Resumo não disponível.

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2011. / Submitted by Gabriela Ribeiro (gaby_ribeiro87@hotmail.com) on 2011-06-27T19:04:23Z No. of bitstreams: 1 2011_WenerPassarinhoCella.pdf: 817931 bytes, checksum: e30e1315643fc5e4bf590c836c330fe8 (MD5) / Approved for entry into archive by Guilherme Lourenço Machado(gui.admin@gmail.com) on 2011-07-15T12:46:05Z (GMT) No. of bitstreams: 1 2011_WenerPassarinhoCella.pdf: 817931 bytes, checksum: e30e1315643fc5e4bf590c836c330fe8 (MD5) / Made available in DSpace on 2011-07-15T12:46:05Z (GMT). No. of bitstreams: 1 2011_WenerPassarinhoCella.pdf: 817931 bytes, checksum: e30e1315643fc5e4bf590c836c330fe8 (MD5) / O Eletrovisuograma Axonal (EVA) foi inicialmente descrito como um teste eletrofisiológico capaz de detectar um potencial de ação do nervo óptico. Para definir sua utilidade na propedêutica eletrofisiológica e padronizar sua técnica e seus parâmetros, foram avaliados indivíduos normais (grupo controle) e indivíduos portadores de afecções neuro-oftalmológicas (grupo de estudo), tais como atrofia bulbar, retinose pigmentar e atrofia óptica glaucomatosa. A técnica de exame foi baseada na estimulação monocular por flash luminoso com intensidade de 0 dB a uma frequência de 1,4 Hz. Eletrodos com cúpula de ouro foram colocados na região temporal da rima palpebral (eletrodo ativo), no lobo da orelha ipsilateral (eletrodo de referência) e na região frontal (eletrodo terra) e conectados a um pré-amplificador através de um canal de entrada para registro elétrico, sendo analisada a média de 100 traçados obtidos após rejeição de artefatos. O traçado normal do EVA consistiu de uma onda positiva inicial (P1, com amplitude média de 2,0 μV e tempo de culminação médio de 23,1 ms) seguida de uma onda negativa (N1, com amplitude média de -3,9 μV e tempo de culminação médio de 41,4 ms). Nos indivíduos normais não foram observadas diferenças significativas entre os sexos e entre os olhos direito e esquerdo, mas o tempo de culminação de P1 e de N1 aumentou com a idade. Nos olhos com atrofia bulbar observou-se diminuição na amplitude de ambas as ondas. Nos olhos com retinose pigmentar observou-se diminuição na amplitude de ambas as ondas e aumento do tempo de culminação de N1. Nos olhos com atrofia óptica glaucomatosa observou-se aumento na amplitude de ambas as ondas e no tempo de culminação de N1. Baseado nas suas características elétricas, sugere-se que P1 é originada do nervo óptico e que N1 é originada da retina interna. Além disso, pela sua constância e reprodutibilidade, valida-se o EVA como um teste eletrofisiológico a ser utilizado na investigação de lesões neurorretinianas. _________________________________________________________________________________ ABSTRACT / Axonal Electrovisogram (AxEv) was initially described as an electrophysiological test capable of register optic nerve potentials. To define its clinical usefullness among electrophysiology tests and standardize its techniques and parameters, a control group was formed. In addition, individuals with phthisis bulbi, retinitis pigmentosa and optic atrophy due to advanced glaucoma were enrolled as a study group. It was established that the technique is based on monocular flash stimulation (prechiasmatic) with a flash intensity of 0 dB at a frequency of 1.4 Hz. Golden cup electrodes were placed on the temporal side of lateral canthus (active electrode), on the ipsilateral earlobe (reference electrode) and on the forehead (ground electrode). Electrodes were connected to a one-channel pre-amplifier and electrical signs were averaged by a means of 100 after artifacts rejection. Normal AxEv waveforms consisted of an inicial positive wave (P1, with mean amplitude of 2.0 μV and mean implicit time of 23.1 ms) followed by a negative wave (N1, with mean amplitude of -3.9 μV and mean implicit time of 41.4 ms). In normal controls, there was no difference between eyes or genders, but P1 and N1 implicit times increased with age. In phthisis bulbi eyes, there was a statistically significant amplitude reduction in both waves. In retinitis pigmentosa eyes, there was a statistically significant amplitude reduction in both waves and an increased N1 implicit time. In glaucomatous optic atrophy eyes, there was a statistically significant amplitude increase in both waves and increased N1 implicit time. Based on AxEv electrical characteristics, we suggest that P1 arises from optic nerve and N1 from inner retinal layers. In addition, wavelets were constant and reproducible, allowing test validation as an electrophysiology procedure clinically indicated to investigate neuroretinal disorders.

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2011. / Submitted by Tania Milca Carvalho Malheiros (tania@bce.unb.br) on 2012-02-09T11:43:28Z No. of bitstreams: 1 2011_AlexandreAugustoCabralMelloVentura.pdf: 2024176 bytes, checksum: d4dc4800039f100dc7145f37d914d030 (MD5) / Rejected by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br), reason: on 2012-02-09T11:45:42Z (GMT) / Submitted by Tania Milca Carvalho Malheiros (tania@bce.unb.br) on 2012-02-09T12:08:58Z No. of bitstreams: 1 2011_AlexandreAugustoCabralMelloVentura.pdf: 2024176 bytes, checksum: d4dc4800039f100dc7145f37d914d030 (MD5) / Approved for entry into archive by Marília Freitas(marilia@bce.unb.br) on 2012-02-16T11:12:38Z (GMT) No. of bitstreams: 1 2011_AlexandreAugustoCabralMelloVentura.pdf: 2024176 bytes, checksum: d4dc4800039f100dc7145f37d914d030 (MD5) / Made available in DSpace on 2012-02-16T11:12:38Z (GMT). No. of bitstreams: 1 2011_AlexandreAugustoCabralMelloVentura.pdf: 2024176 bytes, checksum: d4dc4800039f100dc7145f37d914d030 (MD5) / INTRODUÇÃO: Doenças oculares relacionadas com neovascularizações são a principal causa de cegueira irreversível atualmente na população adulta mundial. A busca por novas drogas e terapias para o tratamento das doenças oculares e a manutenção da visão continua sendo um desafio. OBJETIVOS: Avaliar a toxicidade sobre a retina neurossensorial (RNS) e epitélio pigmentado da retina (EPR) da injeção intra-vítrea (IV) de Octreotida (Sandostatin®) em olhos de coelhos não albinos; avaliar se o aumento súbito do volume vítreo após a injeção IV de 0,1ml de solução salina balanceada (BSS) no olho do coelho leva a danos histológicos na RNS e EPR; e avaliar as complicações clínicas pós-operatórias após a injeção IV em olhos de coelhos. MÉTODOS: 20 coelhos não albinos (40 olhos) foram distribuídos em 4 grupos. O Grupo Controle (5 coelhos – 10 olhos), o qual não recebeu injeção IV, foi sacrificado no início do estudo. Os trinta olhos dos 15 coelhos restantes foram distribuídos em 3 grupos (1:1:1): Grupo Placebo (injeção IV de 0,1ml de BSS); Grupo 1 (Grupo em que foi injetado 0,1ml da apresentação de 0,1mg/ml de Octreotida intra-vítreo); e Grupo 2 (Grupo em que foi injetado 0,1ml da apresentação de 0,5mg/ml de Octreotida intra-vítreo). Os coelhos foram acompanhados por um período de 90 dias após o procedimento, quando então foram submetidos a eutanásia. Todos os coelhos tiveram seus olhos enucleados e avaliados histologicamente. Foram realizadas avaliações clínicas pré e pós-operatória (inspeção do segmento anterior, tonometria de aplanação e oftalmoscopia binocular indireta) e avaliação histológica da morfologia e da espessura das camadas da RNS e EPR. RESULTADOS: Não foram observadas complicações clínicas pós-operatórias significantes. A morfologia histológica e espessura das camadas da RNS e EPR não apresentou diferença significante entre os grupos controle e placebo, grupo placebo e grupo 1 e grupo placebo e grupo 2. CONCLUSÕES: A injeção IV de 0,1ml de Octreotida nas apresentações de 0,1mg/ml e 0,5mg/ml não leva a alterações histológicas tóxicas na RNS e EPR, nem a complicações clínicas pós-operatórias importantes em olhos de coelhos não albinos. A injeção IV de 0,1ml de BSS não leva a danos histológicos ao RNS e ao EPR em olhos de coelhos não albinos. ______________________________________________________________________________ ABSTRACT / BACKGROUND: Eye diseases associated with neovascularizations are the leading cause of irreversible blindness in the adult population worldwide nowadays. The search for new drugs and therapies for the treatment of eye pathologies and vision maintenance remains a challenge. OBJECTIVES: To evaluate the toxicity of the neurosensory retina (NSR) and retinal pigment epithelium (RPE) of Octreotide (Sandostatin®) intra-vitreous (IV) injection in non-albino rabbit eyes; also evaluate whether the sudden increase of vitreous volume after 0.1ml of balanced salt solution (BSS) IV injection into rabbit eyes leads to histological damage for the RPE and NSR and to evaluate the clinical postoperative complications after IV injection in rabbit eyes. METHODS: 20 non-albino rabbits (40 eyes) were divided into four groups. The control group (5 rabbits-10 eyes), which received no IV injection, was sacrificed at baseline. Thirty eyes of the 15 remaining rabbits were distributed into 3 groups (1:1:1): Placebo group (BSS; 0.1ml IV injection), Group 1 (Octreotide 0.1mg/ml; 0.1ml IV injection) and Group 2 (Octreotide 0.5mg/ml; 0.1ml IV injection). The rabbits were followed during a 90 days period after the procedure and sacrificed. All rabbits had their eyes enucleated and histologically examined. Evaluations were made before and after surgery (anterior segment inspection, applanation tonometry and indirect ophthalmoscopy). Histological examination of the NSR and RPE were performed and their morphological features and layer thickness were analyzed. RESULTS: No significant postoperative clinical complications were observed. The histological morphology and thickness of the NSR and EPR showed no significant difference between the control and placebo group, the placebo group and group 1 and between the placebo group and group 2. CONCLUSIONS: Octreotide 0.1mg/ml and 0.5mg/ml; 0.1ml IV injections does not lead to histological toxicity changes in NSR and EPR, nor important postoperative clinical complications in non-albino rabbit eyes. The BSS; 0.1ml IV injection also does not lead to histological damage to the NSR and the RPE in non-albino rabbit eyes.

Made available in DSpace on 2014-06-12T16:26:09Z (GMT). No. of bitstreams: 2 arquivo5416_1.pdf: 666645 bytes, checksum: f9abe5fc8a78f951bc519dde8ca099fd (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2003 / O presente estudo teve como objetivo avaliar a camada de fibras nervosas da retina com laser confocal polarizado em portadores de esquistossomose mansônica na forma hepatoesplênica. Realizou-se um estudo do tipo caso-controle com 50 olhos de 25 indivíduos esquistossomóticos da forma hepatoesplênica (15 masculinos e 10 femininos) com idades variando entre 37 e 73 anos (média de 51,6 +/- 9,6 anos) foram comparados com 46 olhos de 23 indivíduos que não apresentavam hipertensão porta (14 masculinos e 9 femininos) com idades variando entre 37 e 76 anos (média de 50,2 +/- 11,2 anos). Todos os pacientes, para serem incluídos no estudo, apresentavam ao exame oftalmológico: acuidade visual com melhor correção igual ou melhor que 20/40 (0,5); pressão ocular igual ou menor que 18mmHg; relação escavação / disco igual ou menor que 0,4 e com simetria da relação escavação / disco igual ou menor que 0,2, entre os discos ópticos. Utilizouse o aparelho GDxTM Glaucoma Scanning system&#63720; (Laser Diagnostic Technologies, Inc., San Diego, CA, USA). Foram obtidas duas imagens consecutivas, após ajuste de foco e brilho e centralização do disco óptico. A espessura da elipse foi determinada a uma distância de 1,75 diâmetro de disco para cada quadrante, na região peripapilar e, uma imagem média foi criada. Foram avaliados todos parâmetros emitidos pelo relatório do aparelho. Todos os parâmetros que representam médias: Média da espessura (p = 0,01), Média da Elipse (p = 0,01), Média superior (p < 0,01) e Média inferior (p = 0,05) e o que representa uma integral: Integral superior (p = 0,05), apresentaram diferenças estatisticamente significantes, entre os grupos. Todos os outros parâmetros, que representam razões (Simetria, Razão superior, Razão inferior, Razão superior/nasal), modulações (Modulação máxima e Modulação da elipse) e cálculo especial (Number), não apresentaram diferenças estatisticamente significantes. Estes resultados demonstram haver, no grupo de estudo, uma diminuição homogênea na camada de fibras nervosas da retina, em todos os setores da região peripapilar. É possível que às mudanças hemodinâmicas causadas pela hipertensão porta sejam responsáveis pela perda difusa da camada de fibras nervosas da retina, nos pacientes esquistossomótico da forma hepatoesplênica, avaliados neste estudo

During embryonic development, the Hedgehog (Hh) signaling pathway plays an important role in the growth and patterning of numerous tissues and organs. In the developing retina, Hh signaling regulates the proliferation and differentiation of retinal progenitor cells (RPC) through mechanisms that are not completely understood. The principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3), which regulate the expression of target genes responsible for the effects of the Hh pathway on RPC. The network of genes targeted by this pathway in neural progenitor cells however, remains unknown. The objective of this thesis was to identify and characterize novel targets of Hh/Gli during retinal development. Using a computation approach, 390 genes were identified as having at least one conserved Gli binding motif within the vicinity of the coding sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5 retinal explants and that the induction of 25 of these were significantly different between the two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp, two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to determine a role for Sox8 during retinal development which could reflect compensation by the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient and required for Hh mediated induction in progenitor cell proliferation and cell fate determination. Therefore, in this thesis Hh target genes have been identified which could provide some insight into the mechanisms that are responsible for the cellular outcome of a response to the pathway.

Verwendung multimodaler Netzhautbilder (einschließlich quantitativer Fundusautofluoreszenz (QAF)) für die spektrale optische Kohärenztomographie (SD-OCT)-basierte Bildregistrierung und Ausrichtung. Für jede Altersdekade gesunder Erwachsener wurden normative QAF-Netzhautkarten erstellt und erweiterte Methoden zur QAF-Bildanalyse angewendet. / To use multimodal retinal images (including quantitative fundus autofluorescence (QAF)) for spectral-domain optical coherence tomography (SD-OCT)-based image registration and alignment. For each age decade of healthy adults, normative QAF retinal maps were generated and advanced methods for QAF image analysis were applied.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The glial cells of the retina aid in function and maintenance of the retina. The macroglia, Muller cells and the retinal astrocytes, become reactive following injury or disease in the retina, a response that is characterized by hypertrophy, dedifferentiation, loss of functionality, proliferation, and remodeling of tissue and extracellular matrix (ECM). The microglia which are the resident macrophages, also respond to injury/disease becoming activated, undergoing characteristic molecular and morphological changes, which include regulation of secreted factors, changes in inflammatory response and increased phagocytosis. Reactivity in Muller glia is thought to be the result of secreted signals, such as epidermal growth factor, ciliary neurotrophic factor, and broblast growth factor, which are released at the injury site to interact with quiescent glial cells. Furthermore, microglia and macroglia have been shown by some studies to interact following activation. While BMPs are known to be upregulated following injury in the CNS, little information is available concerning their role in reactive gliosis in the retina. We hypothesize that BMP7 indirectly triggers Muller gliosis by activating microglia. Using RT-qPCR, immunofluorescence and western blot, we assessed changes in gliosis markers in the mouse retinal glia following treatment with BMP. Our results showed that BMP7 was able to trigger Muller cell gliosis in the retina in vitro and in vivo. Furthermore, ablation of microglia lead to a subdued gliosis response in the mouse retina following BMP7 exposure. Thus, BMP7 triggers activation of retinal microglia in addition to the Muller glia. IFN-gamma and IL6 could play a role in microglia mediated activation of Muller glia, following exposure to BMP7. We also assessed the role of the Hippo/YAP pathway in the regulation of gliosis in the retina. We demonstrated that YAP was localized to the nucleus of the Muller cells of the retina and was upregulated in IFN-gamma induced gliosis in the mouse retina.

BACKGROUND: Epiretinal membranes are a common disease that can either be idiopathic, meaning no cause can be detected this is usually caused by aging, or secondary which is caused by injury disease or surgery. Despite current treatment methods, there are still persistence of this disease in some rare cases. Methotrexate although traditionally used to treat cancer and rheumatoid arthritis has been being explored as a treatment option in the field of ophthalmology for use against proliferative and migrating cellular diseases coupled with inflammation. Methotrexate has been reported in a few ocular diseases to reduce or stop cell migration and proliferation due to this finding a case study was conducted with this recurrent ERM patient to test its effectiveness against this disease. PURPOSE: To investigate a potential new treatment method for recurrent epiretinal membranes. After a visually significant epiretinal membrane develops there would be an epiretinal membrane (ERM) peel performed. Traditionally if there is recurrence of epiretinal membranes post ERM peeling the patient will be treated with an internal limiting membrane (ILM) peel. For most cases, this will resolve the issue. In the rare instances where an ILM peel doesn’t resolve recurrence, like in this case, we sought to test whether a series of methotrexate injections could help prevent ERM re-proliferation. CASE REPORT: Reporting on a case of a 65-year-old woman with a recurrent recalcitrant epiretinal membrane. This membrane was treated with a pars plana vitrectomy and ERM peeling. The membrane grew back and was met with an ILM peel in hopes of resolution. With continuing recurrence, the patient was treated with another ERM and ILM peel and 12 weekly intravitreal methotrexate (MTX) injections. METHODS: A patient with persistent recurrent epiretinal membranes underwent three surgeries in an attempt to cure the ERM. At every clinical visit, best corrected distance visual acuity was assessed with a Snellen Vision Test and the retina was imaged using optical coherence tomography. Measurements were taken using the machines built in analysis technology to measure retinal thickness and retinal volume at each visit. These were graphed alongside visual acuity to determine complimenting trends. RESULTS: At the first visit the patient began treatment at a visual acuity of 20/200 and a central macular thickness of 676. Seven months after the final methotrexate injection the patient was at a visual acuity of 20/80 and a central macular thickness of 328. The overall results were that visual acuity and central macular thickness significantly improved without ERM recurrence at seven months after treatment. CONCLUSION: When an ERM is significantly impacting the patient’s visual acuity surgery is usually performed in the form of an ERM peel or ILM peel. Although treatment of recurrent epiretinal membranes is well maintained by these procedures there are a small percentage of cases where recurrence is still found post ILM surgery. This case represents the first documented use of MTX to treat recurrent ERM and it suggests great potential for its use in otherwise treatment resistant cases. More research is required to better understand the true potential of this treatment option as well as associated risks.

Las enfermedades neurodegenerativas de la retina, como la Retinitis Pigmentosa (RP), se caracterizan por la pérdida de las neuronas fotorreceptoras por apoptosis, que conduce a la ceguera. Pese a la extensa investigación realizada no se ha podido avanzar en su tratamiento; sin embargo, la posibilidad de regeneración neuronal y la identificación de células progenitoras multipotentes (stem cells) en la retina (Reh y col., 1998; Tropepe y col., 2000), permiten proponer la combinación de dos enfoques simultáneos para el tratamiento de estas enfermedades: impedir la apoptosis de los fotorreceptores y estimular la proliferación de progenitores neuronales para restaurar las neuronas perdidas. La aplicación de este nuevo enfoque requiere un conocimiento a nivel molecular de los mecanismos de regulación del ciclo celular y de aquellos que posibilitan inducir la diferenciación de progenitores indiferenciados en el tipo neuronal deseado. En el laboratorio de Neurobiología del INIBIBB se ha establecido que dos factores tróficos participan activamente en la regulación del ciclo celular de neuroblastos precursores de fotorreceptores: el Factor Neurotrófico Derivado de la Glia (GDNF), que aumenta el número de precursores multipotentes y estimula el ciclo celular, y el Acido Docosahexaenoico (DHA) que induce la salida del ciclo celular de los fotorreceptores y estimula su diferenciación (Rotstein y col., 1996; Rotstein y col., 1997; Rotstein y col., 1998; Politi y col., 2001a; Politi y col., 2001b; Insua y col., 2003). Esto indicaría que en conjunto, ambos factores tróficos estarían actuando para definir el número final de fotorreceptores en la retina, a través del control de la proliferación, diferenciación y supervivencia. Trabajos realizados en el laboratorio nos han permitido establecer por primera vez que la ceramida actúa como segundo mensajero en neuronas de retina de rata, activando la apoptosis inducida por estrés oxidante (German y col., 2006b). Determinamos también que el DHA disminuye los niveles de ceramida para proteger a los fotorreceptores de dicho estrés, modulando la actividad y/o niveles de la glucosil ceramida sintetasa. Estos hallazgos sugieren que otras enzimas de esta vía, y por consiguiente otros esfingolípidos bioactivos podrían intervenir en el control de la supervivencia, proliferación y diferenciación neuronales. La esfingosina-1-fosfato (S1P) es un esfingolípido con importantes funciones biológicas; actúa como molécula señal extra e intracelular, regulando procesos biológicos críticos, como la proliferación, diferenciación, y supervivencia. La ceramida-1-fosfato (C1P) es otro esfingolípido cuyas funciones como regulador de la supervivencia y proliferación celular han sido descubiertas recientemente. La información existente sobre el rol de la S1P y C1P en el sistema nervioso y en la retina es muy escasa. Por ello fueron objetivos centrales de este trabajo de tesis investigar si la S1P y la C1P tienen un papel regulatorio en la proliferación y diferenciación neuronal en la retina. Existen numerosos modelos animales de las enfermedades neurodegenerativas de la retina que posibilitan evaluar los mecanismos moleculares involucrados en la degeneración, así como terapias para el tratamiento de las mismas. El ratón rd1 es uno de los animales más usados como modelo de estas enfermedades, ya que presenta una pérdida selectiva, irreversible y rápida de los fotorreceptores. Por estos motivos, elegimos este modelo para estudiar el proceso de degeneración in vitro de los fotorreceptores y el efecto del DHA sobre dicho proceso. El hallazgo de que los fotorreceptores de ratones rd degeneran por apoptosis durante el desarrollo temprano in vitro, y la reciente identificación en nuestro laboratorio de la ceramida y la esfingosina como mediadores claves en la apoptosis inducida por daño oxidante o por ausencia de factores tróficos de los fotorreceptores de retina de rata (German y col., 2006b; Abrahan y col., 2009a), nos llevaron a investigar también si estos esfingolípidos tendrían un papel en la apoptosis de los fotorreceptores rd durante el desarrollo temprano in vitro. El sistema de cultivo in vitro utilizado en nuestro laboratorio posibilita obtener cultivos neuronales puros enriquecidos en neuronas fotorreceptoras y amacrinas a partir de retinas de ratas y ratones de 0 a 2 días de nacidos, y mantenerlas por una a dos semanas en medio químicamente definido. La utilización de estos cultivos permitió analizar los mecanismos moleculares que rigen la proliferación, diferenciación y supervivencia de las neuronas fotorreceptoras y la modulación de dichos procesos por diversos factores. Uno de los objetivos de este trabajo de tesis fue evaluar los efectos de la S1P sobre la proliferación de progenitores de fotorreceptores. Utilizando cultivos neuronales de retina de ratas de día 0, determinamos que la S1P aumentó diversos parámetros indicadores del avance del ciclo celular, como la incorporación de BrdU y la cantidad de figuras mitóticas observadas, lo que sugiere que promueve la proliferación de los progenitores de fotorreceptores en cultivo. Analizamos además el papel de la S1P en la diferenciación de los fotorreceptores, una vez avanzado el desarrollo in vitro. Utilizando inmunocitoquímica y Western Blot establecimos que la S1P promovió la expresión de dos proteínas específicas de este tipo celular, la opsina, que forma parte del pigmento visual, y la periferina, proteína de los discos de los segmentos externos. La estimulación de la expresión de opsina y periferina fue independiente del transporte retículo endoplasmático-Golgi, ya que no se vio afectado por el tratamiento con Brefeldina A (BFA), que bloquea dicho transporte. La S1P también estimuló la formación de rudimentos de los segmentos externos, denominados procesos apicales, y promovió la localización de opsina y periferina en dichas estructuras, proceso bloqueado por la BFA. Estos resultados indican que la S1P tendría una acción a nivel transcripcional, sobre la expresión de opsina y periferina, no alterada por el agregado de BFA, pero su efecto sobre la formación de los procesos apicales requeriría del tráfico de estas proteínas al extremo de los cilios para su localización en los segmentos externos. Investigamos después si la S1P podría actuar como mediadora de los efectos de diversos factores tróficos sobre los fotorreceptores. En otros tipos celulares, se ha establecido que distintos factores tróficos aumentan los niveles de la S1P. Nuestro hallazgo previo de que el DHA modula la actividad de la glucosil ceramida sintetasa y la similitud de los efectos ejercidos por la S1P con los de GDNF y DHA nos llevó a investigar si estos factores tróficos podrían modular los niveles de la S1P para ejercer sus acciones en las neuronas fotorreceptoras. Determinamos que tanto el GDNF como el DHA requieren de un aumento en los niveles de S1P para producir sus efectos. La inhibición de la esfingosina quinasa 1 (SphK1), enzima involucrada en la síntesis de S1P, con un inhibidor competitivo, el DHS, bloqueó los efectos del GDNF y del DHA sobre la proliferación y diferenciación de las neuronas fotorreceptoras. Evaluamos entonces a través de qué mecanismos el GDNF y DHA afectaban la síntesis de S1P. Para ello, investigamos su efecto en la expresión de la SphK1, mediante inmunocitoquímica y Western Blot. Demostramos que ambos factores tróficos aumentaron la expresión de la enzima y promovieron su localización en la membrana plasmática, mecanismo promovido por otros factores tróficos para activar a esta enzima (Toman y col., 2004). Los resultados obtenidos indican que la S1P sería un mediador de los efectos de los factores estudiados, para promover la proliferación y diferenciación de los fotorreceptores. Otro de los objetivos de este trabajo de tesis fue evaluar los efectos de la C1P en las neuronas fotorreceptoras de retina. El agregado de C1P a los cultivos neuronales obtenidos utilizando retinas de ratas de día 0 generó un aumento en la proliferación de los progenitores de fotorreceptores. Observamos también que la C1P promovió la diferenciación de estas neuronas a tiempos más avanzados del desarrollo in vitro, estimulando la expresión de opsina y periferina, favoreciendo la formación de procesos apicales y promoviendo la localización de dichas proteínas en los mencionados procesos. Estos efectos de la C1P son muy semejantes a los establecidos para la S1P, e indican que también la C1P podría estar implicada en la regulación del desarrollo de las neuronas fotorreceptoras. Otro de los objetivos de mi trabajo de tesis fue investigar el avance del proceso de degeneración in vitro de los fotorreceptores de ratones rd1 y el efecto del DHA sobre dicho proceso, comparándolo con el desarrollo de fotorreceptores obtenidos de retinas de ratones salvajes (wt). Determinamos que los fotorreceptores de ambas cepas murinas sufren un proceso de degeneración similar, con un aumento progresivo en la apoptosis. Esta apoptosis ocurre por la vía mitocondrial, ya que se observó una disminución en el número de fotorreceptores que presentaron depolarización mitocondrial. El DHA tuvo un efecto protector sobre los fotorreceptores de los ratones rd, disminuyendo la progresión de la apoptosis de los fotorreceptores durante el desarrollo temprano in vitro. En los fotorreceptores wt se observó un comportamiento similar. Investigamos también el proceso de diferenciación de los fotorreceptores en ambas cepas y el efecto del DHA sobre dicho proceso. Determinamos que a medida que trascurrió el tiempo de cultivo se evidenció un aumento en los parámetros de diferenciación de los fotorreceptores rd. El DHA estimuló esta diferenciación aumentando la expresión de opsina, la formación de procesos apicales y promoviendo la localización de esta proteína en dichos procesos. Un efecto semejante fue observado en los fotorreceptores wt. Investigamos también la posible participación de la ceramida como mediador en la apoptosis de los fotorreceptores rd. Inhibiendo la síntesis de ceramida con Fumonisina B1 determinamos que el bloqueo de dicha síntesis previno la muerte de los fotorreceptores, disminuyendo su apoptosis. Este resultado sugiere que el aumento en los niveles endógenos de ceramida sería necesario para la activación de la apoptosis de los fotorreceptores rd. Para discernir si la ceramida y/o el producto de su hidrólisis, la esfingosina, actúan como disparadoras de esta muerte, evaluamos el efecto de un inhibidor de la ceramidasa alcalina, el MAPP, sobre dicha muerte. Establecimos que, al contrario de lo que ocurre en la rata, la inhibición de la síntesis de esfingosina no disminuyó la apoptosis de los fotorreceptores, sino que por el contrario, generó un ligero aumento en dicha apoptosis. Estos resultados permiten proponer que la ceramida, y no la esfingosina, sería un mediador clave en la activación de la apoptosis de los fotorreceptores rd. En conclusión, los principales resultados obtenidos en este trabajo de tesis son:  La S1P estimula la proliferación de neuroblastos progenitores de fotorreceptores de rata en cultivo.  La S1P promueve la diferenciación de las neuronas fotorreceptoras in vitro, incrementando los niveles de expresión de proteínas de los segmentos externos y estimulando la formación de procesos apicales.  La formación de los procesos apicales inducida por la S1P y el DHA requiere del tráfico de opsina y periferina al extremo de los cilios para su localización en los segmentos externos.  El GDNF y el DHA aumentan la expresión de la SphK y promueven su localización en la membrana plasmática, incrementando su actividad para generar un aumento en los niveles de S1P.  La S1P actúa como un mediador esencial para los efectos del GDNF y DHA sobre los fotorreceptores.  La C1P estimula la proliferación de los progenitores de fotorreceptores.  La C1P promueve la diferenciación de las neuronas fotorreceptoras en cultivo.  El DHA incrementa la supervivencia de las neuronas fotorreceptoras de ratones rd y wt durante el desarrollo temprano in vitro.  El DHA estimula la diferenciación de las neuronas fotorreceptoras de ratones rd y wt.  La apoptosis de los fotorreceptores de ratones rd ocurre durante el desarrollo temprano in vitro requiere de la síntesis de ceramida pero no de la de su metabolito esfingosina. / Neurodegenerative diseases of the retina, such as Retinitis Pigmentosa (RP), are characterized by the apoptosis of photoreceptors, leading to blindness. In spite of extensive research, treatments for these pathologies are still lacking; the accomplished knowledge about neuronal regeneration and the identification of stem cells in the retina (Reh y col., 1998; Tropepe y col., 2000), let us propose the combination of two simultaneous strategies in order to restore lost neurons: to stop photoreceptor apoptosis and stimulate neuronal progenitors proliferation. This approach requires a comprehensive knowledge on the molecular mechanisms that control cell cycle, and the subsequent differentiation of neuronal progenitors into the required neuronal type. It has been established in our Laboratory of developmental neurobiology in the INIBIBB that two trophic factors actively participate in cell cycle regulation of photoreceptor progenitors: Glial Derived Neurotrophic Factor (GDNF), which promotes multipotent progenitors proliferation; and docosahexaenoic acid (DHA), which induces photoreceptor cell cycle exit and promotes their differentiation (Rotstein y col., 1996; Rotstein y col., 1997; Rotstein y col., 1998; Politi y col., 2001a; Politi y col., 2001b; Insua y col., 2003). Therefore, both trophic factors would define the final number of retinal photoreceptors, through the regulation of their proliferation, differentiation and survival. Previous work from our laboratory established that ceramide acts as a second messenger in oxidative stress-induced apoptosis in rat retinal neurons (German y col., 2006b). We also determined that DHA decreases ceramide levels to protect photoreceptors from this stress, modulating the activity and/or levels of glucosylceramide sinthase. These findings suggest that other enzymes and bioactive sphingolipids could also participate in the regulation of neuronal survival, proliferation and differentiation. Sphingosine-1-phosphate (S1P) is a sphingolipid with important biological functions; it acts as an extra- and intra-cellular signaling molecule, regulating critical biological processes such as proliferation, differentiation and survival. Ceramide-1-phosphate (C1P) is another sphingolipid whose functions as a survival and proliferation regulator have been recently discovered. However, information regarding the roles of S1P and C1P in the Central Nervous System and in the retina, in particular, is very scarce. Hence, a purpose of this work was to investigate the roles of S1P and C1P in retinal neuronal proliferation and differentiation. There are many animal models of retinal neurodegenerative diseases that allow studying the molecular mechanisms involved in degeneration and also evaluate possible therapeutic treatments. The rd1 mice are among the most popular animal models used for these purposes, since they present a selective, irreversible and rapid loss of photoreceptors. Hence, we chose the rd mice model to study photoreceptor degeneration in vitro and the effect of DHA on this process. The finding that rd photoreceptors degenerate by apoptosis during early development in vitro and our recent identification that ceramide and sphingosine act as mediators of oxidative stress- or lack of trophic factors- induced photoreceptor apoptosis in rat retina (German y col., 2006b; Abrahan y col., 2009a), prompted us to investigate if these sphingolipids might have a role in rd photoreceptor apoptosis during early development in vitro. The in vitro culture system used in our laboratory allow us obtain pure neuronal cultures enriched in photoreceptor and amacrine neurons, from 0 to 2 days old- rat and mouse retinas, in which cells survive up to two weeks in a chemically defined medium. This culture system provided a useful tool to analyze the molecular mechanisms regulating photoreceptor neurons proliferation, differentiation and survival, and investigate the role of diverse factors on these processes. One of the purposes of this thesis was to evaluate S1P effects on the proliferation of photoreceptor progenitors. Using PN0 rat retinal neuronal cultures we determined that S1P increased several markers of cell cycle progression, such as BrdU uptake and the amount of mitotic figures, suggesting that S1P promotes proliferation of photoreceptor progenitors in culture. We also analized the role of S1P in photoreceptor differentiation. Using immunocytochemistry and Western Blot we established that S1P promoted the expression of two photoreceptor specific proteins, opsin, which forms the visual pigment; and peripherin, a structural protein of outer segment discs. Enhancement of opsin- and peripherin- expression was independent of endoplasmatic reticulum-Golgi transport, since it was not affected by Brefeldin A (BFA), which blocks this transport. S1P also stimulated the formation of rudimentary outer segments, named apical processes, and promoted opsin and peripherin localization in these structures, a process that was blocked by BFA. These results indicate that S1P might act at the transcriptional level to promote both opsin and peripherin expression, which is not altered by BFA, but its effect on apical processes formation, would require the traffic of these proteins to the end of the cilia, to build outer segments. In different cell types, several trophic factors have been shown to increase S1P levels. Our recent finding showing that DHA modulates glucosylceramide synthase activity (German y col., 2006b), and the similarity of S1P effects with those of GDNF and DHA, led us to investigate if these trophic factors could modulate S1P levels to signal their actions in photoreceptor neurons. We determined that GDNF and DHA required an increase in S1P levels to produce their effects. The inhibition of Sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, with a competitive inhibitor, DHS, blocked GDNF and DHA effects on proliferation and differentiation of photoreceptors, respectively. Then we evaluated through which mechanisms GDNF and DHA affected S1P synthesis. For this purpose, we investigated their effects on SphK1 expression by immunocitochemistry and Western Blot. We demonstrated that both trophic factors increased SphK1 expression and promoted its localization on plasma membrane, a mechanism already demonstrated for other trophic factors to enhance SphK1 activity (Toman y col., 2004). These results suggest that S1P is a mediator of GDNF and DHA to promote photoreceptors proliferation and differentiation. We also evaluated C1P effect on retinal photoreceptors. C1P addition to neuronal cultures from PN0 retinas generated an increase in the proliferation of photoreceptor progenitors. We also observed that C1P promoted photoreceptor differentiation at later time of development in vitro, stimulating opsin and peripherin expression, favoring apical processes formation, and promoting the localization of these proteins in these apical processes. C1P effects are similar to those established for S1P, and suggest that C1P could also be implicated in the development of photoreceptors. One of the purposes of my thesis work was to investigate the progression of the degeneration process in rd1 mouse photoreceptors in vitro and the effect of DHA on this process, and compare it with what occurs in photoreceptors from wild type (wt) mice. We determined that photoreceptors from both animal strains suffer a similar degeneration process, with a progressive increase in apoptosis. This apoptosis occurs through the mitochondrial pathway, since less photoreceptors preserving their mitochondrial membrane potential were observed as their time in vitro increased. DHA had a protective effect on rd photoreceptors, decreasing apoptosis progression during early development in vitro; a similar effect was observed in wt photoreceptors. We also investigated photoreceptor differentiation in both mouse strains and the effect of DHA supplementation on this process. We established that during an increase in rd photoreceptors differentiation parameters was observed with time in culture. DHA stimulated this differentiation: it increased opsin expression, promoted the formation of apical processes and the localization of opsin in these structures. A similar effect was observed in wt photoreceptors. We also investigated the involvement of ceramide as a mediator of apoptosis in rd photoreceptors. By inhibiting ceramide synthesis with Fumonisin B1 we determined that blocking this synthesis prevented photoreceptor death, decreasing apoptosis. This result suggests that an increase in ceramide endogenous levels would be necessary to trigger apoptosis in rd photoreceptors. To discern if ceramide by itself or together with its metabolite sphingosine were responsible for this death, we evaluated the effect of an alkaline ceramidasa inhibitor, MAPP. We established that inhibiting sphingosine synthesis did not decrease photoreceptor apoptosis, as it occurs in rat photoreceptors; on the contrary, this inhibition slightly increased cell death. These results let us propose that ceramide, and not sphingosine, might be a key mediator of apoptosis in rd photoreceptors. The most important results obtained in this thesis are: S1P stimulates the proliferation of rat photoreceptor progenitors in culture.  S1P promotes photoreceptor differentiation in vitro, increasing the expression of outer segment- proteins and stimulating the formation of apical processes.  Formation of apical processes induced by S1P and DHA required the traffic of opsin and peripherin to the end of the cilia, for them to be further localized in the developing outer segments.  GDNF and DHA increase SphK expression and promote its translocation to the plasma membrane, increasing its activity to produce S1P.  S1P acts as an essential mediator for GDNF and DHA effects on photorreceptors.  C1P stimulates proliferation of photoreceptor progenitors.  C1P promotes differentiation of photoreceptors in culture.  DHA increases the survival of rd and wt photorreceptors during early development in vitro.  DHA stimulates differentiation of rd and wt photorreceptors.  Induction of apoptosis in rd photoreceptors during early development in vitro requires ceramide synthesis but not of its metabolite sphingosine.