at risk diagnosed with biomarkers, do not express NOX4 have less oxidative this medicine be capable of delaying disease stress and less neuroinflammation which results in an improvement in the cognitive NON CONVENTIONAL TARGETS FOR tests. For our second target, we are looking THE TREATMENT OF ALZHEIMER´S for compounds that inhibit the interaction DISEASE. Keap-1 (NRF2 repressor protein) and NRF2. López M.G. 1, Luengo E. 1, Trigo P. 1, To do this, we have performed an in-silico Fernández-Mendivíl C. 1, Franco F. 1, del screening of large libraries using docking and Sastre E. 1, Cuadrado A. 2, Rodriguez-Franco molecular dynamics, as well as the synthesis M. I. 3 y León R. 1 4. of new compounds. In the latter case, we 1.Instituto Teófilo Hernando. Departamento want to obtain multitarget compounds with de Farmacología. Universidad Autónoma complementary activities to the induction of

69 NRF2, with the aim of interfering on different age in both control and transgenic mice. nodes of Alzheimer’s pathophysiology. In this Finally, we found an increase in calcium project we are following a sequential screening currents in 3xTg-AD mouse with age that was protocol based on studying the Nrf2 induction, not observed in wild type mice. These findings antioxidant, anti-neuroinflammatory and suggest that throughout the development of neuroprotective properties of the compounds. 3xTg-AD mice and as Alzheimer’s disease is As a last step, those compounds with a more established there is a change in chromaffin cell favorable toxicological, pharmacokinetic and excitability, which causes neurotransmission pharmacodynamic profile will be evaluated in to accelerate. These alterations could have in vivo models of AD. an impact on the response that the organism offers in a stressful situation. ALTERATIONS IN NEUROTRANSMISSION RELATED SYNTHESIS AND TO THE PROGRESSION OF PHARMACOLOGICAL EVALUATION ALZHEIMER’S DISEASE. OF NEW COMPOUNDS DIRECTED TO Nanclares, C., Colmena, I., Baraibar, A.M., PP2A, A PROMISING THERAPEUTIC Muñoz-Montero, A., García, A.G and Gandía, TARGET FOR ALZHEIMER’S Ll. DISEASE. Instituto Teófilo Hernando, Depto. R. López- Arribas1, L. Viejo de Navas1, 2, C. Farmacología, Facultad de Medicina, de los Ríos1, 2. Universidad Autónoma de Madrid, Madrid, 1. Instituto Fundación Teófilo Hernando. Spain. Departamento de Farmacología y Terapéutica, Dpto de Farmacología, Facultad Alzheimer´s disease (AD) is the most common de Medicina, Universidad Autónoma de form of dementia, being aging the main Madrid. C/ Arzobispo Morcillo, 4, Madrid. 2. risk factor for the development this disease. Instituto de Investigación Sanitaria Hospital The alteration of several neurotransmitter Universitario de la Princesa. C/ Diego de systems has been reported in AD, which could León, 62, Madrid. be correlated with changes in the synthesis, storage or release of these neurotransmitters. Introduction: Alzheimer’s disease (AD) is the In this study, we tested how aging affects most common cause of dementia. Nowadays, ionic currents, cell excitability and last there is no cure for AD or a way to stop or steps of exocytosis under physiological and slow its progression. Main histopathological pathological conditions. For this purpose, we hallmarks of AD are senile plaques, and used a triple transgenic model of AD (3xTg-AD) neurofibrillary tangles, generated by that contains mutations in the gene encoding aggregation of the microtubule associated the amyloid precursor protein (APPSwe), protein tau. Over the past two decades, presenilin-1 (PS1M146V) and tauO301L, most scientific efforts in the development which mimics the development of the disease of new drugs to treat AD have focused on on Alzheimer’s patients, and B6129SF inhibiting the degradation of acetylcholine mice (wild type). By using amperometric and avoiding amyloidogenesis. However, less techniques, we have found significant changes interest has aroused the therapeutic approach in the exocytosis of catecholamines occurring consisting in preventing neurofibrillary in mice of 6 and more than 12 months of age, death by inhibiting the abnormal where the pathology is already established, hyperphosphorylation of tau. In this sense, when compared with prepathological mice pharmacological strategies have been almost (2 months), in particular, an increase of the completely oriented to inhibit the activity of number of amperometric spikes, although tau kinase enzymes, with discouraging results. the quantal catecholamine content on Our research group proposes to address the individual spikes is lower. Kinetic analysis aberrant phosphorylation of tau by restoring of secretory spikes shows that as the disease the activity of its main phosphatase enzyme, progresses amperometric spikes are faster protein phosphatase 2A (PP2A), which is in triggering and shorter in duration. Patch- decreased in the brains of patients with AD, clamp technique was also used to measure mainly due to the increase in the expression the different ionic currents involved in the of the endogenous inhibitors I1PP2A and physiological release of catecholamines. We I2PP2A/SET . Hypothesis: The study of the observed a decrease in sodium currents and structure-activity relationship of okadaic an increase in potassium currents in 3xTg-AD acid (OA), a toxin with selective inhibitory compared with controls. Nicotinic currents activity of PP2A, has allowed us to design exhibited a similar pattern throughout the and synthesize new analogue molecules of

70 OA that lack such inhibitory capacity. In this medicinal chemistry in the neurochemistry sense, our starting hypothesis states that field, through the design of novel ligands these compounds, due to their binding to the functioning as multitarget agents in catalytic subunit of PP2A, would be able to Alzheimer’ disease (AD). In this proposal, we compete with the endogenous inhibitors of describe the synthesis and in vitro biological PP2A, and thus, to restore the compromised evaluation of novel indole derivative as single phosphatase activity in AD. Material and chemical entities to simultaneously modulate results: Our molecules are able to reduce OA- multiple targets which comprises i) binding- induced neurotoxicity and some of them also affinity and potential agonist properties of present a good profile in a model of oxidative serotonin 5-HT4R ii) acetylcholinesterase stress in SH-SY5Y cells and in a model of inhibition, iii) serotonin transport re-uptake excitotoxicity in cortical neurons. The new inhibition and iv) inhibition of β-amyloid compounds maintained serine/threonine deposition. phosphatase activity, depressed by the action of two PP2A inhibitors: OA and cytostatin. DESIGN OF NEW BENZIMIDAZOLES Molecular docking studies indicated that the WITH BETA-3 ADRENERGIC compounds studied are capable of binding AFFINITY. PP2A in a similar manner to OA, but does Mella J. not interact with the catalytic site, confirming Laboratorio de Química Medicinal, Instituto our initial hypothesis. Conclusions: Our de Química y Bioquímica, Facultad de compounds have a potential indication for Ciencias, Universidad de Valparaíso. the treatment of neurodegenerative diseases based on the maintenance of PP2A activity, The human receptor β3-adrenergic has which avoids tau hyperphosphorylation. been the target of recent studies due to its potential to modulate various physiological 6. MEDICINAL CHEMISTRY: aspects of the organism involved in numerous RATIONAL DESIGN AND STRUCTURE pathologies such as diabetes, hypertension, ACTIVITY RELATIONSHIP, A overactive bladder, heart problems, SYNTHETIC APPROACH OF depression, and cancer, among others. In this NEW BIOLOGICALLY ACTIVE context, our efforts are focused on the design, SUBSTANCES. synthesis and biological evaluation of new heterocyclic compounds capable of binding to SYNTHESIS OF NEW INDOL the human receptor β3-adrenergic. Since the DERIVATIVES AND THEIR beta-3 receptor is not crystallized, we have ACTIVITY ON CHOLINERGIC AND performed extensive studies based on ligands SEROTONERGIC SYSTEMS AND IN (3D-QSAR, CoMFA, and CoMSIA), which BETA-AMYLOID DEPOSITION. A have allowed us to generate a pharmacophoric MULTIFUNCTIONAL APPROACH TO model that we use as a basis for the rational ALZHEIMER’S DISEASE. design of our compounds. The routes of Pessoa-Mahana P. Departamento de Química synthesis of the heterocycles proposed by our Orgánica y Fisicoquímica, Facultad de Ciencias group follow a similar route to that used to Químicas y Farmacéuticas, Universidad de obtain Mirabegrón, the only drug currently Chile. 3, Centro de Investigación Biomédica available that acts on the beta-3 receptor y Aplicada (CIBAP), Escuela de Medicina, indicated in the treatment of overactive Facultad de Ciencias Médicas, Universidad bladder. de Santiago de Chile. NEONICOTINOIDS, SEARCHING FOR Alzheimer’s disease is the most diffuse form NICOTINIC RECEPTOR LIGANDS OF of senile dementia, and is among the most ALPHA4BETA2 NACHR SUBTYPE devastating brain disorder an individual can AND ITS APPLICATION AS NEW face. It involves progressive and irreversible ANTI-ADDICTIVE SUBSTANCES. decline in cognitive functions including Iturriaga-Vasquez, P. memory, judgment, decision-making, Laboratorio de Farmacología Molecular y orientation to physical surroundings and Síntesis Orgánica, Depto. Cs. Químicas y language. Despite substantial efforts in drug Recursos Naturales, Fac. de Ingeniería y development and an increased understanding Ciencias, Universida de La Frontera, Temuco. of the underlying pathology of Alzheimer’s disease, no effective treatment has yet been Addiction is a chronic and compulsive drug achieved. The main goal of this research seeking, producing detrimental consequences, is to contribute to the knowledge of the and long-lasting changes in the brain. It is

71 considered a brain disorder and a mental and development of new antimicrobial drugs, illness. Addiction is the most severe form of since classic or conventional antibiotic drugs substance use disorders, caused by repeated have been proposed to become obsolete in a misuse of a substance. Neuronal Nicotinic few decades from now. This scenario is not Acetylcholine Receptors (nAChR) are exclusive for bacterial infections, since the involved in nicotine addiction and emerging lack of new clinically effective drugs is also a evidence suggests that nAChR could be acts problem for fungal and parasite infections. In as pharmacological target to be considered this context, natural products have emerged in alcohol abuse. Two of the most common as a valid alternative for the discovery of new addictive substances used and accepted by the antimicrobial agents with new mechanisms society. Nicotinic ligands have been designed, of action and in some cases even in absence synthesized and tested on nicotinic receptor of current resistance mechanisms. Plants for decades, but the focus of the design has are affected by several microorganisms, so been full and partials agonists with good they count with high content of secondary therapeutics results on nicotine addiction metabolites with antibacterial, antifungal (i.e. cytisine and varenicline). However, there and antiparasitic effects such as flavonoids, are little evidences indicating that nicotinic tanins, terpenoids and alkaloids. However, antagonist could expert anti-addictive effects herbal extracts can vary among the same over nicotine addiction and alcoholism. In species due to different extraction methods, our lab, we had designed and synthesized different geographical location and even simple nicotinic analogues with agonist or season collection. So, it is important to count antagonist properties on alpha4beta2 nAChR with adequate characterization methods subtype and using zebrafish as a behavioural in order to achieve reproducible results model we have identified a new nicotinic and standardized extracts respect to their antagonist, named UFR2709 that revert the chemical composition and the proportion of effect of nicotine using a homologous CPP for active principles that can be related to their zebrafish. Additionally, we tested UFR2709 antimicrobial activity. on Wistar-derived University of Chile alcohol- preferring UChB rats a well-known model to REVEALING ANTIMICROBIAL evaluate ethanol consumption. Our results ACTIVITY OF NATURAL PRODUCTS show that UFR2709 are able to decrease USING CHEMICAL SUBTRACTION the ethanol intake using a two-bottle choice AS NEW STRATEGY TO PREPARE paradigm assay. UFR2709, an alpha4beta2 KNOCK-OUT AND KNOCK-IN nAChR antagonist shows an anti-addictive EXTRACTS. effect on nicotine addiction and ethanol Pastene-Navarrete E.1,2. consumption and open a new way for drug 1 Laboratory of Pharmacognosy, Faculty design and the treatment of nicotine and of Pharmacy, Universidad de Concepción, ethanol addictions. Concepción, Chile; 2 Laboratory of Synthesis and Biotransformation of Natural Products, 7. ANTIMICROBIAL ACTIVITY OF Department of Basic Sciences, Universidad HERBAL EXTRACTS AGAINST del Bio-Bio, Chillán, Chile. CLINICALLY RELEVANT PATHOGENS Chilean plants have biased and incomplete chemical-pharmacological studies. The main NATURAL EXTRACTS AND THEIR reason for that has been the low availability ROLE IN THE SEARCH FOR NEW of enough quantities to make biological THERAPEUTIC ALTERNATIVES TO tests and structural elucidation. Moreover, TREAT INFECTIONS. the isolation of specific constituents often Molina-Berríos A. omits the residual complexity existing in Laboratorio de Farmacología, Instituto de plants, in which it is not uncommon to find Investigación en Ciencias Odontológicas, highly active compounds. In this work is Facultad de Odontología, Universidad de presented the application of a new strategy to Chile, Santiago, Chile. investigate antimicrobial activity of medicinal and food plants. This tool unifies different In May 2015 the World Health Assembly pharmacological/phytochemical approaches adopted a global action plan against using the liquid-liquid methodology named antibiotic resistance, since deaths related to Centrifugal Partition Chromatography (CPC) multidrug resistant bacteria have increased to obtain DESIGNER extracts. These extracts in alarming speed in the last decades. One of could be “knock-out” (selective removal of the goals of this plan is to support research one or a group of compounds) or “knock-

72 in” (selective addition of one or a group of on the adhesion ability through crystal violet compounds). In the first example, we prepare assay and the antibiofilm effect through the “knock-out” from propolis and Buddleja viability of biofilm formation and scratch globosa (Matico) and assess their antimicrobial assay. The MIC was able to inhibit adhesion activity. Propolis without caffeic acid phenyl and biofilm formation in an abiotic surface for ester (CAPE) shown similar antimicrobial the resistant strains assayed (ATCC 10231). In activity compared to raw extract, suggesting conclusion, this study demonstrates that this that other compounds present in its residual essential oil from Lavandula dentata could be complexity are responsible for such activity. a promising strategy against biofilms from On the other hand, Matico “knock-out” resistant Candida albicans strains. Since (selective removal of verbascoside), displayed phytodrugs present many active compounds, minimal antimicrobial activity. In this last who makes then difficult to generate example, the re-incorporation of verbascoside resistance, they can be used in conjunction recovered the biological activity. Finally, we with conventional antifungal, sensitizing the perform a double knock-out in Peumus boldus pathogens and decreasing its adhesion and extract, removing cytotoxic compounds (e.g. later formation of biofilms. ascaridole) and isoquinoline alkaloids (e.g. boldine). This Boldo DESIGNER extract PARASITES AND PLANTS: reduce significantly cell injury in H. pylori- ELUCIDATING THE ANTIPARASITIC infected AGS cells without the cell toxicity ACTIVITY OF CICHORIUM INTYBUS observed in the raw extracts. To confirm the (CHICORY). protective properties of this extract in vivo, Peña-Espinoza M. we used a continuous liquid-liquid separation Instituto de Farmacologia y Morfofisiologia, by True Moving Bed system (TMB-500). Facultad de Ciencias Veterinarias, Hence, a dose of 100 mg/kg/day of Boldo Universidad Austral de Chile. DESIGNER extract was able to prevent H. pylori SS1 infection in Mongolian gerbils. Parasitic helminths and protozoa affect billions of people worldwide and are amongst PHYTOPHARMACEUTICALS AND the most prevalent infections in livestock. ANTIMICROBIAL RESISTANCE IN Due to increasing parasite drug resistance VETERINARY MEDICINE. towards the limited therapeutic arsenal Müller-Sepúlveda, A. available, novel antiparasitics are urgently Instituto de Ciencias Agronómicas y needed. Plants with reported antiparasitic Veterinarias. Universidad de O´Higgins. activity have been traditionally used and may provide new lead compounds. One There is an increase in fungal infections antiparasitic plant increasingly investigated owing to the appearance of resistant fungi to is chicory (Cichorium intybus; Asteraceae), different drugs. Candida albicans is part of the a perennial herb distributed worldwide and resident microbiota of the oral cavity but is commonly cultivated as crop for human and also the most frequent fungal pathogen, whose livestock consumption. Chicory has attracted biofilms formation represents one of the main research interest for its effects against resistance mechanisms. In the oral cavity, parasitic nematodes in livestock, which have Candida albicans biofilms are extremely been linked with its content of sesquiterpene resistant to antifungals and together with the lactones (SLs). Previous in vivo studies have absence of new, effective and safe antifungals, confirmed that chicory-fed animals have the search for pharmacological alternatives a reduced parasite burden, but detailed is warranted. It has been described that identification of responsible compounds has essential oils from Lavandula dentata, and not been, until recently, thoroughly explored. endemic plant in Chile, possess antimicrobial By integrating parasitological studies and and antifungal activity against several metabolomic analyses, we have investigated microorganisms including Candida albicans. the anthelmintic activity and phytochemical We described the antifungal and antibiofilm profile of SL-extracts from chicory material effect of Lavandula dentata essential oil on the sampled in different geographical regions. inhibition of Candida albicans Fluconazole- The in vitro activity of chicory SL-extracts was resistant strain (ATCC 10231), to adhere to first evaluated using the free-living nematode abiotic surfaces and to form biofilms. After the Caenorhabditis elegans model and further chemical characterized of the essential oil by confirmed in the parasitic pig nematode Gas Chromatography and the determination Ascaris suum, which is closely related of minimal inhibitory concentration (MIC), with the human parasite A. lumbricoides. we evaluated the effect of this essential oil Marked differences in anthelmintic potency

73 were observed between SL-extracts from between several scientific and technological different chicory material. Bioactivity-based competences for the development of molecular networking analyses suggest that industrially viable L-Asparaginase production some but not all SLs are linked with the process. This new proposal is a continuation anthelmintic activity of chicory. In addition, of the FAPESP Thematic Project (2013 / we have explored the antiprotozoal activity 08617-7) that has provided promising results, of chicory against Trypanosoma cruzi, the since it enabled the development of new etiological agent of Chagas disease. Chicory recombinant strains of bacteria and yeast SL-extracts induced potent concentration- with the capacity to produce L-asparaginases dependent trypanocidal activity against T. with longer half-life, greater stability, lower cruzi trypomastigotes at concentrations that toxicity, and lower side effects in comparison are not toxic to mammalian cells. Isolation to the biopharmaceuticals currently in and testing of individual chicory SLs are clinical use not just in Brazil, but worldwide. undergoing to evaluate their antiparasitic As a continuation of the previously initiated mechanisms. studies, this thematic project proposes the development of processes for the 8. BIOTECHONOLOGY ASPECTS production, under GLP and GMP of 4 OF ASPARAGINASE CLINICAL AND antileukemic biopharmaceuticals with INDUSTRIAL DEVELOPMENT different characteristics and with important potential to be produced nationally and PRODUCTION OF EXTRACELLULAR even for export, including: 1) Escherichia L-ASPARAGINASE: FROM coli BL21 ( DE3) – a recombinant wild- BIOPROSPECTING TO type E. coli ASNase, overexpressed in THE ENGINEERING epichomal vector pet28a with a resistance OF AN ANTILEUKEMIC marker for kanamycin; 2) Escherichia coli BIOPHARMACEUTICAL. BL21 (DE3) – a recombinant wild-type Farias J. A.1; Pessoa A.2; Monteiro G.2; Effer, ASNase from Erwinia chrysanthemi ASNase, B.1. 1. overexpressed in epimasomal vector pet28a 1. Departamento de Ingeniería Química with a resistance marker for kanamycin; 3) Universidad de La Frontera, Chile; 2. Escherichia coli BL21 (DE3) – a recombinant Biochemical and Pharmaceutical Technology E. coli ASNase resistant to human serum Department, School of Pharmaceutical proteases - overexpressed in epichomal- Sciences, University of São Paulo, Brazil. vector pet28a with a resistance marker for kanamycin; 4) Recombinant Pichia pastoris In 2013 the production of L-Asparaginase, – a recombinant wild-type E. chrysanthemi a biopharmaceutical widely used in crisantaspase with humanized glycosylation the treatment of acute lymphoblastic (expressed in pJAG-s1 in the Superman5- leukemia (ALL), was suspended by the Glycoswitch yeast from Biogrammatics™). To foreign manufacturer who supplied the improve aspects of stability, bioavailability, drug (Elspar®) to Brazil since the 1980s. toxicity and hyperallergenicity, which The interruption of this supply led to life are problems observed with current threatening delays in treatment which formulations, nanotechnological approaches forced the Brazilian Ministry of Health such as pegylation and encapsulation in to find emergency alternatives, including polymer vesicles will be used. The project importation of a more expensive alternative aims to develop a production process (Aginasa®). Later, in 2017 and following from optimization of microbial cultures to an international public tender, MS started purification, pegylation and final formulation importing the medicine Leuginase®, from (lyophilized product), in sufficient quantity China. Such frequent changes in the supply to carry out subsequent preclinical studies. of L-Asparaginase has provoked intense The entire study will be accompanied by and controversial debate in Brazil regarding technical and economic evaluations (Quality the quality of the imported medicine. This by Design). motivated our group to develop a technology platform for the production of L-asparaginase with more advantageous characteristics than the imported formulations. Brazil is considered a weak player on the World stage in biopharmaceutical discovery, development and production of biopharmaceuticals, and the present project proposes a union

74 DEVELOPMENT OF those that are in clinical use in the World. BIOTECHNOLOGICAL PROCESS A recombinant E. coli with the capacity to FOR THE PRODUCTION produce L-asparaginase resistant to two OF THE ANTILEUKEMIC plasma proteases was obtained and the BIOPHARMACEUTICAL toxicity studies shows important potential for L-ASPARAGINASE (ASNASE) starting preclinical studies. A recombinant USING GENETICALLY MODIFIED P. pastoris strain with the ability to produce MICROORGANISMS. L-asparaginase with humanized glycosylation Pessoa, A. was also obtained, with great potential Faculty of Pharmaceutical Sciences, to reduce to immunogenic reactions and, University of São Paulo, Brazil. therefore, safer for patients. Pegylation and nanoencapsulation studies of the novel There is a strong global tendency to find L-asparaginases are being conducted and alternative ways to produce pharmaceutical the results have shown that site-directed active principles from biotechnology process. pegylation has the potential to generate a In this scenario, Latin American countries biopharmaceutical with better characteristics show a small expression in both research than the pegylated form on the market. In and production. Worsening the situation, addition, polymer encapsulation studies international suppliers of biopharmaceuticals have been conducted with promising results, to this region are losing interest in the especially since it is a new alternative in the market and discontinuing production, nanobiotechnology process. The project is especially those related to onco-hematologic underway with the development of processes treatment. In this context, the union of of production, in GLP (good laboratory different scientific and technological skills practice) and GMP (good manufacturing have joined to achieve a viable industrial practice), of new L-asparaginases with higher process to biotechnologically produce characteristics and with important potential L-Asparaginase, a biopharmaceutical broadly to be produced nationally and even for used in the treatment of leukemia. Two major exportation. research fronts are being studied with the objective of finding a promising antileukemic ASPARAGINASE ENGINEERING IN biopharmaceutical: the optimization of THE OBTAINMENT OF BIOBETTERS endogenous and heterologous production OF THIS ANTITUMOR ENZYME. processes of the enzyme, with bioprospecting Costa I.M. 1; Costa-Silva T.A. 1; Effer B. 1,2; groups of fungi of the most varied biomes; Meira-Lima G. 1; Biasoto H.P.1; Silva C.1; and the rational engineering of proteins Pessoa A.1; Rangel-Yagui C.1; Farias J. A.2; that will utilize as scaffold the S. cerevisiae Monteiro G.1. and E. coli L-Aparaginases for comparative 1. Biochemical and Pharmaceutical studies with the bacterial isoforms currently Technology Department, School of employed in therapeutics. To improve Pharmaceutical Sciences, University of São aspects of stability, bioavailability, toxicity Paulo, Brazil; 2. Departamento de Ingeniería and allergenicity, problems observed Química Universidad de La Frontera, Chile. with bacterial formulations, several nanotechnological approaches are being Asparaginase (ASNase), an enzyme used, such as pegylation and encapsulation biotechnologically produced in bacteria, in polymeric vesicles, and the project aims is one of the most important compounds to generate a biopharmaceutical to be in the polychemotherapy to treat acute produced industrially. Fungi from different lymphoblastic leukemia (ALL) in children. biomes, such as cerrado, caatinga, marine There are only three options available as environment, and Antarctica, have been medicine: native enzyme from Erwinia isolated and several of them have been chrysanthemi (ErA) or extracted from evaluated for the production of the enzyme Escherichia coli (EcA) and formulated as in shaker and in 3- or 7-Liter bioreactors, and native or PEGylated (PEG-EcA). However, by solid state cultivation. Biochemical and these options yet present some problems in kinetic characteristics are being determined patients, such as to elicit hypersensitivity for all isolated L-asparaginases. The studies and allergenic reactions, neurotoxicity, aim at obtaining recombinant E. coli and and hyperammonemia. Aiming to avoid Pichia pastoris to produce of L-asparaginase some of these problems, our research group with potentially improved characteristics has developed several different mutant (longer half-life, higher stability, lower proteoforms, expressed in bacteria and yeast, toxicity and lower side effects) in comparison in periplasmic or secreted to extracellular

75 space; with improvement in specific activity, MINISYMPOSIA kinetic parameters, and stability; different oligomerization states, glycosylated or not, 1. CARDIOVASCULAR AGING through engineering of genes from E. coli and E. chrysanthemi. We obtained mutants PREVENTING PREMATURE from E. coli ASNase more resistant to human ENDOTHELIAL CELL SENESCENCE: proteases and less immunogenic. In relation THE ROLE OF ANGIOTENSIN-(1-7). to E. chrysanthemi enzyme, our mutants Peiró, C. present higher asparaginase activity than the Departamento de Farmacología, Facultad de native form, with improved kcat. In addition, Medicina, Universidad Autónoma de Madrid. we obtained strains of Pichia pastoris that express glycosylated ASNases from bacteria. Vascular aging is a complex multifaceted Our results suggest several biobetters options process displaying functional and structural developed in this study. alterations that ultimately favour vascular disease and atherosclerosis. Endothelial PRODUCTION OF NOVEL cell senescence, which may be induced by a GLYCOSYLATED L-ASPARAGINASE wide variety of extracellular stressors, is one AS AN ALTERNATIVE AGAINST of the major mechanisms contributing to ACUTE LYMPHOBLASTIC vascular aging. The senescent endothelial cell LEUKEMIA. acquire a phenotype characterized by growth Effer, B. arrest and the acquisition of a senescence- Departamento de Ingeniería Química, associated secretory phenotype (SASP), that Facultad de Ingeniería y Ciencias, Universidad promotes the release of pro-inflammatory de La Frontera. mediators and the onset of sterile age- related inflammation. In this context, the L-asparaginase (L-ASNase) is an important identification of pharmacological tools to bacterial enzyme used as biopharmaceutical to interfere with endothelial senescence may help treat acute lymphoblastic leukemia (ALL). Its retarding vascular aging and its complications. use as medicine has important side effects such Angiotensin (Ang)-(1-7) is a heptapeptide as pancreatitis, abnormalities in coagulation, belonging to the so-called protective arm of the hepatosplenomegaly, immunogenicity, renin-angiontesin system (RAS). Ang-(1-7) is a among others. It has been counteracted by ligand for the G-protein-coupled receptor Mas. PEGylation; however, immunogenicity has In the vascular system, Ang-(1-7) has been been observed in PEG. Here we explore the acknowledged as a physiological antagonist production of recombinant L-ASNase from for angiotensin II (Ang II), since it displays D. chrysamthemi glycosylated like-mammals vasorelaxant, anti-proliferative and anti- in a Glycoswitch® Pichia pastoris strain as inflammatory actions, among other. Here, we an alternative to PEGylation. In our results, tested the capacity of Ang-(1-7) to act as an the recombinant Erwinase occurred in three anti-senescence molecule. In human cultured extracellular, glycosylated and biologically endothelial cells, Ang-(1-7) was capable to active variants; two of them tetramerics attenuate the pro-senescence actions driven by (Erw240) and (Erw160) with specific activity Ang II in terms of DNA damage, senescence- of 15.71 and 302.02 U mg-1 respectively; and associated beta-galactosidase (SA-beta-gal) one new monomeric version (Erw40) with activity and SASP-related cytokine release. 48.45 U mg-1. The lightweight tetramer and Importantly, Ang-(1-7) also attenuated the the monomer showed catalytic efficiency of endothelial cell senescence induced by non- 7.7 x 105 and 1.05 x 106 respectively. Mass RAS stressors, such as the pro-inflammatory spectrometry analysis of the more active cytokine interleukin (IL)-1beta. These tetrameric and monomeric versions showed protective actions of Ang-(1-7) were mediated mainly an oligosaccharide GlcNAc2Man7, by Mas receptors since they were blunted by bound to Asn170, which is part of a predicted the Mas antagonist drug A779. Furthermore, immunogenic T-cell epitope. ELISA assay we demonstrated that Ang-(1-7) exerted its in vitro showed a significant reduction anti-senescence actions by activating two of antibody recognition in the Erw160, cytoprotective systems, i.e., the Nrf2/heme- suggesting the oligosaccharide bound to oxygenase axis and the anti-ageing protein L-ASNasa had a cloaking effect against klotho. Overall, the Ang-(1-7)/Mas receptor antibodies. The new L-ASNase versions axis may a valuable pharmacological target to reported here could provide an alternative for attenuate endothelial senescence and to delay the treatment of ALL. vascular aging induced by a variety of stressors.

76 ROLE OF NLRP3 INFLAMMASOME CARDIAC FIBROBLAST ROLE IN VASCULAR DAMAGE INDUCED BY ON INFLAMMATORY PROCESS: ADIPOKINES. INTERACTION WITH IMMUNE Sánchez Ferrer C.F. CELLS. Departamento de Farmacología, Facultad de Díaz-Araya, G. Medicina, Universidad Autónoma de Madrid. Molecular Pharmacology Laboratory and FONDAP ACCDiS, Universidad de Chile. We have demonstrated that these adipokines promotes vascular inflammation and The abundance and strategic location of cardiac endothelial dysfunction. Moreover, our fibroblasts and also macrophages in cardiac data suggest that vascular deleterious tissue damage, suggest the possibility of a effects evoked by visfatin/eNampt or sDPP4 highly coordinated interaction between both may involve the activation of the NLRP3 cell types, in order to orchestrate the different (nucleotide-binding domain, leucine-rich- stages of cardiac remodeling. In particular containing family, pyring domain-cotainin-3) macrophage is able to adapt their phenotype inflammasome. Indeed, evidence from our and activity according to the cytokine milieu laboratory demonstrates the activation of present in the local cardiac environment. NLRP3 inflammasome by visfatin/eNampt, This phenomenon, known as macrophage while the adipokine-evoked endothelial polarization, contributes to the accumulation dysfunction is prevented by inhibiting its of pro-inflammatory M1 macrophages enzymatic activity with FK866, as well as during the onset of cardiac remodelling, by the antagonism of toll-like receptors-4 while also explaining the high levels of anti- (TLR4) with CLI-095, the interference inflammatory/profibrotic M2 macrophages of NLRP3 inflammasome assembly with found in the later stages of cardiac repair. While MCC950, or the IL-1 receptor blockade with the effects of macrophages on cardiac fibroblast anakinra. Therefore, we propose that visfatin/ activity have been extensively studied, the eNampt induces vascular damage by a ability of cardiac fibroblasts to modulate TLR4-mediated pathway, leading to NLRP3- macrophage behavior is less understood. LPS, inflammasome activation and the paracrine and Heparan sulfate as pro-inflammatory production of IL-1beta. On the other hand, stimulus, triggers on cardiac fibroblast ICAM- we have shown that sDPP4 induces vascular 1 and VCAM-1 expression levels, which allow alterations by activating proteinase-activated spleen mononuclear cells and neutrophils receptors-2 (PAR-2) and upregulating adhesion. LPS triggers high TNF-α/IL-10 thromboxane-A2 (TXA2) release. Moreover, ratio, whereas, TGF-β1 a profibrotic stimulus the endothelial dysfunction evoked by sDPP4 triggers an increase on ICAM-1 and VCAM-1 is also dependent on its enzymatic activity, expression levels, but low TNF-α/IL-10 ratio. being attenuated by its inhibitors K579 and Consequently, cardiac fibroblast under LPS- linagliptin, as well as by the specific PAR- treatment promote monocytes-macrophages 2 antagonist GB83 and the TXA2 receptor M1 polarization. By contrast, cardiac blocker SQ-29,548. Interestingly, this fibroblast under TGF-β1 promote monocytes- pathway also leads to NLRP3 inflammasome macrophages M2 polarization. Our results activation, while the sDPP4-evoked demonstrate that cardiac fibroblasts interact endothelial damage is reduced by interfering with immune cells and contribute to monocyte NLRP3 inflammasome with MCC950. We recruitment and induce their differentiation to conclude that vascular NLRP3 inflammasome M1 or M2 macrophages. activation can be a common pathway for different pro-inflammatory adipokines. 2. NOVEL MOLECULAR PATHWAYS Indeed, targeting NLRP3 inflammasome and FOR SCHIZOPHRENIA some receptors linked to this pathway (TLR4, PAR-2, or IL-1) may represent therapeutic DYSREGULATION OF THE AMYLOID strategies to treat and/or prevent obesity- PRECURSOR PROTEIN AND IRON IN related vascular dysfunction. SCHIZOPHRENIA. Opazo, C. The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC, Australia, Melbourne Neuropsychiatry Centre, Department of Psychiatry, The University of Melbourne & Melbourne Health, Carlton South, VIC, Australia.

77 Schizophrenia (Sz) is a debilitating mental schizophrenia. UPS is a master regulator of illness that disrupts the functioning of the neural development and the maintenance mind. Impairments in certain cognitive of brain structure and function. It functions are core features of Sz, which influences neurogenesis, synaptogenesis are not addressed for existing drug targets and neurotransmission by determining the and remain a crucial unmet therapeutic localization, interaction and turnover of need. Our hypothesis is that schizophrenia scaffolding, presynaptic and postsynaptic is a complex disease resulting from a loss- proteins. Although links between UPS of-function of key pathways that govern dysfunction and neurodegenerative neurodevelopment, neurotransmission and disorders have been known for some time, synaptic connectivity. The Amyloid Precursor only recently have similar links emerged Protein (APP), which we have extensively for neurodevelopmental disorders, such as investigated in relation to Alzheimer’s schizophrenia. In this presentation, we will disease, is a key regulator of brain structure review the components of the UPS that are and function. Our data indicate that iron is reported as dysregulated in schizophrenia elevated in autopsy orbitofrontal cortex from by our group and others, and we will individuals with Sz relative to age- and sex- discuss specific molecular changes to these matched controls. We hypothesize that these components that may explain the complex changes are mediated by the downregulation aetiology of this mental disorder as a of APP, which also occurs in prefrontal syndrome. cortex region of individuals with Sz. Our group have characterized the age-dependent accumulation of iron in the brain of global DR. JORGE MARDONES APP knockout mice. Remarkably, global APP RESTAT AWARD KO display features of Sz, including agenesis of corpus callosum and increased seizure 1. USE OF NPSI-ΒCD COMPOSITE activity. Therefore, we propose that down MICROPARTICLES FOR THE regulation of APP function may represent a CONTROLLED RELEASE OF CAFFEIC common lesion that leads to inappropriate ACID AND PINOCEMBRIN, TWO neurotransmission, synaptic pruning and MAIN POLYPHENOLIC COMPOUNDS synaptic function that are involved in the FOUND IN A CHILEAN PROPOLIS. clinical manifestation of Sz. Guzmán-Oyarzo D.1; Plaza T.2; Recio- Sánchez G.2,3; Abdalla D.S.P.4; Hernandez- THE UBIQUITIN PROTEASOME Montelongo J.2; Salazar L.A.1. SYSTEM IN SCHIZOPHRENIA. 1 , Center of Molecular Biology and Luza, S. Pharmacogenetics, Scientific and The Florey Institute of Neuroscience and Technological Bioresource Nucleus Mental Health, The University of Melbourne, (BIOREN), Universidad de La Frontera; Parkville, VIC, Australia, Melbourne 2, Bioproducts and Advanced Materials Neuropsychiatry Centre, Department of Research Center (BioMA), Faculty of Psychiatry, The University of Melbourne Engineering, Universidad Católica de Temuco; & Melbourne Health, Carlton South, VIC, 3, Department of Physical and Mathematical Australia. Sciences, Faculty of Engineering, Universidad Católica de Temuco; 4, Department of The aetiology of schizophrenia remains Clinical and Toxicological Analyses, Faculty unknow. It has been linked to abnormalities of Pharmaceutical Sciences, Universidade de in dopaminergic, glutamatergic, GABAergic São Paulo, Brazil. and serotoninergic neurotransmission as well as signalling pathways critical for Propolis is widely recognized for its brain growth and maturation. A common various therapeutic properties, which consequence of these pathway abnormalities are attributed to its rich composition in in schizophrenia is a loss in proteostasis of the polyphenols. Polyphenols exhibit multiple key components of these extra/intracellular biological properties, such as antioxidant, pathways. Proteostasis requires the control anti-inflammatory, anti-angiogenic, and of protein synthesis, folding, conformational others. Despite of its multiple benefits, maintenance, protein-protein interaction, oral administration of polyphenols results trafficking and degradation. The ubiquitin- in bioavailability at the site of action. An proteasome system (UPS) is central to alternative to face this problem is the use proteostasis, suggesting it likely plays a of biomaterials at nano-micro scale due to pivotal role in the onset and progression of its high versatility as carriers and delivery

78 systems of various drugs and biomolecules. total hepatic lipids were extracted. HF treated In that sense, the aim of this work is to rats showed a significant body weight gain determine if microparticles of nanoporous only until the end of the treatment compared silicon conjugated with a beta cyclodextrin to control diet rats. Regarding fat tissue, polymer to form the nPSi-βCD composite are perigonadal fat was 77% higher in rat fed an available material for the controlled release of HF diet and the percentage level of lipids in the two main polyphenols of Chilean propolis, the liver increasing up to 8%. These results caffeic acid and pinocembrin. Moreover, together suggest that a HF diet generates it was studied their cytocompatibility with important physiological changes in the HUVECs. Using different physicochemical animal, producing in a short period a state of techniques, it was demonstrated that nPSi- adiposity consistent with pre-obesity in rats. βCD microparticles successfully retained and controlled release caffeic acid and 3. INDOMETHACIN IMPAIRS pinocembrin. Furthermore, nPSi-βCD POLYAMINE METABOLISM IN LUNG microparticles presented cytocompatibility CANCER CELLS: A KRAS MUTATION- with HUVECs culture at concentrations ASSOCIATED FEATURE?. of 0.25 mg/ml. These results suggest that López-Contreras F1, , Muñoz-Uribe M1, nPSi-βCD microparticles can be safely used Perez-Lainez J1, Ascencio-Leal L1, Rivera- to improve the bioavailability of caffeic Dictter A1, Martin-Martin A1, Burgos Aguilera acid or pinocembrin –and eventually R1, Alarcon Uribe P1, López-Muñoz R1. other polyphenols– in the target site, thus 1 Instituto de Farmacología y Morfofisiología, enhancing its therapeutic effect for the Facultad de Ciencias Veterinarias, treatment of different diseases. Universidad Austral de Chile.

2. EARLY EFFECT OF A HIGH-FAT Non-small cell lung cancer (NSCLC) is the DIET ON PERIGONADAL AND most lethal and prevalent type of lung cancer. HEPATIC FAT IN RATS. NSCLC patients carrying mutations in the López-Aguilera A.1; Eyzaguirre-Velásquez Kirsten rat sarcoma viral oncogene homolog J.1; Escobar-Luna J.1; Bravo J.A1; Julio- gene (KRAS) still lack targeted therapies. Pieper M.1 Also, the levels of polyamines (putrescine, 1. Grupo de NeuroGastroBioquímica, spermidine, and spermine) are increased Laboratorio de Bioquímica de Sistemas. in cancer, playing a pivotal role in tumor Instituto de Química, Facultad de Ciencias, proliferation. Indomethacin increases the Pontificia Universidad Católica de Valparaíso, levels of the polyamine-catabolic enzyme Valparaíso, Chile. spermidine/spermine-N1-acetyltransferase (SSAT). Consequently, the aim of this study Obesity is a multifactorial disease of great was to compare the effect of indomethacin impact in Chile and worldwide. Its causes in the polyamine metabolism of two NSCLC are heterogeneous and there are no totally cell lines, with different KRAS mutation effective therapeutic interventions; therefore status. A549 and H1299 NSCLC cells (KRAS- the study and advances in this subject are mutated and wild-type, respectively) were of great relevance. The dramatic increase exposed to indomethacin. Evaluations in the levels of obesity in the population is included SSAT expression and protein levels, related to a progressive change to sedentary and metabolic analysis of cells by CG-MS lifestyles and excessive consumption of highly metabolomics. Moreover, the difference in caloric foods, such as high-fat diets (HF). polyamine synthesis enzymes among cell lines These factors lead to excessive accumulation and the synergistic effect of indomethacin of adipose tissue, and in the long term may combined with inhibitors of these enzymes result in ectopic fat storage. One way to study were investigated. Indomethacin increased this disease and its comorbidities is the use of the expression and levels of SSAT in both rodents fed with HF diet, as a model of diet- cell lines. In A549 cells, indomethacin induced obesity. The aim of this work was significantly impairs polyamine metabolism. to study the early effects of a HF diet on the However, in H1299 cells, the impact of accumulation of adipose tissue. For this, male treatment on the polyamine pathway was Sprague-Dawley rats were fed a HF diet (62% non-significant. Evaluation of the levels of the of calories from fat) from postnatal day 30 for polyamine synthesis enzymes showed that either 15, 30 or 60 days, and were compared ornithine decarboxylase (ODC) is increased in to age-matched rats fed with a control diet A549 cells, whereas S-adenosylmethionine- (14% calories from fat). At the end of each decarboxylase (AMD1) and polyamine treatment, perigonadal fat was weighed and oxidase (PAOX), are increased in H1299

79 cells. Finally, indomethacin demonstrated pharmacological modulation in this pathway a synergistic effect with the PAOX inhibitor could contribute to reducing the harmful MDL72527 in A549 cells, whereas in H1299 effects of adrenergic stimulation in cardiac had a synergistic effect with the AMD1 fibrosis. inhibitors SAM486. Collectively, these results indicate that indomethacin alters polyamine 2. THE EFFECT OF THE ALLOSTERIC metabolism in NSCLC cells and enhances INHIBITOR OF RIPK1 (NEC-1) ON the effect of polyamine synthesis inhibitors OVARY FUNCTION: IMPORTANCE such as MDL72527 or SAM486. However, OF NECROPTOSIS IN FOLICULAR this effect varies depending on the basal DEVELOPMENT. metabolic fingerprint of each type of NSCLC Cuevas, F., Lara, H.E. cell. FONDECYT-1160807. Laboratorio de Neurobioquímica, Departamento de Bioquímica y Biología Molecular, Centre for Neurobiochemical INCORPORATIONS Studies in Endocrine Diseases. Facultad de Ciencias Químicas y Farmacéuticas, 1. AMPK ACTIVATION ON Universidad de Chile. CARDIAC FIBROBLASTS: ROLE IN AUTOPHAGY AND CELL The ovarian follicle develops between PROLIFERATION INDUCED BY proliferation and cell death process. Three CATECHOLAMINES. types of cell death have been reported: Pachecho N.; Portales J.; Aránguiz P. apoptosis, phagocytosis and necrosis. A Escuela de Química y Farmacia, Facultad fourth type of cell death, necroptosis, has de Medicina, Universidad Andres Bello, been recently associated to ovarian function. 2520000 Viña del Mar, Chile. However, the physiological relevance of necroptosis or its involvement in follicular Activation of the adrenergic system is development it is not yet well understood. In commonly associated with cardiac fibrosis the present work we will use pharmacological and remodeling, and cardiac fibroblasts are tools (allosteric inhibitor necrostatin-1), key players in these processes. Interestingly, to study the role that Necroptosis would adrenergic stimulation activates both, cardiac have in follicular development and ovarian fibroblasts autophagy and cell proliferation, function in vivo. We used two groups of however, the underlying mechanisms have animals: Sham and NEC-1. Adults rats were not been elucidated. In the present study, we hemiovariectomized and implanted with a assessed the effects of adrenergic stimulation miniosmotic pump with NEC-1 (20 µM), for 28 on autophagy and cell proliferation in days, or remains without drug administration cultured adult rat cardiac fibroblasts, (sham). At the end of the procedure, rats which were treated with beta-adrenergic were euthanized and the ovaries and plasma agonists and antagonists. Autophagy were collected. The ovaries were fixed for was determined by electron microscopy, morphometric analyses. Plasma levels of subcellular distribution and protein levels of steroid hormones were measured by EIA. LC3-II, and the signaling pathways involved We found that necroptosis inhibition did not in its activation after stimulation with affect the number of secondary follicles, but catecholamines were evaluated by western increased total antral follicles by accumulating blot. Our results suggest that AMPK plays atretic antral follicles. The number of type III a key role in the induction of autophagy, precystic follicles was increased while the cyst through the inhibition of mTOR activity. number did not change. Corpus luteum didn’t Indeed, the AMPK pharmacological inhibitor, change in number but decreased the new compound c, prevents the autophagy induced (bigger size) CL. An increase in testosterone by adrenergic agonists, acting downstream of plasma levels was found. In conclusion, NEC- AKT in the beta2-adrenergic receptor/AKT/ 1 treatment by blocking necroptosis in vivo, mTOR pathway. AMPK activation was also favored cyst formation and the permanence necessary for ERK1/2 phosphorylation and of old corpus luteum thus necroptosis could cell proliferation. In addition, the increase be involved in luteolysis and in the transition in autophagy correlates with intracellular to follicular cyst in the ovary. In vivo models collagen degradation. In summary, here help to describe new pharmacological targets we show that beta2-adrenergic stimulation to regulate follicular development and hence activates AMPK and this protein governs fertility. both processes, autophagy and proliferation in cardiac fibroblasts, therefore, a

80 3. TLR4, BUT NEITHER DECTIN-1 recognition to activate APCs. TLR4 role was NOR DECTIN-2, PARTICIPATES confirmed through a decrease in IL-12p40 IN THE MOLLUSK HEMOCYANIN- and IL-6 secretion induced by FLH when a INDUCED PROINFLAMMATORY TLR4 blocking antibody was used; a reduction EFFECTS IN ANTIGEN-PRESENTING was also observed in DCs from C3H/HeJ CELLS FROM MAMMALS. mice. Additionally, IL-6 secretion induced by JM Jiménez 1, ML Salazar 1, S Arancibia 1, J FLH was abolished in macrophages deficient Villar 1, F Salazar 1,2, GD Brown 2, EC Lavelle for TLR4. We further showed that KLH and 3, L Martínez-Pomares 4, J Ortiz-Quintero 5, FLH induced ERK1/2 phosphorylation. Our S Lavandero 5, A Manubens 6 and MI Becker data showed the involvement of TLR4 in 1,6. the hemocyaninmediated proinflammatory response in APCs, which could cooperate with 1Fundación Ciencia y Tecnología Para el MR in innate immune recognition of these Desarrollo (FUCITED), Santiago, Chile; glycoproteins. 2Medical Research Council Centre for Medical Mycology, University of Aberdeen, 4. DISCOVERY OF NOVEL TASK-3 Aberdeen, United Kingdom; 3Trinity POTASSIUM CHANNEL BLOCKERS. Biomedical Sciences Institute, Trinity Ramírez D.; Zuñiga L.; Kiper A.; Rinné S.; College Dublin, Dublin, Ireland; 4School Decher N.; Caballero J.; González W. and of Life Sciences, University of Nottingham, Caballero J. Nottingham, United Kingdom; 5Facultad de Computational Biophysics, Bioinformatics Ciencias Químicas y Farmacéuticas, Facultad & Drug Design Lab, Instituto de Ciencias de Medicina, Universidad de Chile, Santiago, Biomédicas - Universidad Autónoma de Chile; 6Biosonda Corporation, Santiago, Chile. Chile TASK-3 is a two-pore domain potassium Mollusk hemocyanins have biomedical (K2P) channel highly expressed in uses as carriers/adjuvants and nonspecific hippocampus, cerebellum, and cortex. immunostimulants with beneficial clinical TASK-3 has been identified as an oncogenic outcomes. Hemocyanins have a multivalent potassium channel and it is overexpressed in nature as highly mannosylated antigens. different cancer types. For this reason, the We have shown that hemocyanins are development of new TASK-3 blockers could internalized by antigenpresenting cells (APCs) influence the pharmacological treatment of through receptor-mediated endocytosis by cancer and several neurological conditions. innate immune receptors, such as mannose In the present work, we search for novel receptor (MR). However, the contribution TASK-3 blockers by using a virtual screening of other pattern recognition receptors to (VS) protocol that includes pharmacophore the proinflammatory signaling pathway modeling, molecular docking, and free triggered by hemocyanins is unknown. Thus, energy calculations (MM/GBSA). With this we studied the roles of Dectin-1, Dectin-2, protocol, 19 potential TASK-3 blockers were and Toll-like receptor 4 (TLR4) in the identified. These molecules were tested in hemocyanin activation of murine APCs, both TASK-3 using patch clamp, and one blocker in dendritic cells (DCs) and macrophages, (DR16) was identified with an IC50 = 56.8 ± using hemocyanins from Megathura 3.9 μM. Using DR16 as scaffold we designed crenulata (KLH), Concholepas concholepas DR16.1, a novel TASK-3 inhibitor with an (CCH) and Fissurella latimarginata (FLH). IC50 = 14.2 ± 3.4 μM. Our finding takes on The results showed that these hemocyanins greater relevance considering that not many bound to chimeric Dectin-1 and Dectin-2 inhibitory TASK-3 modulators have been receptors in vitro. However, hemocyanin- reported in the scientific literature until today. induced proinflammatory effects in APCs These two novels TASK-3 channel inhibitors from Dectin-1 knock-out (KO) and Dectin-2 (DR16 and DR16.1) are the first found using a KO mice were independent of both receptors. pharmacophore-based virtual screening and Moreover, the phosphorylation of Syk rational drug design protocol. kinase was not detected after hemocyanin stimulation. On the other hand, we confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin

81 5. IMMUNOLOGICAL BASIS OF 6. ANTI-STEROIDOGENIC EFFECT OF AUTISM: COGNITIVE EFFECTS OF THE RFRP-3 NEUROPEPTIDE AND ITS AUTOANTIBODIES FROM AUTISTIC PARTICIPATION IN THE FOLLICULAR CHILDREN IN MEMORY AND DYNAMICS IN THE RAT. LEARNING PROCESSES. Squicciarini V.1, Bentley GE.2 y Lara HE.1; Sandoval R.1, Rossi G.1,; Cobarrubias A.1; 1, Centre for Neurobiochemical Studies for Arancibia M.1; Araya G.1; Uribe F.1; Gámiz Endocrine Diseases, Facultad de Ciencias F.2; Pancetti F.1; Gonzalez-Gronow M.1 Químicas y Farmacéuticas, Universidad 1, Environmental Neurotoxicology laboratory, de Chile. 2, Laboratorio de Reproductive Department of biomedical Sciences, Faculty Neuroendocrinology, UC Berkeley, California. of Medicine, Universidad Católica del Norte: 2, Universidad de Granada. Ovarian function is highly regulated, either by hormones, autonomic nerves and paracrine Autism spectrum disorders (ASD) involve signals produced by the same ovarian cells. a range of complex neurodevelopmental Changes of these signals modified the normal disorders, characterized by social functioning of the ovary. Gonadotrophin impairments, communication difficulties, inhibitor hormone (GnIH) is a neuropeptide and restricted, repetitive and stereotyped that block the GnRH secretion at hypothalamic patterns of behavior. ASD exerts a significant level and therefore the gonadotropic axis physiological, emotional and financial regulating the ovary. The recently described burden on the families of the individual receptor (GPR147 or NPFF1, homologous and society as a whole. Recently, beside the in mammals) and RFRP-3 (mammalian knowledge about genetic factors involved in homologous peptide of GnIH) in the ovary this pathology, there is new evidence related open the possibility to suggest that the local to immunological causes of ASD. Therefore, presence of the peptide and its receptor it is of outmost importance to elucidate the could participates as regulator of ovarian molecular and physiological mechanisms function. We studied whether RFRP-3 and of ASD pathology. Taking this into account, NPFF1 receptor are present in the rat’s ovary we hypothesized that ASD autoantibodies and the local effect of this neuropeptide on generates autoimmune-related cognitive hormone production and follicular dynamics. impairment characteristic of ASD pathology. We determine the presence of RFRP-3 in To achieve this aim, we used ex vivo the rat’s ovary. RFRP-3 was mainly in the experiments using hippocampal slices and a granulose cells of antral follicles and corpora rat model where mothers were injected with lutea. Then, we studied the effect of 10 ng/mL ASD autoantibodies during pregnancy and/or RFRP-3 on the production of ovarian steroids breast milk period and the breeding was tested ex vivo. RFRP-3 inhibited the hCG-induced after that period using learning and memory ovarian progesterone and testosterone test together with electrophysiological secretion. In order to know if the chronic and immunohistochemical studies. Our presence peptide in the ovary modified the results have shown that normal young rat follicular development and its function, we hippocampal slices incubated with purified designed a local chronic treatment in vivo IgA autoantibodies from ASD patients and with RFRP-3. After 4 weeks of treatment breeding rats from pregnant mothers injected there was a decrease in serum testosterone with the same antibodies, impairs LTP as and an increase in size and number of corpora well as disrupts learning and memory. We lutea suggesting the appearance of new also found that both LTP and learning and corpora lutea and hence increased ovulation. memory were significantly impaired in female No changes appeared in secondary, antral, but not male breeding rats and this alteration cyst or atretic follicles. Data indicate a local are correlated with the presence of ASD effect of RFRP-3 that positively affect ovarian autoantibodies in hippocampal slices. These steroidogenesis and follicular dynamics. This results demonstrate that ASD autoantibodies study opens new pharmacological targets, cross the transplacental barrier and also are such as neuropeptides, to treat disorders in ingested through breeding milk, crossing ovarian function. both intestinal and blood-brain barrier and impairs learning and memory in a sex- preference fashion. The pharmacological implicances of this research involve new mechanisms and possible therapeutical targets for ASD pathology

82 ORAL COMMUNICATIONS 2. D-LACTATE INDUCES NEUTROPHIL EXTRACELLULAR 1. THE N-ACETYLCYSTEINE- TRAPS (NET) RELEASE BY INDUCED REDUCTION OF CHRONIC DISTURBING CELLULAR ALCOHOL COMSUMPTION IS METABOLISM. ASSOCIATED TO THE ACTIVATION Quiroga J.1,2; Manosalva C.3; Alarcón P.1,2; OF THE NRF2/ARE PATHWAY IN Teuber S. 1,2; Ramírez1 F.; Carretta1 M.D.; HIG-ALCOHOL-DRINKING UCHB Hidalgo A.1, Conejeros I4; Hermosilla C4; RATS. Burgos R.A. 1,2 Alvarado-Rosas, R1, Morales P1,2, Farfán N1, 1. Laboratory of Inflammation Pharmacology, Herrera-Marschitz M1, Israel Y1, Quintanilla Faculty of Veterinary Sciences, Institute ME1 of Pharmacology and Morphophysiology, 1 Molecular & Clinical Pharmacology Program, Universidad Austral de Chile, Valdivia, ICBM, 2Department of Neuroscience, Faculty Chile. 2. Laboratory of Immunometabolism, of Medicine University of Chile. Faculty of Veterinary Sciences, Institute of Pharmacology and Morphophysiology, Current evidence suggests that Universidad Austral de Chile, Valdivia, Chile. neuroinflammation and oxidative stress are 3 Faculty of Sciences, Institute of Pharmacy, associated to chronic alcohol consumption Universidad Austral de Chile, Valdivia, and relapse, suggesting that the modulation Chile 4. Institute of Parasitology, Faculty of of oxidative stress induced by chronic alcohol Veterinary Medicine, Justus Liebig University drinking can be a therapeutic target in Giessen, 35392, Giessen, Germany. alcoholism. There is evidence that oxidative stress activates the Nrf2 (Nuclear factor D-lactate is produced during acute ruminal erythroid 2-like), that translocates into acidosis (ARA), a well-known fermentative nucleus where it promotes the transcription of disorder in cattle. Recently, we demonstrated antioxidant genes containing the Antioxidant- that heifers with ARA show aseptic Response-Element (ARE) including neutrophilic synovitis, characterized by the hemoxigenase 1 (HO-1) and NAD(P)H presence of D-lactate, abundant neutrophils, dehydrogenase quinone 1 (NQO1). Previously NET, and metabolic disturbances in synovial we have demonstrated that N-acetylcysteine fluid. It has been described that D-lactate (NAC), a cysteine precursor, with antioxidant entry is required to induce NET-release. action, inhibits alcohol consumption, Since D-lactate is slowly metabolized by neuroinflammation and alcohol-induced mammalian cells, we hypothesized that oxidative stress in chronic drinking rats (UChB). D-lactate induces metabolic disturbances However, the mechanism of the antioxidant in neutrophils, and so could induce NET- action of N-acetylcysteine it is not clear. The release. Blood neutrophils isolated from present study determinates whether the NAC- 5 healthy heifers were treated with 5 mM reduction of chronic alcohol consumption is D-lactate in vitro. First, we performed a associated to the activation of the Nrf2 /ARE GC-MS untargeted metabolomic analysis. pathway in high-alcohol-drinking rats. Chronic D-lactate altered galactose metabolism, alcohol drinking (61days) female UChB rats starch and sucrose metabolism, nucleotide were administered for nine consecutive days sugars metabolism and glycolysis. Using either (i) NAC (100mg/kg/day, per os); (ii) JC-1 probe we observed by flow cytometry NAC + all-trans-retinoic acid (ATRA, a Nrf2 that D-lactate reduced Δψm. In addition, pharmacological inhibitor (10 mg/kg/day D-lactate favored the glycogen degradation, ip); (iii) ATRA+ saline); (iv) Saline. After and increased glucose-1-P and glucose-6-P determining the rates of alcohol consumption, intracellular levels. Also, D-lactate increased all groups were euthanized for hippocampal the AKT and GSK-3β phosphorylation. The histological and Western blot analyses. It inhibition of theses pathways with LY294002 was found that (i) N-acetylcysteine inhibits and CHIR99021, respectively, interfered chronic alcohol intake (ii) N-acetylcysteine the decrease of glycogen and NET release. induced Nrf2 nuclear translocation (ii) Our results suggest that D-lactate induces N-acetylcysteine-induced inhibition of chronic NET release by disturbing cellular metabolic alcohol intake was prevented by ATRA a Nrf2 pathways, involved in glycogen degradation. pharmacological inhibitor. In conclusion these results support the idea that Nrf2 activation is the mechanism by which NAC inhibited chronic alcohol consumption and relapse.

83 3. CHANGES PRODUCED BY production of lactic acid and syneresis over PREBIOTIC FIBERS IN THE time (1 to 4 weeks). Conclusions. The results SURVIVAL OF LACTOBACILLUS showed that although there was a decrease in CASEI, SUBSP CASEI DURING THE the survival of L. casei in the two fibers under SHELF LIFE OF A NUTRACEUTICAL study (apple and potato), they preserved its DAIRY DRINK. probiotic properties (1x108 CFU / mL), while Gómez-Pliego R. 1; Espinosa-Raya J. 2; the physical and chemical changes observed Morán-Díaz J.R. 3, Guevara-Salazar J.A. over time (1-4 weeks) did not modify the 3; Rios-Guerra H. 4; Morales-Ríos E.I. 2; sensory properties of fermented dairy drinks. Quintana Zavala D 4. 1 Laboratorio de Microbiología Industrial, 4. SYNTHESIS, CHARACTERIZATION, Sección de Ciencias de la Salud, THEORETICAL STUDY AND IN VITRO Departamento de Ciencias Biológicas, 4 EVALUATION OF BETA-LACTAMIC Sección de Química Orgánica, Departamento COMPOUNDS AND IMINES WITH de Ciencias Químicas, Facultad de Estudios POTENTIAL ANTIBACTERIAL Superiores Cuautitlán, Universidad Nacional ACTIVITY. Autónoma de México, Av. 1 de Mayo s/n, Morán Díaz J.R. 1; Ávila Melo J.L. 2; Santa María las Torres, Cuautitlán Izcalli, Quintana Zavala D. 3; Gómez Pliego R. 4; 54740, Edo., México, México; 2 Farmacología Jiménez Vázquez H.A. 2; Guevara Salazar Conductual, Sección de Estudios de J.A. 5; Trujillo Ferrara J.G. 1 Posgrado e Investigación, Departamento 1. Laboratorio de Investigación de Bioquímica, de Farmacología. México, D.F., México; 3 Escuela Superior de Medicina, Instituto Departamento de Farmacología, Escuela Politécnico Nacional, 2. Departamento de Superior de Medicina, Instituto Politécnico Química Orgánica, Escuela Nacional de Nacional, Plan de San Luis y Díaz Mirón, S/N, Ciencias Biológicas, Instituto Politécnico 11340, México, D.F., México; 5 Laboratorio de Nacional, 3. Laboratorio de Química Orgánica, Química Orgánica, Centro de Investigación Centro de Investigación en Ciencia Aplicada en Ciencia y Tecnología Avanzada Unidad y Tecnología Avanzada, Instituto Politécnico Legaria, Instituto Politécnico Nacional, Nacional, 4. Laboratorio de Microbiología Legaria No. 694, 11500, México, D.F., México. Industrial L-502-Anexo, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Introduction. The accelerated current Autónoma de México, 5. Laboratorio de lifestyle has generated important changes Farmacología, Escuela Superior de Medicina, in the population’s diet at global level. Instituto Politécnico Nacional. There are reports that indicate that the use of prebiotics and probiotics are important One of the most serious problems worldwide factors in the modulation and restoration of is the resistance of the main pathogenic gastrointestinal tract microbiota (MTGI), bacteria to the antibiotics used today. The since by decreasing intestinal permeability evolution of the resistance seen in the light and gastrointestinal inflammation modify of the Darwinian and Lamarckian theories of homeostasis of immunity related to glucose adaptation gives rise to the understanding of and lipid absorption, decreasing factors the causes of resistance and if the causes are correlated with diseases such as obesity known, solutions can be proposed, otherwise, and overweight (Fukuda et al., 2014). what will be achieved is to amplify the The formulation and development of new problem to such an extent that hospitals will nutraceutical foods fermented with probiotic become the repertoire of microbial infections microorganisms and added prebiotic fibers resistant to any chemotherapeutic treatment. such as apple and potato could be a viable With the current biochemical knowledge, a alternative in the treatment of emerging and rational design of antibiotics with in silico high impact diseases such as those mentioned experiments of chloromonobactams was above (Mishra et al., 2019; Gibson et al., 2017). proposed from imines p-substituted with Objective. In the present work, the changes stereochemistry (E), which demonstrated produced by prebiotic apple and potato that both sets of molecules comply with fibers on the survival of Lactobacillus casei the Lipinski rule of 5, which offers a viable subsp. casei were evaluated, as well as the pharmacokinetics towards the organism. The physicochemical changes produced during synthesis of chloromonobactams was carried the shelf life of nutraceutical dairy drinks. out in two phases. The first is the synthesis of Results. Dairy drinks fermented showed p-substituted imines with (E) configuration; changes in the survival of L casei, and in the the second is a [2+2] Staudinger physical-chemical properties (viscosity, pH, cycloaddition to obtain chloromonobactams.

84 The characterization of all the synthesized extracellular traps (NETs) release, which was compounds was performed by physical reduced by 2-DG and CCCP, but increased tests (determination of Rf, melting point, by oligomycin. These results suggest that and solubility tests), and spectroscopy (UV- PAF triggers respiratory burst and NETs visible and IR spectrophotometry, 1H and release through glycolysis and mitochondrial 13C NMR spectroscopy, and high-resolution metabolism-dependent mechanisms. mass spectrometry). The evaluation of the in vitro antibacterial activity was carried out by Multitarget drug design for the treatment of the disk diffusion method.The study strains Alzheimer’s Disease. were S. aureus sensitive to dicloxacillin, E.coli and P. aeruginosa sensitive to aztreonam. The 1,2Rafael León, 1,2Patrycja Michalska, results obtained so far show that the imines 1,2Pablo Duarte, 1,2Paloma Mayo, 1,2Izaskun have antibacterial activity against the bacteria Buendia, 1,2Enrique Crisman y 1,2Manuela under study, with the p-iodo imine and the G. López beta-lactam without substituents showing an Instituto de Investigación La Princesa. activity similar to aztreonam on P. aeruginosa. Hospital Universitario La Princesa. Madrid, España. 5. ROLE OF MITOCHONDRIAL Instituto Teófilo Hernando del Medicamento. METABOLISM IN OXIDATIVE Departamento de Farmacología. Facultad de RESPONSE AND NETS RELEASE Medicina Universidad Autónoma de Madrid. INDUCED BY PAF IN BOVINE España. NEUTROPHILS. Quiroga J. 1 ; Alarcón P. 1; Manosalva C. 2; Neurodegenerative diseases (NDDs) are Teuber S. 1; Carretta M. D. 1; Hidalgo M. A. 1; currently considered a worldwide pandemia Burgos R. A. 1 with a prevalence of about 47 million people. 1, Laboratory of Inflammation Pharmacology, It is estimated that, in 2050, two billion Institute of Pharmacology and people will be over 60 years old, thus the Morphophysiology, Faculty of Veterinary number of people affected is expected to Sciences, Universidad Austral de Chile; 2, triple. Therefore, the search for effective Faculty of Sciences, Institute of Pharmacy, drugs capable of controlling neuronal cell Universidad Austral de Chile. death is one of the great challenges of this century. Neutrophils (PMN) constitute the main line AD is associated with several neuronal of cellular defense in the innate immune abnormalities in energy metabolism such response. Since they obtain energy primarily mitochondrial dysfunction, a decline through glycolysis, it is assumed that they do in glucose uptake, dysfunction in Ca2+ not produce ATP by oxidative phosphorylation. homeostasis. It has been shown that However, mitochondrion of PMN maintains a oxidative damage occurs before the onset transmembrane potential, which is normally of significant Aβ plaque formation. For associated with respiratory chain and instance, the free radical theory of ageing oxidative phosphorylation for ATP synthesis. implies progressive ROS cell damage with PMN were isolated from healthy heifers and age, leading to enhanced mitochondrial DNA stimulated in vitro with platelet activating mutations, futile mitochondrial Ca2+ cycling factor (PAF), a key biochemical mediator in with excess ATP consumption and ensuing various inflammatory conditions. Incubation mitochondrial dysfunction. On the other with PAF 100 nM increased mitochondrial hand, it is now increasingly recognized that transmembrane potential and mitochondrial inflammation also strongly contributes to reactive oxygen species (mtROS) production. extensive oxidative stress found in AD brains. While mtROS levels were reduced using We therefore hypothesize that mitochondrial rotenone 10 uM (mitochondrial complex I dysfunction could be the potential link between inhibitor), these were increased by oligomycin neuroinflammation and neurodegeneration. 10 uM (mitochondrial complex V inhibitor). Another common characteristic is the PAF 100 nM also stimulated respiratory burst interconnection between these pathways in PMN, which was reduced not only with that causes feedback pathological loops 2-deoxy-D-glucose 2 mM (2-DG, glycolysis that accelerates the advance of the disease. inhibitor), but also with rotenone 10 uM, Therefore, their therapeutic approach must oligomycin 10 uM and carbonylcyanide- be directed to several pathological nodes, as 3-chlorophenylhydrazone 5 nM (CCCP, the design of multitarget drugs capable of oxidative phosphorylation uncoupler). stopping different pathological pathways at Finally, PAF 1 uM induced neutrophils the same time.

85 In this sense, the intrinsic cellular defense Fat Diet (HF) and High Fat Diet / Calafate pathway, the Nrf2-ARE pathway, has been (HFC). The mice were subjected to indirect proposed as a therapeutic alternative for the calorimetry. Post-euthanasia was evaluated: development of effective drugs. Therefore, we gene and protein expression of UCP-1, PGC- are developing new multitarget compounds 1alpha, OPA1 (fusion), DRP1 (fission) (qPCR, that combine the Nrf2 induction activity with western blot or immunofluorescence), other specific targets capable of reducing mitochondrial Oxygen Consumption Rate oxidative stress, neuroinflammation and (OCR) (XF24 Seahorse), HSP70 (amount of the formation of protein aggregates, besides mitochondria) and mitochondrial activity activating neuronal survival pathways that (with MTO). The consumption of calafate could be of potential therapeutic relevance extract produced an increase in energy to afford neuroprotection in Alzheimer’s expenditure and a decrease in respiratory disease. quotient. The treatment presented differences Acknowledgments: We thank IS Carlos at the level of mitochondrial function, with an III (Ref: PI17/01700), Fundación la Caixa increase in thermogenesis (UCP-1) a recovery (CaixaImpulse CI17-00048), Fundación of OCR, and a significant effect on the MTO FIPSE (FIPSE-12-00001344), BAYER AG / HSP70 ratio. It did not substantially (T4D-2015-03-1282) and Comunidad de modify the mitochondrial morphology. The Madrid y Fondos estructurales de la UE ref: consumption of a calafate extract rich in S2017/BDM-3827 polyphenols increases energy expenditure and improves mitochondrial function in obese 6. THE CONSUMPTION OF A mice. Additional studies on mitochondrial CALAFATE EXTRACT MODULATES dynamics are required to complement these THE ENERGY EXPENDITURE, hypothesis. FUNCTION AND MITOCHONDRIAL DYNAMICS OF BROWN ADIPOSE 7. FROM HOMO SAPIENS TO HOMO TISSUE OF OBESE MICE. TECHNOLOGICUS, BIOETHICAL Ramírez L.A.1; Quezada J.2; Elorza A.2; Cruz CHALLENGE OF TRANSHUMANISM. G.3, R. Bravo-Sagua R.4; Garcia-Diaz D.1 Rifo F. L. 1, Laboratorio de Bioquímica, Departamento Instituto Superior de Bioética, Facultad de de Nutrición, Facultad de Medicina, Medicina, UCSC Universidad de Chile; 2, Bioenergética Experimental, Departamento de Biología, Transhumanism, an empiricist thesis whose Facultad de Ciencias Biológicas; 3, Laboratorio anthropology dispenses with metaphysics. de Alteraciones Reproductivas y Metabólicas, It explains the human dynamism from Instituto de Fisiología, Facultad de Ciencias, functionalist neurobiologicism, which Universidad de Valparaíso; 4, Laboratorio de underlines the human to the functioning, Nutrición Básica y Epidemiología Genética, of its neural connections, and that seeks Instituto de Nutrición y Tecnología de los its sustenance in scientific perfection. In Alimentos, Universidad de Chile. the bioethical field it is founded is liberal utilitarianism. There are authors who Obesity is a public health problem of global argue that we are in the last stage of the concern. In its pathogenesis, the White development of homo sapiens, and in the era Adipose Tissue has a crucial role. There of homo technologicus, it has the possibility of is a mitochondrial dysfunction and lower continuing the evolution of the human species oxidative capacity in adipocytes of obese towards a superior, better and happier, using individuals, with modifications in their technology to its scope. Transhumanism morphology. In contrast, Brown Adipose raises many questions, among others. Has Tissue (BAT) has a thermogenic function neurobiological physicalism been proven? through UCP-1. A new approach proposes Who tells me that the more perfect I am to increase energy expenditure through diet. physically and psychically, that the more Our objective in this work was to evaluate the capacities I have, I will be happier? What is effect of a calafate extract rich in polyphenols happiness? What does it mean to be better on mitochondrial energy, function and or more perfect, who determines it? We try dynamics (fusion / fission) of obese mice. The a response in the moral and ontological field. analyzes were performed on adult C57BL / Then there are issues of a practical nature 6J male mice, which were subdivided (n = 10 when implementing the transhumanist plan: each) into 4 dietary regimens / treatments: embryonic selection and eugenic elimination Control Diet (C), Control / Calafate (extract: of embryos and fetuses with defects, problems 50 mg total polyphenols / kg weight; CC), High derived from nanotechnology applied to the

86 brain and neuroethics, cryopreservation 9. DEVELOPMENT OF A problems, use of drugs that change RECOMBINANT VACCINE personality, resource distribution problems, CANDIDATE AGAINST HANTAVIRUS. etc. Starck-Méndez M.F. 1,2; Neira P.J. 2, Varas This study aims to address the ethical and N.M.J. 1,2; Toledo J.R. 1,3; Acosta J. 1,3; anthropological challenge that underlies Sanchez O. 1,2 transhumanism. 1,Center for Biotechnology and Biomedicine Spa., Concepción, Chile. 2, Department of 8. SYNTHESIS AND EVALUATION Pharmacology, School of Biological Sciences, OF INDOLYL-BENZAMIDO- Universidad de Concepción. 3, Department PIPERAZINES AS POTENTIAL of Physiopathology, School of Biological MULTI-TARGET-DIRECTED LIGANDS Sciences, Universidad de Concepción. IN ALZHEIMER´S DISEASE. Rodriguez-Lavado, J. 1, Mallea, M. 1, Andes virus is the main causative agent of Gallardo-Garrido, C. 2, Osorio, R. 1, Chung, H. Hantavirus cardiopulmonary syndrome 2, Pessoa- Mahana, C. 2, Iturriaga-Vásquez, (HCPS) in South America. There are currently P. 3, Saitz-Barria, C. 1, Pessoa-Mahana, H. 1 no vaccines or treatments against Andes 1, Facultad de Ciencias Químicas y virus. However, there are several evidences Farmacéuticas, Universidad de Chile. suggesting that antibodies against Andes virus envelope glycoproteins may be enough Alzheimer’s disease (AD) is a chronic, to confer full protection against HCPS. The progressive and fatal neurodegenerative main goal of the present work was to develop disorder affecting cognition, behavior, and a vaccine candidate against Hantavirus, function, being one of the most common based on the surface glycoproteins Gn and causes of mental deterioration in elderly Gc. With this purpose, the sequence encoding people: Around 50-60% of the overall the extracellular domains of both antigens dementias correspond to AD. World Health was introduced into the methylotrophic Organization estimates that about 46.8 yeast Pichia pastoris. After induction million of people worldwide currently suffer with methanol, the recombinant antigens from AD, thus becoming a major public health accumulated intracellularly as insoluble concern as the world’s population ages. (World aggregates. After cell disruption, the Alzheimer’s report, 2018). Development of recombinant antigens were solubilized and Multi-Target Directed Ligands (MTDLs) has purified by metal-ion affinity chromatography. emerged as a promising approach for targeting The immunogenicity of both antigens was complex etiology of Alzheimer’s disease determined in immunization assays in both (AD). Following these approach, and given mice and Syrian hamsters. In both species it our interest in the search and development was possible to detect the presence of specific of novel drugs displaying affinity acting as antibodies against Gn and Gc. Part of these promiscuous ligands. In the present work, antibodies showed neutralizing activity. The a novel series of indolylpropylpiperazinyl results obtained to date suggest that the Gn piperazinebenzamides were synthesized and and Gc antigens from Andes virus, produced biologically evaluated as multifunctional in P. pastoris, have the potential to become ligands in the following targets: the first commercial vaccine against HCPS. acetylcholinesterase, SERT, and beta- amyloid peptides The synthesis involved 10. ADHERENCE TO connection between Piperazine benzamides ANTIHYPERTENSIVE with N-Boc substituted piperazine derivative. PHARMACOLOGICAL TREATMENT IN Boc cleavage and further coupling with ELDERLY PEOPLE FROM HUALPEN indolylpropyl tosylates was achieved in two SUBMITTED TO A TRANSMEDIAL step-one pot reaction, obtaining the final PSYCHOEDUCATIONAL PROGRAM. compounds with good to excellents overall yields. Finally, the obtained compounds Sepúlveda, M.J.1*; Pinto, R.1; Iturra, R.1; were evaluated in its capabilities for AChE Müller, H.2; Chamblás, I.3; Victoriano, M.4; inhibition, SERT- affinity, β-amyloid Casanova, M.P.5; Guevara, P.6; Aguilera, R.7; inhibition, and cell toxicity (viability) with Cid, P.8 very promising results. 1 Departamento de Farmacología, Facultad de Ciencias Biológicas. 2 Departamento de Medicina Interna, Facultad de Medicina. 3 Departamento de Trabajo Social, Facultad

87 de Ciencias Sociales. 4 Departamento POSTERS de Nutrición, Facultad de Farmacia. 5 Departamento de Estadística, Facultad 1. Poster retirado” de Ciencias Físicas y Matemáticas. 6 Departamento de Ingeniería Eléctrica, 2. SEARCH FOR ANALOGS OF M554 Facultad de Ingeniería. 7 Departamento de AND M890 FOR INTERACTION Economía, Facultad de Ciencias Económicas WITH THE GΒΓ DIMER IN REGIONS y Administrativas. 8 Departamento de INVOLVED IN THE REGULATION OF Fundamentos de Enfermería y Salud Pública, THE GLYCINE RECEPTOR. Facultad de Enfermería. Universidad de Argel A. Y.; Guzmán G. L.; Jiménez C. V. Concepción, Concepción, Chile. Molecular neurobiology laboratory, physiology department, biological Sciences In Chile, the number of elderly people Faculty, Universidad de Concepción. has steadily increased, with 20% of the population projected by 2025. This change Ethanol is the drug with highest consumption associated with a sedentary lifestyle is levels, with effects at different levels of the linked with an increased prevalence in Central Nervous System. Accute consumption chronic pathologies such as Hypertension. at high levels can induce coma and death. The national prevalence is 27.6%. The lack This molecule modulats the activity of Glycine of pharmacological adherence constitutes receptor (GlyR), a ligand gated ion channel one of the main problems in the control that belong to the cys-loop subfamily of ion of the disease, considering that only channels. Recently it has been identified that approximately 50% of the patients adhere Gbg protein as a modulator of the channel properly. Given this problem, a Transmedial interacting with the cytoplasmic domain Psychoeducational Program (PST) was and potentiating the activity of the channel. developed to support primary care treatment, With determined structure of Gbg and GlyR focused on knowledge of the disease, as well cytoplasmic domain, it has been able to as promoting the benefits of pharmacological identify chemical entities to inhibit the etanol treatment and a healthy lifestyle. effects. Initially, peptides were designed and The PST was evaluated in two CESFAM in then peptidomimetic small molecules were the commune of Hualpén with three levels developed, like M554 and M890. These of intervention: Group A (n = 104) through a molecules interact with Gbg and inhibit mobile application “AFAM-Health”, Group B etanol effects in vitro and in vivo. After that (n = 97) using video capsules and Group C (n new molecules were designed applying = 98) as control. bioisosteric changes in the original molecules The average age of the 299 elderly was 72 M554 and M890. Through bioinformatic ± 7.6 years, 83.7% have as their source of technics like docking , molecular dynamics income the retirement salary, 88.6% lives, and free energy calculations (MM-GBSA), accompanied, 74.3% attend their health it has been identified the derivatives checks alone and 68.6% of them walk. (R,S)-M554_3, (S)-M554_13, (R)-M554_13, After one year of intervention, group A was M890_4 y M890_5 which interact in the Gbg significantly more adherents (Morisky-Green hotspot surface with conserved aminoacids. test) over time with values of 52, 73, 64 and Finally, cytotoxicity assays were performed 64% of adherents at the beginning, third, in HEK cells determining that the molecules sixth and twelfth month, respectively. Group were not toxic for cells. A had 4.5 ± 1.8 medications / day, 43.4% corresponded to antihypertensives, Losartan 3. BIOGUIDED ISOLATION OF is the main one. SECONDARY METABOLITES From these results, it is determined that older PRESENT IN THE MEDICINAL adults who use the “AFAM-Health” App as a SPECIES CALDCLUVIA PANICULATA support increase pharmacological adherence (CUNONIACEAE) WITH INHIBITORY unlike those who only receive the traditional ACTIVITY IN VITRO ON THE ENZYME primary care treatment. Α-GLUCOSIDASE. Astudillo A. 1; Céspedes C. 3; Alvear M. 4; Massri M. 4; Iturriaga P. 1,3; Schalchi H. 1; Hormazábal E. 1,2. 1, Centro de Excelencia en Investigación Biotecnológica Aplicada al Medio Ambiente, Universidad de La Frontera. 2, Laboratorio Química Ecológica, Departamento Ciencias

88 Químicas y Recursos Naturales, Universidad 4. DETECTION AND IDENTIFICATION de La Frontera. Temuco. 3, Laboratorio OF ANTIBACTERIAL COMPOUNDS de Farmacoquímica y Síntesis Orgánica, IN LIQUID FERMENTATIONS OF Departamento Ciencias Químicas y Recursos FUNGUS STEREUM SP. BY HPTLC- Naturales, Universidad de La Frontera. BIOASSAY-MS. Temuco. 4, Laboratorio Bioquímica de J. Avendaño-Godoy 1, 2; P. Aqueveque; M. Suelos, Departamento Ciencias Químicas Aranda 2; K. Henríquez-Aedo 1. y Recursos Naturales, Universidad de La 1 Laboratory of Biotechnology and Genetics Frontera. Temuco. of Food. Department of Food Science and Technology, Faculty of Pharmacy, University Diabetes mellitus (DM) is a metabolic of Concepcion, Barrio Universitario s/n, disease characterized by an increase in Concepcion, Chile; 2 Laboratory of Advanced blood sugar levels. According to the World Research on Foods and Drugs. Department Health Organization, 422 million adults of Food Science and Technology, Faculty worldwide had DM by 2014. There are of Pharmacy, University of Concepcion, studies based on ethnobotanical knowledge Barrio Universitario s/n, Concepcion, Chile; that support the use of medicinal plants with 3 Laboratory of Microbiology and Applied hypoglycemic activity, ascribing their activity Mycology. Department of Agroindustries, to the presence of phenolic compounds. Faculty of Agricultural Engineering, Mapuche medicine suggests the consumption University of Concepción, Campus Chillan, of “Tiaca” (Caldcluvia paniculata) as a Chile. hypoglycemic treatment. The objective of the research was to evaluate in vitro the Basidiomycetes belonging to higher fungi, inhibitory activity of secondary metabolites offer an exciting field to obtain new structures present in C. paniculata on a-glucosidase. For with high potential for medical applications. this purpose, the plant material was defatted Higher fungi have an important advantage as and macerated in a hydroalcoholic solution producers of bioactive secondary metabolites: for 7 days. The hydroalcoholic extracts were they release them to liquid media. Then, dried, resuspended (MeOH:H2O - 70:30) the objective of this work was to detect and and partitioned by liquid extraction, with identify compounds with antibacterial activity solvents of increasing polarity. The activity of in liquid fermentations of fungus Stereum sp. the partitions was determined by inhibition Pure mycelial cultures were produced from on a-glucosidase: Aqueous solutions of impressions of spores of fruiting bodies, the dry extract were prepared at different which were then cultured in YMG medium concentrations (1-100 ug/mL), using (glucose, malt extract, yeast extract and agar) p-nitrophenyl-(1,4)-a-D-glucopyranoside as a previously sterilized. For liquid fermentation, substrate and acarbose as positive inhibition the following was performed: small sections control. The positive control of inhibition (15-10) of 5 mm diameter plug were cut on the enzyme (acarbose) presented an under sterile conditions and transferred to IC50 of 1288 ug/mL. Ethyl acetate partition an Erlenmeyer flask containing liquid YMG presented the lowest IC50, reaching values medium. The flasks were incubated at 20-22 of 13.6 and 14.5 ug/mL for leaf and stem °C on a Shaker orbital shaker with constant respectively. The most active partition was shaking. The cultures were stopped when fractionated by column chromatography abundant mycelia were observed, the glucose using silica gel and eluted with solvents of source was emptied, and the pH was about increasing polarity. The 15 groups obtained 7. The liquid culture was filtered to separate were evaluated for their inhibitory capacity the broth and the mycelium. Bioactive on a-glucosidase. Group G. 9 presented better compounds were extracted with ethyl acetate inhibition with an IC50 of 20.2 ug/mL. The from culture media. The total extract was chromatographic profiles of leaves and stems concentrated to dryness in a rotary evaporator were analyzed by HPLC, observing similar (45 °C), weighed and stored at 4 °C. The profiles and the presumptive presence of extract dissolved in methanol was seeded on phenolic compounds. The results obtained HPTLC plates silica gel 60 F254. Separation are conclusive regarding the hypoglycemic was performed using the mixture of toluene- property of C. paniculata. ethyl acetate (3.15 : 1.85 v/v) as a mobile phase. The extract was seeded in triplicate by dividing the HPLTC plate into three sections: the first section was used for the bioassay (direct bioautography), the second section for the chemical derivatization and

89 the third section for the mass spectrometry dextran 4.4kDa (FD4) was evaluated ex analysis (MS). After chromatography, the vivo for 120 and 180 min by everted gut sac first section was dried and a buffer solution technique. Results: Gut permeability to FD4 was atomized. The plate was immersed in is increased in animals exposed for 24h to 10 Bacillus subtilis bacteria suspension and ppm of NaAsO2 in comparison to controls. incubated at 37 °C for 2 hours. Subsequently, In addition, the metalloid concentration was the plate was atomized with a solution of higher in every studied tissue of exposed rats, methylthiazolidiphenyl-tetrazolium bromide in comparison to controls. In the brain, As was (MTT), incubated at 37 °C for 30 min and found in hypothalamus and cerebral cortex. finally dried completely on a heating plate This data suggest that As is able to cross the at 50 °C for 5 min. A zone of inhibition was BBB and increases gut permeability in the rat, detected on the HPTLC plate as a colorless an effect that might lead to alterations in BBB. zone/band on a purple background. Using the In conclusion, a toxic pollutant such as As third section of the plate (dried previously), might cause alterations in the brain-gut axis, this bioactive/inhibitory zone was directly effects which gives a novel approach in the eluted by means of the TLC-MS interface study of As toxicity. coupled to the electrospray ionization source (ESI) of a triple quadrupole mass 6. TWO CONSERVED ALPHA- spectrometer. Full scan mass spectra (m/z HELICES IN CORTICOTROPHIN 100-1000) were recorded in positive (ESI+) RELEASING FACTOR BINDING ionization mode. The bioactive compound PROTEIN CONTAINING A tentatively corresponds to Himanimide C. HYDROPHOBIC PATCH DETERMINES ITS SORTING TO THE 5. EFFECTS OF ARSENIC (AS) REGULATED SECRETORY PATHWAY. EXPOSURE ON BLOOD-BRAIN Bastías C.P.1; Blanco E.H.2; Lagos C.F.3; BARRIER AND COLONIC Gysling K.1 PERMEABILITY IN HEALTHY YOUNG 1: Depto de Biología Celular y Molecular, RATS. Facultad de Ciencias Biológicas, Pontificia Barrera-Bugueño, C.1,2; Heresmann, I.1; Universidad Catolica de Chile. 2: Universidad Quiroz, W.2; Julio-Pieper, M.1; Bravo, J.A.1. de Antofagasta. 3: Chemical Biology & 1Grupo de NeuroGastroBioquímica, Drug Discovery Lab, Escuela de Química y Laboratorio de Química Biológica. Instituto Farmacia, Facultad de Medicina y Ciencia, de Química, Facultad de Ciencias, Pontificia Universidad San Sebastián. Universidad Católica de Valparaíso, Valparaíso. Chile 2Laboratorio de Química Corticotrophin releasing factor binding Analítica Ambiental, Instituto de Química, protein, (CRF-BP) is a 37 kDa glycoprotein Facultad de Ciencias, Pontificia Universidad that binds CRF with high affinity. CRF- Católica de Valparaíso, Valparaíso, Chile. BP in the periphery controls CRF levels in plasma during pregnancy. In the central Arsenic (As) is a toxic metalloid, which has nervous system, CRF-BP facilitates the traffic become a health burden worldwide. Growing of CRFR2alfa acting as an escort protein. evidence indicates that As has harmful effects Previously, it has been shown that CRF-BP on the central nervous system (CNS), as this enters the regulated secretory pathway (VSR). metalloid crosses the blood-brain barrier However, the sorting signal(s) are presently (BBB). On the other hand, there is little evidence unknown. We decided to determine the sorting suggesting that loss of intestinal permeability signal(s) of CRF-BP to VSR. We used NPS @, might affect BBB permeability. The aim of an in silico secondary structure prediction this study is to evaluate if oral exposure to As tool and PEPWHEEL to draw predicted alpha affects intestinal and BBB permeability, as helixes and the in silico modeling of CRF-BP this pollutant might have an impact on what protein structure. Additionally, we did studies now is known as the brain-gut axis. Methods: of sorting of chimeras containing the putative Female Sprague–Dawley rats (PND35) where sorting signals in PC12 cells over-expressing given 10 ppm of NaAsO2 in the drinking water the selected chimeras. In silico analysis and for 24h (n=5), and compared to control rats modeling of CRF-BP protein structure showed (n=6). At 24h the following samples were the presence of three alpha-helix domains, collected: brain, colon, lung, stool and liver (50-74), (128-149), (229-251). The alpha- tissues. Each sample was lyophilized and then helixes domain (50-74) and (229-251) in microwave digested in order to determinate CRF-BP is highly conserved among different total As concentration by HPLC-HG-AFS. mammalian species and has a hydrophobic Additionally, colonic permeability to FITC- patch characteristic of other sorting domains

90 to the VSR. The results show that the alpha- Endocrinas, Facultad de Ciencias Químicas y helix domain (50-74)-CRFBP is capable Farmacéuticas, Universidad de Chile. of restore the sorting of a chimeric variant of proCART precursor, without its sorting The ovary is an endocrine organ which is domain to the VSR. Furthermore, the presence regulated by hormonal and neural signals. of the alpha-helix domain (50-74)-CRF-BP in It is well known that noradrenaline controls the chimeric variant of proCART allowed its ovarian steroidogenesis and folliculogenesis. secretion triggered by a depolarizing stimulus. Its source comes from sympathetic neurons Our results show that the preserved alpha- that innervate the ovary. In addition, evidence helix domain (50-74)-CRF-BP, present in the suggests that acetylcholine enhances follicular amino terminal of CRF-BP, is responsible for development and its intraovarian production its destination to the VSR. Further studies are would be in granulosa cells which express needed to evaluate if the other alpha-helix choline acetyltransferase (ChAT), vesicular domains also play a role in the sorting of CRF- acetylcholine transporter (VAChT) for storage BP to the VSR. and acetylcholinesterase. Besides, cholinergic muscarinic receptors (M1, M3, M5) are 7. TTAGP 1.0: A COMPUTATIONAL expressed in ovarian follicles. However, it is TOOL FOR THE SPECIFIC not known how this intraovarian cholinergic PREDICTION OF TUMOR T CELL system is regulated. In vitro studies had PEPTIDES. shown that human granulose cells incubated Beltrán J.F.; Herrera L.; Farías J.G. with neuronal growth factor (NGF), a Department of Chemical Engineering, Faculty neurotrophin produced by ovary, increased of Engineering and Science, Universidad de ChAT. The objective of the present work was La Frontera. to determine if NGF enhances acetylcholine production in the rat ovary. 26 days old rats Nowadays, cancer is considered a global ovaries were incubated with 100 ng/mL NGF pandemic and millions of people die every year during 3 and 24 hrs. 7 days old rats were because this disease remains a challenge for treated with 50 mg/Kg guanethidine during the world scientific community. Even with the 3 weeks to induce a chronic endogenous NGF efforts made to combat it, there is a growing increment and, after 3 months, ovaries were need to discover and design new drugs and obtained. We measured mRNA levels by qRT- vaccines. Among these alternatives, antitumor PCR, acetylcholine levels by fluorometric peptides are a promising therapeutic solution assay, NGF and noradrenaline by ELISA to reduce the incidence of deaths caused by kit, and NGF by western blot. After 3 hrs, cancer. In the present study, we developed NGF produced an increment in ChAT and TTAgP, an accurate bioinformatic tool VAChT mRNA levels, but a decrease in that uses the random forest algorithm for acetylcholine in medium. After 24 hrs, we antitumor peptide predictions, which are found a modest but constant increase in presented in the context of MHC class I. acetylcholine production. Guanethidine The predictive model of TTAgP was trained treatment didn’t induce an endogenous NGF and validated based on several features of increment despite that noradrenaline levels 922 peptides. During the model validation decreased. Altogether these data suggest that we achieved sensitivity = 0.89, specificity NGF regulates intraovarian acetylcholine = 0.92, accuracy = 0.90 and the Matthews production and storage ex vivo. Further correlation coefficient = 0.79 performance research is needed to elucidate if a longer measures, which are indicative of a robust time of NGF stimulation is needed to better model. TTAgP is a fast, accurate and intuitive visualize the neurotransmitter. software focused on the prediction of tumor T cell antigens. 9. AMPHETAMINE AND TEMPOL MODULATE EXTRACELLULAR 8. NGF INCREASED CHOLINE CONCENTRATION OF DOPAMINE ACETYL TRANSFERASE AND THE AND THE PHOSPHORYLATION VESICULAR ACETYLCHOLINE LEVEL OF THE DOPAMINE TRANSPORTER EXPRESSION IN RAT TRANSPORTER. OVARY EX VIVO. Blanlot C.1; Zegers J.A.1; Yarur H.E.1; Gysling Benitez A. 1; Lara H.E. 1. K.1. 1, Laboratorio de Neurobioquímica, Pontificia Universidad Católica de Chile. Departamento de Bioquímica y Biología Molecular, Centro de Estudios Amphetamine (AMPH) is a highly Neurobioquímicos para Enfermedades reinforcing, widely abused stimulant drug

91 that increases dopamine extracellular levels essential players in the physiology of the in the mesocorticolimbic system, in neurons central nervous system and impairment of its projecting from the Ventral Tegmental function underlie many neurological diseases, Area to Nucleus Accumbens and Prefrontal including epilepsy, autism, chronic pain, Cortex. AMPH-stimulated efflux of dopamine anxiety and schizophrenia among others. through the dopamine transporter (DAT) only The function of GlyRs containing the alpha1 happens if DAT is previously phosphorylated. or alpha2 subunits, can be competitively Some kinases that act on DAT are PKC, ERK inhibited by colchicine independently of and PKA and their activity is modulated by microtubule depolymerization. Interestingly, different signaling pathways. The increase a recent report showed that colchicine binds in the production of reactive oxygen species directly to the GlyRs containing the alpha3 (ROS) after the intake of AMPH, besides subunit, suggesting that the alpha3 GlyRs producing brain damage, can modulate the mediated the suppression of the inflammatory action of these kinases. Scavenging ROS pain exerted by colchicine. However, the may be a way to avoid the toxic effects of functional effects on the alpha3 GlyRs function oxidative stress induced by AMPH. Tempol elicited by colchicine are still undefined. is an antioxidant that has neuroprotective Using electrophysiological techniques and activity, diminishing the presence of oxidative molecular docking simulations, here we markers in the brain. It has been shown that show that colchicine is an inhibitor of the Tempol interferes with the development of alpha3 GlyR function. Colchicine, elicited behavioral sensitization induced by cocaine. concentration-dependent inhibitory effects We evaluated the effect of Tempol and AMPH on alpha3 GlyRs at micromolar range. in the phosphorylation level of DAT using Single-channel recordings show that the rat Nucleus Accumbens synaptosomes that colchicine inhibition is associated with a were stimulated with AMPH, Tempol and decrease in the open probability of the ion Tempol before AMPH. We also measured the channel. Molecular docking assays suggest concentration of dopamine in the medium that colchicine preferentially bound to the where the synaptosomes were incubated by orthosteric site in an agonist-free, closed state microdialysis and electrochemical detection. of the ion channel. Our results thus define the Western Blot analysis showed that AMPH pharmacological modulation of colchicine on increased the phosphorylation of DAT and alpha3 GlyRs. that the presence of Tempol avoided this increase. The extracellular concentration of 11. CHARACTERIZATION OF THE dopamine in the presence of AMPH alone NEUTROPHIL/LYMPHOCYTE RATIO was significantly increased. No changes were IN A SAMPLE OF PATIENTS WITH observed in the presence of Tempol alone. RHEUMATOID ARTHRITIS. Interestingly, AMPH in the presence of Cáceres B. 1; Flores D. 1; Saez K. 2; Ormazabal Tempol induced a lower increase in dopamine V. 3; Castro I. 4,5; Nova-Lamperti E. 1; concentration. Taken together, these findings Lamperti L.1. 1Facultad de Farmacia, suggest a role of ROS in the mechanism 2Facultad de Ciencias Físicas y Matemáticas, by which AMPH increases dopamine 3Facultad de Ciencias Biológicas, 4Facultad extracellular levels in Nucleus Accumbens. de Medicina, Universidad de Concepción y 5Hospital Guillermo Grant Benavente, 10. COLCHICINE COMPETITIVELY Concepción. ANTAGONIZES THE ALPHA 3 SUBUNIT OF THE GLYCINE Introduction: Rheumatoid arthritis (RA) is RECEPTORS. a systemic disease, still unknown etiology Burgos C.F. ; Lara C.O. ; Riquelme C. ; San and autoimmune character characterized Martín V. ; Flaig D. ; Soto P. ; Aguayo L.G. by chronic inflammation in the synovial ; Fuentealba J. ; Castro P.A. ; Guzmán L. membrane. The activity of the disease in RA is ; Muñoz-Montecino C. ; Yévenes G.E. ; determined with DAS28 index, which allows Moraga-Cid G. evaluating the efficacy of the drug therapy Department of Physiology, Faculty of administered. Monitoring the evolution of the Biological Sciences, University of Concepción, disease activity is essential to avoid disability Chile. in long term. The increase in the neutrophil/ lymphocyte ratio (NLR) has been described as Glycine receptors (GlyRs) are anion-selective a parameter of inflammation associated with neurotransmitter-gated ion channels, a predominance of neutrophils and a decrease member of the pentameric Ligand Gated in lymphocytes. This NLR relationship has Ion Channels (pLGICs) family. GlyRs are proven to be a good predictor of inflammation

92 in chronic diseases such as diabetes and Also, we quantified the NO production elicited cancer. Objective: To characterize the NLR by anthocyanidins by chemiluminescence. relationship in patients with RA. Methods: Both and D3G elicit concentration-dependent 11 AR patients and 11 controls of similar vasodilation as potent as acetylcholine used ages and same sex were recruited and signed as a standard control and 10 to 100 times an informed consent approved by Ethical more potent than 17-beta-estradiol (E2) and Scientific Committee. A blood sample was genistein respectively. The anthocyanins performed on a Sysmex XS-1000i device. The activity is dependent on the endothelium absolute values of neutrophils were divided and eNOS enzymatic activity (97-98%, p by lymphocytes. The values of DAS28 and <0.001). G36 significantly reduced the erythrocyte sedimentation rate (ESR) were anthocyanins vasodilation (p <0.01) such as analyzed. Statistical tests were used for G-1, GPER agonist (p <0.001). However, the variables. Results: The NLR was higher in anthocyanins response was not blocked by RA patients, 2.72±0.68 versus controls with fulvestrant, which decreased E2 activity by 1.53±0.38 p<0,0001. The classification of 28%. The perfusion of the mesenteric vascular disease activity according to DAS28 shown bed with 100 nM or 1 µM of delphinidin that NLR was 3.69 ± 0.25 for high activity, and D3G, or its application to endothelial 2.49 ± 0.29 for moderate and 2.23 ± 0.32 cells culture, increased NO production for low and the coefficient of HSV-NLR compared to the controls. We conclude that correlation was 0.34 and DAS28-NLR was anthocyanins, glycosylated or not, induce 0.815. Conclusion: The NLR was higher in AR a potent vasodilator response mediated by patients than in the control group. It is shown NO production that may be linked to GPER that NLR correlates positively with DAS28 activation at the vascular level. In addition, and HSV. In addition, the NLR was higher for the anthocyanin mechanism is rapid and non- patients with high disease activity, compared genomic in nature. to moderate and low. 13. MOLECULAR 12. GPER ANTAGONISM OF CHARACTERIZATION OF HEAD AND THE POTENT ANTHOCYANIN- NECK SQUAMOUS CELL CARCINOMA INDUCED VASODILATION AND NO (HNSCC) 3D MODEL AND PRODUCTION IN A VASCULAR BED METASTATIC CAPACITY EFFECTS OF AND ISOLATED ENDOTHELIAL OLD DRUGS WITH NEW ANTITUMOR CELLS. ACTIVITY. Calfío C.; Huidobro-Toro JP. Carrasco J.1; Martínez D.1; Jara J.1. Laboratorio de Farmacología, Facultad de 1, Laboratory of Pharmacology, Faculty of Química y Biología y Centro de Nanociencia y Dentistry, Institute for Research in Dental Nanotecnología de la Universidad de Santiago Sciences (ICOD), Universidad de Chile. de Chile, Santiago, Chile. Head and neck squamous cell carcinoma Anthocyanins are colored water-soluble (HNSCC) has an incidence in worldwide of flavonoids present in red-bluish berries, more than 350000 people and the mortality fruits and vegetables. Flavonoids conserve associated with this cancer is 50%. Although structural similarities with other molecules the treatment for these patients generally such as steroids. Genistein is recognized as a consists of surgical, pharmacological and natural phytoestrogen with potent estrogenic radiation therapies, survival at 5 years is only activity. Considering the potent vasodilation 53%. One of the reasons for high mortality elicited by the glycosylated or anthocyanin and low survival is due to the presence aglycones is strictly endothelium-dependent, within tumor mass a subset of cells called we proposed that the anthocyanin-induced cancer stem cells (CSCs), which represent vasodilation is mediated by the estrogen the most tumorigenic subpopulation. This receptor coupled to a trimeric G protein population grows like spheroids and in (GPER) or estrogen receptor alpha (ERa), its center has hypoxic cells that are highly both associated to a rapid, non-genomic resistant to chemotherapies. Here, the goal mechanism. We evaluated the vascular was to obtain in vitro spheroid CSC from response induced by the anthocyanin HNSCC (HNCSCs) and determine expression delphinidin and its 3-O-glucoside derivative protein level involving on metabolism, (D3G) in pre-contracted mesenteric vascular hypoxia, autophagy, and antitumor target, in beds in the presence or absence of 1µM-G36, a addition to assess the effects of Itraconazole purported GPER receptor antagonist, or 1µM and hydroxychloroquine on invasion capacity fulvestrant a recognized ER α/β antagonist. on spheroid cultures. Spheroid formed from

93 Cal27 and HEp-2 cell lines using culture environment (deferoxamine) improves their selecting conditions and protein expression effectiveness. The aim of this study was to levels was determine by immunoblotting. determine whether intranasal administration Spheroid was treated with Itraconazole and of secretome derived from preconditioned hydroxychloroquine and then seeded over human MSCs prevents the (i) behavioural matrigel to Boyden chamber 3D invasion development, (ii) motor and (iii) cognitive assays. Successful formation of spheroid disabilities resulting from PA. PA was induced from the Cal27 and Hep-2 cell lines, where by immersing foetuses-containing uterine needed 4500 and 3500 cells respectively to horns into a water bath at 37 °C for 21 min. obtain sizes greater than 300 µm. Spheroids Two hours after birth and at postnatal day (P)7 were also subjected to hypoxic conditions MSCSs (6 ug/16 ul, obtained from 1x10⌃6 with oxygen concentrations below 5%, preconditioned-MSCs) or 16 ul of vehicle VDAC, PDK1, Hexokinase II, Hif-1 and LC3B were administered intranasally to asphyxia- protein expression levels were different exposed or control rats. Neurobehavioral between cell line and monolayer or spheroid development was evaluated by monitoring cultures. HNCSCs exposure to Itraconazole the righting reflex (at P1, P4, P7 and P14); and hydroxychloroquine modulate invasive negative geotaxis (P7, P14 and P21), and capacities, compared with control conditions. cliff aversion (P7, P14 and P21). Locomotor Here we showed that protein expression activity (P7) and motor coordination (P60) patterns are different between monolayer were evaluated by open field and rotarod, and spheroid cultures and the effects of two respectively. Anxiety (P30), by open field and different drugs on metastatic capacity of novel object recognition memory (P30). All HNCSCs. the PA induced effects were positively affected by MSCSs treatment, including improvements 14. MESENCHYMAL STEM in: (i) the remarkable developmental delay in CELL SECRETOMES (MSCSS) the performance of righting and cliff aversion ADMINISTRATION IMPROVES reflexes; (ii) the decrease in locomotor BEHAVIOURAL DEVELOPMENT, activity and deficits in motor coordination MOTOR AND COGNITIVE and balance; (iii) an increase in anxiety and IMPAIRMENTS INDUCED BY novel object recognition memory deficits. PERINATAL ASPHYXIA. Carril J.1; Obrecht S.2; Contreras N.2; Araya 15. INHIBITORY EFFECT OF M.1; Monzón E.1; Farfán N.1; Alvarado R.1; COUMARINS DERIVATIVES ON Tapia-Bustos A.1;Vásquez R.1; Bustamante THE VIABILITY OF HELICOBACTER D.1; Quintanilla M.E.1; Ezquer F.3; Israel PYLORI ATCC 43504 AND Y.1; Valdés J.L.2; Herrera-Marschitz M.1; RECOMBINANT CARBONIC Morales P.1,2. ANHYDRASE ACTIVITY. 1Molecular & Clinical Pharmacology Program, Carvajal, R.C. 1,2,3.; García, A1.; Zúñiga, ICBM, 2Department of Neuroscience, Faculty F3.; Alarcón, J5; Ormazábal, V.4 & Pastene- of Medicine University of Chile. 3Center for Navarrete, E. N.5. Regenerative Medicine, Faculty of Medicine- 1Laboratorio de Patogenicidad Bacteriana, Clínica Alemana, Universidad del Desarrollo, Departamento de Microbiología, Facultad Santiago, Chile. de Ciencias Biológicas, Universidad de Concepción. 2Laboratorio de Farmacognosia, Perinatal asphyxia (PA) induces deficits Departamento de Farmacia, Facultad de in neurological reflexes, development, Farmacia, Universidad de Concepción. motor coordination, emotional behaviour 3Departamento de Biología Clínica e and cognition. At present, no treatment Inmunología, Facultad de Farmacia, significantly attenuates or prevents these Universidad de Concepción. 4 Departamento sequelae. Mesenchymal stem cells (MSCs) de Farmacología, Facultad de Ciencias have been proposed as a therapeutic tool for Biológicas, Universidad de Concepción. several CNS diseases, since MSCs display 5Laboratorio de Síntesis y Biotransformación remarkable antioxidant, anti-inflammatory de productos Naturales, Departamento and repairing features (neurogenesis, de Ciencias Básicas, Facultad de Ciencias, synaptogenesis, myelination). MSCs exert Universidad del Bio-Bío. paracrine effects by secreting a combination of nano-vesicles and soluble factors Helicobacter pylori (Hp) is a Gram negative (referred to as secretomes). Preconditioning pathogen that affects more than 50% of the MSCs with pro-inflammatory cytokines world population, and it is responsible for (TNFalfa plus INFgamma) or hypoxia-like different gastric pathologies. Since increasing

94 antibiotics resistance cause serious failure in of several genes. Potential HIF-1a activated Hp eradication therapy, new pharmacological genes were identified in the rat genome targets need to be identified to affect the following hypoxic conditions, by extracting viability of this bacteria. Carbonic anhydrase promoter sequences of Rattus Norvegicus (CA, EC 4.2.1.1) is an interesting new Hp from the UCSC database Genome Browser. target since it is involved in neutralizing the 8762 genes with the HIF-1a binding sequence acid pH of the human stomach cooperatively (5`-RCGTG-3`) were identified using the “R” with urease. It has been described that software. These genes were introduced to the specific coumarin derivatives can inhibit CA. Gen Ontology platform for performing an Besides, several coumarins exhibit a strong enrichment analysis, selecting the following antibacterial activity. Objective: to clone processes linked to PA: (i) Hypoxia (865 and characterize carbonic anhydrase from genes); (ii) Glucose Metabolism (330 genes); the highly aggressive strain Hp ATCC 43504 (iii) Neurogenesis (1243 genes); (iv) Apoptosis and to determine the coumarins derivatives (814 genes); (v) Angiogenesis (165 genes), effects on both, the recombinant protein and and (vi) Regulation of Gene Expression Hp viability. Methodology: a-CA was cloned, (2076 genes). 865 hypoxia-associated genes expressed in cell line HEK 293 and purified. were further selected and compared with Recombinant α-CA was characterized by experimental data by ChIP-Seq, with 772 and esterase activity and protonography. We 98 genes from human and zebrafish genomes, determined the inhibitory effect of coumarins respectively, identifying 79 genes for the derivatives against recombinant α-CA by three species. The 8762 genes were then esterase activity and on Hp to determine its analysed by the Kyoto Encyclopedia of Genes MIC and MBC. Results: A 50 kDa functional and Genomes (KEGG) platform, selecting recombinant Hp a-CA was cloned that forms a the HIF-1 pathway, identifying 47 genes. dimer structure in solution. The recombinant The 79 genes filtered for human, zebrafish Hp a-CA it is closely related to F78 strain, and rat were compared with the 47 genes with a 99.1% of identity. Esterase activity was obtained by the KEGG platform, yielding determined, complemented by protonography 12 genes. Finally, 12 genes were compared analysis, and kinetic parameters. Overall, six with 47 genes referred by the literature to coumarins showed inhibitory activity against be associated to PA, identifying 5 genes: (i) Hp, with MIC ranging from 125 ug/mL to 15 Bcl2; (ii) Hif-1a; (iii) Ldha; (iv) Pdk1, and ug/mL, and two coumarins were inhibitors (v) Vegfa. The pharmacological inhibition of of the recombinant protein. Conclusion: The HIF-1a to establish the gene expression levels characterization of this highly aggressive Hp of candidate genes in an in vivo model of PA CA strain and the determination of inhibitory is studied, providing a proof-of-principle for effects of coumarins, makes them a potential the participation of HIF 1a on the regulation candidate for Hp therapy and eradication of gene expression following PA.

16. IDENTIFICATION OF POTENTIAL 17. DEVELOPMENT AND CANDIDATE GENES RELATED TO CHARACTERIZATION OF A HYPOXIA INDUCIBLE FACTOR 1 NANOPARTICULATE SYSTEM ALPHA (HIF-1 ALPHA) INVOLVED IN FOR NASAL ADMINISTRATION OF PERINATAL ASPHYXIA IN RATS. CURCUMIN DERIVATIVE. Emmanuel Casanova 1; Pablo Baéz 2, Luis Salas M.J.1; Castillo E.2; Gómez C.1; von Valenzuela 2, Rodrigo Assar 2, Andrea Tapia Plessing C.1. 1 Departamento de Farmacia, 1, Paola Morales 1, Katherine Marcelain Facultad de Farmacia, Universidad de Cubillos 2, Mario Herrera-Marschitz 1. Concepción, Concepción, Chile. 2 Facultad 1 Programme of Molecular & Clinical de Medicina y Ciencia, Universidad San Pharmacology, ICBM, Medical Faculty, Sebastián, Concepción, Chile. University of Chile and 2 Cancer Genomics Laboratory, Basic-Clinical Oncology In the present work, the nanoparticles (NPs) Department, Medical Faculty, University of of chitosan were elaborated and characterized Chile, Santiago, Chile. by means of the ionic gelation method, using for this the magnetic stirring and as a Perinatal asphyxia (PA) is characterized crosslinking agent sodium tripolyphosphate by interruption of oxygen bioavailability at (TPP) and / or a derivative of thiamine. The birth. Hypoxia implies HIF-1 alpha (HIF-1a) optimization of the NPs of the chitosan the activation, a key sentinel protein, which, upon response has been carried out by means of translocation to the nucleus, binds to response a central composite design, in which the elements (HREs), promoting transcription different parameters have been evaluated as

95 the relationship between the mass between that the CF-to-CMF differentiation increases the chitosan/derivative of thiamine/TPP, ALX/FPR2 protein levels, and consequently, different speeds and times of agitation, pH ALX/FPR2 activation by RvD1 enhances of the solutions and order of addition of the collagen-1 synthesis. Methods: Secondary components, with respect to the average culture of adult rat CF was starved for 24 diameter, polydispersity and zeta potential hours, stimulated with TGF-b1 (10 ng/mL) of the NPs. The physical characteristic of the for 72 hours on fetal bovine serum (FBS) best formulation gave spherical shapes, with 1% to induce CMF differentiation and then an average diameter of 136,12 ± 3,60 nm, a treated with RvD1 at different intervals polidispersion index of 0,42 ± 0,02 and a zeta in presence and absence of PD98059, potential of +31,06 ± 0,97 mV for white NPs. LY294002 (ERK ½ and AKT inhibitors) and The NPs loaded with curcumin have a stability WRW4 (ALX/FPR2 antagonist). The proteins of 90 days at 5 °C ± 3 °C and a stability of 6 levels of p-EKR ½, p-AKT and collagen-1 days at 25 °C ± 2 °C with 60 % ± 5 % relative were measured by western blot. Results: After humidity. A cell viability study was carried CMF differentiation, it was found an increase out, using the THP-1 cell line, the results of ALX expression. On CMF, RvD1 activated indicated that the NPs with a concentration the ERK ½-AKT pathways and enhanced the of 11 μg/mL curcumin equivalents, the expression of collagen-1. These effects were viability is higher than 80 %. Finally, the blocked by PD98059, LY294002 and WRW4. encapsulation efficiency of curcumin in the On the other hand, RvD1 did not decrease final NPs formulation was 24,4 ± 4,48 %, with α-SMA protein levels, which suggests that a load of 455 ± 0,06 ug%, with a size, PDI and it does not reverse CMF differentiation. zeta potential of 218,3 nm, 0,134 and +31,9 Conclusions: Our results suggest that the CF- mV, respectively. Keywords: nanoparticles, to-CMF differentiation increases the protein chitosan, curcumin, cell viability. levels of ALX/FPR2, and that RvD1 enhances collagen-1 synthesis through ERK ½ and AKT 18. RESOLVIN D1 INCREASES pathways. COLLAGEN-1 SYNTHESIS ON RAT CARDIAC MYOFIBROBLAST 19. PROCYANIDINS-RICH EXTRACT THROUGH ALX/FPR2 RECEPTOR NANOEMULSIONS O/W AS A ACTIVATION. POTENTIAL ANTITUMORAL TOOL: Esteban Castro-Carrasco1; José Miguel Lillo1; EVALUATION IN MELANOMA CELL Guillermo Díaz-Araya1,2. LINE. 1Laboratory of Molecular Pharmacology, Cerda-Opazo P. 1,2,3; Gotteland M. 4; Department of Pharmacological and Oyarzun-Ampuero F. 2, 3; Garcia L. 1, 3. Toxicological Chemistry, Facultry of Chemical 1, Depto. Bioquímica y Biología Molecular, and Pharmaceutical Sciences, University of Facultad de Ciencias Químicas y Chile, Santiago, Chile. 2Advanced Center Farmacéuticas, Universidad de Chile, for Chronic Diseases (ACCDiS), FAculty of Santiago, Chile; 2, Depto. Ciencia y Tecnología Chemical and Pharmaceutical Sciences & Farmacéutica, Facultad de Ciencias Químicas Faculty of Medicine, University of Chile, y Farmacéuticas, Universidad de Chile, Santiago, Chile. Santiago, Chile; 3, Advanced Center for Chronic Diseases (ACCDiS), Santiago, Chile; Background: Cardiac fibroblast (CF) to 4, Depto. de Nutrición, Facultad de Medicina, cardiac myofibroblast (CMF) differentiation Universidad de Chile, Santiago, Chile. is mainly mediated by TGF-b1 released from immune cells by angiotensin II (Ang II) effect, During the last 10 years, the incidence of or in vitro conditions due to mechanical melanoma in Chile has incremented 27.2%. stress. CMF are able to produce extracellular Approximately 80% of all skin cancer-related matrix proteins, mostly collagen, in the deaths are attributed to melanoma and long- scar formation proccess. Also, in the CF-to- term survival of patients is only 5%. This CMF process it has been demonstrated the type of cancer is considered a multifactorial upregulation of Kinin-B1 and AT1R receptor, disease related to environmental interaction among others. However, the presence of and genetic susceptibility that trigger a Resolvin D1 (RvD1) receptor, ALX/FPR2, constitutive activation of several signaling has not been elucidated. RvD1 is an anti- pathways, promoting proliferation and inflammatory lipidic mediator that regulates survival of tumoral cells. Current therapies matrix proteins like a-SMA and collagen in cause a considerable number of side effects, various cell types, yet there is not evidence of still representing a global human health issue. this effect on CMF. Purpose: To demonstrate Recently, the use of polyphenols has been

96 reported for both prevention and treatment nutrition on other brain areas remains of melanoma. Some evidence demonstrates elusive. Recently, it has suggested that that different extracts of polyphenols (grape, fetal exposure to maternal obesity causes pomegranate, among others) are high decreased neurogenesis and impaired potential candidates to treat various types hippocampal learning. The hippocampus is of cancer. Of note, the main limitations of important for learning and memory, and its these molecules are the low bioavailability development is sensitive to the metabolic and the necessity of a high concentration environment in utero. In a model of maternal dose to cause a biological effect. The aim of obesity induced by a high-fat diet (HFD, this study was to utilize the nanotechnology 60Kcal in fat) consumption we study whether to encapsulate procyanidins and to evaluate this adverse prenatal environment impairs its antitumoral activity in B16F10 melanoma the hippocampal synaptic transmission. In cell line. To achieve the proposed objective, offspring mice, during adolescence, using an avocado peel procyanidin-rich extract was electrophysiological recordings in the CA1 used and cellular viability, proliferation and area of mice hippocampus, we observe migration in B16F10 cells were evaluated. that maternal HFD consumption increases Nanoemulsions were elaborated by solvent the frequency and amplitude of Inhibitory evaporation and were in a nanometric range postsynaptic currents. This increase in of 170 ± 2 nm and showed low polydispersity inhibition level onto pyramidal neurons (between 0.1 and 0.2). The nanoformulations could have important consequences in the showed negative zeta potential (−44 ± 4 mV), excitation/inhibition balance, being able to realizing a stable system. The administration modify the hippocampal cognitive function in of procyanidin-rich extract nanovehicles was juvenile offspring mice. more efficient in reducing cellular viability, proliferation and migration compared to free 21. PHARMACOKINETIC extract. Altogether, our results suggest that VARIABILITY IN PATIENTS WITH encapsulation of procyanidins significantly KIDNEY TRANSPLANTATION, improves its effect on melanoma therapy. TREATED WITH CYCLOSPORINE. Cerpa L.1,4; Rodríguez M.S.2; Corvalán F.1; 20. MATERNAL HIGH-FAT Contreras S.1; Cayún JP.1,4; Llull G. 1,3; DIET CONSUMPTION DURING Sandoval C.1; Farías N.2; Alvarez C.2; Plubins PREGNANCY AND LACTATION L.2; Ñuñez G.2; Castro L.2; Espinoza R.2; IMPAIRS THE INHIBITORY Chavez R.2; Goic I.2; Mur P.2; Cordero C.2; SYNAPTIC TRANSMISSION OF CA1 Acuña P.2; Moya C.2; Varela N.1,4; Quiñones REGION OF HIPPOCAMPUS OF THE L.1,4. YOUNG OFFSPRING. 1. Laboratory of Chemical Carcinogenesis Cerna C.1, Valero V. 1,2, Santander O.1,3 and Pharmacogenetics (CQF), Department García F.1,3, Guiffa F.1,4, Cruz G. 1 and of Basic and Clinical Oncology, Faculty Fuenzalida M. 1* of Medicine, University of Chile. 2. Adult 1Centro de Neurobiología y Fisiopatología Nephrology Unit, San Juan de Dios Hospital. Integrativa (CENFI), Instituto de Fisiología, 3. Clinical Laboratory, San Juan de Dios Facultad de Ciencias, Universidad de Hospital. 4. Red Latinoamericana para Valparaíso, Valparaíso, Chile. 2Programa de la Implementación y Validación de Guías Doctorado en Ciencias e Ingeniería para la Clínicas Farmacogenómicas (RELIVAF- Salud, Universidad de Valparaíso, Valparaíso, CYTED). Chile. 3Programa de Doctorado en Ciencias Mención neurociencia, Universidad de Introduction: Cyclosporine (CsA) is an Valparaíso, Valparaíso, Chile.4Programa de immunosuppressive drug, used to prevent Magister en Ciencias, mención neurociencia, organ rejection in transplant patients. Universidad de Valparaíso, Valparaíso, Chile. However, this drug is characterized by having a narrow therapeutic range and high Before birth and early in life the brain inter and intra-individual pharmacokinetic development is acutely sensitive to its variation. There are some genetics variants environment. Experimental and clinical involved in pharmacokinetic variability, data indicate that maternal obesity can both at the level of cyclosporine absorption predispose the offspring to suffer metabolic and metabolization. However, genetic and neuronal alterations. Most studies variants of relevant impact are not yet have focused on the functional relationship identified. Objective: to evaluate the genetic between maternal obesity and hypothalamus variants involved in the pharmacokinetics of alterations. However, the impact of maternal cyclosporine and to establish an association

97 between the pharmacogenetic profile, and the (OE-O) is effective and a therapeutic option safety and efficacy of the treatment during on bacterial biofilms, but its activity on fungal the first three months after transplantation. biofilms is unknown. Methods. The minimum Methodology: One hundred and seven inhibitory concentration (MIC) was patients (107) with kidney transplants from determined on reference strains for C.Albicans San Juan de Dios Hospital (Project No. (fluconazole sensitive and resistant) and 028-13) were retrospectively included and non-albicans strains (C.tropicalis, C.krusei were genotyped for the genetic variants and C.glabrata). The antibiofilm effect was CYP3A4 *1B rs2740574, CYP3A4*22 evaluated by: a) morphogenesis inhibition rs25599367, CYP3A5*3 rs776746, POR*28 assay: an inoculum was incubated for 5 hours rs1057868, MDR1 3435 rs1045642, MDR1 at 37°C in the presence of the OE-O MIC, 2677 rs2032582 and MDR1 1236 rs1128503. then the percentage of filamentous cells was Genotypic frequency were associated with counted using a Neubauer chamber; and b) blood concentrations of cyclosporine (CsA), inhibition of biofilm adhesion: an inoculum creatinine value and blood pressure within the was incubated in a 96-well plate in the first three months post-transplant. Results: presence of the CIM of OE-O for 4 hours at Heterozygous patients for CYP3A4*1B had a 37°C, the adhered biofilm was stained with lower creatinine value from the second post- crystal-violet and absorbance was measured transplant week. In relation to cyclosporine in microplate reader. Fluconazole and levels, an increase was observed, in patients nystatin were used as controls. Results. AE-O with at least one altered allele for CYP3A5*3 significantly inhibited both biofilm adhesion from the second post-transplant week. and morphogenesis of the strains studied Conclusions: The results show that genetic compared to controls. Conclusion. These variants can account for variations in the results indicate that OE-O has significant pharmacokinetic parameters of cyclosporine, antibiofilm activity in both C. albicans and which affect the efficacy and safety of non-albicans strains. cyclosporine treatment. It is expected that, based on the results found, a genetic 23. EFFECTS OF MODAFINIL predictive panel of response to CsA will be ADMINISTRATION ON SOCIAL setted-up to be used before transplantation. PLAY BEHAVIOUR AND DOPAMINE TRANSMISSION IN JUVENILE RATS. 22. ANTIFUNGAL AND ANTIBIOFILM Cid-Jofré V.1,2; Gárate M.1; Sotomayor- EFFECT OF OREGANUM VULGARE Zárate R.3, Cruz G.2; Renard GM.1. ESSENTIAL OIL AGAINST CANDIDA 1 Laboratorio de Conductas Sociales y ALBICANS AND NON-ALBICANS. Adictivas, Escuela de Medicina, Centro de Cid, C.1; Mûller, A.; Díaz, M. 2; Jara, J.; investigaciones Biomédicas y aplicadas, Molina-Berríos, A.1. Facultad de Medicina, Universidad de 1, Laboratorio de Farmacología, Instituto Santiago de Chile. 2 Laboratorio de de Investigación en Ciencias Odontológicas, Alteraciones Reproductivas y Metabólicas, Facultad de Odontología, Universidad de Centro de Neurobiología y Fisiopatología Chile. 2, Laboratorio de Nanobiomateriales, Integrativa (CENFI), Instituto de Fisiología, Instituto de Investigación en Ciencias Facultad de Ciencias, Universidad de Odontológicas, Facultad de Odontología, Valparaíso. 3Laboratorio de Neuroquímica y Universidad de Chile. Neurofarmacología, Centro de Neurobiología y Fisiopatología Integrativa (CENFI), Oral candidiasis is the most common fungal Instituto de Fisiología, Facultad de Ciencias, infection in humans. The most frequent Universidad de Valparaíso. causative agent isolated is Candida albicans (C. albicans), but the number of resistant strains Modafinil (MOD) is a stimulant used to such as Candida krusei (C. krusei), Candida enhance wakefulness and vigilance. The tropicalis (C.tropicalis) and Candida Glabrata mechanism of action of MOD has not (C.glabrata) has increased. It is treated locally been completely elucidated but a blockage with miconazole and nystatin; however, of dopamine (DA) and norepinephrine patients present high recurrence rates due transporters has been observed. Some clinical to the formation of biofilms. Biofilms are trials are testing MOD for the treatment of biological communities adhered to a surface attentional deficit disorder (ADD). In view of (oral mucosa, dental prostheses) with high a reported over diagnostic of ADD, evaluating resistance to antifungals, which has driven the effects of MOD in healthy individuals is the search for natural alternative therapies, important. Herein, we evaluate the effects such as essential oils. Oregano essential oil of MOD on social play behaviour (SPB) and

98 DA extracellular levels in juvenile rats. 35 controlling feeding behavior. In addition, LH juvenile male Sprague-Dawley rats were projects glutamatergic/orexinergic neurons treated from PND25 with MOD (75 mg/ to the ventral tegmental area (VTA), which kg i.p.) or vehicle for 14 days. Locomotor it sends dopaminergic projections to the and social exploration were tested 24 hours nucleus accumbens and LS. This circuit is after de last injection. Nucleus Accumbens very important for the intake of rewarding (NAc) and ventral tegmental area (VTA) foods, but it is not known the effects of were dissected to measure DA content by chronic exposure to high-fat diet (HFD) on HPLC coupled to electrochemical detection. LS neurotransmission. For this work we We observed a decrease in the “pinning used 2 groups of male Sprague-dawley rats events” (responses to play behaviour) and exposed from weaning to postnatal day 60 tendency to decrease in the pouncing latency (PND 60) to chow diet (control) and HFD. (the events of solicitation to play) in the At PND 60 the animals were euthanized MOD group. Also, there was a decrease in and the LS was microdissected to measure DOPAC/DA and DOPAC content in VTA. No by western blot key proteins involved in differences in social exploration time and DA LS dopaminergic neurotransmission. Our content were observed. SPB is fundamental results demonstrate that exposure to HFD to establish social and cognitive development results in a significant weight gain at the end in highly social animals like rats and humans. of the experimental period together with These preliminary results show that MOD an increase in retroperitoneal fat levels. In could affect SPB by altering the rewarding LS the chronic exposure to HFD resulted in effects of socialization. Importantly, DA levels an increase in the expression of the type 2 are almost the same in both NAc and VTA, dopamine receptor (D2) and the dopamine although there is a tendency for lower levels transporter (DAT) compared to control rats. in MOD group. More studies are needed to These results suggest that these proteins unravel the effects of stimulants, specially on functionally reduce the dopaminergic tone in young population, over important social skills LS, which would affect their inhibitory control like playing, social interactions and memory over LH activity. However, the implication of these results will be evaluated in subsequent 24. HIGH-FAT DIET EXPOSURE experiments. INCREASES THE EXPRESSION OF KEY PROTEINS IN DOPAMINERGIC 25. BOLDINE PREVENTS TGF-Β1- NEUROTRANSMISSION OF RAT INDUCED DIFFERENTIATION LATERAL SEPTUM. OF CARDIAC FIBROBLASTS BY Collio, V.1; Martínez-Pinto, J.2; Cruz, G.2; INHIBITING FOXO1. Bonasco, C.2; Renard, G.M.3; Sotomayor- Contreras A. 1, Anfossi R. 2., Suarez C. 2, Zárate, R.2. 1Programa de Magíster en Cárdenas, S. 1., Vivar, R 2. Ciencias Biológicas mención Neurociencias, 1.- Department of Biology, Faculty of Facultad de Ciencias, Universidad de Basics Sciences, Metropolitan University of Valparaíso, Valparaíso, Chile. 2Centro de Educational Sciences; 2.- Faculty of Medicine, Neurobiología y Fisiopatología Integrativa University of Chile. (CENFI), Instituto de Fisiología, Facultad de Ciencias, Universidad de Valparaíso, Diabetes and myocardial infarction promote Valparaíso, Chile. 3Centro de Investigación the development of cardiac fibrosis. Normally, Biomédica y Aplicada (CIBAP), Escuela de cardiac fibroblasts (CF) are responsible for the Medicina, Facultad de Ciencias Médicas, synthesis and maintenance of extracellular Universidad de Santiago de Chile, Santiago, matrix components (ECM), whereas in Chile. pathological conditions CFs differentiate into cardiac myofibroblasts, generating Obesity is a global pandemic that must be an imbalance in the secretion of the ECM studied from many points of view, such as proteins. TGF-beta1 plays a crucial role in the social, preclinical, clinical, economic, etc. development of cardiac fibrosis by regulating At the level of the central nervous system, the expression of FoxO1, a transcription factor there are several structures involved in that is involved in functions such as apoptosis, the control of food intake, being one of the oxidative stress and cell differentiation. On most important to promote the feeding the other hand, boldine, a natural alkaloid, the lateral hypothalamus (LH). The main has been shown to exert antifibrotic effects in brain area that controls the neural activity experimental models of diabetes. Therefore, of LH is the lateral septum (LS), which it this work attempted to demonstrate the sends GABAergic projections towards LH, antifibrotic effect of boldine in a model

99 of cardiac fibrosis in vitro. To respond to effects, and generation of polluting waste this hypothesis, the differentiation of adult or expensive raw material. Therefore, due Sprague-Dawley rats CF by TGF-beta1 was to their nature and structural diversity they used as in vitro model of cardiac fibrosis. The could have great potential for cosmetic use, differentiation of CF was determined by the for example, as hair dyes. To characterize expression of alpha-SMA and CTGF, through the fungal pigments and evaluate their western blot (WB) and immunocytochemistry antioxidant effect, Penicillium murcianum (ICQ). On the other hand, the activation of species was selected. This fungus is an eco- FoxO1 was evaluated by analyzing phospho- type found in Chilean native forests, which FoxO1 (WB) and nuclear location of FoxO1 was cultivated under conditions previously (ICQ). AS1842856 a FoxO1 activity inhibitor optimized for the production of brown- was used to evaluate the role of FoxO1. TGF- yellow pigments. To facilitate the chemical- beta1 10ng/ml increased the expression of biological analysis, the extract obtained alpha-SMA and CTGF at 48h, which was from the culture broth was fractionated corroborated by an ICQ against alpha-SMA. by Centrifugal Partition Chromatography In addition, TGF-beta1 increased the activity (CPC) with a phase system of ethyl acetate: of FoxO1, which was determined by decreased butanol: water. The above allowed to purify phosphorylation of FoxO1 and increased several fractions, which were evaluated for nuclear localization, whereas inhibition of their antioxidant activity in the DPPH assay. FoxO1 prevented the differentiation of CF These fractions recorded an inhibition close induced by TGF-beta1. Finally, boldine 50uM to 90% and an IC50 value of 4.64 mg / mL for and 100uM abolished the differentiation of the crude extract. In the case of the purified CF and the activation of FoxO1 induced by fractions, two were selected with the highest TGF-beta1. Collectively ours results suggest antioxidant activity corresponding to an IC50 that boldine prevents TGF-beta1-induced CF of 1.17 mg / mL and 0.708 mg / mL, which were differentiation by inhibiting FoxO1. subsequently analyzed by HPLC / PAD / MS. Our preliminary results revealed the presence 26. CHEMICAL-BIOLOGICAL of azafilones such as monashexenone, CHARACTERIZATION OF monankarin and monaphilol and the ANTIOXIDANT PIGMENTS PURIFIED anthraquinoids sterigmatocystin, endocrocin FROM EXTRACTS AND FRACTIONS and flavokermesic acid, which would be OF PENICILLIUM MURCIANUM responsible for the yellow pigmentation of the AND ITS POTENTIAL COSMETIC extract. These compounds have bibliographic APPLICATIONS. antecedents related to antioxidant and Contreras-Machuca P.1, Avello M.1, Pastene antimicrobial activities, among others, E.1,6, Machuca A.2, ArandaM.3, Hernández which highlights a great opportunity for V.4 & Fernández M.5. future research and applications for these 1 Laboratorio de Farmacognosia, metabolites of P. murcianum. Departamento de Farmacia, Facultad de Farmacia, Universidad de Concepción, 2 27. IN VITRO PROPAGATION Laboratorio de Biotecnología de Hongos, OF RODOPHIALA PRATENSIS Departamento de Ciencias y Tecnología AND ITS TOXICITY IN VITRO ON Vegetal, Universidad de Concepción, Campus EPITHELIAL CELLS OF GASTRIC Los Ángeles, 3 Laboratorio de Espectrometría ADENOCARCINOMA (AGS). de Masas, Departamento de Ciencia y Correa D.I. 1; Pastene-Navarrete E.R. 1,2; Tecnología de los Alimentos, Facultad de Bustamante L. 2; Baeza M. 3; Alarcón-Enos Farmacia, Universidad de Concepción, 4 J.I 4. Centro de Biotecnología, Universidad de 1Laboratorio de Farmacognosia, Dpto. de Concepción, 5 Laboratorio de Tecnología Farmacia, Facultad de Farmacia, P.O. Box Farmacéutica, Departamento de Farmacia, 237, Universidad de Concepción, Concepción, Universidad de Concepción, 6 Laboratorio Chile; 2Dpto. de análisis instrumental, de Síntesis y Biotransformación de Productos Facultad de Farmacia, Universidad de Naturales, Universidad del Bío-Bío. Concepción, Concepción, Chile; 3Dpto. Botánica, Facultad de Ciencias Naturales y In order to find new sources of active Oceanográficas, Universidad de Concepción, metabolites as functional ingredients for Concepción, Chile; 4Laboratorio de Síntesis the cosmetic industry, the idea of studying y Biotransformación de Productos Naturales, filamentous fungi that produce natural Dpto. Ciencias Básicas, Universidad del Bio- pigments arises. These compounds meet the Bio, Chillan, Chile. requirements of having low toxicity, adverse

100 Amaryllidaceae is a family of bulbous toxicity and many side effects. Therefore, plants, producers of alkaloids which are new therapies directed to the metastatic cells biogenetically related and exhibit high of the CRC are urgently needed, with high pharmacological activity. Lycorine and pharmacological efficacy, reducing the side homolycorine alkaloids have been studied as effects and treatment costs. In recent years, potent antitumor agents. However, the study neoplastic mitochondria are an attractive of these molecules is difficult due to the low pharmacological target for cancer therapy, availability and production in the plant in the since they have higher mitochondria potential. wild, so the objective of this study is by in vitro In our laboratory, gallic acid derivatives linked propagation to obtain biomass of R. pratensis to an aliphatic chain of ten carbons associated in an efficient and sustainable way, to identify with triphenylphosphonium (TPP+C10), a the type of Alkaloids that are produced and lipophilic cationic molecule that induces the assess their cytotoxic potential on tumor uncoupling of the electron transport chain cells. Methodology: Rhodophiala prantesis was synthesized and evaluated in CRC cells bulbs, were sterilized and cut into twin-scales, as well as gentysic derivative (GA-TPP+C10). to sow them in Murashige-Skoog growth The objective of this study is to evaluate the medium, supplemented with sucrose and synergistic effect of the compounds, GA- different combinations of naphthalenacetic TPP+C10 and TPP+C10 in combination acid and 6-benzylaminopurine, the alkaloid with conventional drugs for the treatment analysis was performed by CG-MS. AGS of CRC, 5-FU and oxaliplatin, using the cells were cultured in DMEM medium COLO 205 metastatic human CRC cell line. supplemented with SBF (10%), antibiotic Through the MTT assay, the cytotoxicity of (1%). The assay was performed in 96-well the combinations was evaluated after 48 h of plate using resazurin at 6 and 24 hours after treatment and by flow cytometry the synergy exposure of the alkaloid extract. R. pranthesis of the combinations inducing apoptosis was callus were obtained in in vitro culture in evaluated. The observed results showed semi-solid medium. In addition, 25 alkaloids that bothTPP+C10 and GA-TPP+C10 are were identified in the bulb’s alkaloid extract synergistic with low concentrations of 5-FU which decreased the viability of AGS cells and Oxaliplatin, inducing greater apoptosis of at 6 and 24 hours of exposure. Conclusion: the colorectal cells. In conclusion, the results Hormonal combinations were evaluated for suggest that these new compounds could the production of callus of R. pratensis, in synergize the effects of conventional therapy addition the alkaloids have cytotoxic activity against colorectal cancer. on AGS as a function of exposure time. 29. CHANGES IN DOPAMINE 28. SYNERGISTIC EFFECT RECEPTOR TYPE 2 EXPRESSION IN OF GENTISIC AND GALLATE PREFRONTAL CORTEX INDUCED DERIVATIVES WITH STANDARD BY EARLY-LIFE INTESTINAL THERAPY FOR COLORECTAL DYSBIOSIS. CANCER CELLS. Covarrubias MJ.1; González-Arancibia C.2,3; Cortés, G. 1,2, Ramírez, D.1,2, Rojas, D. 1, Martinez-Pinto J.2; Sotomayor-Zárate R.2; Escobar, B.1,3, Catalán, M.1. Julio-Pieper M.1; Bravo JA.1. 1 Laboratory of Biochemistry, Metabolism 1Grupo de NeuroGastroBioquímica, and Drug Resistance, ICBM, Facultad de Laboratorio de Química Biológica. Instituto Medicina, Universidad de Chile, Santiago, de Química, Facultad de Ciencias, Pontificia Chile. 2 Department of Biology, Faculty of Universidad Católica de Valparaíso, Basic Sciences, Metropolitan University Valparaíso. Chile. 2, Laboratorio de of Education Sciences, Santiago, Chile. Neuroquímica y Neurofarmacología, Centro 3 Pharmacology Laboratory, Research de Neurobiología y Fisiopatología Integrativa Institute of Dental Sciences (ICOD), School (CENFI), Instituto de Fisiología, Facultad of Dentistry, University of Chile, Santiago, de Ciencias, Universidad de Valparaíso. 3, Chile. Programa de Doctorado en Ciencias mención Neurociencias, Facultad de Ciencias, Colorectal cancer (CRC) is the third leading Universidad de Valparaíso. cause of cancer death in the world. The standard drugs currently used for the Intestinal microbiota has been shown to treatment of CRC are 5-fluouracil (5- modulate central nervous system function. FU), oxaliplatin and irinotecan. These For instance, a reduction in the gut’s chemotherapeutic agents are effective in the microbiota richness and diversity through early stages of the disease, presenting high the use of antibiotics, sensitizes mice to drug

101 seeking behaviors. Furthermore, this behavior development of gut, immune and brain is driven by changes in dopamine receptor physiology. We have previously shown that expression within the mesocorticolimbic exposing pregnant Sprague-Dawley dams system, which strongly supports the to an oral cocktail of wide-spectrum non relevance of the microbiota-gut-brain axis in absorbable antibiotics (neomycin, bacitracin, the development of addictive behaviors. In vancomycin all three at a dose of 100 mg/ most animals, early-life gut colonization by kg and pimaricin at 5microg/kg) from microbial symbionts occur begins at birth, embryonic day 18 until post-natal day (PND) with bacteria coming from the mother, and 7, lowers intestinal microbial richness and is a process that happens in parallel with diversity in the male offspring at PND35. early stages of brain development. Therefore, Additionally, early-life exposure to antibiotics changes in maternal gut microbiota lowers dopamine receptor 1 (D1) expression richness and diversity would impact on the in key areas of the mesocorticolimbic circuit development of neuropsychiatric diseases of male offspring when compared to age later in life, including addiction. To test this, matched controls that were not exposed to we administered pregnant Spraque-Dawley antibiotics perinatally. This suggest that dams a cocktail of oral wide-spectrum early-life exposure to antibiotics, which antibiotics (neomicyn, bacitracin, vancomycin affects gut microbial ecology, impacts on and pimaricin) from embryonic day 18 till dopaminergic circuits related to reward. post-natal day (PND) 7, and then evaluated However, another question was raised: dopamine receptor 2 (D2) expression in the what happens in substantia nigra (SN), prefrontal cortex (Pfx) of female and male another major source of dopamine that is offspring at PND60, and compared with the also involved in reward and motor function. offspring of pregnant dams given saline. Thus, we evaluated D2 expression in the SN The results show that Pfx D2 expression of males (PND35) from Sprague-Dawley in female offspring of antibiotic exposed dams exposed to the aforementioned cocktail dams is reduced (although not significant) of antibiotics, and compared it with control when compared to age matched controls, rats. Immunohistochemical analysis revealed but no differences were observed in the that early-life exposure to antibiotics does not male offspring. These results suggest that a affect D2 expression in comparison to control reduction in the diversity and richness of gut rats. This result suggest that reduction of microbes during early-life provokes changes microbial diversity and richness in early life, in PFx D2 expression in females, but not affects specifically the mesocorticolimbic males, and furthermore, this changes might circuit, with no effects on the D2 expression impact the female’s drugs seeking behaviors. in the SN. Such specific effect further suggests that within the microbiota-gut- 30. EARLY-LIFE EXPOSURE TO brain communication, there are very specific ORAL WIDE-SPECTRUM NON- pathways that may in part underlie the ABSORVABLE ANTIBIOTICS AND basis of neuropsychiatric disorders, such as THEIR EFFECTS ON DOPAMINE addiction. RECEPTOR 2 EXPRESSION IN THE SUBSTANTIA NIGRA OF SPRAGUE- 31. MECHANICAL STIMULATION DAWLEY RATS. INCREASES EXTRACELULLAR Da-oliveira, Y. M.1; Urrutia-Piñones, J.1; ATP AND NO SECRETION TO Martinez -Pinto, J2; Sotomayor-Zarate, R2; THE MEDIA OF CELL CULTURES; Julio-Pieper, M.1, Bravo, JA.1. PHYSIOLOGICAL IMPLICATIONS. 1Grupo de NeuroGastroBioquímica, Donoso F.; Donoso M.V.; Huidobro-Toro J.P. Laboratorio de Química Biológica. Instituto Laboratorio de Nucleótidos, Departamento de Química, Facultad de Ciencias, Pontificia de Biología, Facultad de Química y Biología, Universidad Católica de Valparaíso, y Centro de Nanociencia y Nanotecnología, Valparaíso. Chile. 2, Laboratorio de CEDENNA, Universidad de Santiago de Chile. Neuroquímica y Neurofarmacología, Centro de Neurobiología y Fisiopatología Integrativa As a requirement of many protocols it is (CENFI), Instituto de Fisiología, Facultad common to add drugs to cell cultures to de Ciencias, Universidad de Valparaíso, investigate cell processes in the presence Valparaíso. Chile. of a determined pharmacological agent. However, if this procedure is not well Microbial gut colonization begins at birth, controlled by appropriate standards, it may where the transfer of intestinal microbes cause additional experimental variability. from mother to infant is key in early-life Our working hypothesis proposed that

102 pipetting/agitation of the culture medium buffer at 37°C, 95% O2-5% CO2. Muscle causes extracellular ATP secretion, which in tension was recorded by a Grass polygraph the case of endothelial cells is also related coupled to a transducer; contractions were to NO secretion. Endothelial cells of the induced by electrical field depolarization rat mesentery, fibroblasts, and oocytes of or with chemical stimuli. The muscular Xenopus Laevis were used. In endothelial cell contraction induced by the exogenous cultures, 100 uL of Tyrode buffer is gently application of 100 uM norepinephrine applied; extracellular fluid sample is collected decreased following epithelium removal to measure ATP and NO production. ATP (1.5±0.2g versus 0.8±0.1g, n =10, p<0.0038), and metabolites are quantified as fluorescent but not those elicited by ATP or KCl. ethene purines; separated and quantified by Exogenous ATP, at concentrations that do HPLC procedures. NO was determined by a not induce contractions (1-100 uM), reduced fluorescent probe as the [DAF-NO] complex. the motor effect induced by electric trains Buffer application increases medium ATP of 0.15 Hz. Epithelial removal reduced this from 117 ± 75.7 to 317 ± 25 pmoles ATP/ ATP effect (p <0.05). ATP reduces muscle mg protein (p <0.001); the signal peaked contraction induced by 4 Hz trains, in the by one minute, thereafter, ATP decays. phasic component, epithelial removal causes This stimulus also rapidly and significantly a greater inhibitory effect of ATP (p <0.05). increases NO production (p <0.0001). This The inhibition of NO synthesis with 100 uM increase is blunted by 150 µM L-NAME, an N-omega-nitro-l-arginine does not modify eNOS inhibitor, and by apyrase (4 U/mL), the inhibitory effect of ATP on the electrical suggesting the participation of extracellular stimulus. In contrast, indomethacin increases ATP. The agitation of fibroblast culture the inhibitory effect of ATP on 0.15 Hz in the medium in culture increases 4-fold ATP absence of epithelium, and on 4 Hz, both secretion, a transient effect that decreased in the presence and absence of epithelium. in minutes. Likewise, Xenopus oocytes Altogether, present results suggest that a agitation by a variable inclination agitator non-identified arachidonic acid derivative, increased 3.8 times extracellular ATP (p but not NO, sensitizes the motor response of <0.01). In conclusion, different mechanical the duct, demonstrating that the epithelium stimuli secrete extracellular ATP in different participates and modulates its motor activity. cell types, suggesting that cells respond to chemical and sensory stimuli by increasing 33. CHANGES IN THE nucleotide secretion. The nucleotide surge EXTRACELLULAR LEVELS may cause unexpected variations in the final OF DOPAMINE IN NUCLEUS cellular response due to indirect or direct ACCUMBENS OF ADULT RATS purinoceptor activity. NEONATALLY EXPOSED TO SEX HORMONES: STUDIES OF 32. MODIFIED SYMPATHETIC BRAIN MICRODIALYSIS USING NERVOUS TERMINALS AND METHYLPHENIDATE. ENDOTELIAL CELLS FROM THE Elgueta-Reyes, M.1,2; Renard, G.M.3; MESENTERIAL ARTERIAL BED Sotomayor-Zárate, R.1. BY STREPTOZOTOCIN-INDUCED 1 Laboratorio de Neuroquímica y DIABETES. Neurofarmacología, Centro de Neurobiología Donoso, M.V., Huidobro-Toro, J.P. y Fisiopatología Integrativa (CENFI), Laboratorio de Farmacología, Departamento Instituto de Fisiología, Facultad de Ciencias, de Biología, Facultad de Química y Biología, Universidad de Valparaíso. 2 Programa de CEDENNA, Universidad de Santiago de Chile. Magíster en Ciencias Biológicas mención Neurociencia, Facultad de Ciencias, Respiratory, intestinal and / or vascular Universidad de Valparaíso. 3 Laboratorio epithelial cells modify the activity of its de Neurobiología de Conductas Sociales y adjacent smooth muscle layer. We studied Adictivas, Escuela de Medicina, Centro de whether the prostatic vas deferens epithelium Investigaciones Biomédicas y Aplicadas, modulate the motor response induced by Facultad de Medicina, Universidad de electrical stimulation by releasing an epithelial Santiago, Chile. messenger. To this aim, the isometric muscular contraction of the prostatic segment Sex hormones produce several effects of the rat vas deferens intact or mechanically in reproductive and non-reproductive denuded of its epithelium was recorded using tissues. In this sense, brain expresses sex a force displacement transducer. The tissues hormones receptors in cortical and limbic were placed in a super fusion bath with Tyrode areas. Nigrostriatal and mesocorticolimbic

103 pathways are modulated by sex hormones, (Ab) peptide. Experimental evidence has affecting the expression of key dopaminergic shown that the oligomeric form is able to induce proteins in adult animals. However, few reactive oxidative species (ROS). Ascorbic studies have been focused in long-term acid (AA) is an essential micronutrient with effects produced by early exposure to sex a preponderant role in oxidative stress and its hormones. Last time, our lab has been shown main transport system in the brain is SVCT2, some neurochemical and behavioral changes that incorporates the reduced form of vitamin produced by neonatal administration of sex C (ascorbic acid, AA), which is the prevalent hormones in brain dopamine areas such as form in vivo. Recent works show the relevance higher levels of dopamine (DA), expression of AA in AD’s progression by the inhibition of tyrosine hydroxylase and addictive-like of SVCT2, nevertheless, these studies didn’t behaviors induced by morphine. Last time, analyze how vitamin C is acquired by neurons we published that adult rats exposed during and compartmentalized within organelles. first hours of postnatal life to sex hormones In this work, we studied the subcellular had a lower locomotor activity induced by localization of the SVCT2 transporter in methylphenidate (MPD) than control rats primary cultures of mouse hippocampal and this effect was associated with a lower neurons, and the changes in localization and expression of the dopamine transporter expression levels associated to oligomeric (DAT) in nucleus accumbens (NAcc). Ab treatment. Material y methods: 18 days Therefore, the aim of this work was studied embryos hippocampus of C57BL/6 mice were the basal and stimulate (MPD: 5 mg/kg) dissociated and plated. Were cultured in vitro extracellular levels of DA, Glutamate and by 9 days until its treatment with oligomeric GABA in NAcc of adult rats exposed during Ab. At 24 and 48 hours of treatment the the first hours of postnatal life to estradiol subcellular localization of SVCT2 was valerate (EV: 0.1 mg/50μL). Our results evaluated, though immunofluorescence. showed a lower NAcc DA release induced by Results: It was observed that hippocampal MPD in EV rats compared to control rats. In neurons show expression of SVCT2 which control rats we observed in NAcc a reduction is located mainly at the mitochondria. in extracellular levels of GABA after MPD Treatment with Ab oligomers increases administration, however this effect was not the colocalization of SVCT2 with this observed in EV rats. In addition, basal and organelle. Discussion: Our results suggest stimulate glutamate levels in NAcc were that mitochondrial AA might be relevant for not different between experimental groups. neuronal survival in response to ab damage These results suggest that neonatal exposure and suggest that this transporter would be a to EV affect the neurochemical response to new therapeutic target to treat this disease. psychostimulants in adulthood, which could be a vulnerability factor to increase the doses 35. NOVEL CAFFEIC ACID of abuse drugs. DERIVATIVES AGAINST ORAL CANCER CELLS. 34. MITOCHONDRIAL EXPRESSION Escobar B.1, Rojas D.2, González D.2, Cortés OF SVCT2 IN HIPPOCAMPAL G.2, Jara JA.1, Catalán M.2. NEURONS TREATED WITH 1.-Laboratory of Pharmacology, ICOD, OLIGOMERIC AB. Facultad de Odontología, Universidad Escobar-Acuña K.1; Panes J.1; Saavedra de Chile 2.- Laboratory of Biochemistry, P.1; Moraga G. 1; Fuentealba J.1; Rivas C.2; Metabolism and Drug Resistance, ICBM, Muñoz-Montesino C.1. Facultad de Medicina, Universidad de Chile. 1, Laboratorio de Neuropatología Molecular, Departamento de Fisiología, Facultad The cancer is a cellular process characteristic de Ciencias Biológicas, Universidad de by uncontrolled growth and dissemination, Concepción. 2, Laboratorio de Antioxidantes, being the second cause of death worldwide. Departamento de Fisiopatología, Facultad Likewise, the oral cancer is one of the ten de Ciencias Biológicas, Universidad de most common neoplasm in the world, Concepción. mainly concern the tongue. The gold standard therapies are surgery, which can Introduction: Alzheimer’s disease (AD) is a be complemented with radiotherapy and/or neurodegenerative disorder characterized chemotherapy. The latter, represent the first- by progressive memory impairment and line therapies, involving decreasing tumor size cognitive dysfunction. The most distinctive and abolishing microscopic disease. However, phenomenon correlating with AD is the this cancer has small average of survival, presence of aggregates form of the β-amyloid several side effects and drug resistances.

104 Therefore, new drugs are currently being 25 mM fructose at different times. The mRNA studied, such as novel caffeic acid derivatives levels of hypertrophic markers (Beta-MHC, reporter before with antitumoral effect ANP, BNP and RCAN1.4) were determined in breast cancer. We evaluated these new by qRT-PCR. Phosphorylated -AMPK, total- compounds by measurements of cytotoxic AMPK and Beta-MHC protein levels were effect by MTT assay, mitochondrial effects by determined by inmunowestern blot (WB). evaluation of mitochondrial-transmembrane The results showed that fructose increased potential by flow cytometry and ATP levels mRNA levels of the hypertrophic markers through luminescence assay. We showed and Beta-MHC protein levels. Further, it was that the derivates have cytotoxic effect in observed that metformin was able to increase human cancer cell lines Cal27 and Hep-2. AMPK activation even in the presence of We found that the compounds generated the fructose which could prevent the progression decrease in cellular ATP levels and decrease of cardiomyocyte hypertrophy induced by the measuring ATP levels on human cancer this carbohydrate. cell line Cal27 and Hep of the mitochondrial- transmembrane potential. In addition, we 37. INDUCTION OF CELLULAR evaluated the selectivity of these compounds SENESCENCE IN PRIMARY ADULT by exposing normal epithelium cells to their MOUSE CARDIAC FIBROBLASTS. action by evaluation of cytotoxicity. Our Espitia-Corredor, J.A.1,2; Peiró-Vallejo, C.2; results demonstrated the novel caffeic acid Díaz-Araya, G.A.1. derivates exert a selective cytotoxic effect by 1, Laboratory of Molecular Pharmacology, mitochondrial mechanism. Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical 36. CARDIOMYOCYTES and Pharmaceutical Sciences, University HYPERTROPHY INDUCED BY of Chile. 2, Laboratory L-5, Department CHRONIC STIMULATION WITH of Pharmacology, Faculty of Medicine, FRUCTOSE: BENEFICIAL EFFECT OF Autonomous University of Madrid. METFORMIN. Escorcia L.A.1; Muñoz-Rodríguez C.1; Introduction: Cellular senescence – a Labraña P. 1; Catalán M. 2; Olmedo I.1. hallmark of aging – is related with the 1, Programa de Farmacología, Instituto de biomarkers appearance and up-regulation, Ciencias Biomédicas, Facultad de Medicina, such as senescence-associated beta- Universidad de Chile. 2, Programa de galactosidase activity (SABG), activation of Farmacología Molecular y Clínica, Instituto de tumor suppressor proteins (p53, p21CIP1) Ciencias Biomédicas, Facultad de Medicina, and increase of DNA damage associated Universidad de Chile. proteins (gammaH2A.X). Recent studies suggested that interleukin-1beta (IL-1b) and Fructose intake has been increased in doxorubicin (Dox) promote cardiac aging recent decades. Studies have indicated through an inflammatory process. Cardiac that high fructose consumption would be fibroblast (CF) keeps the extracellular matrix associated with an increase in the incidence homeostasis and actively participates on of cardiovascular diseases (CVD) such as damage-associated inflammatory and scaring cardiac hypertrophy. Cardiac hypertrophy processes that are altered in cardiac aging. is an adaptive response to chronic work Few studies have evaluated the increase of overload imposed on the heart, where these biomarkers on CF by pro-inflammatory cardiomyocytes undergo numerous molecules such as IL-1b and Dox as morphological and functional changes. potential inducers of cardiovascular damage. According to literature, fructose is able to Objective: The objective is to evaluate IL-1b decrease cell redox defenses and reduce and Dox effects upon SABG, levels of tumor adenosine monophosphate-activated protein suppressor- and DNA damage-associated kinase (AMPK) activation. AMPK activation is proteins as biomarkers of cellular senescence. related to cardioprotective effects, and during Methods: CF were isolated from 8-10-week- hypertrophy allows the heart to change its old C57BL/6 male mice. CF were serum- metabolism. The objective of this work was to starved by 24 hours prior to stimulation with study the effect of fructose on cardiomyocyte IL-1b (2,5 ng/mL) and Dox (10 nM) for 24 hypertrophy and AMPK activation in the hours. SABG was measured by microscopy presence of metformin, a cardioprotective with a commercial kit (Cell Signaling drug widely used in the treatment of type TECHNOLOGY®) and protein levels of p53, 2 diabetes mellitus. Cultured neonatal rat p21CIP1, and gammaH2A.X by immune- cardiomyocytes (1-3 days) were treated with blot. Results: IL-1b and Dox increase the

105 percentage of SABG positive cells as long threshold. Taken together, the activation of as the protein levels of p53, p21CIP1, and this receptor within MSNs of the Nacc has gammaH2A.X. Conclusions: IL-1b and Dox effects on excitability parameters of these show an inductor effect of cellular senescence cells and also a possible modulation of – featured by increased SABG and p53, serotonin and dopamine transmission within p21CIP1 and gammaH2A.X levels – on CF. this nucleus.

38. NEUROPHYSIOLOGIC 39. MESENCHYMAL STEM MODULATION OF NUCLEUS CELL SECRETOME (MSCSS) ACCUMBENS BY THE ACTIVATION ADMINISTRATION REDUCES

OF SEROTONIN RECEPTOR 5-HT2A OXIDATIVE STRESS AND Estay, C.1,2; Bonansco, C.2; Fuenzalida M.2; NEUROINFLAMMATION INDUCED Sotomayor-Zárate R.2. BY PERINATAL ASPHYXIA IN RAT 1Programa de Doctorado en Ciencias HIPPOCAMPUS. mención Neurociencias, Facultad de Ciencias, Farfán N1, Araya M1, Monzón E1, Carril J1, Universidad de Valparaíso, Valparaíso, Chile. Alvarado R1, Tapia-Bustos A1, Vásquez R1, 2 Centro de Neurobiología y Fisiopatología Santapau D3, Bustamante D1, Quintanilla Integrativa (CENFI), Instituto de Fisiología, ME1, Ezquer F3, Valdés JL2, Israel Y1, Facultad de Ciencias, Universidad de Herrera-Marschitz M1, Morales P1,2. Valparaíso, Valparaíso, Chile. Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas, Serotonin is a neurotransmitter implicated Facultad de Medicina, Universidad de Chile. in most processes related to mood, sleep regulation, sexual behavior and cognitive Perinatal asphyxia (PA) is an important modulation, being important in synaptic obstetric risk occurring at the time of delivery. transmission as well. While, serotonin Surviving children develop long-lasting transmission is not completely, new motor and cognitive deficits. PA implies a evidences showed an important role for primary energy crisis resulting from oxygen serotonin receptor activation in prefrontal interruption, followed by a secondary insult cortex (PFC), dorsal raphe nucleus (DRN) linked to the required re-oxygenation, leading and ventral tegmental area (VTA), specifically to oxidative stress, neuroinflammation the activation of serotonin receptor 5HT2A. and cell death, affecting basal ganglia Electrophysiological studies in PFC showed and hippocampus. Neuroinflammation that serotonin has effects on excitability activates NF-kappa b signalling, which profiles of pyramidal cells of layer V implies p65 nuclear translocation and pro- through the activation of 5HT2AR where inflammatory gene expression, resulting its expression pattern is very high. These in glial activation and apoptosis. Oxidative cells project to nucleus accumbens (Nacc; stress activates Nrf2, the antioxidant defense another nucleus with high expression of master regulator, promoting antioxidant 5HT2AR), where the specific role of 5HT2AR gene transcription, including hemoxigenase activation is not known regarding synaptic 1 (HO-1) and NAD(P)H dehydrogenase transmission. To address this issue, we used quinone 1 (NQO1). MSCs have been proposed electrophysiological whole cell patch clamp as potent agents to treat several conditions and current clamp techniques to record associated with neuroinflammation and in acute coronal slices from Nacc of adult oxidative stress. Preconditioning of MSCs Sprague Dawley rats, MSNs in presence of with pro-inflammatory cytokines or hypoxic serotonin and TCB-2 (agonist of 5-HT2AR). conditions improve their effectiveness. The experiments were made in presence of The aim of this study was to determine picrotoxin and tethrodotoxin to determine whether a single intranasal administration and characterize the specific activation of of secretome derived from preconditioned 5-HT2AR in MSN only of the excitatory human MSCs to asphyxia-exposed rats inputs. Our results show a decrease in the activate (i) the Nrf2 pathway, (ii) reducing amplitude of mEPSCs (miniature excitatory oxidative stress, (iii) neuroinflammation postsynaptic currents), which is related with and (iv) cell death in rat hippocampus. a postsynaptic event, and no significant Two hours after birth MSCSs (6 ug/16 ul, changes in the frequencies of mEPSCs. obtained from 1x10-6 preconditioned- Also, we observed changes in firing rate and MSCs) or 16 ul of vehicle were administered action potential threshold parameters in the intranasally to asphyxia-exposed or control presence of TCB-2, augmenting the firing rats. Animals were euthanized at day P7. rate and diminishing the action potential Oxidative stress was monitored by the GSSG/

106 GSH ratio, Nrf-2 nuclear translocation and methods are computational requirement HO-1 and NQO1 protein levels by Western extensive. We develop a Abpred, an algorithm blots (WB) and immunofluorescence (IFI). based in protein interfaces features and Neuroinflammation by microglial reactivity machine learning selection to propose a (anti-Iba-1, IFI) and p65 nuclear translocation point-mutations combinatory to optimize the (WB). Cell death by cleaved caspase-3 protein affinity. We train Abpred using an artificially- level (WB). The administration of MSCSs: balanced dataset derived from SKEMPI-2.0; (i) lowered the PA increased GSSG/GSH 1392 single-point mutations on 50 Ab-antigen ratio, and (ii) increased both Nrf2 nuclear complexes. Evaluation on blind-test (20% of translocation and HO-1, NQO1 levels, (iii) dataset) achieved an RMSE of 1.66 kcal/mol, reduced the microglial reactivity and nuclear and 0.593 correlation. Abpred enable the P65 and (iv) reduced cleaved caspase levels. affinity optimization in silico to accelerate the design of news TAbs. 40. A COMPUTATIONALLY GUIDED ANTIBODY AFFINITY OPTIMIZATION 41. SELECTION OF MOLECULES FOR METHOD BASED ON MACHINE THE FUNCTIONALIZATION OF GENE- LEARNING SINGLE-POINT MUTANTS CHARGED-GOLD-NANOPARTICLES SELECTION. THAT ACHIEVE TARGETING OF Fica-León V.1; Garrido, J.L.3; Barría, M.I.2; PROSTATE CANCER CELLS. Salas-Burgos, A.1. Fleitas-Salazar N.; Godoy E.; Pedroso- 1, Nanocell Laboratory, Pharmacology Santana S.; Cerro R.P., Oliva R.; Fernández Department, Biological Science Faculty, K, Gonzalez I.; Toledo J.R. Concepción University. 2, Virology Universidad de Concepción. Laboratory, Microbiology Department, Biological Science Faculty, Concepción Metal nanoparticles (NPms) have attracted University. 3, Ichor Biologics LLC, New York, the interest of biomedical researchers due to NY 10065, USA. their potential application in the diagnostic and treatment of diseases like cancer. These Antibodies are the biomolecules with NPs can enter the cell depending on their therapeutic applications (TAbs) with highest size, charge, and surface functionalization. growth and great effectivity. Currently, we NPs between 10 to 100 nm have a large know 78 monoclonal antibodies approved surface area that can be functionalized with by the FDA, and many others are in last different molecules for targeting (antibodies, clinical stages. The applicability of antibodies ligands of cell surface proteins), detection depends on two properties: specificity, and (fluorescent probes) and treatment (drugs, affinity. Antibody affinity maturation isa proteins, nucleic acids) of affected cells. In natural process and can be experimentally this way, NPs could allow detection of target emulated from diversity libraries and epitope cells and be a therapy, at the same time. Such selection, process called optimization. This NPs has been described as ¨theranostic NPs¨. selection is affected by external factors Among molecules used to successfully target and unexplored conformational states, the cancer cells are folic acid, RGD peptides, and discovered sequences have variable affinities EGF‑receptor antibodies. In this work, we with values we can improve. On the other show the formulation and characterization hand, the next generation sequencing, the of gold nanoparticles designed for gene molecular three-dimensional modelling, and delivery treatment of prostate cancer cells. molecular docking have enabled perform Selectivity for prostate cancer cells of NPms rational antibody design without protein functionalized with folic acid or anti-LOX-1 crystal complex. This process is assisted by antibody were compared. These NPms were computers and complex algorithm based on tested in four prostate cancer cell lines: biophysical properties of the intermolecular LnCap, Du-145, C4-2B, and PC3 while RWPE- forces that drive the binding energy, the 1 was used as a control cell line. Molecules that interface between the epitope on the antigen, mediate cell targeting of NPs are determinant and the complementary determinants in the success of theranostic nanoparticles. region on the antibody are dependent of These NPs could favor cancer early detection, the residues type and position in this no improving treatment response and/or continuous loop. To improve and optimize achieving a complete recovery. the affinity we need explored the residues substitution and evaluate the energy free changes. The combinatory is about 10^9 unique sequences, while the free energy

107 42. INFLUENCE OF DIVERSE 43. CYTOTOXICITY OF FACTORS ON ADHERENCE TO ORAL NITROFURANS AND C-5 ANTIDIABETIC MEDICATION IN SUBSTITUTED FURANS. MEXICAN PATIENTS WITH TYPE 2 EVIDENCE OF NITROREDUCTION- DIABETES. INDEPENDENT CYTOTOXIC Flores-Durán D. M.; Guzmán-Trujillo M.; EFFECTS. Briones- Aranda A. Gallardo C.A. 1,2, Pessoa-Mahana H. 2, Facultad de Medicina Humana de la Faúndez M. 1. Universidad Autónoma de Chiapas, México. 1, Laboratorio de Farmacología y Toxicología Molecular, Facultad de Química, Several factors can affect adherence to Pontificia Universidad Católica de Chile. 2, pharmacological treatment in patients with Departamento de Química Orgánica y Físico type 2 diabetes (T2D), including gender, Química, Facultad de Ciencias Químicas y the time of evolution of the disease, and Farmacéuticas, Universidad de Chile. the beliefs of the patient about health. The aim of the present study was to examine the Nitrofurans constitute a family of drugs that main factors that influence adherence in T2D have antibacterial and antiparasitic effects. patients affiliated with a public hospital in The consumption of these causes various Chiapas, Mexico. A cross-sectional study was adverse effects which are mainly attributed to carried out on a sample of 200 patients being the monoelectronic reduction of the 5-nitro treated with oral hypoglycemic agents, of group mediated by host enzymes. This triggers which 50% were women and the average age the generation of nitroanion radical which, of the women was 61.2 ± 7.7 years and of the upon entering redox cycling with molecular men 65.2 ± 1.4 years. The patients were asked oxygen, results in the formation of superoxide to provide the most recent result of fasting anion radical and the regeneration of the glycemia and to fill out a sociodemographic nitro derivative. The generated oxidative questionnaire, the Morisky-Green test and stress allows to explain, at least in part, the the Beliefs about Medicines Questionnaire adverse effects attributed to nitrofurans. (BMQ). The possible associations among the However, not all side effects are attributable variables were analyzed by the Chi-squared to that mechanism. In order to demonstrate test, while the comparison of the results of the that the cytotoxic effects of nitrofurans BMQ between compliant and non-compliant are not solely due to the reduction of the patients was examined with the Mann- 5-nitro group, derivatives of 3 commercial Whitney U test. The results show that 77% of nitrofurans (nitrofurazone, nitrofurantoin the women and 85% of the men presented a and nifuroxazide) were synthesized in lack of adherence to medication. The habit which the 5-nitro group was substituted by of smoking had a significant association Methyl, Bromo or Hydrogen, maintaining the with the lack of adherence. The men and parental skeleton. The synthesis was carried women who were compliant mentioned a out in aqueous solution through ultrasound, greater need for the medication (U1=369.50, mixing the aldehydes with the corresponding U2=553; p<0.005). However, only the hydrazines. Subsequently, the effect of these compliant women had levels of glycemia derivatives on cell viability and their ability significantly lower than the group of non- to generate intracellular ROS was evaluated. compliant patients (t=757; p<0.05). Hence, it The results show that the cytotoxic effect is possible that the responses of the men were of furan derivatives is dependent on the more prone to falsehoods than those of the presence of electron withdrawing groups women. This could justify a future revision of in position 5 of the furan ring; while the the instruments used to test the adherence of generation of intracellular ROS does not men. depend exclusively on the presence of the 5-nitro group. This suggests new toxicity mechanisms independent of oxidative stress induced by redox cycling of nitrofurans.

108 44. ELEPHANT BLACK Aβ+BG: 76±7%) and also the decrease on GARLIC EXTRACT PREVENTS transient intracellular calcium induced by SO- MITOCHONDRIAL DYSFUNCTION Aβ (54±5%). Finally, our results suggest that INDUCED BY BETA-AMYLOID the bioactive compounds present in BG could PEPTIDE IN MOUSE HIPPOCAMPAL be new pharmacological tools to treat the SO- SLICES. Aβ toxicity. Gavilán J.1; Panes J.1; Salgado G.1; Godoy P.A.1; Muñoz N.2; Ramírez-Molina O.1; Varas 45. ROLE OF ROCK-1 AND ROCK-2 P.4; Pérez C.3; Yévenes G.2; Fuentealba J.1. IN ADHESION AND MIGRATION OF 1, Laboratorio de Screening de Compuestos TRYPANOSOMA CRUZI- ACTIVATED Neuroactivos, Departamento de Fisiología, MACROPHAGES TREATED WITH Facultad de Ciencias Biológicas, ATORVASTATIN. Universidad de Concepción. 2, Laboratorio González-Herrera, Fabiola 1; Cerutti, de Neurofarmacología, Departamento de Camilla2; Clayton, Natasha2; Guzmán- Fisiología, Facultad de Ciencias Biológicas, Rivera, Daniela1; Vivar, Raúl1; Ridley, Anne2; Universidad de Concepción. 3, Laboratorio Maya, Juan Diego1. de Química de Productos Naturales, 1, Programa de Farmacología Molecular Departamento de Botánica, Facultad de y Clínica - ICBM, Facultad de Medicina, Ciencias Naturales y Oceanográficas, Universidad de Chile. 2, School of Cellular and Universidad de Concepción. 4, Agrícola Molecular Medicine, University of Bristol. Melimei, Manao, Ancud, Chiloé. Chagas Disease (CD) is caused by protozoan Soluble oligomers of the beta-amyloid Trypanosoma cruzi (T. cruzi). The most peptide (SO-Aβ) are central elements in the severe CD clinical manifestation is Chronic pathogenesis of Alzheimer’s Disease (AD). Chagas cardiomyopathy (CCC). Currently It has been demonstrated that SO-Aβ forms treatment is benznidazole, a trypanocidal pores in the plasma membrane which leads therapy, ineffective against CCC. Therefore, to Ca2+ influx and leakage of large molecules, is interesting to develop new therapeutic such as ATP. Cytosolic Ca2+ overload induces pharmacologic strategy. It has been observed mitochondrial dysfunction and synaptic in autopsies of patients with CCC that present failure resulting in cell death. Some studies an important leukocytes infiltration in cardiac suggest that common black garlic (Allium tissue, and more of 50% are macrophages. sativum) prevents the toxicity of SO-Aβ due to The macrophage infiltration needs the its enriched composition of sulfur metabolites. macrophages adhesion and migration from However, there are no studies on the biological the vascular endothelium to damage site. activity of elephant black garlic (Allium Rho-associated protein kinase (ROCK) 1 and ampeloprasum), and recently in our laboratory 2 are serine-threonine kinases activated by we identified new sulfur compounds that are small GTPase RhoA. ROCK phosphorylate not present in common garlic. The objective myosin light chain (MLC), promoting cellular of this work was to evaluate neuroprotective contraction and generating focal adhesion. properties of elephant black garlic extract (BG) On the other hand, ROCK phosphorylate against SO-Aβ toxicity. In mouse hippocampal cofilin, increasing actin polymerization. slices, BG (20 µg/mL) prevented the decrease Therefore, ROCK activation affects cellular in cellular viability induced by SO-Aβ (2.5 migration and adhesion. In our laboratory µM) by 60±6%. In parallel, BG kept the we have observed, T. cruzi activates mitochondrial membrane potential stable ROCK in human macrophages, leading to compared with SO-Aβ (SO-Aβ: 67±3%; SO- proinflammatory phenotype and atorvastatin Aβ+BG: 96±5%), suggesting a direct effects inhibited ROCK, changing the phenotype of on mitochondrial function; additionally, the this cells. However, it has not been studied intracellular (SO-Aβ: 70±6%; SO-Aβ+BG: ROCK-1 and ROCK-2 role in T. cruzi effect 110±9%) and extracellular ATP levels (SO- and atorvastin over macrophages adhesion Aβ: 180±23%; SO-Aβ+BG: 125±16%), were and migration. Human macrophages U937 recovery when BG was present. We also were used, with a knockdown for ROCK-1 and observed that BG normalized the levels of the ROCK-2 and with both proteins constitutively mitochondrial fusion protein MFN1 (SO-Aβ: active by nucleofection. T. cruzi-activated 49± 10%; SO-Aβ+BG: 91±13%). On the other macrophages were treated with atorvastin hand, our results showed that BG preserved and adhesion process to endothelial cells the synaptic structure and prevented the (HUVEC) were determined by microscopy decrease in SV2 (SO-Aβ: 64±4%; BG: 97±9%) and western blot. Also, migration was studied and PSD95 proteins (SO-Aβ: 45±8%; SO- by microscopy time lapse.

109 It was observed that both isoforms are involved of NMDAR that impair the GABAergic in adhesion and migration increase promoted interneurons maturation and function, by T. cruzi and is inhibit by atorvastin. These decrease the GABA release, which can reverse results allow us to propose atorvastin as a the temporal dependence of STDP-LTD therapy to decrease macrophages infiltration modifying the synaptic plasticity and function in CCC. of mPFC network in adulthood mice.

46. IMPAIRMENT OF SPIKE 47. CHARACTERIZATION OF THE TIMING-DEPENDENT PLASTICITY ADIPOGENIC PROTEIN E4ORF1 IN PREFRONTAL CORTEX IN A FROM ADENOVIRUS 36 THROUGH KETAMINE TREATED MICE. AN IN-SILICO APPROACH. Guiffa F1,2, Morales K1, Sotomayor-Zárate Gutierrez A. 1, Monteiro G.F. 2.3, Machuca J. R1, Bonansco C1 and Fuenzalida M1. 4, Venthur H. 4, Feres F. 3, Hirata M.H. 2.3, 1- Centro de Neurobiología y Fisiopatología Hirata R. 2.3, Cerda A. 1.5. Integrativa (CENFI), Instituto de Fisiología, Centro de Excelencia en Medicina Facultad de Ciencias, Universidad de Traslacional, CEMT-BIOREN, Temuco, Chile. Valparaíso, Valparaíso, Chile. 2- Magíster en Ciencias Biológicas mención Neurociencia, Adenovirus 36 (Ad-36) is related to human Facultad de Ciencias, Universidad de obesity due to its adipogenic activity mediated Valparaíso. by the Early 4 Open Reading Frame 1 (E4orf1) protein. Mechanisms underlying adipogenic The medial prefrontal cortex (mPFC) is a effect of E4orf1 are not completely understood; key structure involved in cognitive functions however, it has been characterized the like working memory and decision-making. increased proliferation and differentiation Long-term changes in synaptic plasticity, i.e: of fat cells through the activation of the long-term potentiation (LTP) and long-term Phosphatidyl Inositol 3 Kinase pathway by depression (LTD) have been proposed as the binding proteins containing PDZ-domains. cellular substrate of these cognitive processes. We aimed to characterize E4orf1 structure During the postnatal development, the and analyze its interactions with PDZ-domain maturation of mPFC circuits depends largely containing proteins in order to recognize on the network of inhibitory interneurons, important residues with pharmacological which modulate the pyramidal neurons purpose. In-silico approaches such as (PYN) function. The mPFC interneurons homology modeling, molecular dynamics and are especially vulnerable to injury during molecular docking between E4orf1 and five adolescence and the impairment of GABAergic PDZ-domains from different proteins (PDZ- interneurons function is involved in several 1 and 2 from Disk Large Homolog 1; PDZ-3 neuropsychiatric diseases, including from Membrane Associated Guanylate Kinase schizophrenia (SZ). However, it is still 1; and PDZ-7 and 10 from Multy PDZ-Domain unknown how the disruption of GABAergic Protein 1) were performed. Mutagenesis interneurons development during adolescence of selected residues was performed to can affect the long-term synaptic plasticity in evaluate its importance in the stabilization the adult brain. Using electrophysiological and of E4orf1:PDZ complexes. We predicted the pharmacological approaches, we evaluate the first 3D model of E4orf1, which suggests a efficacy of synaptic transmission and spike- key role of residues at c-termini region (114 timing-dependent plasticity in mPFC in an SZ to 125), demonstrating its importance in mice model based in the adolescence treated initial stabilization. The complex formed by with non-competitive NMDAR antagonist predicted E4orf1 and PDZ10 was more stable ketamine (Ket). Through recording in PYN of than others. Moreover, residues at “core” the layer II / III of mPFC slices in adulthood, we region (residues 80 to 85) in E4orf1:PDZ10 found that the frequency of spontaneous and complex were important in stabilization as miniature inhibitory post-synaptic currents demonstrated by its electrostatic interactions. (sIPSC and mIPSC) was lower in Ket treated Mutagenesis highlighted residues 80-85, animals than control. Also, we observe that demonstrating its importance in complex the paired-pulse ratio of eIPSC was higher in stabilization. In conclusion, E4orf1 forms a Ket mPFC slice than control. Using the STDP stable complex with PDZ10 domain, being protocol we found that while the protocol in residues 80-85 of particular importance. t-LTP induced a similar potentiation that in Characterization of E4orf1 interactions control, the t-LTD protocol was unable to provides a first approach in discovering induce depression, conversely it can induce druggable targets for Ad-36 induced obesity. LTP. These data suggest that hypofunction

110 48. INTERFERON AS THERAPEUTIC where well-characterized morphological and CANDIDATE FOR CANINE VIRAL functional alterations occur. At present there DISEASES. is no specific and early urinary marker that Gutierrez N. 1; Espinoza F. 1; Hidalgo A. 1; allows determining the risk of developing Lamazares E. 1;Toledo J.R. 1. diabetic nephropathy once diabetes is 1, Laboratorio de biotecnología y biofármacos, established. Therefore, the objective of this Facultad de Cs Biologicas, Universidad de work was to evaluate SGLT-2 as a specific Concepción. urinary marker of renal damage in a murine model with early diabetic nephropathy. Type Chilean dog population is estimated in 3,2 1 diabetes was induced in Wistar rats by million, with a continue growing in pet market administration of streptozotocin (60 mg / kg, associated with care products. In health ip). Diabetic rats were sacrificed 21 days after expenditure, vaccines reach 25% of total cost induction. Proteinuria and creatinine were in pet’s care. Among pathologies that affects determined as renal function tests. Western pets, viruses are the main source of infectious blotting analyzed the expression of SGLT-2 and diseases, including distemper, parvovirus β2-microglobulin. An increase in the urinary and tracheobronchitis. Vaccines against excretion of SGLT-2 was found in the third parvovirus are currently available, however week of damage with diabetic nephropathy still is considered one of the most important and the presence of β2-microglobulin in the disease worldwide, with a high prevalence urine as a marker confirmed the presence level in our country. Nowadays, there is of damage. These results suggest SGLT- not a large spectrum and specifics antiviral 2 as a new urinary marker of diabetic drugs, having to use human antivirals or nephropathy that allows predicting the risk of from another species. Specifically, cytokines developing diabetic nephropathy. Keywords: used as therapeutics activates immune Diabetic nephropathy, streptozotocin, β2- system and helps to defense against virus, microglobulin, SGLT-2 preventing replication and infection. In this work we developed a prototype drug for 50. TMA-6(2,4,6- canine specifically antiviral treatment, based TRIMETHOXYAMPHETAMINE) in a recombinant dog-derived interferon. The MODULATES NEUROPLASTICITY molecule was produced in a E. coli expression IN THE PREFRONTAL CORTEX system. Besides, specific interferon activity AND ENHANCES HEAD-SHAKES was measured by OAS-2 and PKR mRNA BEHAVIOR IN SPRAGUE-DAWLEY quantification, in Madin Darby Canine RATS. Kidney (MDCK) cell line and dog lymphocytes Hernández, A.1; Klink, A.2,3; Castro-Castillo, primary cultures stimulated with Canine V.4; Barra, R.5,6; Sáez-Briones, P.2,5. recombinant interferon was obtained with 1 Laboratory of Neurobiology, Faculty purity around 88% and the antiviral markers of Chemistry and Biology, Universidad were enhanced in canine cells, hence the de Santiago de Chile. 2 Laboratory of cytokine could be an option for antiviral Neuropharmacology and Behavior, Faculty therapy in canine population. of Medical Sciences, Universidad de Santiago de Chile. 3 Studiengang Biophysik, Johann 49. THE GLUCOSE TRANSPORTER Wolfgang Goethe Universität (Frankfurt am (SGLT-2) AS A POSSIBLE URINARY Main, Germany). 4 Department of Organic MARKER OF EARLY DIABETIC Chemistry and Physical Chemistry, Faculty NEPHROPATHY. of Chemical and Pharmaceutical Sciences, Hernández- Carmona Luis A.¹, Rodríguez- Universidad de Chile. 5 School of Medicine, Muñoz Rafael², Namorado-Tónix María del Faculty of Medical Sciences, Universidad Carmen² Graciela-Cervantez Luz Graciela² de Santiago de Chile. 6 CIBAP, Faculty of and Reyes-Sánchez José Luis² Medical Sciences, Universidad de Santiago de 1) Pharmacology Dept., 2) Physiology, Chile. Biophysics and Neurosciences Dept. Center for Research and Advanced Studies, National The search for new analogs of the Polytechnic Institute (CINVESTAV-IPN) entactogen MDMA (3,4-methylenedioxy- methamphetamine, “ecstasy”) is of great Hyperglycemia is characterized by high blood relevance for the development of new glucose concentrations (≥126 mg / dL) and drugs useful in the treatment of prevalent over time these glucose levels damage renal neuropsychiatric diseases. While the microvasculature. Diabetic nephropathy central effects described for MDMA is the final complication of this pathology, clearly differ from other psychotropic

111 compounds, it has been proposed that the which consists of the covalent binding of presumed low potency hallucinogen TMA- polyethylene glycol (PEG) chains to the 6 (2,4,6-trimethoxyamphetamine) might enzyme, at specific or random sites. This allows induce MDMA-like effects at low doses reducing the recognition of the enzyme by in humans. Since both hallucinogens and the immune system and its early elimination MDMA might exert different effects on from the bloodstream. In this work, we cognitive processes, one may anticipate performed the N-terminal PEGylation of differential effects with respect to E.coli ASNase, with methoxy polyethylene neuroplasticity at key sites of the central glycol-carboxymethyl N hydroxysuccinimidyl nervous system. Here we compared the ester (mPEG-NHS 10kDa) in 100 mM PBS acute effects of TMA-6 with MDMA on (i) at pH 7.5 and PEG: ASNase ratio of 25:1. As in vivo induction of long-term potentiation a result, we obtained the monoPEGylated (LTP) in the prefrontal cortex of the rat, and ASNase with an activity of 134 ± 11.5 IU/ (ii) induction of paroxysmal head rotations mg and acceptable kinetic parameters. termed “head-shakes”, which is considered MonoPEGylated ASNase also showed more a behavioral proxy in rodents for human stability at different pH, temperatures and hallucinogenic-like effects. The results against human serum proteases than the obtained showed that 20 mg/kg TMA-6 not native enzyme, demonstrating its potential as only prevented the induction of LTP in the a less immunogenic biopharmaceutical in the prefrontal cortex but turns it into a long- treatment of ALL. term depression-like event. In addition, TMA-6 significantly increased the number 52. CHARACTERIZATION OF CA125 of head-shakes, verifying the hallucinogenic ANTIGEN EXPRESSED IN HUMAN nature of this compound. In contrast, 10 CERVICAL CARCINOMA CELLS. mg/kg MDMA significantly increased the Hidalgo A.1; Meza, C.1; Benavente, B.1; Leiva, prefrontal cortical LTP but fully abolished MJ.1; Montesino, R.1; Toledo J.R1. the number of head shakes. Taken together, 1, Laboratorio de Biotecnología y Biofármacos, and unlike the presumption based on the Dpto. de Fisiopatología, Facultad de Ciencias subjective interpretation of its effects in Biológicas, Universidad de Concepción. humans, the inverse electrophysiological/ behavioral profile of TMA-6 referred to Epithelial ovarian cancer is the seventh MDMA suggests that the former seems death cause related to cancer in women to lack entactogenic-like effects, rather worldwide. Due to the absence of early supporting the hallucinogenic essence of this stage clinical symptoms, patients are usually drug. diagnosed when decease had spread further than ovary with an unfavorable prognostic. 51. SITE-SPECIFIC PEGYLATION OF Cancer antigen 125 (CA125) is a serum L-ASPARAGINASE: AN ALTERNATIVE marker extensively used in gynecology for FOR THE TREATMENT OF ACUTE monitoring epithelial ovary cancer patients. LYMPHOBLASTIC LEUKEMIA. It’s a repetitive peptide antigen of the Herrera L. 1,2; de Oliveira C.2; Beltrán J.F. 1; membrane glycoprotein MUC16, whose Pessoa A.2 and Farías J.G. 1. over-expression in ovarian cancer has been 1 Department of Chemical Engineering. linked with both pro-metastatic and pro- Universidad de La Frontera, Temuco. Chile; 2 tumorigenic properties. In the present study, Department of Biochemical-Pharmaceutical we characterized the N-oligosaccharides Technology. University of São Paulo, São bonded to the C-terminal region of MUC16, Paulo. Brazil. expressed in cervical carcinoma cell culture by adenoviral transduction. Enzymatic L-asparaginase (ASNase) is a therapeutic deglycosylation, HPLC, and MADI-TOF enzyme considered a cornerstone in the analysis showed mainly complex type of treatment of Acute Lymphoblastic Leukemia oligosaccharide N-linked, with bi-antennary, (ALL), the most common cancer in children mono-sialylated and mono-focusylated worldwide. Four formulations of bacterial core structures, predominantly. It has been origin are available in the market for the previously reported that N-glycan profiles treatment of ALL. However, despite their from cancer patients shows an increase of effectiveness, they generate immunological these structures, compared to healthy groups. reactions, decreasing the action of the drug Furthermore, these N-glycan structures have and damaging patient safety. PEGylation been described as part of innate and adaptive is one of the strategies adopted to reduce immune response recognition by CA125 the immunogenicity of asparaginase, antigen. Concluding, the glycosylation status

112 of the CA125 may provide specific biomarkers in order to study steric effect into the and therapeutic target for gynecologic cancer. binding site. Our results indicate that, some compounds are able to displace radioligands 53. DESIGN, SYNTHESIS AND from nicotinic receptor and MAT, showing a BINDING AFFINITIES OF promiscuous behavior. However, the ranges CYCLOALKYLAMINES AND of affinities were in the micromolar order. PIPERIDINES ESTERS DERIVATIVES In addition, docking experiments were ON ALPHA4BETA2 NICOTINIC performing in order to rationalize the binding RECEPTOR AND MONOAMINE mode and the similar interaction between TRANSPORTERS (HSERT AND nAChR, SERT and DAT with our compounds. HDAT). Hodar, M.1; Guerra-Diaz, N.2 ; Pessoa- 54. RATIONAL DESIGN AND Mahana, H.2; Reyes-Parada, M.3; Iturriaga- BIOLOGICAL EVALUATION OF Vásquez, P.1. TRIAZOLOPYRIDINES AGAINST T. 1, Laboratorio de Farmacología Molecular CRUZI. y Síntesis Orgánica, Depto. Cs. Químicas Lapier M.1-2; Ramos-Aguayo A.1; Quintero y Recursos Naturales, Fac. de Ingeniería H. 3; Maya J.2. y Ciencias, Universida de La frontera. 2 1Laboratorio de Antioxidantes y radicales Departamento de Química Orgánica y libres, Departamento de Química Inorgánica Fisicoquímica, Facultad de Ciencias Químicas y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile. 3, y Farmacéuticas, Universidad de Chile. Centro de Investigación Biomédica y Aplicada 2Laboratorio de Bioquímica, Metabolismo (CIBAP), Escuela de Medicina, Facultad de y Resistencia a Fármacos, Instituto de Ciencias Médicas, Universidad de Santiago Ciencias Biomédicas ICBM, Facultad de Chile. de Medicina, Universidad de Chile. 3Laboratorio de Productos Naturales, The monoaminergic and cholinergic Departamento de Química Farmacológica y neurotransmitter systems exhibit, in the Toxicológica Facultad de Ciencias Químicas y central nervous system (CNS), a wide range Farmacéuticas, Universidad de Chile. of functional interactions and mutual regulations. Furthermore, acetylcholine Since a while ago, Azole-containing (ACh) actions mediated by nicotinic receptors compounds have been recognized for their (nAChRs), as well as monoamines such as antifungal features and with well-known serotonin (5-HT) and dopamine (DA), are applications in current clinical field. Some of involved in the modulation of several brain features highlighting azole heterocycle come functions, including (but not limited to) from its specific metabolism, those related cognition, voluntary movement, motivation to its high binding affinity to heme-containg and reward, mood, attention and learning, proteins such as cytochrome p450. The p450 as well as in the physiopathology of a complex has the function of metabolizing variety of diseases. In addition, most of the drugs, exogenous and endogenous molecules drugs currently used for the treatment of that could render in cell toxicity. Lanosterol neurological and neuropsychiatric disorders 14α demethilase (CYP51) is the key enzyme such as Parkinson`s and Alzheimer`s in sterol biosynthesis in Trypanosoma cruzi diseases, depression, drug addiction, (Tc), agent that causes Chagas disease. schizophrenia, etc., have mechanisms of The inhibition of this enzyme will induce action associated to the regulation of one or accumulation of toxic metabolites that cause more of these systems. Indeed, there are some the death of the parasite. Recently, we have examples of therapeutically useful drugs, synthesized and characterized derivatives which act through simultaneous interactions of triazolo pyridines, based on a rational with SERT/DAT and nAChRs. Therefore, study (structure-activity), where we found it seems attractive to search/formulate that compound 1 has antiproliferative effect ligands that show such a promiscuous profile against the replicative form. From this fusing structural aspect of nicotinic ligands structure, we obtained 24 molecules with and antidepressants. Here, we design and variations in positions 3 and 7 in the ring synthesize cycloalkylamines and piperidines [1,2,3] triazolo [1,5-a] pyridine with different esters derivatives. Binding experiments were electrophiles such as pyridines, thiophenes, assessed for alpha4beta2nAChR on brain benzenes and pyrazines. Studies (structure/ synaptosomes and hSERT and hDAT from activity) of this new series have shown that specific cell lines. The ester moieties were the compound 2 increased the trypanocidal acetyl, propionyl and benzoyl derivatives potency. On the other hand, preliminary

113 docking studies showed that the association Through a systematic analysis of 26 analogs energy of the compound to CYP51 are similar of 2,6-DTBP, the present work allowed the to the binding energy values of fluconazole identification of novel a3GlyRs PAMs with to CYP51, our approximations have shown improved efficacy. In addition, the generation that both the azole fraction and pyridine of a pharmacophore based on these findings are potentially related to the coordination expand the possibilities for further design with hemo. This has led to design of a new and development of new GlyRs PAMs. proposal of antichagasic drugs with a possible action on CYP51. 56. ATORVASTATIN AND ROSUVASTATIN TREATMENT 55. EXPLORING NOVEL ALLOSTERIC CONTRIBUTES TO THE DECREASE MODULATORS OF ALPHA3 GLYCINE OF PROLIFERATION IN HUMAN RECEPTORS: STRUCTURE-ACTIVITY UMBILICAL ARTERY SMOOTH RELATIONSHIP OF 2,6-DI-TERT- MUSCLE. BUTYLPHENOL DERIVATES. Leal, K. 1 ; Saavedra, K. 1 ; Salazar, L.A. 1. Lara, C.O. 1; Burgos, C.F.1, San Martin V.P.1, 1 Centro de Biología Molecular y Marileo A.M.1, Sazo, A.E.1, Moraga-Cid, G.1; Farmacogenética, Universidad de La Yévenes, G.E. 1. Frontera, Temuco. 1 Department of Physiology, Biological Sciences Faculty, University of Concepción. The treatment of choice for hypercholesterolemia is the use of statins. Glycine Receptors (GlyRs) are pentameric Enzymes that inhibit the enzyme 3-hydroxy- anion-permeable ligand-gated ion channels methylglutaryl coenzyme A reductase, highly expressed in spinal cord and brain which is a limiting factor in the biosynthesis stem, where mediate processes such as motor of liver cholesterol. Despite this, clinical coordination and sensorial processing. The studies have revealed that statins can exert modulation of glycine receptors composed atheroprotective effects, beyond the decrease by alpha3 subunit (a3GlyRs) by positive of serum cholesterol. These effects have been allosteric modulators (PAMs) has been called pleiotropic effects and include anti- associated to generation of analgesia in proliferative and anti-migratory properties chronic pain models. Previously, we shown in vascular smooth muscle cells, which are that the non-sedative propofol analog a key cell type in atherosclerotic plaque 2,6-di-tert-butylphenol (2,6-DTBP) is a development. For this reason, Human PAM of a3GlyRs. Here, we analyzed series Umbilical Artery Smooth Muscle cultures of 2,6-DTBP analogs using bioinformatics were stimulated with 10 ng/ml of platelet- and electrophysiological methods. Whole- derived growth factor and treated with 20 cell recordings shown that 2,6-DTBP (0,1 μM atorvastatin and 20 μM rosuvastatin mM) potentiate a3GlyRs-evoked currents for 24 hours. The proliferative effect was in a 171±21%. The first set of experiments subsequently evaluated for 48 hours by evaluated the impact of the tert-butyl group spectrophotometry using the CellTiter 96® positions around the phenolic core. These AQueous One Solution Reagent colorimetric studies concluded that the presence of two assay. The migratory effect was also evaluated tert-butyl groups at the position 2 and 4 for 4 hours by optical microscopy using around the phenolic core (i.e. 2,4-DTBP) transwell with 8 µm pores. Migrated cells were sufficient to generate an a3GlyR PAM were counted by means of ImageJ software with a significantly higher efficacy. A second with automated macros function commands. set of molecules evaluated the impact of It was observed that 20 μM statins treatment additional chemical groups on the 2,4-DTBP reduces cell proliferation in a 48-hour period scaffold (i.e. methyl, ethyl, oxime, amine, (p=0,05). However, there is no decrease in carboxylic, sulfonamide groups). Whole- migratory cell capacity after being treated cell recordings shown that ≈30% of these with atorvastatin (p=0,05) and rosuvastatin compounds displayed improved efficacy in (p=0,05). For this reason, atorvastatin and comparison to 2,6-DTBP (≈3-4 fold of a3GlyR rosuvastatin treatments might contribute to potentiation). Bioinformatics analysis shown the proliferation reduction in smooth muscle that all the compounds evaluated have an cells, which are involved in atherosclerotic ADME profile compatible with drugs-like plaque formation. Financial support: molecules. Pharmacophore modeling shown FONDECYT 1171765 & DIUFRO DI19-2018 that the localization of polar, hydrophobic and charged groups within the 2,4-DTBP is pivotal for the a3GlyR modulation.

114 57. EXPRESSION AND Immunology Department, Pharmacy Faculty, CHARACTERIZATION OF A NOVEL Concepción University. SINGLE-CHAIN ANTIBODY AGAINST VASCULAR ENDOTHELIAL GROWTH Type II Diabetes Mellitus (T2DM) have an FACTOR (VEGF) IN GOAT MILK. estimated 8.5% of the world adult population Leiva-Carrasco M. J1., Parra N2., Montesino affected in 2014 and a projection of reaching R1., Sánchez O2., Macaya-Zapata L2., Toledo 17.9% in 2060. Many different approaches J.R1. have been developed for tackling the no- 1, Laboratorio de Biotecnología y Biofármacos, insullinic management of hyperglycemia in Departamento de Fisiopatología, Facultad the diabetic patient, exploring the diverse de Ciencias Biológicas, Concepción. 2, variety of targets that the multifactorial Laboratorio de Biofármacos Recombinates, nature of T2DM offers, which includes Departamento de Farmacología, Facultad classicals insulin sensitizers like metformin, de Ciencias Biológicas, Universidad de and also new pharmacological developments. Concepción. One new approach to control T2DM is the use of sodium-glucose transporter 2 Tumor angiogenesis is a hallmark of cancer (SGLT2) inhibitors. While SGLT1 is involved and plays a significant role in establishing in intestinal glucose intake, SGLT2 is a vascular supply within the tumor located in the proximal tubules of kidneys, which is essential for tumor growth and and is responsible for most of the glucose metastasis. The vascular endothelial growth reabsorption (>90%). Inhibition of SGLT2 factor (VEGF) plays an important role in in combination with traditional treatments angiogenesis process promoting endothelial have shown improvements in the control cell proliferation, migration, and invasion. of glycemia and a concomitant reduction VEGF is overexpressed in many solid cancers in cardiovascular risk. Challenges in the including lung cancer, one of the most development and extension of the chemical common cancers in the world. According space associated with the inhibitory activity to this, different approaches have been of SGLT2 are the high costs in time and developed to suppress tumor angiogenesis, money of the in vitro research and the lack of including anti-VEGF monoclonal antibodies the human transporter atomic coordinates, as part of the cancer immunotherapy strategy. due to the difficulties to crystallizing However, high production costs limit the membrane proteins. These two problems widespread access to this treatment. In this can be addressed through a methodology study, we designed a novel single-chain that includes an exploration of molecular monoclonal antibody (anti-VEGF) that can dynamics from the protein transporter and bind to VEGF. This antibody can be efficiently the structure-based virtual screening. We expressed in the mammary gland of goats by have developed a robust pipeline perform a adenoviral transduction and purified from virtual screening through massive molecular their milk. Results showed that anti-VEGF docking against a diversity library of was able to avoid cells migration in a wound molecules for the exploration of the chemical healing test and suppressed VEGF-induced space for the discovery of new inhibitors. microvessel sprouting in rat aortic ring assay. We use SGLT2 and known inhibitors to Furthermore, in vivo efficacy was evaluated perform a sequential refinement to optimize on a xenograft lung tumor model where anti- the identification from a not supervised VEGF treatment had an inhibitory effect methods and extend the methodology to the on tumor growth. Our findings suggest the other potential candidates transporter with therapeutic potential of anti-VEGF as an anti- therapeutics applications. tumor agent correlated with suppression of angiogenesis. 59. CHARACTERIZATION OF THE INTESTINAL MICROBIOTA OF 58. DIVERSITY LIBRARY PEOPLE TREATED WITH INFUSIONS SEQUENTIAL SCREENING TO OF ACAENA SPLENDENS AND IDENTIFY COMMON DESCRIPTORS ITS ASSOCIATION WITH THE OF THE SGLTS INHIBITORS. INFLAMMATORY STATUS. Lema J.M.1; Ormazabal, V.A.1,2; Zuñiga, Tabilo B.; Pino J.A.; Vieytes M.P. F.A.2; Salas-Burgos, A.1. Biomedical Sciences Laboratory, Faculty of 1, Nanocell Laboratory, Pharmacology Medicine, University of Atacama. Department, Biological Science Faculty, Concepción University. 2, Exosome Given the importance and usefulness of Laboratory, Clinical Biochemistry and medicinal plants as therapeutic agents,

115 the Chilean Ministry of Health, under the of the G protein (Gβγ). Through bioinformatic guidance of the world health organization studies, molecule M554 was selected, which (WHO), has been working in recent years binds to Gβγ at the same site of interaction on the regulatory framework of the policy of for GlyR-DC inhibiting the effects of ethanol Complementary Medicine and Traditional in vitro and in vivo. In this project it was Herbal Medicines with clinical evidence studied whether this molecule inhibited the support. Acaena splendens (Amores secos) interaction between these 2 proteins. For this has been used in Chilean traditional medicine objetive, a fusion protein of GlyR-DC and for the treatment of fever and inflammation. Glutathione S-transferase (GlyR-DC-GST) In addition, an infusion of Acaena splendens, was expressed and purified. Comparative similar to that recommended by folk healers, studies of GST pull-down showed that exhibited highly significant antiinflammatory GlyR-DC-GST retained its ability to interact activity (46.7%) in a murine model of with Gβγ. At the same time GlyR-DC was inflammation. Additionally, there is an incubated with cell extracts, and the affinity emerging body of work on the human gut of GlyR-DC with Gβγ in the absence and in microbiome and how it mediates feedback the presence of 200 µM M554 was compared. between the foods we eat and our bodies. The Densitometric analysis allowed to determine gut microbiome is also an important mediator that the interaction between both proteins of inflammation in the gut and systemically. effectively decreased in the presence of this Considering that the mechanism(s) by molecule. Therefore, these results show that which Acaena splendens modulate systemic this molecule decreases the binding capacity inflammation are unknown, we propose that of Gβγ with GlyR-DC, leaving clear that this infusions of Acaena splendens could decrease is the basal mechanism for the inhibition of systemic inflammation through changes in ethanol effects and supporting the projections human gut microbiome. To test this idea that M554 could have a pharmacological thirty healthy volunteers will consume an potential to treat acute ethanol intoxication. infusion prepared from Acaena splendens for six and twelve days and blood/fecal 61. COMPLEX INHIBITION OF samples will be obtained before and after the OXPHOS AND Α-KETOGLUTARATE treatment for microbiome analysis (Illumina DEHYDROGENASE COMPLEX BY 16S rRNA sequencing and Oxford Nanopore GENTISIC ACID-TPP+ INDUCES MinIon) and inflammatory markers analysis CELL DEATH IN BREAST AND LUNG (cytokine array kits and ELISA cytokine kits). CANCER CELL LINES. The results from this proposed study related López-Torres, C.; Fuentes-Retamal, S.; with the effect of medicinal plants on the Palominos, C.; Urra, F.; Catalán, M.; Ferreira, composition of the intestinal microbiota and J. its association with systemic inflammation Clinical and Molecular Pharmacology could provide new therapeutic strategies for Program, Institute of Biomedical Sciences treatment of inflammation-related diseases (ICBM), Faculty of Medicine, University of (e.g. colorectal cancer) a condition of high Chile. prevalence in the Atacama region. Cancer cells have a more hyperpolarized 60. STUDY OF A MOLECULE THAT mitochondrial membrane potential than INTERFERES IN GΒΓ BINDING WITH normal cells, which allows selectively THE CYTOPLASMIC DOMAIN OF guide towards mitochondria small cationic GLYCINE RECEPTOR α1. molecules such as triphenylphosphonium López A.D.E; Guzmán L. (TPP+), which participate as chemical Laboratorio de Neurobiología Molecular, chaperones for pharmacophores moieties. Facultad de Ciencias Biológicas, Universidad This property was used to synthesize from de Concepción. the natural product, gentisic acid, derivatives bound to TPP+ (GA-TPP+C10). This Ethanol is the most widely used drug of abuse mitocondriotropic compound is capable of in the world. Its effects go from desinhibition, produce a time-dependent complex inhibition headaches, nausea, vomiting, even respiratory of mitochondrial bioenergetics characterized depression and death. Recently, the glycine by 1) initial phase of mitochondrial receptor (GlyR) has been identified as one of uptake with uncoupling effect of oxidative the targets in which this drug acts, enhancing phosphorylation, 2) inhibition of complex its inhibitory activity. This mechanism I-dependent respiration and, 3) a late phase of involves the interaction of the cytoplasmic mitochondrial accumulation with inhibition domain of GlyR (GlyR-DC) with the βγ dimer of alfa-ketoglutarate dehydrogenase

116 complex activity. This complex is part of the alpha7beta2. However, Q-01 presented an tricarboxylic acid cycle composed of three inhibition similar to dihydro-beta-eritroidine subunits that oxidizes and decarboxylates (selective antagonist of nAChR containing the alfa-ketoglutarate which is necessary for the subunit beta2); suggesting that Q-01 might be synthesis of aspartate, an essential amino acid more selective for alpha7beta2 nAChR than to proliferation and cell survival. The above EQ-01. To understand the electrophysiological was verified using human breast and lung results, molecular docking studies were cancer cell lines, by the addition of exogenous performed for the compound Q-01 at the permeable metabolites: alfa-ketoglutarate nAChRs α7 and alph7beta2. These studies (dm-alfaKG), aspartate (m-Asp) and pyruvate suggest that Q-01 would be more selective by (pyr). It was shown that dm-alfaKG and subtype alpha7beta2. m-Asp, but not pyr, produce a decrease in cell death caused by GA-TPP+C10. Moreover, the 63. STRUCTURE-BASED VIRTUAL bioenergetic crisis induced triggers a drastic SCREENING STUDIES TO IDENTIFY mitochondrial membrane potential drop, NOVEL POTENTIAL AGONISTS FOR G1-phase cell cycle arrest with a significant SALMO SALAR GHSR1A-LR. increase in ROS. In addition, this blockade of Macaya-Zapata L. 1; Starck-Méndez M. F. 1,3; mitochondrial functions triggers a metabolic Toledo J. R. 2,3; Acosta J. 2; Sánchez O. 1,3. remodeling toward glycolysis and pro-survival 1 Laboratorio de Biofármacos Recombinantes, AMPK activation. Our results describe an Departamento de Farmacología, Facultad anti-cancer mechanism of GA-TPP+C10 that de Ciencias Biológicas, Universidad de induce a complex inhibition of mitochondrial Concepción. 2 Laboratorio de Biotecnología y bioenergetics in a time-dependent manner Biofármacos, Departamento de Fisiopatología, in breast and lung cancer cells that may have Facultad de Ciencias Biológicas, Universidad relevance at therapeutic level. de Concepción. 3 Centro de Biotecnología y Biomedicina Spa. 62. ELECTROPHYSIOLOGICAL RECORDINGS OF 3-(PYRIDIN-3- Ghrelin is a growth hormone (GH) YLMETHOXY)QUINUCLIDINE (Q-01), secretagogue and functions primarily as a A NOVEL SELECTIVE COMPOUND GH-releasing hormone and as an orexigen. It FOR THE Α7Β2 NICOTINIC has also been documented to be involved in ACETYLCHOLINE RECEPTOR. the immune system, stress response, energy López J.J.1; Mejía-Piedras J.2; Hernández- metabolism and growth in fish. Its receptor, Abrego A.2; García-Colunga J.2. Growth hormone secretagogue receptor 1, Department of Organic Chemistry, (GHS-R), is a class A G protein-coupled Faculty of Chemical Sciences, University receptor (GPCR) mostly expressed in the of Concepcion, Concepcion, Chile. 2, hypothalamus. In Atlantic salmon (Salmo 2Departamento de Neurobiología Celular salar) and other salmonids, a ghrelin receptor y Molecular, Instituto de Neurobiología, isoform called GHSR1a-LR has been identified. Universidad Nacional Autónoma de México, This receptor has an unique characteristic: the Querétaro, México. second extracellular loop (ECL2) is notably longer than in others GHS-R. In particular, A novel heteromeric alpha7beta2 nicotinic GHSR1a-LRs have the characteristic that acetylcholine receptor (nAChR) with ghrelin or GH secretagogues treatment functional properties different from those either does not increase intracellular of alpha7 and alpha4beta2 nAChRs, was Ca2+ or requires pharmacological doses recently identified. Although its functions to activate the receptor. Given the high are not known, it appears this nAChR may conservation in folding and topology of class be involved with Alzheimer’s disease. To date A GPCR receptors, a comparative model of there are no synthesis reports of ligands for GHSR1a-LR receptor from Salmo salar was alpha7beta2 nAChR. Our work was based generated. This model was based on the on the design and synthesis of new ligands, crystallographic structure of Neurotensin-1 as well as on establishing their possible receptor in active-like conformation (PDB effects with electrophysiological records in ID: 4XES). Subsequently, a conformational interneurons from the stratum radiatum of exploration was carried out through the rat hippocampal CA1 region. Thus, of accelerated molecular dynamics simulations eleven synthesized ligands, only Q-01 and (aMD). These simulations allowed us to EQ-01 inhibited the Choline-induced ionic obtain diverse conformational variants current: 51 and 100%, respectively; that is to (inactive, intermediate and active), favoring say, these acted as antagonists of alpha7 and the selection of small molecules compatible

117 with the receptor active state. Finally, we that gelsemine did notmodify the fraction searched for potential agonists through of desensitized current or the decay time virtual screening, applying an ensemble constant of receptors. Our results that docking based strategy using multiple gelsemine is able to negatively modulate the snapshots of GHSR1a-LR receptor extracted synaptic activity of cortical neurons. Future from aMD trajectories. A small molecule studies may contribute to shed light on the library, containing 4997 positive or neutral mechanisms underlying the beneficial effects charged compounds obtained from ZINC12 of the Gelsemium alkaloids in the control of database, was filtered under parameters of pathological anxiety through the modulation drug-like properties and possible central of inhibitory receptors. nervous system activity. From this campaign four agonist-potential molecules, based 65. CYTOTOXIC EFFECT OF on predicted affinity in different receptor HYDROXYCHLOROQUINE, structural samples, were identified. ITRACONAZOLE AND CISPLATIN ON SPHEROID CULTURE OF ORAL 64. SYNAPTIC EFFECTS OF THE SQUAMOUS CELL CARCINOMA. ALKALOID GELSEMINE ON Martínez D.; Yévenes S. CORTICAL NEURONS. Universidad de Chile. Marileo, A.M.1; Gavilan, J.1; Lara, C.O.1; San Martín, V.P.1; Burgos, C.F.1; Sazo, A.1; Oral Squamous Cell Carcinoma (OSCC) is Yévenes, G.E.1. the most common type of oral cancer and Universidad de Concepción. Cisplatin is the chemotherapeutic agent most commonly used, which induces cell Several behavior studies have suggested that death by apoptosis. Furthermore, tumor cells the natural alkaloid gelsemine has different (TC) acquire resistance against cytotoxic biological effect, such as analgesia and effect of the drug. Therefore, it is necessary anxiolytic. Nevertheless, until now there is to develop more effective treatments based few information about neurophisyological on the metabolic changes that occur in TC. mechanism, and pharmacological target Hydroxychloroquine and Itraconazole are associated to this biological effect. Early two drugs traditionally used in traditional studies from our lab have shown that medicine, as immunomodulator and gelsemine decreases the frequency of antifungal, respectively. Recently, it has glycinergic and glutamatergic events in the been described that they may have antitumor spinal cord neurons culture that suggest effect, because Hydroxychloroquine may important effect in the synaptic function. inhibit autophagy, a mechanism of adaptation Despite these advances, it is unknown if to metabolic stress, and Itraconazole would gelsemine can modulate GABAARs and disturb energy metabolism. Tumor spheroid GABAergic synapsis, which is relevant in the cultures are an in vitro model suitable pathological anxiety phenomena. Here, we for antitumor activity evaluation because examined the functional effects of gelsemine they can reproduce tumors in vivo main on native sistem using electrophysiological characteristics, such as hypoxia-related techniques. Studies performed on cultured drug resistance and the presence of Cancer neurons was realized to explore the potential Stem Cell (CSC), which may be responsible effect of gelsemine in the synaptic activity of of chemotherapy resistance and tumor cortical neurons, which express the functional recurrence. In this project the expression of component of a GABAergic synaptic. the markers of CSC CD44, CD56 and ALDHA1 Our electrophysiological result show that in spheroidal cultures of Cal-27 (COCE cells) gelsemine 50 mM produced a significative was evaluated through flow cytometry. The reduction of the frequency, but not in the results show greater expression of these amplitud of the GABAergic and glutamatergic markers in spheroidal cultures, compared to synaptic activity. I addittion, Effects of monolayer cultures. In addition, cytotoxicity gelsemine on the agonist sensitivity and on for spheroidal cultures was determined by the desensitization rates of GABAAR native, the MTT assay for Hydroxychloroquine, are evaluated. Analysis of concentration – Itraconazole and Cisplatin. The IC50s were response curves revealed that gelsemine 249, 472 and 577 micromolar at 48 hours and significantly decreased the apparent 272, 298 and 261 micromolar at 72 hours, affinity for GABAAR without changing the respectively. A viability decrease induced by maximal current amplitudes. Analyses of these drugs was observed in a concentration- the GABA-activated currents stimulated by dependent manner. At present is being saturating agonist concentrations indicated evaluated if the drug combinations have

118 a synergy effect reducing the individually 67. SIMVASTATIN AND 15-EPI- required concentration. LIPOXIN A4 INDUCE CARDIAC REPAIR THROUGH NOTCH 1 66. NEONATAL EXPOSURE TO ACTIVATION IN CHRONIC CHAGAS TESTOSTERONE PROPIONATE CARDIOMYOPATHY. INDUCES AN INCREASED Guzmán-Rivera; D.1; González-Herrera; F.1; EXPRESSION OF RGS9-2 AND Lapier; M.1; Carrillo; I.1; Quinteros; H.1; PKCΒ2 IN NACC AND VTA OF ADULT Fuentes; S1.; Pesce; B1.; Castillo; C2.; Liempi; FEMALE RATS. A2.; Kemmerling U2.; Maya; J.D.1. Martínez-Pinto, J.; Müller, E.; Sotomayor- 1Molecular and Clinical Pharmacology Zárate, R. Program, Biomedical Sciences Institute Laboratorio de Neuroquímica y (ICBM), Faculty of Medicine, University of Neurofarmacología, Centro de Neurobiología Chile, Santiago, Chile 2 Program of Anatomy y Fisiopatología Integrativa (CENFI), and Developmental Biology, Biomedical Instituto de Fisiología, Facultad de Ciencias, Sciences Institute (ICBM), Faculty of Universidad de Valparaíso, Valparaíso, Chile. Medicine, University of Chile, Santiago, Chile.

Research in programming is focused on the Chagas Disease, caused by Trypanosoma study of stimuli that alters sensitive periods in cruzi, is endemic in Latin America and development, such as prenatal and neonatal worldwide because of migration. Without stages, that can produce long-term deleterious appropriate treatment this disease progress effects in various organs or tissues such as to a chronic phase that could affects the the brain, affecting brain circuits and related heart. Chronic Chagas Cardiomyopathy behaviors. Previously, we have demonstrated (CCC), the most severe clinical manifestation, that neonatal programming with sex involves a progressive inflammatory hormones affects the mesocorticolimbic myocarditis affecting ventricular wall circuitry, increasing the synthesis and release causing cardiovascular complications due to of dopamine (DA) in striatum and Nucleus diminished cardiac function and heart failure. accumbens (NAcc); also, we have observed a Despite intense research no one drug can reduction of locomotor activity in response to stop or reverse the progressive heart damage. methylphenidate in female rats treated with Simvastatin, a drug that decreases blood testosterone propionate (TP). However, is not cholesterol, has anti-inflammatory effects clear if the alterations observed in our model and inhibits platelet aggregation. Previously, are related to modifications in the signaling we described that simvastatin reduces pathway or DA release/uptake. Interestingly, myocardial inflammation caused by T. cruzi RGS9-2, which is expressed in dopaminergic through 15-epi-lipoxin A4 (15-epi-LXA4) neurons, can inhibit the signal transduction production, a pro-resolutory inflammation of dopamine receptor 2 (D2) and have been molecule. Several reports suggest that related to drug addiction and movement simvastatin activates Notch pathway after a disorders. Also, PKCβ2 can increase the stroke enhancing blood flow by promoting amphetamine-stimulated dopamine efflux angiogenesis. CCC progress with myocardial regulating the Dopamine Transporter (DAT) inflammation, endothelial damage with activity. The objective of this work was to micro focal ischemia and fibrosis. We propose evaluate if the neonatal reprogramming that simvastatin reverts cardiac damage in with Estradiol Valerate (EV) or TP affects the chronic T. cruzi infection by 15-epi-LXA4 the expression of Rgs9-2 and Pkcβ2 in production and Notch 1 pathway activation. NAcc and Ventral Tegmental Area (VTA) BALB/c mice were chronically infected in adult rats using qPCR. The expression of with T. cruzi Dm28c strain and treated with Rgs9-2 and Pkcβ2 was increased in NAcc simvastatin 1 mg/Kg/day and 15-epi-lipoxin and VTA of female rats treated with TP; no A4 25 µg/Kg/day for 20 days. At day 80 significant changes were observed in males post-infection animals were euthanized to under any condition. These results suggest analyze the heart, Notch pathway, fibrosis, that the neonatal exposure to TP modifies and angiogenesis process. In chagasic mice, the expression of Rgs9-2 and Pkcβ2 in female the cardiac function was restored with rats, and this modification can account for the simvastatin and 15-epi-lipoxin A4 treatment. modifications in response to methylphenidate The Notch signaling pathway was active observed in our model. Further analysis using in cardiac tissue, a finding that correlated WB or IHC are needed to depict the functional with drug treatment, the fibrosis process alteration in this model. was decreased, and angiogenesis was also evidenced in this model. Thus, we concluded

119 that simvastatin and 15-epi-LXA4 improve maceration with methanol and subsequently cardiac architecture and function through fractionated, for the isolation. The putative Notch 1 activation by increasing blood flow alkaloid composition of the fraction was and decreasing cardiac remodeling. Thus, analyzed by GC-MS, highlighting the it could be incorporated rapidly in CCC presence of type licorin and crinamine. The treatment. inhibitory activity of the alkaloid extract and the isolated compounds was evaluated 68. INHIBITORY ACTIVITY OF by the Ellman method, finding IC50 values ACETYLCHOLINESTERASE AND (ug / mL) for hexane, chloroform and BUTYRYLCHOLINESTERASE butanol extract of 17.12, 8.89 and 19.09 for FROM AMARYLLIS BELLADONNA acetylcholinesterase and 77.27, 55.44 and ALKALOIDS. 200 for butyrylcholinesterase respectively. Mella M. 1,4; Iturriaga-Vásquez P. 2,4; Quiroz A. 3,4; Moraga F. 3,4; Mutis A. 3,4; 69. USE OF MEDICINES BY Hormazábal E. 3,4. SOUTHERN BRAZIL FARMERS 1, Estudiante de Bioquímica, Universidad AND ITS RELATIONSHIP WITH de La Frontera. 2, Laboratorio de EXPOSURE TO PESTICIDES FROM Farmacoquímica y Síntesis Orgánica, DIFFERENT CROPS. Universidad de La Frontera. 3, Laboratorio Calinca Skonieski Karina Raquel Fagundes Química Ecológica, Departamento Ciencias Matheus Henrique Machado Bento Anderson Químicas y Recursos Naturales, Universidad Joel Martino Andrade Daniel Barbosa de de La Frontera. 4, Centro de Excelencia en Chaves Samara de Cesaro Cavaler Andressa Investigación Biotecnológica Aplicada al Talita Nunes Maiara Grasiela Rossi Fabiana Medio Ambiente (CIBAMA). Elias Dalila Moter Benvegnú. Universidade Federal da Fronteira Sul - Campus Realeza. Alzheimer’s disease, a neurodegenerative disorder characterized by an irreversible and Agriculture in its development model has progressive loss of memory. Traditionally not been addressing issues such as the the pharmacological treatment associated environment. Consequently, countless with the disease in its medium to moderate substances ended up being released into stages, or similar diseases related to a deficit the environment, plenty of them with of the neurotransmitter acetylcholine are the ability to alter the behavior of several mainly guided through inhibitors of the physiological systems, inducing numerous enzyme acetylcholinesterase. Only four pathologies. Concomitantly, several studies compounds have been approved as treatment have demonstrated a growing use of against alzheimer diseases: donepezil, medicines. Therefore, the goal of this study rivastigmine, galantamine and memantine. is to investigate the relationship between In most cases these drugs are well tolerated, the use of medicines by farmers and their however, various side effects such as: exposure to pesticides. Upon approval by the nausea, vomiting, among others may occur. Research Ethics Committee from the Federal That is why it is necessary to search for new University of the Southern Border, farmers molecules with pharmacological potential were randomly selected in two cities: Mafra, for their treatment. A potential source of Santa Catarina and Planalto, Paraná, both acetylcholinesterase inhibitors is Amaryllis in Southern Brazil. The subjects were then belladonna, belonging to the Amaryllidaceae asked to fill up a form for data collection. A family, widely distributed worldwide. total number of 251 farmers participated in Several studies support the presence of the research, being 123 from Mafra and 128 alkaloids in this species with varied biological from Planalto. The average age of the subjects activities. Considering the variation in the in this study was 48,4 ± 14,4 and 114 were production of metabolites reported in this female while 137 were male. Out of these, species, depending on the geographical 23,1% (58) are making use of neuropsychiatric distribution, it is interesting to analyze the drugs, 32,7% (82) of cardiovascular drugs, alkaloids present in the representative of 17,5% (44) of metabolic disorder drugs, this family introduced in our country and its 0,8% (2) of respiratory disorder drugs, 1,6% pharmacological potential. The objective of (4) of gastrointestinal drugs and 2% (5) this research was to evaluate the inhibitory of musculoskeletal purposes drugs. When activity of alkaloids isolated from Amaryllis correlation tests were performed between belladonna on enzymes acetylcholinesterase the type of crops and the drugs used by the and butyrylcholinesterase.The alkaloids respective farmers, the results showed a present in the plant’s bulbs were obtained by greater use of medicines for metabolic (p = 0.014) and musculoskeletal disorders (p =

120 0.025) from wheat crops farmers in Mafra. 71. ANTITUMOR PROTOTYPE BASED Thus, the data suggests that farmers in wheat ON POLYMERIC NANOPARTICLES crops are more likely to make use of drugs for WITH APPLICATION IN GENE metabolism and musculoskeletal disorders, THERAPY. as it might be related to the specific pesticides Ñacato A.1; Cerro R.P.1; Rivas V.2; Rivas C.1; used in this crop. Ramos T. 1; Gómez-Gaete C.2; Toledo J.R.1. 1, Biotechnology and Biopharmaceuticals 70. SUCRALOSE INTAKE IMPAIR Laboratory, Pathophysiology Department, THE HIPPOCAMPAL POSTSYNAPTIC School of Biological Sciences, Universidad de INHIBITORY CURRENTS. Concepción. 2, Laboratory of Pharmaceutical Muñoz-Perez de Arce A. 1,2,3; Bravo J.A. 2; Technology, Department of Pharmacy, School Fuenzalida M. 1. of Pharmacy, Universidad de Concepción. 1, Laboratorio Plasticidad Neuronal, Centro de Neurobiología y Fisiopatología Gene therapy is a therapeutic strategy mainly Integrativa (CENFI), Facultad de Ciencias, focused on correcting altered or mutated Universidad de Valparaíso; 2, Laboratorio genetic sequences, which can induce the Neurogastrobioquimica, Instituto de development of hereditary or acquired Química, Pontificia Universidad Católica de pathologies, such as cancer. Nanoparticles Valparaíso; 3, Programa de Magíster Ciencias based on biocompatible polymers have been Biológicas mención Neurociencia, Facultad used as carriers for therapeutic molecules, de Ciencias, Universidad de Valparaíso. due to their ability to encapsulate labile molecules such as linear or plasmidial DNA Non-caloric sweeteners (NCS) are widely by electrostatic interactions. Thus, it could used in foods with the aim of reducing sugar prevent their degradation by nucleases present consumption and caloric intake. Sucralose in the environment. Safety and effectiveness is the most used NCS worldwide and in of the polymers make feasible in vivo tests Chile, with the current “Labeling Law”, its and future commercial products as described consumption has been increasing. Sucralose and approved by regulatory agencies, such intake causes alterations in the composition as the FDA. The aim of this work was to of the intestinal microbiota, which is design and evaluate a formulation based on closely related to mental health through polymeric nanoparticles as gene therapy the crosstalk communication between gut applied to prostate cancer. Nanoparticles and brain. Considering that microbiota can were elaborated using a double emulsion affect behavior and modulate GABA levels, method, with solvent evaporation, and brain plasticity and cognitive function, we PLGA as the main matrix agent, associated wonder whether NCS affect the integration with a cationic polymer. A model plasmid, and synaptic function in hippocampal CA1 which transcribes a tumor progression pyramidal neurons in adult Sprague Dawley blocker was encapsulated. Physicochemical rats treated with 0.5% sucralose in the characteristics of nanoparticles were analyzed drinking water for a period exceeding 17 by Zetasizer Nano, and their effect was days. Using patch-clamp recordings in whole evaluated in vitro, in a human tumor cell line. configuration we observe that membrane Nanoparticles were obtained in nanometric potential pyramidal neurons NCS treated rats size range, with a polydispersion index (PdI) have a more depolarized value than control less than 0.2, and the surface charge was group and no effect on the trigger threshold positive. Morphologically, nanoparticles were of action potentials. Also, we observe that spherical according to Transmission Electron frequency of the spontaneous inhibitory Microscope images. It was also observed an synaptic currents in PYNs is lower than control increase in the genetic transformation rate slices. The paired pulse protocol did not show for human tumor cells, and an alteration in differences between animals treated with the characteristic tumor progression markers NCS and control, suggesting that NCS intake expression. These results are promising modify the presynaptic and postsynaptic for the development of new therapeutic excitability, no apparent effect on the release candidates based on nano delivery system for of GABA. These results show for the first time complementary treatment in different types that permanent consumption of sucralose of cancer. may have affect GABAergic synaptic efficacy in the central nervous system.

121 72. RESOLVIN D1 PREVENTS 73. ANTIBIOTIC SUSCEPTIBILITY CARDIAC HYPERTROPHY AND PROFILE OF HELICOBACTER PYLORI FIBROSIS IN ANGIOTENSIN II- IN THE ARAUCANÍA REGION. INFUSED C57BL/6 MICE. Oporto M. 1.; Troncoso C.1; Cerda A.1; Olivares-Silva F. 1,2; De Gregorio N. 2; Hofmann E.;2,3, Sierralta A.2,4; Ríos E.2,3; Sánchez-Ferrer C. 3,4; Peiró-Vallejo C. 3,4; Coppelli L.5; Barrientos L.1 Pavez M.1. Díaz-Araya G. 1 Centro de Excelencia en Medicina 1,2 Traslacional CEMT- UFRO, Temuco. 2 1, Laboratorio de Farmacología Molecular, Departamento medicina Interna, UFRO, Departamento de Química Farmacológica y Temuco. 3 Endoscopia CAT, Temuco. 4 Toxicológica, Facultad de Ciencias Químicas Endoscopia HHHA, Temuco. 5 Endoscopía y Farmacéuticas, Universidad de Chile. 2, H. Villarrica. Centro Avanzado de Enfermedades Crónicas (ACCDiS), Facultad de Ciencias Químicas Introduction: Antibiotic resistance is one y Farmacéuticas y Facultad de Medicina, of the main causes of therapeutic failure Universidad de Chile. 3, Departamento in eradication treatments of Helicobacter de Farmacología, Facultad de Medicina, pylori (Hp), which currently and according to Universidad Autónoma de Madrid. 4, Maastricht consensus the standard consists of Instituto de Investigación Sanitaria Hospital a triple scheme of a proton-pump inhibitors Universitario La Paz (IdiPAZ), Madrid, (PPI) +2 antibiotics. In recent years, high España. resistance rates to the main antibiotics used in these treatments have been reported in Background: Resolvin D1 (RvD1) is an several countries. Even varying between endogenous specialized lipid mediator geographical areas of the same country, which enzymatically derived from docosahexaenoic prevents generalizing efficient therapies acid and synthesized locally in acute in certain populations without previous inflammatory processes, where it exerts pro- susceptibility studies Aim: To evaluate the resolving effects demonstrated in diverse susceptibility profile of Hp, in the Araucania pathological models. Angiotensin II (Ang region, against antibiotics used in eradication II), in cardiac tissue, contributes to the therapy. Methods: A descriptive study on development of cardiac hypertrophy and 37 Hp, isolates from gastric biopsy samples fibrosis. To date, there are not studies on the on dyspeptic patients was performed in potential protection that RvD1 may provide main health centers of the Araucania. The at the structural and functional level in an susceptibility profile against amoxicillin, Ang-II infusion model. Purpose: To evaluate clarithromycin, levofloxacin, metronidazole RvD1 effects in Ang II-induced cardiac and tetracycline was performed by agar hypertrophy and fibrosis. Methods: Alzet® dilution. Minimum inhibitory concentration osmotic mini-pumps filled with Ang II (1.5 values were evaluated according European mg/kg/day) were implanted in C57BL/6 Committee on Antimicrobial Susceptibility mice for 14 days, previous basal left ventricle Testing, using Hp ATCC 43504 as a quality (LV) functionality assessment. RvD1 (3 ug/ control strain. Results: All isolates reported day) was injected intraperitoneally. At the resistance at least one antibiotic and 81.08% end of the infusion period, the animals were showed resistance to two or more antibiotics. sacrificed, and functional and histological 13.8% of the Hp isolates were resistant to parameters were studied. Results: 14-day amoxicillin, 45.94% to clarithromycin, 41.66% Ang II infusion increased heart weight/tibia to levofloxacine, 81.08% to metronidazole length ratio, LV thickness, ejection fraction, and 16.66% to tetracycline. Conclusion: shortening fraction and collagen deposition The resistance rates to metronidazole, at the interstitial and perivascular area. clarothromycin and amoxicillin were higher Treatment with RvD1 significantly prevented to reported in Chile and there are not previous LV dysfunction, hypertrophy and collagen reports to LVZ. These results show the need deposition in both areas. Conclusions: RvD1 of future studies of therapeutic efficacy in the prevents Ang II-induced cardiac hypertrophy Araucanía as well a new review of current and fibrosis demonstrating cardioprotective eradication strategies. properties. Further studies will be performed to elucidate the possible mechanisms of action of RvD1. Ethics approval: The Institutional Animal Care and Use Committee of the University of Chile (CICUA) approved the protocol (CBE2018-12).

122 74. ANALYSIS OF THE FUNCTIONAL 75. INHIBITORY ACTIVITY ON SPECIFICITY IN THE SUGAR GLYCOGEN PHOSPHORYLASE A OF PORTER FAMILY TO IDENTIFY NEW PHENOLIC EXTRACTS FROM LEAVES INHIBITORS OF GLUT1. AND FRUITS OF 8 UGNI MOLINAE Oppliger M.1; Ormazabal, V.A.1,2; Zuñiga, TURCZ GENOTYPES. F.A.2; Salas-Burgos, A.1. Ordóñez, J.L.1; González, J.1; Pérez, R.; 1, Nanocell Laboratory, Pharmacology Bugueño, I.1; Guzman, P. 1; Seguel, I.2; Department, Biological Science Faculty, Delporte, C.1. Concepción University. 2, Exosome 1, Laboratorio de Productos Naturales, Laboratory, Clinical Biochemistry and Departamento de Química Farmacológica y Immunology Department, Pharmacy Faculty, Toxicológica, Facultad de Ciencias Químicas Concepción University. y Farmacéuticas, Universidad de Chile; 2, Instituto de investigaciones agropecuarias The movement of glucose and other sugars and (INIA) Carillanca. polyols with essential functions in the energy metabolism of living beings through biological One of the strategies used for the discovery of membranes is carried out by transporter natural products with hypoglycemic activity proteins belonging to the family of sugar is the analysis of species used by traditional transporters (SP family, TC code 2.A.1.1), medicine, such as Ugni molinae, Myrtaceae, where we find the human GLUT transporters, popularly known as murtilla. In this context, the hexoses transporters in yeasts, among the analysis of the inhibition on the activity of others. There are highly specific transporters Glycogen phosphorylase A (Gpa), an enzyme for a physiological substrate, and there are expressed in brain, muscle and liver, which has others much more promiscuous. There is major role on post-prandial hyperglycemic an increasing interest in pharmacology to peaks in diabetic patients, could account for design GLUTs inhibitors, whose development potential candidates for the development has risen since the determination of the of new treatments. Therefore, the aim of crystallographic structure of human GLUT1. this work was to demonstrate and compare, For glucose homeostasis diseases that through an in vitro spectrophotometric occur with hyperglycemia and cancer, the methodology, the Gpa inhibitory activity of therapeutic use of these molecules has been phenolic-rich extracts obtained from leaves proposed. In the first case, its potential use and fruits of 8 murtilla genotypes from the is in the downregulation of the incorporation INIA-Carillanca germplasm bank, which of glucose to the blood, while in cancer the were cultivated at the same edaphoclimatic aim is to avoid the dispensation of glucose conditions. Compared to caffeine (IC50 = to cells with active proliferation. Several 5,3 ug / mL), the leaves ethanolic extracts inhibitors of transporters are designed from were more potent (EETs; IC50 between 1,03 substrate/solute modifications. To advance – 3,52 ug / mL; p ≤0,05), while the fruit in the understanding of the molecular acetonic extracts were less potent (EACs; bases that explain the varied selectivity of IC50 between 27,9 – 86,1 ug / mL; p ≤ 0,05) the SP family, we carried out the present than the reference substance, as well as less work, which consists of the identification potent than the leaves extracts. Based on our and characterization of sequence profiles results, the leaves and fruits of U. molinae related to the specificity of substrates for could be a potential source of bioactive the SP family. To achieve this goal, we first phenolic compounds for the treatment of characterized the functional diversity of the hyperglycemia, through the development of family by identifying functional subclasses functional foods or phytopharmaceuticals. using supervised methods that build the On the other hand, based on the results functional classification based on empirically from this work, INIA-Carillanca will be able obtained transport activities reported for to classify the genotypes for their potential the transporters from databases. From hypoglycemic effects, which will allow the the analysis of the specificity determinant future to promote the cultivation of murtilla positions (SDPs), we build structures to find for its agronomic and commercial value, as inhibitors in a pharmacophore-like mode to well as for its medicinal properties. accelerate the identification of new inhibitors and predict the selectivity of other GLUT1 orthologues.

123 76. EVALUATION OF ANTICANCER 77. ANXIOGENIC EFFECT OF POTENTIAL OF DHA + P1G10 IN CELL AMPHETAMINE ON ZEBRAFISH LINES DERIVED FROM CANCERS USING A NOVEL TANK DIVING TEST WITH HIGH INCIDENCE IN CHILE. AND MONOAMINE TRANSPORTER Ortega L.A.1,2; Reyna-Jeldes M.A.1; Lobos L. GENES EXPRESSION. 3; Schnaiderman A.2; Coddou C.1. Paillali, P.1; Viscarra, F.1 ; Reyes-Parada, 1, Laboratorio de Señalización Purinérgica, M.2; Iturriaga-Vásquez, P.1. Departamento de Ciencias Biomédicas, 1, Laboratorio de Farmacología Molecular Facultad de Medicina, Universidad Católica y Síntesis Orgánica, Depto. Cs. Químicas del Norte, Coquimbo, Chile; 2, Schnaiderman y Recursos Naturales, Fac. de Ingeniería y Abraham & Cía; 3, Centro de Medicina Ciencias, Universida de La frontera. 2, Centro Regenerativa, Universidad del Desarrollo, de Investigación Biomédica y Aplicada Santiago, Chile. (CIBAP), Escuela de Medicina, Facultad de Ciencias Médicas, Universidad de Santiago Cancer is one of the most frequently diagnosed de Chile, Chile. diseases worldwide, being the second most frequent cause of death. Previous studies have Monoamine Transporters regulate established that omega-3 fatty acids, mainly neurotransmission via the reuptake of DHA (docosahexaenoic acid), have protective dopamine, serotonin and norepinephrine in effects against various types of cancer, among the brain and regulate the neurotransmitters these, gastric cancer, which is one of the homeostasis. This class of protein are target most common cancers in the world, with for a wide number of compounds including one of the highest mortality rates in Chile. antidepressants, drugs for neuropsychiatric On the other hand, Carica papaya protein and neurodegenerative disorders, and fraction P1G10 also has proven anti-cancer substance of abuse, such as amphetamine. properties due to its proteinase activity. Our This drug of abuse has been described that data indicate that gastric adenocarcinoma produce anxiogenic effect on rodents and it cells (AGS) are more sensitive to DHA than is well known that interacts with monoamine non-tumor gastric epithelium cells (GES-1), transporters inducing monoaminergic determining, through MTT, an IC50 of 40.47 release. In our group we have used zebrafish µM in AGS. Through Hoechst/Annexin V/ as models for behaviour using different IP was found that DHA promotes apoptosis drugs that acts over nicotinic receptors and in AGS cells, but not in GES-1. It was also monoamine transporters. The novel tank determined that DHA decrease procaspase-3 diving test has been used as a model for to protein levels in AGS cells only. In vivo assays test anxiolytic behaviour on zebrafish, the in BALB/c NOD/Scid mice conclude that time spending on the bottom of the tank has DHA treatment for 6 weeks significantly been describe as anxiogenic-like behaviour in decreases the volumes of tumors generated this model. This work shows the anxiogenic- by AGS cells xenografts. To assess the effects like effects produced by amphetamine on the of DHA+P1G10 in vitro, we determined cell novel tank diving test. Additionally, we design proliferation through MTT, treating cell lines the primers and detect the genes expression derived from the main types of cancer in our of Crebs, DAT, NET and SERTa, SERTb by country: gastric, lung, gallbladder and breast. PCR. Also we measure the expression changes To observe cell apoptosis/necrosis visually, using qPCR for this monoamine transporter we will stain treated cells with Hoechst/ using a chronic dose of amphetamine. Our Annexin V/PI solution, and to gain further results indicate that, amphetamine induce insight into the mechanism of DHA+P1G10 anxiogenics-like effects of diving behaviour -induced apoptosis, we will examine protein and increase genes expression of MAT´s at levels of procaspase-3 and caspase-3/7 different level. activity using Western blot and luminescence assay, respectively. Thus, this research seeks to determine and validate the joint use of DHA and the protein fraction P1G10, on cell lines of the main types of cancer in Chile.

124 78. MITOCHONDRIOTROPHIC with a consequent decreased of the maximal POLIHYDROXY-BENZOATES respiration. In addition, concomitant use DERIVATIVES AGENTS MODIFY of GA-TPP+C10 and doxycycline is able THE EXPRESSION OF METASTATIC to generate a selective synergic cytotoxic BIOMARKERS IN HUMAN effect on the activation of apoptotic COLORECTAL METASTATIC CELLS processes in BC cells, which suggest that this IN VITRO. combined strategy based on the blockage Palominos, C.1, Ruz, D.1, Fuentes-Retamal, of mitochondrial bioenergetics inhibition- S.1, Jara, J.A.2, Castro-Castillo, V.3, Vivar, induced adaptive response may have R.1, Ferreira, J.1, Catalán, M.1. therapeutic relevance in breast cancer. 1 Programa de Farmacología Molecular y Clínica, ICBM, Facultad de Medicina, 79. AMYLOID BETA OLIGOMERS Universidad de Chile, Santiago, INDUCE MITOCHONDRIAL Chile. 2 Programa de Farmacología y DYSFUNCTION BY ITS Farmacogenética, Instituto de Investigación DIRECT INTERACTION WITH de Ciencias Odontológicas (ICOD), MITOCHONDRIAL MEMBRANES ON Facultad de Odontología, Universidad de HIPPOCAMPAL SLICES. Chile, Santiago, Chile. 3 Departamento de J. PANES-FERNÁNDEZ1, J. GAVILAN 1, Fisicoquímica y Química, Facultad de Ciencias P.A. GODOY1, O. RAMIREZ-MOLINA1, T. Químicas y Farmacéuticas, Universidad de SILVA-GRECCHI, N. MUNOZ-MOLINA, C. Chile, Santiago, Chile. MUNOZ-MONTECINO1, J. FUENTEALBA1. Department of Physiology, Faculty of The metabolic plasticity of cancer cells is Biological Sciences, University of Concepción, the main limiting factor in the research of Concepción, Chile. effective pharmacologic treatments, for development of drug resistance, being one of Alzheimer’s disease (AD) is a the major obstacles in the clinical treatment, neurodegenerative disorder characterized as a promising target for new anti-cancer by impaired learning and memory loss. drugs therapies. Mitochondria have been Amyloid beta peptide (Aβ) plays a key role the main factor in the metabolic plasticity. in the pathogenesis of AD, especially soluble This organelle also participates promoting oligomers (SO-Aβ) because can reproduce the metastasis, tumor-initiating cells and survival major aspects of the disease. In vitro studies and propagation of cancer stem cells, which have associated mitochondrial dysfunction transforms it into an attractive therapeutic with an early role in the AD; however, the target. Previously reports have demonstrated molecular events are not understood with that GA-TPP+C10 triggered a mitochondrial precision. In this work, we have studied the dysfunction, characterized by an inhibition intracellular effects of SO-Aβ treatments, on of electron transport chain (ETC) and mitochondrial morphology and mitochondrial AKGDH complex inhibition which triggers potential (ΔΨm). We found that the degree cell death. In order to increase this effect, of colocalization between Aβ and TOM20 we have analyzed mitochondrial resistance was increasing at 24 h of SO-Aβ treatments, mechanism generated by the action of with a Manders coefficient (0.640 ± 0.1). this compound, highlighting an increased Furthermore, we evaluated the ΔΨm using expression of PGC1α and ETC components- the JC-1 probe, we observed that at chronic related genes encoded by mitochondrial DNA. treatments (24h), SO-Aβ shown a decrease on In order to inhibit the resistance generated by ΔΨm near to 50% of the control conditions. this inhibitory mechanism of action, we have Additionally, at the same times (SO-Aβ, 24h) incorporated a second agent, doxycycline, strong changes were observed in the size of the which demonstrated inhibits the synthesis mitochondrial network in primary cultures, mitochondrial proteins by blockage of only displacing the equilibrium towards a more mt-ribosome activity. This effect inhibits the granular pattern in mitochondria that present adaptive survival response generated to the a positive colocalization with Aβ. Secondly, action of GA-TPP+C10 evidenced by inhibition the intracellular distribution of SO-Aβ of ETC-related protein. Interestingly, the (2.5uM) in a mouse hippocampal slices model combined therapy increments significatively was evaluated by immunohistochemistry the mRNA levels, both ETC-components and electron transmission microscopy and mitochondrial biogenesis signaling (TEM), where we observed the presence factors (PGC1a-TFAM-NRF1-NRF2), which Aβ targeted with gold nanoparticles in suggests a greater mitochondrial damage, an intramitochondrial zone. On the other evidenced by a decreased mitochondrial mass hand, it was observed that ΔΨm showed

125 a progressive decrease in time manner on 81. ANTIMICROBIAL under SO-Aβ treatments (JC-1590/520 C: SUSCEPTIBILITY TESTS OF 1.01 ± 0.01; SO-Aβ 3h: 0.78 ± 0.04). This HELICOBACTER PYLORI ISOLATES study suggest a new pathogenic mechanism FROM PATIENTS IN THE BIOBÍO in AD, where cytotoxic effects of SO-Aβ are REGION: COMPARISON OF AGAR related with their direct interaction with the DILUTION AND DISK DIFFUSION. mitochondria, and reveals a novel therapeutic Parra-Sepúlveda C. 1; Sánchez-Alonzo K.1; strategies for neuroprotection. Arellano L. 1; Olivares J. 1; Manríquez C. 2; González C.1; Garcia A.1. 80. CYTOTOXIC EFFECT OF 1, Laboratorio de Patogenicidad Bacteriana, COMBINATIONS OF ITRACONAZOLE, Departamento de Microbiología, Facultad HYDROXYCHLOROQUINE AND de Ciencias Biológicas, Universidad de CISPLATIN IN HEAD AND NECK Concepción. 2. Departamento de Obstetricia CARCINOMA IN LOW GLUCOSE y Puericultura, Facultad de Medicina, CULTURES. Universidad de Concepción. Pardo A.; Vidal D.; Jara J. Laboratorio de Farmacología, Facultad de Antimicrobial susceptibility testing Odontología, Universidad de Chile. for Helicobacter pylori is increasingly important due to resistance to the most Head and neck cancer (HNC) is the sixth most commonly used antimicrobial agents. The common malignancy in the world, corresponds Gold Standard proposed by the CLSI is the to 6% of cancer cases and is responsible for agar dilution method, but it is difficult to 1-2% of deaths worldwide. The most prevalent perform routinely. The objective of this work HNC subtypes are laryngeal cancer and oral was to determine the concordance of disc squamous cell carcinoma. These pathologies diffusion in comparison to the agar dilution are very aggressive and have poor prognosis method for: clarithromycin, metronidazole, and recurrences, which can be caused by a levofloxacin, amoxicillin and tetracycline, possible resistance to chemotherapy. Cisplatin using 44 strains of H. pylori from patients is the chemotherapeutic most used to treat in the BioBío region. Univariate analysis was these pathologies, however it has been high performed, and the Kappa test was applied for rates of resistance. This resistance may be concordance using the Stata V.14 program. caused partially by “Cancer stem cells”, which The resistance rates were for clarithromycin are resistant to stress stimuli such as starvation 29.5% and 25.0%; metronidazole 45.4% and low oxygen levels. It has been described and 56.8%; Levofloxacin 31.8% and 25.0; in some studies that there are drugs, such Amoxicillin 2.2% and 0% respectively by as Itraconazole and hydroxychloroquine, an disk diffusion and agar dilution. Tetracycline antifungal drug that acts at the mitochondrial showed no resistance with any of the 2 membrane of the tumor cell by inhibiting methods used. Clarithromycin presented a the VDAC1 receptor, and an antimalarial/ considerable degree of concordance with a k immunosuppressive that has been described = 0.6571 (p <0.0001). Metronidazole did not with antineoplastic potential by inhibiting show concordance for the techniques under autophagy, respectively. In this way it is study (p=0.1586). Levofloxacin presented an proposed that the combination of these drugs almost perfect concordance with a k=0.8333 sensitize the effect of Cisplatin. Cell viability (p<0.0001). On the other hand, the Kappa tests were performed with the compounds at test was not calculated for amoxicillin 24, 48 and 72 hours under normoxia conditions and tetracycline, since 97.3% and 100% with low glucose medium (1,0 g/L) in two concordance were obtained respectively. cell lines, laryngeal squamous cell carcinoma The disc diffusion method presented a high (HEp-2) and squamous tongue carcinoma degree of agreement with the Gold Standard (CAL-27), using as a control oral dysplastic for clarithromycin, levofloxacin, amoxicillin cells (DOK). This will be carried out in order and tetracycline. This is an easy method to to obtain the IC50 of each compound and assess susceptibility to H. pylori especially if determine their cytotoxic effect. A cytotoxic it is performed routinely. For metronidazole effect has been observed for all compounds there was a high degree of disagreement assessed on tumor cells, highlighting the with agar dilution, which has already been efficacy of Hydroxychloroquine over Cisplatin reported. Finally, other studies with a greater and Itraconazole. The combination of number of isolations are necessary to assess hydroxychloroquine or itraconazole with whether the method of disk diffusion, which cisplatin, improve the cytotoxic effects on is simpler and cheaper, can be continued tumor cells. routinely in our region.

126 82. POLYMERIC BIOCOMPATIBLE 83. LEUKEMIA INHIBITORY NANOCARRIERS FOR DRUG FACTOR, A NEW MODULATOR DELIVERY APPLICATIONS SHOWS OF THE OVARIAN CHOLINERGIC PRESERVED BIOLOGICAL ACTIVITY SYSTEM IN SUBFERTILE RAT. OF LOADED PROTEINS, IN VITRO Peña S., Vargas C. Rubio M. and Paredes A.H. AND IN VIVO. Laboratory of Neurobiochemistry, Center Pedroso-Santana, S.1; Fleitas-Salazar, N.1; for Neurobiochemical Studies of Endocrine Gancino-Guevara, M.1,2; Lamazares, E.1; Diseases. Faculty of Chemical and Gómez-Gaete, C.3; Toledo, J.R.1. Pharmaceutical Sciences, University of Chile, 1. Biotechnology and Biopharmaceuticals Chile. Laboratory, Pathophysiology Department, School of Biological Sciences, Universidad Leukemia Inhibitory Factor (LIF) is a de Concepción, Chile. 2. School of proinflammatory cytokine that participates Biological Sciences and Engineering, the regulation of ovarian functions. LIF YachayTech University, Ecuador. 3. participation in the subfertility period has Pharmacy Department, School of Pharmacy, not been described and the mechanism of Universidad de Concepción, Chile. action is unknown. In vitro studies have shown that LIF increase the acetylcholine In the search for new and more effective (ACh) synthesis, choline acetyltransferase therapies, polymeric nanoparticulate (ChAT) expression and its activity in the systems which protect the drugs and increase upper cervical ganglion. In our laboratory, bioavailability have been developed. The use an intrinsic ovarian cholinergic system has of biocompatible and biodegradable polymers been determined recently, which participates could guarantee the harmless character of in the regulation of ovarian function. The aim the formulation while allows the controlled was to evaluate the LIF/LIF Receptor (LIFR) release of the active principle. Using ionotropic levels and its effect on the ovarian cholinergic gelation method, we synthesized chitosan- system in subfertile rats. We measured LIF TPP nanoparticles loading recombinant and and LIFR mRNA and protein levels by qRT- model proteins, in a reproducible way. This PCR and western-blot at 3 (fertile) and 9 nanoparticulate system showed a peak of months old (subfertile) Sprague-Dawley protein released around the fourth day, in rats. To evaluate the LIF effect on the vitro, and promoted the internalization of ovarian cholinergic system, rat ovaries were loaded BSA-FITC conjugates by Hep-2 cells incubated in vitro for 3 and 8 h with LIF after 24 hours of incubation. Cytotoxicity (100ng/ml) and buffer Krebs (vehicle). ACh assay evidenced the benign character of the production and the mRNA content of the formulation, while experiments of biological genes encoding the ChAT and AChE enzymes activity in vitro and in vivo, showed a specific it was determined by fluorometry and qRT- biological response due to the system loading. PCR respectively. The results show increase Visualization of the nanoparticles was in LIF protein levels and increase of LIFR possible thanks to transmission electronic mRNA in ovaries in fertile period in subfertil microscopy. This procedure proved to be an period. Incubation with LIF increases ACh effective method to formulate proteins, and, in incubation medium, without observing potentially, other molecules, in a safe way, changes in ovarian ACh levels. The ChAT and while the release of the active principle can be AChE mRNA content enzymes significantly delayed over time. This type of systems can decrease at 3h of incubation (40% and 50%, be used in drug delivery applications in which respectively). In contrast, in ovaries incubated pharmacological interaction with the cell is for 8 h, LIF does not affect ovarian ACh levels required. nor in the incubation medium. These results suggest that LIF regulates ovarian cholinergic function during reproductive aging, pending as LIF and the cholinergic system regulate follicular development at this period.

127 84. IDENTIFICATION OF RESIDUES and promotion of DA efflux. Thus, this study INVOLVED IN THE DOPAMINE provides a starting point for a further detailed TRANSPORTER-GBETAGAMMA characterization of the DAT-Gbetagamma PHYSICAL/FUNCTIONAL interaction and a better understanding of its INTERACTION. contribution to DAT-mediated efflux. Pino J.A. 1; Nuñez-Vivanco G. 2; Hidalgo G. 3; Quiroz M. 4; Reyes-Parada M. 5; Torres 85. AMYLOID BETA OLIGOMERS G.E. 4. INTERRUPT NUCLEAR CA2+ 1, Biomedical Sciences Laboratory, Faculty TRANSIENTS AND GENE of Medicine, University of Atacama. 2, EXPRESSION INDUCED BY Center for Bioinformatics, Simulations GABAZINE IN HIPPOCAMPAL and Modelling, Universidad de Talca. 3, NEURONS. Department of Pharmacology & Therapeutics, Lobos P. 1; Vega I. 1; Bruna B. 1; Henriquez N. School of Medicine, University of Florida. 1; Hidalgo C. 1 , 2; Paula-Lima A. 1,3. 4, Department of Molecular, Cellular & 1, Laboratorio de Señales Mediadas por Biomedical Sciences, City College of the City Calcio, Instituto de Neurociencia Biómedica University of New York. 5, School of Medicine, (BNI), Facultad de Medicina, Universidad de Faculty of Medical Sciences, Universidad de Chile.; 2, Laboratorio de Señales Mediadas Santiago de Chile. por Calcio, Instituto de Ciencias Biomedicas (ICBM), Departamento de Neurociencia The dopamine transporter (DAT) plays Facultad de Medicina, Universidad de Chile. ; a crucial role in the regulation of brain 3, Laboratorio de Biología Celular y Molecular dopamine (DA) homeostasis. Through re- , Instituto de Ciencias Odontologicas (ICOD), uptake of DA, DAT serves two important Facultad de Odontología, Universidad de functions: the termination of synaptic Chile. transmission at dopaminergic terminals, and the replenishment of vesicular DA pools. In Ca2+ signals are essential mechanisms addition to uptake, DAT can also function to that regulate neuronal plasticity. Nuclear release DA. This process, which is referred to Ca2+ transients generated by neuronal as DAT-mediated efflux, is the mechanism activity induce changes in gene expression used by potent and highly addictive and in dendritic spine remodeling, which psychostimulants, such as amphetamine and are mediated by the rapid activation and its analogues, to increase extracellular DA expression of transcription factors. Among levels in motivational and reward areas of the them, Npas4 is known for inducing distinct brain. It has long being recognized that DA activity-dependent gene programs that neurons release DA through exocytotic and regulate the expression of neurotrophic non-exocytotic processes. However, the exact factors and antioxidant enzymes. Thus, mechanism by which physiological signals Npas4 may provide a molecular link between or psychostimulants, such as amphetamine, neuronal activity and the activation of induce DA efflux through DAT still remains memory and neuroprotection signaling a complex and not completely understood pathways. Amyloid-beta oligomers (A-beta area of research. Recently, we discovered Oligomers) are synaptotoxins that induce that the G protein betagamma subunits aberrant Ca2+ signals and promote Reactive bind to the intracellular carboxy-terminus Oxygen Species (ROS) generation,leading to of DAT and regulate transporter activity. synaptic plasticity disruption. In this work, More importantly, we have observed that we studied the effects of AβOs in nuclear activation of Gbetagamma promotes DAT- Ca2+ signals production and gene expression mediated DA efflux. However, the amino acid induced by Gabazine, a GABA(A) receptor residues involved in Gbetagamma interaction blocker that functions as an inductor of site(s) in DAT and their role in transporter synaptic activity. To this aim, we transfected regulation remain largely unknown. Here, primary hippocampal neuronal cultures with we used a combination of bioinformatics, a genetically encoded Ca2+ indicator with mutagenesis, immunoprecipitations, and nuclear destination (GCaMP3-NLS). We pre- functional assays to identify the Gbetagamma incubated these cultures with AβOs for 6 h and binding site on DAT and its role in transporter applied Gabazine at the microscope stage, to regulation. Preliminary functional studies record nuclear Ca2+ signals by live-imaging. are consistent with previous biochemical We also performed immunocytochemistry evidence indicating that the sequence FREKL to evaluate CREB phosphorylation and RT- located in the carboxy-terminus of DAT plays qPCR to evaluate the mRNA expression of a role in Gbetagamma interaction with DAT Npas4, BDNF and of the antioxidant enzymes

128 Glutamate-Cysteine-Ligase (GCL) and induction of epileptogenic behavior using NADPH-Quinone-Oxidoreductase (Nqo1) Pentylenetetrazol to evaluate CNS health in these conditions. Our results indicate after neurula-treatments. We observe a that neurons treated with A-beta Oligomers decrease of almost ~50% in the seizure showed reduced nuclear Ca2+signals and latency onset necessary for epileptogenic diminished Npas4, GCL and Nqo1 mRNA behavior vs not treated controls. Our results expression levels in response to GBZ. In suggest that, glutamate could be released summary, the present results indicate that using vesicles proteins and the expression A-beta Oligomers altered the activation of of AMPAR don’t participate in the normal signaling pathways induced by gabazine, neural tube development but could regulate leading to a disruption of neuroprotective additional later process important for the gene expression pathways essential to CNS establishment on Xenopus laevis. memory and learning processes, which are affected in neurodegenerative diseases. 87. TRIPANOCIDAL ACTIVITY OF CASTANEDIA SANTAMARTENSIS 86. EVALUATION OF SINAPTIC (ASTERACEAE) AGAINST COMPONENTS DURING TRYPANOSOMA CRUZI. NEURULATION OF XENOPUS LAEVIS Quintero H.1; Lapier M2; Carbonó, E3; Torres EMBRYOS. O4; Liempi A5; Maya J2; Delporte C1. Ingrid Pinto Borguero1, Nicolás Zúñiga S.1, 1,Laboratorio de Productos Naturales, Claudio Retamal U.1, Patricio Castro M.1. Departamento de Química Farmacológica y 1 Laboratory of Physiology and Pharmacology Toxicológica, Facultad de Ciencias Químicas for Neural Development, Department of y Farmacéuticas, Universidad de Chile. Physiology, Faculty of Biological Sciences, 2,Laboratorio de Bioquímica, Metabolismo y Universidad de Concepción, Concepción, Resistencia a Fármacos, Instituto de Ciencias Chile. Biomédicas ICBM, Facultad de Medicina, Universidad de Chile. 3,Herbario UTMC, In chordates, neurulation and neural tube Universidad del Magdalena, Colombia. formation is the first step in the central 4,IDEFARMA, Programa de Regencia en nervous system (CNS) development. Failures, Farmacia, Facultad de Ciencias de la Salud, by genetic or environmental alterations, Universidad de Córdoba. 5,Laboratorio in this process may induce neural tube de mecanismos de infección parasitaria, defects (NTDs). It has been described in Instituto de Ciencias Biomédicas ICBM, the literature, that the use of anti-epileptic Facultad de Medicina, Universidad de Chile. drugs (AEDs) during pregnancy, increase the probability of NTDs by mechanisms not Chagas disease (CD) an endemic disease from completely identified. Recently, glutamate Latin America, is caused by Trypanosoma signaling through NMDAR has been proposed cruzi infection. More than 7 million people to participate in neurulation process. Here are infected. Currently, there are two drugs we hypothesized that glutamate would be derived from nitro compounds for the released by a vesicular-related mechanisms treatment of CD with important side effects, which contribute to cellular responses that cause the treatment to be abandoned, necessary for normal neural tube formation. furthermore, the effectiveness in the chronic For evaluate this, we use Xenopus laevis phase is still controversial. Therefore, is embryos, collected in different neurulation necessary the search for new drugs that are stages (9 - 20 hours post-fertilization more effective and better tolerated. Castanedia (hpf)) for obtain RNA transcripts. Then we santamartensis R. M. King & H. Rob, is performed PCR and qPCR for assess relative known for their properties to treat skin sores. expression studies, focalized in evaluate the The objective of this study was to evaluate the presence of vesicular release-related and trypanocidal activity of an ethanolic extract synaptic receptor proteins. We observe the (ETE) of C. santamartensis and its fractions. presence of vesicle related proteins, such as Air-dried and powdered leaves, were SNAP25, VAMP2, Syntaxin and VGLUT1, extracted at room temperature with ethanol as well as glutamate receptor MGLuR2 and and concentrated and dried by evaporation AMPAR during neurulation. Then, to evaluate at reduced pressure. The fractionation was the functionality of AMPAR in neurulation, obtained by chromatographic separation we perform pharmacological studies, using methods, using solvents of different polarity. the antagonist CNQX. We observe that CNQX The in vitro trypanocidal activity of the ETE don’t provoke any evident alteration in neural and the fractions was determined against tube formation. Finally, we performed an T. cruzi trypomastigotes (Dm28 strain)

129 using MTT and flow cytometry techniques. 89. NEW LIPOPHILIC CATIONS Nifurtimox was used as a reference drug. DERIVED FROM CAFFEIC ACID The IC50 (concentration that produce INDUCE CYTOTOXIC EFFECT IN a 50% parasitic death) was calculated HUMAN COLORECTAL CANCER using the least squares method. The ETE CELLS. presented trypanocidal activity (IC50 of Ramírez D1,2, Rojas D1, Cortez G1,2, Escobar 197.3 micrograms/mL). The CS200; CS300 B1,3, Jara JA3, Catalán M1. and CS400 fractions, presented trypanocidal 1 Laboratory of Biochemistry, Metabolism activity with IC50 values of 91.2; 63.9 and 15,4 and Drug Resistance, ICBM, Facultad de microgramos/mL respectively. The IC50 of Medicina, Universidad de Chile, Santiago, the reference drug was 5.7 micrograms/mL. Chile. 2 Department of Biology, Faculty of The results indicate that C. santamartensis Basic Sciences, Metropolitan University contains secondary metabolites with activity of Education Sciences, Santiago, Chile. against T. cruzi. 3 Pharmacology Laboratory, Research Institute of Dental Sciences (ICOD), School 88. EFFECTS OF P2X2R of Dentistry, University of Chile, Santiago, OVEREXPRESSION IN CELL LINES Chile. AND ITS IMPACT IN SIGNALING PATHWAYS ASSOCIATED TO AMPK/ One of the deadliest pathologies worldwide CAMKII. is cancer. Colorectal cancer is the third Ramírez-Molina, O; Godoy, PA; Silva- most common type of cancer. A few drugs Grecchi, T; Mennickent, D, Gavilán J; Panes- are provided for treatment this disease, like Fernández, J, Muñoz-Molina, N; Cuevas, ME; 5-fluorouracil, oxaliplatin and irinotecan, Fuentealba, J. as standard therapy. However, this Departamento de Fisiología, Facultad therapy several times failed due to high de Ciencias Biológicas, Universidad de drug resistance and side effects, leading Concepción, Concepción, Chile. cancer progression. There are several risk factors both exogenous and endogenous One of the main toxic agents in Alzheimer’s that increase the incidence of this disease Disease (AD) are the soluble oligomers of in the organism, hence the importance of the Aß peptide (OS-Aß). Chronic treatments characterize the cancer cells from cytological, with OS-Aß have been shown to increase the metabolic and molecular aspects. These expression of the P2X2 receptor (P2X2R) features give them the differences between in PC12 cells and rat hippocampal cells, normal epithelium cells, becoming with high participating in increasing intracellular proliferative rates in an uncontrolled manner. calcium and allowing a leak of ATP to the In this sense, the mitochondria appear as extracellular environment. AMPK protein a new target for new molecules against kinase has several roles on protein, energy and cancer, since they have high mitochondrial- mitochondrial metabolism and is regulated transmembrane potential than normal cells, by changes in the levels of intracellular capable to accumulate cationic compounds. Ca2+ and AMP/ATP ratio. AMPK is This work is focused in the evaluation of a capable of phosphorylate PGC-1a, which is new set of molecules derivatives from caffeic a transcription co-activator that, when is acid attached to a different size length of phosphorylated, is activated and translocates triphenylphosphonium-aliphatic chain and to the nucleus, promoting mitochondrial their effect on human colorectal cancer cells. biogenesis. Using PC12 cells to overexpress We evaluated cytotoxic effect by MTT assay, P2X2R, the effect of its activation was the decrease of mitochondrial potential by assessed by ATP treatments, on AMPK activity flow cytometry and the decrease of cellular and the subcellular distribution of PGC- of ATP levels by luminescence. The results 1a. From functional experiments (calcium showed that the compounds were cytotoxic in microfluorimetry and electrophysiology), colorectal cell lines (HCT-15 and COLO 205), immunocytochemistry and Western blot, decreasing mitochondrial-transmembrane it was concluded that overexpression and potential and cellular ATP levels. In activation of P2X2R by ATP, prevents an conclusion, these new compounds may increase in AMPK activity and generates induce cytotoxic effect by a mitochondrial changes in the subcellular distribution of mechanism, inducing bioenergetics stress, PGC-1a, which suggests that P2X2R would suggesting the importance of studying new be related to the toxicity generated by the pharmacological agents taking advantage of Aß peptide and the intracellular calcium the cellular singularities like mitochondrial overload. metabolism.

130 90. BIOLOGICAL EFFECT OF NOVEL understanding the mechanisms that trigger TRIAZOLOPYRIDINES AGAINST abnormal proliferation rates and subsequent MACROPHAGES MURINE. tumor migration in gastric epithelia. Between Ramos-Aguayo A.1; Lapier M.1-2; Olea-Azar these possibilities, purinergic signaling C.1; Maya J.2. emerges as promising pathway that regulate 1Laboratorio de Antioxidantes y radicales cell growth, proliferation and migration libres, Departamento de Química Inorgánica according to the expression rates of its many y Analítica, Facultad de Ciencias Químicas receptor classes and subclasses. Among these, y Farmacéuticas, Universidad de Chile. P2Y2 receptor (P2Y2R) is widely known by its 2Laboratorio de Bioquímica, Metabolismo contribution to cell invasion and metastasis in y Resistencia a Fármacos, Instituto de prostate, colorectal and colon cancer; which Ciencias Biomédicas ICBM, Facultad is in contrast to the antiproliferative effects de Medicina, Universidad de Chile. reported for P2X4 receptor (P2X4R) on cancer 3Laboratorio de Productos Naturales, models in vitro. Despite all this background, Departamento de Química Farmacológica y purinergic signaling involvement in gastric Toxicológica Facultad de Ciencias Químicas y cancer remains unknown. For this reason, Farmacéuticas, Universidad de Chile. our investigation was focused to characterize the expression profile of P2Y2R and P2X4R, Recently, in our group we have synthesized and in terms of protein levels by western blot and characterized triazolo pyridine derivatives, gene expression by qPCR, in cell lines derived based on a rational study (structure-activity), from primary gastric adenocarcinoma where we found that compound 1 has an (AGS), moderately and mildly differentiated antiproliferative effect against the replicative metastatic gastric adenocarcinoma (MKN- form of the Trypanosome cruzi parasite. 74 and MKN-45, respectively) and healthy However, to evaluate the cytotoxic activity gastric epithelia (GES-1). Moreover, to of the new [1.2.3] triazolo [1.5a] pyridine assess P2Y2R and P2X4R contribution in a murine cell model, which is the first to gastric cancer growth and invasion, we defense system against T. cruzi, we find evaluated the effect of different agonists and the macrophages. Viability results by MTT antagonists on cell proliferation by Resazurin indicate that compound 1 and 2, have cytotoxic assay, and stablished metastatic potential activity in this system. On the other hand, by by transepithelial electrical resistance flow cytometry and propidium iodide, we (TEER) measurements in the gastric cell have found that compound 2 can stop the cell lines described above after overexpressing cycle, consequently, stop cell proliferation or silencing P2Y2R and P2X4R. Our results and induce apoptosis death processes. This provide preliminary insights on gastric cancer effect is observed in the literature with classic pathophysiology that can be used as future sterols synthesis inhibitors. This indicates pharmacological approaches for treatment. that they are possibly affecting an enzyme of the p450 complex in mammalian cells. 92. INTRACELLULAR AMYLOID- BETA OLIGOMERS DECREASE 91. P2Y2R AND P2X4R EXPRESSION EXCITABILITY AND AMPA PROFILE AND ITS ROLE IN MEDIATED CURRENT IN NUCLEUS PROLIFERATION AND METASTATIC ACCUMBENS NEURONS. POTENTIAL IN GASTRIC CANCER N.O. Riffo, E.J. Fernandez and L.G. Aguayo. CELL LINES. Laboratory of Neurophysiology, Department Reyna-Jeldes M.A.; Cerda-Barraza D.C.; of Physiology, University of Concepcion. Coddou C. Laboratorio de Señalización Purinérgica, Alzheimer disease (AD) is a progressive Departamento de Ciencias Biomédicas, neurological disorder that causes dementia in Facultad de Medicina, Universidad Católica an increasingly aging worldwide population. del Norte, Coquimbo, Chile. Despite the current dogma of AD, where extracellular aggregates of amyloid beta Gastric cancer is considered a major health peptide (Aß) initiates neurotoxicity, growing concern due to its unspecific symptomatology evidence shows synaptic dysfunction and on early stages and complex pathophysiology loss of limbic functions in early stages that hinders any attempts for targeted before amyloid plaque deposition, where the therapeutic approaches. This disease has presence of intracellular Aß has been reported. high incidence and mortality rates worldwide, The nucleus accumbens (NAc), a central being the third cancer-related cause of death integrative brain area of the limbic system, in Chile, which focuses scientific work in is particularly affected in AD in humans and

131 transgenic mice models. However, the effect the glycine-activated current through that intracellular Aß may have on neuronal recombinant alpha1-GlyRs and alpha3-GlyRs function has still not been examined. by ≈35%. Molecular docking studies based Therefore, in this study we analyzed the on the alpha3-GlyR crystal structure suggest effects of intracellular Aß oligomers (iAßo) that koumine interacts with the orthosteric on acutely dissociated NAc neurons. To pocket of the receptor, favoring a closed state evaluate if iAßo could modulate components of the ion channel. Ongoing biochemical of the neurotransmission, we used a modified studies will determine whether koumine whole-cell patch clamp technique to dialyze directly interacts whit the extracellular Aßo intracellularly through the recording domain of alpha3-GlyRs. Overall, these electrode. The effects of iAßo were study results demonstrate the actions of koumine on the maximum evoked current (Imax) on the GlyR function. These results, together where under control conditions, the AMPA with ongoing studies, may contribute to current was 149 ± 18 pA and decreased to 73 understand the mechanisms underlying the ± 15 pA after the application of iAßo 1 µM. koumine-induced analgesia and anxiolysis. Interestingly, GABA and GLY currents were not affected. Furthermore, iAßo was able 94. PHARMACOLOGICAL to decrease accumbal neurons excitability, INHIBITION “IN VIVO” OF OVARIAN diminishing the number of action potential ACETILCOLESTERASE REVERTS spikes and its amplitude. Overall, these POLIQUISTIC OVARY PHENOTYPE IN findings showed that iAßo inhibited the RAT. amplitude of AMPA receptors in accumbal Riquelme R., Ruz F., Lara HE. neurons and also decreased neuronal Laboratorio de Neurobioquímica, excitability. These effects support the notion Departamento de Bioquímica y that iAβo is able to impair neurotransmission Biología Molecular, Centro de Estudios in limbic areas. Neurobioquímicos para Enfermedades Endocrinas, Facultad de Ciencias Químicas y 93. FUNCTIONAL MODULATION Farmacéuticas, Universidad de Chile. AND MOLECULAR INTERACTION OF THE ALKALOID KOUMINE WITH Ovarian function is subject to endocrine and GLYCINE RECEPTORS. nerve regulation. An increase in sympathetic Riquelme, C.R., Sazo, A.E., Lara, C.O., tone by cold stress (CS) induces a phenotype Marileo, A.M., San Martín, V., Petermann, A., like polycystic ovarian condition (PCO). On Flaig, D., Soto, P., Pineda, B., Moraga-Cid, G., the other hand, a local cholinergic system has Yévenes, G.E. been described in rat ovary, in which we find Departamento de Fisiología, Universidad de acetylcholine (ACh), the muscarinic receptor Concepción. M1, and the enzyme acetylcholinesterase (AChE). Chronic treatment with the AChE Koumine is one of the main alkaloids of the inhibitor Huperzine A (Hup-A) has been Gelsemium genus plants. Behavioral studies reported to increase the fertility on the rat. have reported that the administration of In this context, the purpose of this study is koumine exerted analgesic and anxiolytic to determine whether the long-term changes effects. The mechanisms underlying these induced by CS on ovarian function can be beneficial effects are not well defined. reversed by increasing ACh chronically by However, behavioral studies have shown that administering Hup- A. In this study, Sprague- the analgesic and anxiolytic effects of koumine Dawley rats were subjected to CS subsequently are inhibited by strychnine, a selective hemiovarioectomized and implanted with a antagonist of inhibitory glycine receptors miniosmotic pump with Huperzine A (10 μM) (GlyRs), which are chloride-permeable or subjected to the procedure but without the pentameric ligand-gated ion channels implantation of miniosmotic pump (Sham). expressed in the central nervous system. To 28 days after the procedure the ovary and date, whether koumine is able to modulate the serum were collected to measure steroid the function of GlyRs is unknown. Here, by hormones Testosterone (T), Progesterone using biochemical, electrophysiological and (P4) and Estradiol (E2) by enzyme bioinformatics approaches, we studied the immunoassay and the follicular development potential modulation of GlyRs by koumine. by morphometry. A second group of rats were Our electrophysiological studies showed used to measure the fertility after mate with that koumine negatively modulates the GlyR males of proven fertility. The results show function. For example, the acute application that CS generates a polycystic phenotype with of 25 micromolar of koumine inhibited cysts, hyperandrogenism and low fertility.

132 The administration of Hup-A reverses the of the release kinetics showed that the alterations in follicular development and encapsulation of ACPV-56 within MPs delays hyperandrogenism produced by CS but not its release at least 10 times when compared to increase the fertility. The pharmacological the free drug. As a conclusion, the developed potential of these findings gives to the microparticles represent a promising cholinergic local system relevance in the alternative for treatment of RA. treatment of PCO. 96. DEVELOPMENT OF CHEMICALLY 95. DEVELOPMENT OF ACPV-56 CROSS-LINKED HYDROGELS LOADED MICROPARTICLES, FOR WITH POTENTIAL BIOMEDICAL THE TREATMENT OF RHEUMATOID APPLICATIONS. ARTHRITIS. PRELIMINARY STUDY. Rivas B. 1; Pedroso S. 1; Fleitas N. 1; Sandoval Riquelme C.1 ; Gómez-Gaete C.1 ; Torres P.1 ; C. 2; Gómez C. 3; Toledo J.R. 1. Chávez-Santoscoy RA.2 ; Arellano-Villaseñor 1, Laboratorio de Biotecnología y Biofármacos, N.2. Departamento de Fisiopatología, Facultad 1 Departamento de Farmacia, Facultad de Ciencias Biológicas, Universidad de de farmacia, Universidad de Concepción, Concepción; 2, Departamento de Química Concepción. 2 Facultad de Ciencias Químicas Analítica e Inorgánica, Facultad de Ciencias e Ingeniería, Universidad Autónoma de Baja Químicas, Universidad de Concepción; California-Campus Tijuana, México. 3, Departamento de Farmacia, Facultad de Química y Farmacia, Universidad de Rheumatoid arthritis (RA) is a chronic Concepción. disease whose worldwide incidence is increasing. Currently, there are non- Hydrogels based on natural origin polymers pharmacological and pharmacological have shown promising results as scaffolds approaches for the therapeutic management for cell encapsulation and drug delivery in of RA. From the latter, the drug ACPV-56 tissue engineering. In this work, biopolymeric has demonstrated anti-arthritical activity hydrogels, from natural and biocompatible due to its ability to inhibit the proliferation polymers, were produced by two methods, of activated lymphocytes, which results chemical cross-linking with glutaraldehyde in a marked anti-inflammatory effect. and freeze-thawing. The hydrogels were Clinical studies have proven that upon oral characterized using scanning electron administration of therapeutic doses, ACPV- microscopy (SEM) and Fourier-transform 56 causes gastrointestinal adverse effects infrared spectroscopy (FTIR). The SEM that negatively influence the compliance images showed that the structure and to chronic treatments. A strategy to avoid morphology of the hydrogels produced by the adverse gastrointestinal effects of chemical cross-linking differed from those ACPV-56 and increase the dosing time produced by freeze-thawing, while FTIR intervals is by incorporating the drug into analysis also revealed different chemical a controlled release system administered composition between them. Their potential by intramuscular injection. As none of the biomedical application was also assessed. available formulations containing ACPV-56 First, their biocompatibility with HEP-2 cell is intended for parenteral administration, line was tested using an MTT assay. The the objective of this research is to develop a results showed that the chemically cross- drug delivery system based on biodegradable linked hydrogels did not affect the cell viability microparticles (MPs) encapsulating ACPV- compared to the freeze-thawing-produced 56. The MPs, elaborated by spray drying of a hydrogels. We further tested the potential of mixture of anionic polysaccharides, cationic chemically cross-linked hydrogels to retain and phospholipids, were characterized and release bioactive compounds in the cells in terms of its in vitro release kinetics, by loading the hydrogels with BSA protein employing conditions that emulate the conjugated with FITC. Using a fluorescence physiological environment. The formulation microscope, we observed that the HEP-2 cells parameters were optimized in order to obtain were stained green, indicating a successful MPs suitable for injection. The obtained release of the conjugate. These data suggest micro particles were spherical, with a that our chemically cross-linked hydrogels medium diameter close 20 µm, relatively have the potential to be used for drug delivery mono-disperse and with a minor tendency to in tissue engineering applications. aggregation. The incorporation of ACPV-56 did not affect the physicochemical properties of the developed MPs. Preliminary findings

133 97. ANTIDEPRESSIVE LIKE data. Interestingly, noribogaine 40 mg/kg EFFECT INDUCED BY THE ACUTE elicited an antidepressant-like effect per se ADMINISTRATION OF IBOGAINE with a higher potency than fluoxetine. Our AND NORIBOGAINE IN RATS AND data support the possibility that this potent ITS POSSIBLE MECHANISM OF antidepressive-like action could collaborate, ACTION. at least in part, to explain the ibogaine´s Rodríguez P.,1,2 Urbanavicius J.,2 Prieto previous anti-addictive effects. J.P.,2 Fabius S.,2 Reyes A.L.,3 Sames D.,4 Scorza C.2, Carrera I.1. 98. MODULATION OF ANTIOXIDANT 1 Laboratorio de Síntesis Orgánica, ACTIVITY IN DERIVATIVES OF Departamento de Química Orgánica, Facultad AMINOETHYL PHENANTRENE de Química -Universidad de la República, AND HALO- APORPHINES WITH Montevideo – Uruguay; 2 Departamento de ANTINEOPLASTIC ACTIVITY. Neurofarmacología Experimental, Instituto Rodríguez O. 1, Garrido-Ayala L. 2, Salgado- de Investigaciones Biológicas Clemente Camacho C. 1, Santos J.C. 3, Vallejo E. 1, Estable, Montevideo – Uruguay; 3 Centro Georges N. 1, Asencio M. 1. Uruguayo de Imageneología Molecular, 1. Laboratorio de Investigación e Innovación Montevideo – Uruguay; 4 Department of Química, Facultad de Ciencias Naturales, Chemistry, Columbia University, New York – Matemáticas y Medio Ambiente, Universidad United States. Tecnológica Metropolitana. 2 Laboratorio de Tecnología de Alimentos. Universidad de Previous human subjective data and animal Santiago de Chile 3. Laboratorio de Síntesis studies demonstrated that the psychedelic y Química Teórica, Universidad Andrés Bello. alkaloid ibogaine, and its metabolite noribogaine, have potent antiaddictive In the search for new molecules with effects. The biological mechanism through antineoplastic and antioxidant properties both compounds elicit this beneficial derived from the natural product boldine (1), effect remains still unclear. Among several it was proposed to increase the molecular molecular targets, ibogaine and noribogaine lipophilicity of the precursor molecule with inhibit the serotonin transporter (SERT). the expectation of improving the above This action and a longterm increase of brain- mentioned bioactivities. Using (1) as starting derived neurotrophic factor RNAm levels matherial was prepared: secoboldine (N, N- in the rat prefrontal cortex that we found in (methyl) (ethyl) phenanthrene; seco-boldine our previous study after the acute ibogaine (2)) and a series of halo derivatives (halo = i.p. administration, lead us to hypothesize Cl or Br) (3-6), which were characterized by that the anti-addictive property of ibogaine NMR-1H and -13C and the measurement of and noribogaine could be related to a potent their melting point. Boldine (1) has the lowest putative antidepressant-like effect. Consistent dissociation energy in the phenolic group of with this possibility we characterized C-9 with respect to C2-OH which is consistent the behavioral effects (dose and time- with the highest acidity recorded for first dependence) induced by the acute ibogaine phenolic group. The insertion of one halogen (20 and 40 mg/kg) and noribogaine (20 atom (3,4) maintains the energy value to break and 40 mg/kg) administration in rats using the O-H bond, but in compounds that bearing the forced swimming test (FST). Fluoxetine two halogen atom (5,6) the dissociation (40 mg/kg/i.p.) a standard antidepressant energy markedly increases while antioxidant drug, was used as a control. We found that capacity decrease in these compounds. This ibogaine and noribogaine induced a dose- experimental evidence is correlated with the and timedependent antidepressive-like effect. “local softness” (LS) exhibited by boldine To know if the antidepressive-like effect and halogenated compounds. In conclusion, induced by ibogaine was due to an effect the antioxidant activity in these compounds per se or by the presence of its metabolite can be modulated by the insertion of halogen (noribogaine) we intravenously injected atoms in the appropriate positions. animals with ibogaine. Ibogaine 1 and 5 mg/ kg after an i.v. injection on animal behavior 99. GALLIC AND GENTISIC ACID immediately evaluated in the FST. Under DERIVATES INDUCE AUTOPHAGY IN these conditions, ibogaine did not generate MURINE AND HUMAN COLORECTAL an antidepressant effect. Ibogaine seems to CANCER CELL LINES. depend on noribogaine content to induce the Rojas D.1, Ramirez D.1-2, Cortes G.1-2, beneficial effect. All the behavioral responses Escobar B.1-3, Catalán M.1. were consistent with the pharmacokinetic 1 Laboratory of Biochemistry, Metabolism

134 and Drug Resistance, ICBM, Facultad de is a bioactive plant with antiparasitic activity Medicina, Universidad de Chile, Santiago, related with its content of sesquiterpene Chile. 2 Department of Biology, Faculty of lactones (SL). Most antiparasitic studies of Basic Sciences, Metropolitan University chicory have been focused on nematodes, but of Education Sciences, Santiago, Chile. poorly explored against parasitic protozoa. 3 Pharmacology Laboratory, Research The aim of this study was to evaluate the Institute of Dental Sciences (ICOD), School antiprotozoal effects of SL-rich chicory of Dentistry, University of Chile, Santiago, extracts against Trypanosoma cruzi, the Chile. etiological agent of Chagas disease. SL of chicory leaves and roots were extracted Colorectal cancer is the third most common from 5 chicory cultivars (Spadona, Goldine, neoplasm in the world. The standard Larigot, Measeto and Benulite) using treatment consists mainly in surgery and methanol/water and purified by solid-phase the use of three first-line chemotherapies, extraction. The extracts were dissolved 5-fluorouracil, oxaliplatin and irinotecan. in DMSO. SL profiles of the extracts were The high drug resistance, several side effects characterised by UHPLC-MS metabolomics. and high costs of the treatments, give an The cytotoxicity of extracts was tested on opportunity to search for new molecules T. cruzi trypomastigotes (Y strain) and with new pharmacological targets. In the mammalian Vero cells. Trypomastigotes were recent years, cancer cell mitochondria have incubated for 24 h with serial dilutions of become in an interesting pharmacological extracts (100-6.3 µg/mL), and benznidazole target due to their high mitochondrial- was used as positive control. Vero cells were transmembrane potential that allows exposed to extracts for 24 h at 100 and 50 accumulated cationic probes conjugated µg/mL to evaluate cytotoxicity. Cell viability with cytotoxic pharmacophore. In our was evaluated by resazurin reduction test. laboratory, the gallic and gentisic acid Chemical profiling showed that chicory derivatives as conjugated with lipophilic extracts have distinct content of SL among cationic triphenylphosphonium through cultivars and between plant parts. All the an aliphatic chain of ten carbons have been extracts had dose-dependent effect against tested in breast and colorectal cancer cells. isolated trypomastigotes. However, Spadona These compounds triggered a series of events leaf extract was the only with no toxicity that leads cell apoptosis. The objective of against Vero cells at 100 µg/mL, suggesting this work is described how the decrease of a selective trypanocidal activity. Taken ATP levels induces the activation of AMPK, together, these results revealed that chicory which promotes death by autophagy in Spadona leaf warrants deeper exploration colorectal cancer lines. Through western regarding the relationship between its blot and luminescence assay, we observed chemical profile and antiprotozoal activity. that in human and murine colorectal cell Consequently, these results encourage further lines, the derivatives induce the reduction in investigation of chicory as a source of SL with cellular ATP levels followed by the activation antiparasitic therapeutic potential. of AMPK and LC3B, leading to cell death by autophagy. In conclusion, our compounds 101. AUTISTIC AUTOANTIBODIES may be inducing apoptosis by triggering ABSORBED FROM BREAST mitochondrial unbalance, energy stress and MILK GENERATES COGNITIVE autophagy. IMPAIRMENT IN BREEDING FEMALE BUT NOT MALE RATS. 100. ANTIPROTOZOAL ACTIVITY OF Rossi G.1,2; Cobarrubias A.1; Arancibia M.1; CHICORY (CICHORIUM INTYBUS) Araya G.1; Uribe F.1; Gonzalez-Gronow M.1; AGAINST TRYPANOSOMA CRUZI. Sandoval R.1. Romero-Uzqueda Y. 1; Peña-Espinoza, M. 1; 1. Environmental Neurotoxicology Valente, A. 2; Williams, A. 2; Thamsborg, S. Laboratory, Department of Biomedical 2; López-Muñoz, R. 1. Sciences, Faculty of Medicine, Universidad 1, Instituto de Farmacología y Morfofisiología, Católica del Norte; 2, PhD. Program in Facultad de Ciencias Veterinarias, Applied Ecology and Biology, Universidad Universidad Austral de Chile. Católica del Norte.

Chagas disease is an endemic parasitosis in Autism spectrum disorders (ASD) involve Latin America. However, its drug treatment a range of complex neurodevelopmental frequently induces adverse effects. Thus, it disorders, characterized by social is urgent to develop new therapies. Chicory impairments, communication difficulties,

135 and restricted, repetitive and stereotyped MDMA (3,4-methylenedioxymethampheta patterns of behavior. ASD exerts a significant mine, “ecstasy”) is a psychotropic drug that physiological, emotional and financial induces an “open mind” state in healthy burden on the families of the individual humans characterized by heightened self- and society as a whole. Recently, beside the acceptance, openness to communication knowledge about genetic factors involved and a fear threshold decrease known as in this pathology, there is new evidence the entactogenic syndrome. Disregarding related to immunological causes of ASD. its therapeutic potential, direct evaluation Therefore, it is of outmost importance to of MDMA-like effects in animal models elucidate the molecular and physiological remained limited to the pro-social paradigm, mechanisms of ASD pathology. Data from us a model that recreates different stereotyped and others have shown that normal young rat rodent behaviors. In contrast to these classical hippocampal slices incubated with purified pharmacological criteria, helping behavior is a IgA autoantibodies from ASD patients and complex type of pro-social paradigm that has breeding rats from pregnant mothers injected been recently described in rodents. It stands with the same antibodies, impairs LTP as well out because of its pertinence to develop a as disrupts learning and memory. Taking more sophisticated pharmacological model to this into account, we hypothesized that ASD study human-like behaviors, as it may occur autoantibodies are absorbed from breast in rats as a result of the interaction between milk and generates autoimmune-related psychomotor capabilities and the amount of cognitive impairment characteristic of ASD stress experienced by the animal, even in the pathology. To achieve this aim, we used absence of reward. Despite of its relevance, a rat model where mothers were injected the behavioral characterization of the effects with ASD autoantibodies during breast milk of MDMA in this model remains unexplored. period and the breeding was tested after In the present work, a preliminary that period using learning and memory characterization of the effects of MDMA on test together with electrophysiological and helping behavior in male rat pairs (helper rat immunohistochemical studies. We found + victim rat; with/without previous individual that both LTP and learning and memory were housing) after 12 days-administration/ significantly impaired in female but not male training cycles at two different dose levels (5 breeding rats and this alteration are correlated mg/kg; 10 mg/kg i.p.) has been attempted with the presence of ASD autoantibodies using a slightly modified water-trap model in hippocampus and Cortex. These results developed ad hoc. The results obtained demonstrate that ASD autoantibodies are indicated that MDMA might not enhance incorporated from breeding milk, cross both helping behavior compared to controls when intestinal and blood-brain barrier and impairs the acting roles of each pair member has not learning and memory in a sex-preference been interchanged. In contrast, current data fashion. They also give us new knowledge seems to be in agreement with the notion about possible causes of autism and opening that helping assistance may rather depend a new line in pharmacological therapies. on if the rat pair met each other previously or not and/or the experience of being trapped 102. MDMA (3,4-METHYLENEDI in the water trap, at least at the dose ranges OXYMETHAMPHETAMINE) AND evaluated. HELPING BEHAVIOR: PRELIMINARY CHARACTERIZATION IN SPRAGUE- 103. EFFECT OF A LACTOBACILLUS DAWLEY RATS. ADMINISTRATION ON ANXIETY- Vilches-Lagos, M.J.1,4, Albornoz- LIKE BEHAVIORS IN ADULT RATS. Bustamante, J. 1,4, Castro-Castillo, V.2; Salazar-Contreras, C.1, Escobar-Luna, J.1, Hernández, A.3; Sáez-Briones, P. 1,4. Barrera-Bugueño, C.1, Julio-Pieper, M.1, 1 Laboratory of Neuropharmacology and Gotteland M.2, Bravo, JA.1. Behavior, Faculty of Medical Sciences, 1Grupo de NeuroGastroBioquímica, Universidad de Santiago de Chile. 2 Laboratorio de Química Biológica. Instituto Department of Organic Chemistry and de Química, Facultad de Ciencias, Pontificia Physical Chemistry, Faculty of Chemical and Universidad Católica de Valparaíso, Pharmaceutical Sciences, Universidad de Valparaíso. Chile. 2Departamento de Chile. 3 Laboratory of Neurobiology, Faculty Nutrición, Facultad de Medicina, Universidad of Chemistry and Biology, Universidad de de Chile, Santiago. Chile. Santiago de Chile. 4 School of Medicine, Faculty of Medical Sciences, Universidad de There is increasing evidence that gut Santiago de Chile. microbes affect central nervous system

136 (CNS) function. For instance, there are some present study investigated whether a Chilean Lactobacillus strains with proven anxiolytic Humulus lupulus extract, previously selected and antidepressant like effects in rodents. by demonstrate antioxidant properties However, there is also evidence that other (enzymatic and in vitro assays), can elicit Lactobacillus have anxiogenic effects in rats. effects on the central nervous system, using In this regard, our previous findings show various experimental models in rats. that 2 week administration of the potential Methods: a) Treatments: Two different doses probiotic bacteria Lactobacillus casei L-54-2- (low or high) of Humulus lupulus extract 33 to healthy pre-pubescent Sprague-Dawley from a regional variety, or Medi-Drop- male rats, increased anxiety like behaviors, sucralose used as vehicle (control group), while lowering hippocampal expression of were orally administered for 42 days to 5-HT1A receptor. Therefore, we asked if adults male Sprague-Dawley rats. Extracts this anxiogenic-like effect was due to the were administrated one hour before the rat’s young age (post-natal day [PND] 35). behavioral tests performed in this study. To test this, we administered 104 CFU/ml b) Open field was used for the evaluation of of L. casei L-54-2-33 in the drinking water locomotor activity. Anxiety was evaluated of male Sprague-Dawley rats from PND65 by elevated plus-maze test (EPM). Results: till PND76, and compared their anxiety-like No significant differences were observed on behaviors with age and sex matched control the locomotor behavior of rats, following rats fed with vehicle (sucralose and MRS the oral administration of two doses of broth) in the drinking water for the same extract or control, measured by total distance amount of time. Anxiety-like behaviors were travelled and average speed, on the open field then evaluated using the open field (OF) apparatus. But rats treated with low dose of test and elevated plus maze (EPM). Rats fed extract significantly increased (16,75 +5,1 with the bacteria spent significantly less time sec) the time spent on the central zone of the in the central zone of the OF in comparison open field, compared to control (4,7+ 1,7 sec); to controls, while there were no differences this parameter is correlated with statistically between bacteria fed and controls rats in the difference observed with open arm spent time EPM. These results match with our previous on the EPM between control and medium findings in younger male rats, suggesting that dose of extract. Conclusion: These results the anxiogenic effects of L. casei L 54 2 33 are show that the low dose of this Chilean lupulus strain specific, and that these effect do not extract, could exert and anxiolytic effect. depend on the age of the animal. Together our findings suggest that bacteria known to 105. PRODUCTION OF promote changes in the CNS, might exert its RECOMBINANT HUMAN strain-specific effects regardless of the age of INTERLEUKIN-4 EXPRESSED IN the host, which is a novel feature in probiotic ESCHERICHIA COLI AS INCLUSION (or in this case psychobiotic) interventions BODIES. Urrutia J.1,3; Herrera P.1,3; Mansilla R.1,3 ; 104. ANXIOLYTIC EFFECTS OF A Toledo J.2,3 ; Sánchez O.1,3. CHILEAN EXTRACT OF HUMULUS 1, Laboratorio de Biofármacos LUPULUS. Recombinantes, Departamento fr Facultad Godoy, J (1); Sáez, S.(1); Ríos M(2); Silva, de Ciencias Biológicas, Universidad de M.E(2).; Rivera, F(3); Simirgiotis, M.J.(2) Concepcion1; Laboratorio de Fisiopatología, Sánchez-Montoya, E.L.(2). Facultad de Ciencias Biológicas, Universidad (1)Escuela de Química y Farmacia, Facultad de Concepcion2; Centro de Biotecnología y de Ciencias, Universidad Austral de Chile (2) Biomedicina SPA.3 Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile., Universidad Interleukin-4 (IL-4) is a potent lymphoid cell Austral de Chile. (3)Instituto de Anatomía, growth factor that stimulates the growth and Histología y Patología, Facultad de Medicina, survivability of certain B cells and T cells. It Universidad Austral de Chile. exhibits anti-inflammatory responses and participates in immune processes by providing Introduction: Humulus lupulus is broadly protection from intracellular pathogens. IL-4 cultivated in the world both for beer also plays an important role in T helper cells manufacture, but also for its medicinal differentiation. Additionally, it can suppress properties, for the treatment of excitability pro-inflammatory cytokines. In this work, and restlessness. There are important regional a His-tagged recombinant human IL-4 was differences in metabolome composition of overexpressed in Escherichia coli under the plants that influence its response. The the control of a T7 promoter. The resulting

137 inclusion bodies were separated from cellular Here, by using immunocytochemical and debris by centrifugation and solubilized by GST pull-down assays, we reported a direct 8M urea. The denatured IL-4 was refolded association between the a1, a2 and the a3 in a single chromatographic step by gradual subunits of the GlyRs with AKAP79. Confocal removal of denaturant agent. This protocol imaging showed that AKAP79 specifically yielded 4.5 mg of IL-4 from 40g of biomass. co-localized with all the GlyRs subunits. In The refolded protein was highly pure and addition, in pull-down studies we observed subsequent biological activity assay that was that a GST-ICDa3GlyR fusion protein are measured in the human erythroleukemia able to bind AKAP79.Thus, our experimental cell line TF-1 suggested that IL-4 had similar data contributes to the characterization of a activity profile to the commercial produced new intracellular partner of the GlyRs. This protein. The results of this study suggest that open new avenues in the searching of new on-column refolding represent a convenient therapeutic targets for the inflammatory and low-cost process for the refolding of chronic pain treatments. IL-4 and may be a promising candidate for development as commercial reactive for 107. PERINATAL ASPHYXIA INDUCES cancer research. LONG-TERM DEMYELINATION, OLIGODENDROCYTES DAMAGE 106. A-KINASE ANCHORING AND NEUROINFLAMMATION IN RAT PROTEIN AKAP79 INTERACTS WITH BRAIN: PREVENTION BY NEONATAL THE INTRACELLULAR DOMAIN OF MESENCHYMAL STEM CELLS THE GLYCINE RECEPTORS ALPHA TREATMENT. SUBUNITS. Andrea Tapia-Bustos1, Carolyne Lespay- Sazo, A.E.1,2, Lara, C.O.1, Marileo, A.M.1, San Rebolledo1, Ronald Perez-Lobos1, Valentina Martín, V.1, Peterman, A. 1, Flaig, D. 2, Soto, Vio1, Emmanuel Casanova1, Rosario Matte1, P2., Pineda, B2., Riquelme, C.R.2., Moraga- Emilia Licci1, Diego Bustamante1, José Luis Cid, G.2, Yévenes, G.E.1. Valdés1,2, Fernando Ezquer3, Mario Herrera- 1Laboratory of Neuropharmacology, Marschitz1, Paola Morales1,2. 2Laboratory of Structural 1 Programme of Molecular & Clinical Neuropharmacology. Department of Pharmacology, ICBM, 2Department of Physiology, University of Concepción, Neuroscience, Medical Faculty, University Concepción. of Chile. 3Center for Regenerative Medicine, Faculty of Medicine-Clínica Alemana, Glycinergic inhibition is critical for Universidad del Desarrollo, Santiago, Chile. breathing control, muscle tone regulation and nociception. Studies showed that PKA The effect of perinatal asphyxia (PA) was phosphorylation in the residue S346 located evaluated on myelination, oligodendrocytes, in the intracellular domain (ICD) of the neuroinflammation and cell death in rat glycine receptor (GlyRs) containing the alpha telencephalon and hippocampus from 3 subunit. This posttranslational modification postnatal (P)1 up to 14 days, a period produces inhibition of glycinergic function characterized by a spur of neuronal in the spinal cord, which was related to the networking, finding a sustained injury generation of inflammatory chronic pain. that may have profound adverse effects on Noteworthy, the molecular mechanisms neuronal development. The study evaluated associated with the inhibition of a3GlyR by whether that injury could be prevented by phosphorylation are not yet elucidated. In mesenchymal stem cells (MSCs) treatment. this context, the interaction of the A-kinase PA was induced by immersing foetus- anchoring protein (AKAP79) to partners containing uterine horns into a water bath involved in nociceptive pathways has been at 37°C for 21 min. Asphyxia-exposed (AS) recently reported. Specifically, it has been and sibling caesarean-delivered (CS) foetuses observed that disruption of the interaction were resuscitated and nurtured by surrogate between AKAP79 and TRPV1 decreases dams. Animals were euthanized at P1, 7 or sensitization in nociceptive neurons. 14, dissecting samples from telencephalon Furthermore, the direct interaction between and hippocampus to be assayed for (i) AKAP79 and the beta3 and beta2 subunits myelin (MBP and transcriptional factors of the GABAAR promote the PKA-mediated involved in repairing demyelination, phosphorylation of serine residues located Olig-1and 2; immunofluorescence, in the ICD of those subunits. Nonetheless, RT-PCR); (ii) oligodendrocyte whether AKAP79 is able to bind GlyRs and density (immunofluorescence); (iii) modulates its function is still unknown. neuroinflammation (RT-PCR, ELISA,

138 immunofluorescence), and (iv) cell death was to evaluate the MICA expression in gastric (TUNEL). Two hours after delivery, AS adenocarcinoma and their relationship with and CS neonates were injected with either the allelic variants and effect on the regulation 5 μl of vehicle or 5x104 MSCs into the of NKG2D receptor. We study the MICA left lateral ventricle. It was found that PA expression and release in samples of tumor produced: (i) a decrease of MBP density and tissue by flow cytometry and ELISA assay. oligodendrocyte/mm3 at P7 in telencephalon, We isolated DNA genomic to determinate the but not in hippocampus; (ii) an increase of MICA allele by sequence based-typing PCR Olig-1, in telencephalon at P7; (iii) an increase using specific primers. Also, we evaluate the of IL-6 mRNA levels in telencephalon at expression of NKG2D in tumor- infiltrating P7, and of IL-1β mRNA in hippocampus at NK cells by flow cytometry. Our results P14; (v) an increase of cell death, including indicated that the expression of NKG2D oligodendrocyte at P7 in telencephalon; on NK cells was inversely proportional to (vi) MSCs treatment prevented the effect the levels of MICA on tumor cells, and that of PA on demyelination, oligodendrocyte not all patients showed detectable levels of density, neuroinflammation and cell death. soluble MICA (sMICA) in their serum and, It is proposed that PA induces regionally and while the diminished expression of NKG2D developmental-dependent changes in brain on cytolytic cells did not correlate with the regions, and MSCs treatment can prevent concentration of sMICA in the serum of GA the changes induced by PA on myelination, patients, this could be due to the presence oligodendrocyte density, neuroinflammation of MICA in exovesicles as the MICA-STR and cells death. A5.1 variant. In conclusion, we propose that MICA is an immunotherapeutic target 108. MHC-CLASS I POLYPEPTIDE- in gastric adenocarcinoma and the MICA RELATED SEQUENCE A (MICA) AS allelic variants should be considered in the AN IMMUNOTHERAPEUTIC TARGET therapeutic strategies. IN CANCER. Toledo-Stuardo K. 1; Ribeiro C.1; Rodríguez 109. INTERNALIZATION J. 1; Jerez B.1; Tello S.1; Farías C. 1; González MECHANISM OF FOLATE-MODIFIED P. 2; Molina M.C.1. PAMAM DENDRIMERS IS MEDIATED 1, Laboratorio de Anticuerpos BY MORE THAN ONE ENDOCYTOSIS Recombinantes e Inmunoterapia. Centro PATHWAY. de Inmunobiotecnología, Programa Torres J.; Vásquez P.; Vidal F.; Guzmán L.; Disciplinario de Inmunología, ICBM, Alderete, J. Facultad de Medicina, Universidad de Chile. Laboratorio de Neurobiología Molecular, 2, Programa de Genética Humana, Instituto Facultad de Ciencias Biológicas, Universidad de Ciencias Biomédicas, Universidad de de Concepción. Chile. Nowadays, central nervous system (CNS) MICA is a ligand to NKG2D, an activation diseases affect 1.5 billion people worldwide receptor that triggers natural killer (NK) and there is a continuous development of new cells effector functions for early tumor therapies. However, in many cases efficiency elimination; however, the normal function of therapies is low because of biological of MICA/NKG2D axis is compromised in barriers and deficient biodistribution of cancer, including gastric adenocarcinoma drugs. New advances in the nanomedicine (GA). Several mechanisms have been have allowed the creation of nanotransporter proposed to explain this response, including systems. Among them, polyamidoamine the presence of released MICA, either as (PAMAM) dendrimers have demonstrated soluble proteins or in exovesicles, which a great potential in drug delivery to may favor down-modulation of NKG2D in CNS. PAMAM dendrimers are polymeric cytolytic cells, resulting in desensitization structures composed by an ethylenediamine of NK cells as the tumors progress. MICA is core that branches creating layers, called a highly polymorphic molecule that codifies generations, which end in primary amines allelic variants, which have been described protonated at physiological pH and can be to affect NKG2D binding avidity and cell modified with other terminal groups, such cytotoxicity, while some MICA-STR variants as folate. Considering the current difficulty located in the transmembrane domain of delivering drugs to the CNS, we examined promote NKG2D internalization. MICA-STR the internalization mechanism of folate- A5.1 variant acquires a GPI-anchor which is conjugated PAMAM dendrimers mediated recruited in exosomes. The aim in this work by folate receptor α (FRα), a membrane

139 protein overexpressed in choroid plexus observed that the count of viable CaCo-2 cells that once it binds to folate is internalized decreases as the concentration increases. by the caveolae endocytosis pathway, and In the case of HEK cells no changes in the is postulated as a target tissue for drug count are observed. MTT assay only with the delivery to CNS. In this study, we selected gastrointestinal digestion samples observated the HeLa cell line for internalization an effect of inhibition of the metabolic activity experiments, based on confocal and western- in the case of caco-2 cells, in hek cells there is blot results. One unmodified (G4) and two no significant effect. Cytotoxicity assays using folate-modified (PFO25 and PFO50) fourth LDH do not show significant changes in the generation PAMAM dendrimers were used. activity of the enzyme lactate dehydrogenase Confocal images showed that PFO50 was in any case. Finally, it is concluded that the not able to entry HeLa cells, unlike PFO25 samples have positive effect on viable cell and G4, which were visualized after one count and MTT assay, because damage hour incubation. Quantification of Mander’s colorectal cancer cells (caco-2) but not healthy coefficients indicated only a slight increase cells (HEK), while very mild cytotoxicity was of colocalization of PFO25 with FRα than observed at through the LDH assay unmodified G4, which suggests that the internalization pathway of folate-modified 111. ASSESSMENT OF DIFFERENT dendrimers is possibly mediated by more PHARMACOLOGICAL ACTIVITIES than one endocytosis mechanism. OF PEUMUS BOLDUS EXTRACTS USING CHEMICAL SUBTRACTION 110. EFFECT AQUEOU EXTRACT STRATEGY. OBTAINED FROM LEAVES OF U. Torres J. P. 1; Correa D.1; Alarcón J. 3; MOLINAE AND THEIR RESPECTIVE Gómez-Alonso S.2; Silva C.1; Pastene. E.1,3. PRODUCTS OF GASTOINTESTINAL 1 Laboratory Pharmacognosy, Department of DIGESTION ON THE VIABILITY OF Pharmacy, Faculty of Pharmacy, University COLON CANCER CELLS. of Concepción, Concepción, Chile 2 Regional Torres E. R. 1; Avello M. 1; Pastene E. 2. Institute for Applied Scientific Research, 1, Laboratorio de Farmacognosia, Faculty of Chemical Sciences, University of Departamento de Farmacia, Facultad de Castilla-La Mancha, Castilla-La Mancha, Farmacia, Universidad de Concepción. 2, Spain. 3 Laboratory of Synthesis and Laboratorio de Síntesis y Biotransformación Biotransformations of Natural Products, de productos naturales, Departamento de Department of Basic Sciences, University of Ciencias Básicas, Facultad de Ciencias, Bio-Bio, Chillán, Chile. Universidad del Bío-Bío. Peumus boldus Mol., (Monimiaceae) Colorectal cancer is the third most common is a Chilean medicinal three used for diagnosis in men (10%) and second in gastrointestinal and liver diseases. women (9.4%). The use of chemotherapy to Phytochemical profiling of this plant is fight this disease leads side effects, this the based on its aporphine alkaloids, phenolic main reason for investigations of possible compounds and essential oil. However, in antiproliferativity of different natural sources. herbal infusions some authors thought that In that regard, active compounds U. molinae flavonoids are responsible for its antioxidant and their products the gastrointestinal and chemopreventive effects rather than metabolized could act as prophylactic and alkaloids and essential oil. The objective complementary because effcts anticancer of this study was to evaluate different has been reported.The aims is To asses the knock-out extracts prepared by chemical effect aqueou extract obtained from leaves subtraction oriented to selectively remove of U. molinae and their respective products alkaloids and essential oils from crude of in vitro gastrointestinal digestion on the extracts. These extracts were obtained by viability of colon cancer cells. Ugni molinae means of conventional centrifugal partition leaves were used to prepare an aqueous chromatography (CPC) and pH-zone-refining extract, with this a gastrointestinal disgestion CPC. DPPH bleaching test, cytotoxicity in was performed, obtaining also a final residue. AGS cells, DNA damage in monocytes (Comet These samples were evaluated in different assay) and inhibition of Acetylcholinesterase viability tests, such as trypan blue exclusion, were determined for all extracts. Solutions metabolic activity (MTT) and cytotoxicity of the different lyophilized extracts were (LDH), on colorectal cancer cells (CaCo- prepared at different concentrations (1-1000 2) and healthy cells (HEK) for a period ug/mL). The results of DPPH assay indicated of 24 hours. When treating the cells, it is an IC50 of 63.05, 109 and 43.73 ug/ml for

140 total extracts, alkaloids and polyphenols to induce neural tube defects. In addition, respectively, the fraction containing the in silico studies using molecular docking polyphenols having greater antioxidant techniques, we determine the possible site of capacity. In turn, cytotoxicity tests showed cbx and enx association in the protein, located that polyphenols at concentrations lower in the extracellular domain (E2). Taken than 1000 ug/mL protected AGS cells. On together these results, we suggest that Cx 46 the contrary, alkaloid fraction reduced cell and Cx 32 will participates as hemichannel in viability from 400 ug/mL whereas fraction neural tube closure and their blockade results containing essential oil displayed higher in NTDs. toxicity from 125 ug/mL. Only the fraction with alkaloids displayed an expected 113. INHIBITION OF ENDOPLASMIC acetylcholinesterase inhibition. RETICULUM EXIT RESCUES A NIEMANN PICK TYPE C DISEASE 112. PARTICIPATION AND ROLE MODEL. OF CONNEXINS IN THE RELEASE Urbina J.; Astete G.; Milla L.A. OF GLUTAMATE AND ATP IN Centro de Investigación Biomédica y THE NEURULATION PROCESS IN Aplicada, Escuela de Medicina, Facultad de XENOPUS LAEVIS. Ciencias Médicas, Universidad de Santiago Tovar L.M 1; Benavibes C.I 1; Gonzalez A.A 1; de Chile. Castro P.A 1. Laboratorio de fisiología y farmacología Currently, more than 70 lysosomal diseases para desarrollo neural, Departamento de have been identified, accumulating Fisiología, Facultad de Ciencias Biológicas, substrates in lysosomes and late endosomes. Universidad de Concepción. Within this group we find the Niemann- Pick type C (NPC) disease, that generates Neurulation is an important process in aberrant accumulations of cholesterol and the formation and development of CNS. other lipids within cells, resulting in early This event correspond to the first step neuronal death. NP-C1, the most common of neural embryonic development (stg protein showing disease-causing mutations, 12,5–20 in Xenopus laevis) and implicates codes for a transmembrane protein NPC1, different cellular process like, migration and present in lysosomes and late endosomes proliferation. Alterations in the signaling of membranes. NP-C2, is caused by a mutation this period could result in neural tube defects in the NPC2 gene that encodes the NPC2 (NTDs). Several studies has demonstrated protein, which is soluble and present in the the participation of connexins (Cxs) as same organelles. The disease produced by hemichannels in cellular communication loss of function of NP proteins generates through the release of ATP and glutamate, hepatomegaly and splenomegaly. Based on regulating cellular migration and stabilizing preliminary laboratory data, we hypothesize synaptic transmission. In this investigation, that proteins related to the organization we identified the presence of several Cxs of the endoplasmic reticulum (ER) are during neurulation. To evaluate its relative necessary to maintain the disease phenotype. expression, we identified their RNA In order to identify other proteins involved, transcripts in different stages of Xenopus we studied Tango1, a transmembrane ER laevis neural embryonic development such protein that organizes vesicle cargo. Its loss as: stg 10; stg 12,5; stg 14 and stg 20. Our of function produces disorganization and results revealed the presence of transcripts stress of the ER. It was analyzed in a model of Cxs 43, 45, 46, 32 and 26 in different of Drosophila melanogaster where NP-C1 stages of Xenopus laevis development. The is replicated with a knock-down of dnpc1a more important proteins correspond to Cx gene, NPC1 ortholog, through RNAi. Using 46 (GJA3) which has 6 fold expression vs Cx this system, we determined that tango1 45 (GJA7) and Cx 43 (GJA1). In turn, Cx 32 loss of function reverts NP-C1 phenotype, (GJB1) have a significant presence of 3 fold improving Drosophila larval development vs Cx 26 (GJB2), the second more abundant, progression. Also, the effect of the inhibition during neurulation. Later, we decided to of ER secretion was analyzed using Fli- evaluate the functionality of these Cxs as 06, a compound that inhibits exportation. hemichannels through pharmacological We tested a pharmacological model that blockage assays, using inhibitors such as phenocopies NP-C1, completely reverting carbenoxolone (cbx) and enoxolone (enx). We NPC phenotype. The study corroborates that found values of IC50 of ~30 µM and ~20 µM the organization of the secretory pathway is for cbx and enx respectively in their capacity determinant to maintain the phenotype of

141 this disease and that by itself an alteration 115. CLINICAL CHARACTERISTICS in it results in a phenotype equivalent to the OF NEUROLOGICAL PATIENTS deficiency of NPC1. INFECTED WITH HTLV-1 AND DETERMINATION OF THE LOCATION 114. ADDITIVE EFFECT OF OF TAX VIRAL PROTEIN. MODAFINIL AND CAFFEINE ON THE Valenzuela M.A.1; Puente J.1; Cartier L.2; LOCOMOTIVE ACTIVITY OF ADULT Ramírez E.3. 1 Departamento de Bioquímica RATS. Y Biología Molecular, Facultad de Ciencias Urbina, A.1,2 ; Sotomayor-Zárate, R.2. Químicas y Farmacéuticas, Universidad 1Programa de Magíster en Ciencias Biológicas de Chile. 2 Departamento de Neurología, mención Neurociencias, Facultad de Ciencias, Facultad de Medicina, Universidad de Chile. Universidad de Valparaíso, Valparaíso, Chile. 3 Departamento de Virología, Facultad de 2Centro de Neurobiología y Fisiopatología Medicina, Universidad de Chile. Instituto de Integrativa (CENFI), Instituto de Fisiología, Salud Pública (ISP). Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile. Tropical Spastic Paraparesis neuropathogenesis (abbreviated HAM / TSP, Currently, many people are subject to a high “HTLV-1-associated myelopathy / tropical work and academic load that leads them spastic paraparesis”), endemic in Chile, to consume psychotropic substances to be shows by anatomo-pathological studies able to carry out these highly demanding spinal cord injuries due to axonal loss and activities. In Chile, it has been observed demyelination of cortical spinal beams, that some of these substances commonly visualized by NMR. 70% of patients start their used are modafinil and caffeine to promote disease with paretospastic gait. Since 2009 wakefulness and concentration. Concomitant the screening of HTLV-1 in blood banks. use of modafinil and caffeine could trigger The prevalence in donors studied in the ISP anxious symptoms and psychomotor in PBMCs (“Peripheral Blood Mononuclear agitation in humans. Modafinil has different Cells”) containing T-CD4+ lymphocytes, action mechanisms, being the blocking of the main viral reservoir, showed real-time dopamine transporter (DAT) the most known prevalence of 1.2 healthy / 1000 individuals. and relevant. On the other hand, caffeine Neuropathogenesis is attributed to the viral is an adenosine receptor antagonist and protein Tax because the virus does not infect inhibitor of phosphodiesterase. Therefore, neurons and 40% of patients with paraparesis the concomitant use of modafinil and caffeine are seronegative for the virus, but express a tax could promote a higher effect on neural gene. In cerebrospinal fluid (CSF) of patients activity compare to the use of caffeine or we detect Tax (by ELISA) and in isolated cells modafinil alone. The objective of this work (by immunofluorescence) and in plasma (by was to measure the additive effects of the “Western Blot”). Tax secreted from patient administration of modafinil and caffeine on PBMCs agrees with the extracellular role the horizontal and vertical locomotor activity. that we propose, because we know that it To assess the traveled distance and number interacts with semaphorin-4D that triggers of bipedestation, we used rats treated with the disassembly of microtubules and actin caffeine (20 mg/kg, i.p.), modafinil (80 mg/ fibers through Plexin-B1. kg, i.p.) and caffeine plus modafinil. Our preliminary results show that rats treated 116. POLYMERIZATION ACTIVITY with caffeine plus modafinil produce an AND CYTOTOXICITY OF MOLECULES increase on locomotor activity (horizontal WITH AFFINITY FOR LAU/PLA and vertical) compared to the administration BINDING SITE OF TUBULIN AS of caffeine or modafinil alone. The effect NOVEL STABILIZING AGENTS. induced by caffeine plus modafinil was Vásquez Pilar 1, Zúñiga Matías 2, Guzmán additive. To correlate these behavioral effects Leonardo 1, Jiménez, Verónica 3. with an increase in dopaminergic activity in (1) Departamento de Fisiología, Facultad the mesolimbic and nigrostriatal pathways, de Ciencias Biológicas, Universidad de we will measure the dopamine release in Concepción, Concepción, CL (2) Center for striatum and nucleus accumbens using in Bioinformatics, Simulations and Modelling, vivo brain microdialysis and fast scan cyclic Facultad de Ingeniería, Universidad de Talca, voltammetry Talca, CL (3) Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés Bello, Concepcion, CL.

142 The importance of microtubules in cellular has been interested in study how neonatal division set these proteins as pharmacological exposure to sex hormones such as estradiol targets for antimitotic agents, known as valerate (EV) affect the functionality of tubulin binding agents (TBA), which can midbrain dopaminergic neurons of adult promote stabilization or destabilization of male and female rats. The aim of this work tubulin polymerization. The occurrence was to evaluate the release and uptake of adverse drug reaction associated to kinetics of striatal dopamine (DA) induced several of these agents drives the need for by methylphenidate (MPH: 5.0 mg/kg i.p.) the development of new TBAs with a safer of adult female rats exposed during the first pharmacological profile. In this regard, hours of postnatal life to estradiol valerate a combination of computational virtual (EV: 0.1 mg/50 uL of sesame oil s.c.) or screening, molecular dynamics and binding vehicle (50 uL of sesame oil s.c.). Our results free energy estimations was performed by did not show significant differences in our group, based on the stabilizing LAU/PLA the voltammetry parameters such as peak binding site of tubulin. A set of 7 candidates amplitude, area and tau (time constant of were proposed as potential stabilizing agents decay). Despite these results, we cannot rule with affinity for the site. In this work, we out changes in the voltammetry parameters confirm the polymerization capacity for these in nucleus accumbens, a key nucleus of the 7 candidates in vitro at concentrations of 50 reward circuit, where we have previously and 100 µM. Also, we observed an additive observed a reduction in DAT expression of effect of the compounds when co-treating with animals programmed with sex hormones. Taxol, confirming a non-competitive binding with taxane-site binders. Finally, viability 118. CYTOTOXIC EFFECTS CAUSED assays in a cancer cell line were developed BY DELOCALIZED LIPOPHILIC showing a cytotoxic effect of molecules at CATIONS DERIVED FROM 100 µM. These results set a starting point POLYHYDROXY-BENZOIC ACIDS IN of further studies for the characterization of COMBINATION WITH DOXYCYCLINE the novel agents that will open possibilities ON LUNG CANCER CELLS. for the rational screening of new tubulin Vidal D.A. 1; Pardo A. 1; Jara J.A. 1; Ferreira stabilizing agents. J. 2. 1, Laboratorio de Farmacología, Instituto 117. RELEASE AND UPTAKE de Ciencias Odontológicas, Facultad de KINETICS OF DOPAMINE ARE Odontología, Universidad de Chile. 2, PRESERVED IN STRIATUM OF Laboratorio de Bioenergética y Cáncer, ADULT FEMALE RATS EXPOSED Instituto de Ciencias Biomédicas, Facultad de DURING FIRST HOURS OF Medicina, Universidad de Chile. POSTNATAL LIFE TO ESTRADIOL VALERATE. Lung cancer has the highest mortality Velásquez, V.B.1; Escobar, A.P.2; España between all neoplasms, being the second R.A.3; Sotomayor-Zárate, R.1. leading cause of death in Chile. These cancers 1Centro de Neurobiología y Fisiopatología are classified as small cell carcinoma or non- Integrativa (CENFI), Instituto de Fisiología, small cell carcinoma, being Smoking the Facultad de Ciencias, Universidad de main risk factor. Conventional treatments Valparaíso, Valparaíso, Chile. 2Centro are radiotherapy, chemotherapy and surgery; Interdisciplinario de Neurociencias de however, 5-year survival rates remain Valparaíso (CINV), Facultad de Ciencias, extremely poor, due to the development of Universidad de Valparaíso, Valparaíso, Chile. resistance and eventual relapse. The Cancer 3Department of Neurobiology and Anatomy, Stem Cells hypothesis suggests that they are Drexel University College of Medicine, responsible for tumor initiation and growth Philadelphia, USA. and are resistant to conventional treatments. Therefore, it is necessary to develop new Sex hormones play an important role therapies that allow us to effectively eliminate in regulating reproductive and non- resistant tumor cells (TC). Mitochondria reproductive tissues, such as the brain. In the may be considered as a therapeutic target in nervous system, sex hormones are important the treatment of cancer, because it exhibits a in its development and neural plasticity, greater transmembrane potential in TC, being however changes in the sex hormones milieu susceptible to being the target of positively during fetal or neonatal stages affect brain charged molecules. The delocalized lipophilic function and generate persistent changes cations of triphenylphosphonium (TPP+) are until adulthood. During last 7 years our lab molecules synthesized from gallic acid, mono

143 and polybenzoates decyl esters. Therefore, of rat primary culture. In order to confirm we evaluated 4 decyl polyhydroxybenzoate ITH12575 selectivity for NCLX, the exchanger compounds linked to TPP+ as potential was silenced by a siRNA and neuroprotective cytotoxic agents in two lung cancer cell assays were evaluated. Finally, mitochondrial lines (NCI-H727 and NCI-H1299) and in stress assays were performed using the pulmonary fibroblasts (PH) as control. seahorse method, in presence/absence Doxycycline is an antibiotic of the tetracycline of both a toxic stimulus (high potassium group; recently its has been studied this concentration) and the compound. Data from antineoplastic use producing the inhibition of ATP synthesis, mitochondrial respiration and mitochondrial biogenesis in TC. Cell viability respiratory maximal capacity were obtained. assay was performed with the compounds The results show that, by the regulation of at 24, 48 and 72 hours in normoxia and mitochondrial calcium, the mitochondrial hypoxia with 5% oxygen to determine IC50, metabolism is partially recovered thanks to to subsequently perform a combination test ITH12575. of compounds with doxycycline. The results showed that the TCT analyzed are sensitive to 120. FOXO1 MEDIATES HIGH the cytotoxic action of all compounds and this GLUCOSE-CARDIAC FIBROBLASTS effect is increased as time goes by. In addition, DIFFERENTIATION there are no significant differences in IC50 Vivar R 1, Anfossi R 1; Cárdenas S 2; Contreras between hypoxia and normoxia cultures. A 2. 1) Faculty of Medicine, University of Chile; 119. TARGETING MITOCHONDRIAL 2)Department of Biology, Faculty of Basics METABOLISM IN Sciences, Metropolitan University of NEURODEGENERATIVE DISEASES Educational Sciences. THROUGH NCLX BLOCKADE. Viejo L. 1,2; L. Arribas R. 1; Palomino A. 2; Normally cardiac fibroblasts (CF) maintain Egea J. 2; Martinez-Ruiz A. 2; de los Ríos C. the homeostasis of the extracellular matrix 1, 2. (ECM) in the heart, whereas in pathological 1 Instituto Teófilo Hernando, Universidad conditions such as diabetes, become more Autónoma de Madrid, C/ Arzobispo Morcillo, active promoting cardiac fibrosis. High 4, 28029, Madrid, Spain. 2 Instituto glucose (HG) induces CF differentiation, de Investigación Sanitaria del Hospital where TGF-beta1 has a crucial role. TGF-beta1 Universitario de La Princesa, C/ Diego de requires FoxO1 to induce CF differentiation, León, 62, 28006, Madrid, Spain. whereas FoxO1 is deregulated in diabetes, resulting in its hyperactivation, oxidative The loss of mitochondrial function is stress and cell differentiation. Therefore, in part of the almost all neurodegenerative this work we wanted to determine the role diseases. Therefore, there’s a reduction in of FoxO1 in CF differentiation promoted ATP synthesis ending up in a dysfunction of by high glucose. CF obtained from adult neuronal dynamics. At least three proteins Sprague-Dawley rats were incubated in HG, from the Krebs’ circle are calcium sensitive. In as a in vitro model of hyperglycemia. CTGF our laboratory, we’re focus in the design and and alpha-SMA expression was determined synthesis of CGP37157 derivatives, reference by RT-PCR, whereas CTGF and alpha-SMA blocker drug of the mitochondrial sodium/ protein were evaluated by westernblot calcium exchanger (NCLX), which also has (WB). The activation of FoxO1 was analyzed neuroprotective properties. Our aim is the evaluating its phosphorylated forms, its discovering of new pharmacological tools able nuclear localization and the expression of to handle calcium flux between mitochondria FoxO1 specific genes targets (p21cip and and cytosol, lowering the calcium overload p15ink) by RT-PCR. The oxidative stress was descried in the cytosol. Putting together evaluated analyzing the expression of the these two ideas, we wondered if the partial FoxO3a, catalase and SOD2 proteins by WB, blockade of mitochondrial calcium efflux and ROS production by colorimetry. The role could improve not only mitochondrial of FoxO1 was demonstrated using AS1842856 metabolism but also calcium handling. The (FoxO1 inhibitor) and FoxO1 silencing using CGP57137 derivative selected was ITH12575, siRNA. HG increased the protein and mRNA a benzothiazepine with a different aromatic of CTGF and alpha-SMA (CF differentiation substitution. First, the calcium movements marker), whereas HG decreased of AKT were studied by the fluorescent dye Fluo4, activation, decreased phospho-s256-FoxO1 in the human neuroblastoma SH-SY5Y level, increased FoxO1 nuclear localization cell line and in embryonic cortical neuros and increased FoxO1 genes target expression

144 (FoxO1 activation marker). Likewise, HG 122. ACTIVATION OF NMDA decreased FoxO3a, catalase and SOD2 RECEPTORS DURING CHRONIC PAIN protein, and increased ROS production. In RECRUITS PROTEIN SRC KINASES addition CF differentiation induced by HG TO OPEN PANNEXIN1 CHANNEL IN was completely abolished by FoxO1 inhibition NEUROPATHIC RAT MODEL. using AS1845628 and FoxO1 silencing. Zepeda K. D 1; Hernández A. 1; Pelissier T. 1; Collectively these data suggest that FoxO1 is Constandil L. 1. necessary to CF differentiation induced by 1 Laboratorio de Neurobiología, Facultad de high glucose and suggest that FoxO1 would be Química y Biología, Universidad de Santiago a pharmacological target for new treatments de Chile. 2 Prog. Farmacología Molecular against diabetic cardiomyopathy. y Clínica, ICBM, Facultad de Medicina, Universidad de Chile. 121. PHARMACOLOGICAL COMPARISON BETWEEN In experimental pain models the upregulation PREFRONTAL CORTEX AND of NMDA receptor (NMDAR) appears to be NUCLEUS ACCUMBENS crucial in the enhanced responsiveness of NEUROTRANSMITTER LEVELS nociceptive neurons of dorsal horn of the AFTER BASOLATERAL AMYGDALA spinal cord for initiation and maintenance of STIMULATION. pain. In the chronic pain model performed Zegers J.A.1; Yarur H.E.1; Bastías C.P.1; by our laboratory, it was shown that Gysling K.1. intrathecal injection i.t. of 10Panx (Inhibitor Departemt of Cell and Molecular Biology, panx1 channel peptide) prevents the effect Faculty of Biological Sciences, Pontificia of hyperalgesia caused by i.t of NMDA in Universidad Católica de Chile. neuropathic rats suggesting an interaction between Panx1 channel and NMDAR. Our Glutamatergic neurons of the basolateral work is based on elucidating whether the amygdala (BLA) innervate both, the nucleus regulation of the Panx1 channel by post- accumbens (Nac) and prefrontal cortex (PFC) translational modifications is carried out (McDonald, 1991). The relation between BLA by Src kinases in neuropathic rats model. and Nac has been implicated on the control In dye uptake experiments (used to assess of motivated behavior (Stuber et. al., 2011). Panx1 channel opening) of the spinal cord Furthemore, the triade between BLA, Nac slices of neuropathic rats have demonstrated and PFC has also been shown to be critical in that pharmacological inhibition of PP2 (Src the regulation of goal-directed behavior by an tyrosine kinase protein inhibitor), and 10Panx inhibitory control of PFC on Nac dopamine decreased dye uptake in neurons stimulated release during amygdala activation (Jackson by NMDA. Likewise, rats treated with et. al. 2018). BLA has been implicated in 10panx-NMDA or PP2-NMDA decreases the fear, anxiety and stress (Simon et. al., 2014; expression of phosphorylated Panx1 (pPanx1) Janak and Tye, 2015). The stress response and phosphorylated Src527 (pSrc527) by is centered in the corticotrophin releasing western Blotting. Algesymmetric test (Randall factor (CRF) system (Bale and Vale, 2004). Selitto) results have shown that intrathecal There are two major receptors for CRF in administration of 10Panx (300 μM, 10μl i.t ) the brain, type-1 and type-2 CRF receptors -NMDA (0.6 mM, 10μl i.t) and PP2 (3.3 mM, (CRF-R1 and CRF-R2). Several studies have 10μl i.t )-NMDA (0.6 mM, 10μl i.t) inhibit shown a significant role of CRF-R1 in the pain in neuropathic rats compared to control. stress response; however, the role of PFC We conclude that Panx1 or Src inhibition and Nac CRF-R2 in the stress response is prevents nociceptive signaling induced by poorly understood. We studied the role of upregulation of NMDAR in neuropathic rats, CRF-R2 in PFC and Nac neurotransmitter suggesting that the pronociceptive effects levels after BLA stimulation by double in vivo of pharmacological activation of NMDAR microdialysis in PFC or Nac of anesthetized induce the opening of the Panx1 channel adult rats. Local infusion of antisauvagine probably mediated by Src kinase. 30 (CRF-R2 antagonist) in the PFC or Nac significantly increased PFC and Nac dopamine and glutamate extracellular levels induced by BLA stimulation. Our results suggest that there is an inhibitory tone mediated by CRF-R2 controlling dopamine and glutamate extracellular levels in PFC and Nac that dependon BLA stimulation.

145 123. PARTICIPATION OF VGLUT AND 124. INDOMETHACIN IMPAIRS GLUTAMATE SIGNALING DURING POLYAMINE METABOLISM IN LUNG NEURULATION IN XENOPUS LAEVIS. CANCER CELLS: A KRAS MUTATION- Nicolás Zúñiga S.1, Camilo Venegas L.1, ASSOCIATED FEATURE? Patricio Castro M.1. López-Contreras F1, Muñoz-Uribe M1, Perez- 1Laboratory of Physiology and Pharmacology Lainez J1, Ascencio-Leal L1, Rivera-Dictter for Neural Development, Department of A1, Martin-Martin A1, Burgos Aguilera R1, Physiology, Faculty of Biological Sciences, Alarcon Uribe P1, López-Muñoz R1. Universidad de Concepción, Concepción, 1 Instituto de Farmacología y Morfofisiología, Chile. Facultad de Ciencias Veterinarias, Universidad Austral de Chile. Introduction: The development of the nervous system begins with the closure of Non-small cell lung cancer (NSCLC) is the the neural tube, one of the morphological most lethal and prevalent type of lung cancer. changes present in the neurulation process. NSCLC patients carrying mutations in the Several signaling pathways participate in Kirsten rat sarcoma viral oncogene homolog the formation of the neural tube, such as gene (KRAS) still lack targeted therapies. FGF, Wnt, Chordin and glutamate, recently Also, the levels of polyamines (putrescine, described. Failures in this process lead spermidine, and spermine) are increased to the formation of neural tube defects in cancer, playing a pivotal role in tumor (NTDs), the second more prevalent birth proliferation. Indomethacin increases the defect worldwide. Use of antiepileptic drugs levels of the polyamine-catabolic enzyme during pregnancy had shown an increase spermidine/spermine-N1-acetyltransferase in the incidence of NTDs by mechanism (SSAT). Consequently, the aim of this study not fully understood yet. Here, we propose was to compare the effect of indomethacin that glutamate, the principal excitatory in the polyamine metabolism of two NSCLC neurotransmitter of the nervous system and cell lines, with different KRAS mutation specifically its vesicular transporter VGLUT1, status. A549 and H1299 NSCLC cells (KRAS- participate in the normal closure of neural mutated and wild-type, respectively) were tube. Methods: X. laevis embryos were treated exposed to indomethacin. Evaluations with Rose Bengal, a VGLUT1 antagonist, at included SSAT expression and protein levels, early neurula stage (14 hpf, neural plate) and metabolic analysis of cells by CG-MS until end of neurulation (21 hpf). Then, metabolomics. Moreover, the difference in we perform a morphological evaluation of polyamine synthesis enzymes among cell lines neural tube closure and immunofluorescence and the synergistic effect of indomethacin experiments. Additionally, after neurula combined with inhibitors of these enzymes treatments, behavior studies using Xenopus were investigated. Indomethacin increased tadpoles (stg 45-49), were performed to the expression and levels of SSAT in both evaluate epileptic sensibility by measure cell lines. In A549 cells, indomethacin seizure latency onset using Pentylenetetrazol significantly impairs polyamine metabolism. (PTZ). Results: We observe and incomplete However, in H1299 cells, the impact of neural tube closure in embryos treated with treatment on the polyamine pathway was Rose Bengal in a dose-response manner, with non-significant. Evaluation of the levels of the an EC50 of 3.5±0.5μM. Furthermore, we polyamine synthesis enzymes showed that observe a decrease of ~43% (p<0.01, ANOVA) ornithine decarboxylase (ODC) is increased in in the seizure latency onset necessary for A549 cells, whereas S-adenosylmethionine- epileptogenic behavior induced by PTZ and a decarboxylase (AMD1) and polyamine 5-fold increase (0.4±0.3 m vs 5±0.2m) in the oxidase (PAOX), are increased in H1299 distance traveled at 2 minutes after treatment cells. Finally, indomethacin demonstrated (p<0.05, ANOVA). Conclusions: VGLUT1- a synergistic effect with the PAOX inhibitor mediated glutamate signaling participate MDL72527 in A549 cells, whereas in H1299 in normal neural tube development. Partial had a synergistic effect with the AMD1 blocking of this pathway at neurula modifies inhibitors SAM486. Collectively, these results the epileptogenic-induce response in tadpoles, indicate that indomethacin alters polyamine possibly by alter the normal establishment of metabolism in NSCLC cells and enhances the nervous system. the effect of polyamine synthesis inhibitors such as MDL72527 or SAM486. However, this effect varies depending on the basal metabolic fingerprint of each type of NSCLC cell. FONDECYT-1160807.

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