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Departamento De Morfología Y Biología Celular Programa De Doctorado En Biología Molecular Y Celular

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Departamento De Morfología Y Biología Celular Programa De Doctorado En Biología Molecular Y Celular Departamento de Morfología y Biología Celular Programa de Doctorado en Biología Molecular y Celular REGULACIÓN DE LA ACTIVIDAD DE LAS CÉLULAS GANGLIONARES MELANOPSÍNICAS EN LA RETINA DEL RATÓN (Mus musculus). DESARROLLO DE SOFTWARE PARA SU ANÁLISIS. TESIS DOCTORAL FELIPE CONTRERAS GARCÍA 2016 II IV VI VIII RESUMEN DEL CONTENIDO DE TESIS DOCTORAL 1.- Título de la Tesis Español/Otro Idioma: Inglés: REGULACIÓN DE LA ACTIVIDAD DE LAS REGULATION OF MELANOPSIN-CONTAINING CÉLULAS GANGLIONARES MELANOPSÍNICAS GANGLION CELLS ACTIVITY IN MOUSE (Mus EN LA RETINA DEL RATÓN (Mus musculus). musculus) RETINA. DEVELOPMENT OF DESARROLLO DE SOFTWARE PARA SU SOFTWARE FOR ITS ANALYSIS ANÁLISIS 2.- Autor Nombre: FELIPE CONTRERAS GARCÍA DNI/Pasaporte/NIE: Programa de Doctorado: BIOLOGÍA MOLECULAR Y CELULAR (Interdepartamental) Órgano responsable: BIOQUÍMICA Y BIOLOGÍA MOLECULAR RESUMEN (en español) Los seres vivos han evolucionado adaptándose a los cambios ambientales cíclicos, para lo que han desarrollado ritmos fisiológicos que preparan el organismo para tales cambios. Los más estudiados son los circadianos (con un periodo de alrededor de un día) y se manifiestan en BIS - ciclos de actividad/reposo, que se sincronizan con los ciclos externos de 24 horas, 010 - principalmente por la señal lumínica transmitida a través de las células ganglionares intrínsecamente fotosensibles de la retina (ipRGC). Las ipRGC integran las señales que reciben VOA - de conos y bastones con la que ellas mismas generan al captar la luz mediante el fotopigmento melanopsina, enviándola al núcleo supraquiasmático (SCN), el reloj biológico principal. Entre MAT - las ipRGC existen subpoblaciones celulares que se definen por la cantidad de melanopsina que FOR expresan, el tamaño de su soma, la localización de sus dendritas, así como por la expresión de las dos isoformas de la melanopsina y por su regulación. En esta tesis doctoral se han desarrollado métodos de registro y medida de la actividad animal basados en vídeo que, combinados con diversas condiciones experimentales, permiten deducir características del funcionamiento de las ipRGC y del SCN. Los resultados obtenidos evidencian que la ausencia de conos y bastones en ratones con degeneración retiniana rd podría afectar al desarrollo del SCN. Asimismo, se ha observado que el tiempo de postefectos (demora en manifestar el ritmo endógeno) aumenta con la diferencia entre el periodo endógeno del animal y el fotoperiodo ambiental previo. Se ha puesto de manifiesto igualmente la capacidad de la melanopsina para regenerar su propio sustrato, al observarse que en ratones rd la luz azul provoca una modificación progresiva del patrón circadiano. Además, mediante la deleción dirigida de células dopaminérgicas en la retina se ha observado un retraso en su actividad bajo condiciones normales de iluminación, lo cual refuerza la idea de un rol crucial de la dopamina en la fisiología de la retina y, en particular, de las ipRGC. Por otro lado, con el propósito de objetivar las observaciones al microscopio se han construido herramientas informáticas para el estudio avanzado de las preparaciones histológicas. Se ha IX desarrollado software para la fusión de imágenes obtenidas manualmente en 3 dimensiones en un microscopio de campo claro y para la fusión por capas celulares de imágenes procedentes de un microscopio confocal. También se ha creado un programa para la detección y medida automatizada de células mediante la combinación de técnicas de análisis de imagen, inteligencia artificial y arquitectura de software. Todo este conjunto de herramientas se ha utilizado para medir la cantidad de fotopigmento en más de 10000 ipRGC, cuantificándose el inmunomarcaje, en unos casos, de la melanopsina total y, en otros, de sus isoformas. El análisis de los datos, empleando técnicas de data mining, ha permitido describir varias subpoblaciones celulares en función del diámetro de su soma y de la intensidad de inmunomarcaje, que fueron posteriormente confirmadas mediante la observación de las dendritas de células de cada una de ellas. Se ha creado un modelo probabilístico de clasificación sin precedentes para categorizar los tipos de ipRGC en función de los parámetros mencionados. Este modelo ha permitido cuantificar la expresión in vivo e in vitro (en cultivo organotípico de retina) de cada tipo celular en diferentes momentos circadianos y combinaciones de iluminación. Nuestros resultados muestran la existencia de un gran grupo de ipRGC no descrito previamente, así como de subpoblaciones con diversa regulación dentro los tipos de ipRGC. Estas herramientas informáticas, desarrolladas para atender el propósito de la investigación presentada en esta tesis, además de haber sido fundamentales para la obtención de los resultados de la misma, por su carácter innovador y su amplia aplicabilidad, pueden revelarse de gran utilidad en otros campos de investigación. RESUMEN (en Inglés) Living beings have evolved and adapted to cyclic environmental changes, developing physiological oscillations that prepare their organism for such changes. Among these physiological rhythms, those of about one day, circadian rhythms, may be the most widely studied ones. The most observable expression of such rhythms are the sleep/wake cycles, which are synchronized with the external 24-hour cycles mainly due to the luminance signal transferred through the intrinsically photosensitive retinal ganglion cells (ipRGCs). ipRGCs integrate the signal they receive from cones and rods with the one they generate themselves when they capture light thanks to the melanopsin photopigment, sending this signal to the suprachiasmatic nucleus (SCN), the main biological clock and regulator of circadian rhythms. Amidst ipRGCs several subpopulations exist. These are defined, among other characteristics, by the amount of melanopsin they express, their soma size, their dendrite location, as well as the expression of both melanopsin isoforms and their regulation. During this PhD thesis, we have developed video-based animal activity registry and measurement methods which, combined with diverse experimental conditions, enable the X deduction of how ipRGC and SCN features work. Our results show that the absence of cones and rods in retinally degenerate (rd) mice may affect SCN development. Likewise, we have noticed that adaptation time to their own endogenous rhythm, i.e. the duration of post-effects, increases with the difference between the endogenous period of the animal and the previous environmental photoperiod. We have also shown the relevance of the capability of melanopsin to regenerate its own substrate through the observation of the modification of the circadian pattern of rd mice under blue light. Besides, targeted deletion of dopaminergic cells in the retina has resulted in a delay of activity under normal illumination circumstances, which reinforce the idea of a decisive role for dopamine in the physiology of the retina and, particularly, of the ipRGCs. Furthermore, in order to standardize microscope observations, we have developed computing tools for an advanced study of histological preparations. we have developed software to manually stitch images obtained in 3 axes in a bright-field microscope as well as in cellular layers of images originated in confocal microscopy. We have also created a program for automated cell detection and measurement with a combination of techniques from image analysis, artificial intelligence and software architecture. We have used this whole set of tools to measure the amount of photopigment in over 10000 ipRGCs, measuring inmunolabelling of whole melanopsin in some cases and its isoforms in others. Data analysis employing data mining techniques has allowed us to describe various cell subpopulations based on their soma diameter and melanopsin inmunolabelling intensity. We have confirmed these subpopulations later on in our research through the observation of dendrites from representative cells. With this, we have created an unprecedented probabilistic classification model that has allowed us to categorize ipRGCs based on the aforementioned parameters. This model has allowed us to quantify the expressions in vivo and in vitro (in retinal organotypic culture) of each cellular type in different circadian moments and illumination combinations. Our results show the existence of a large ipRGC group which had not been previously described, as well as subpopulations with diverse regulation inside each ipRGC type. The computing tools we have developed were fundamental in order to achieve the research purposes of this PhD thesis. Moreover, thanks to their innovative nature and broad applicability, they may be of great utility to many other research fields. SR. DIRECTOR DE DEPARTAMENTO DE BIOQUIMICA Y BIOLOGIA MOLECULAR XI XII La presente tesis doctoral ha sido elaborada en el Departamento de Morfología y Biología Celular de la Universidad de Oviedo y adscrita al programa de doctorado interdepartamental "Neurociencias" y posteriormente adaptada al programa de doctorado de “Biología Molecular y Celular” del Departamento de Bioquímica y Bilogía Molecular. Felipe Contreras García fue beneficiario del programa "Severo Ochoa" de ayudas predoctorales para la formación en investigación y docencia de la Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (FICYT). Este trabajo ha sido realizado dentro del proyecto de investigación "Desarrollo postnatal y organización del sistema de fotorrecepción no visual de la retina de mamíferos" del
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