La retina de los vertebrados está compuesta por cinco tipos de neuronas: fotorreceptores (FRs, conos y bastones), bipolares, ganglionares, horizontales y amacrinas, y células no neurales entre las que se destacan las células gliales de Müller. Durante el desarrollo, estas neuronas se originan a partir de células progenitoras que pasan a través de una serie de estados de competencia determinados por factores genéticos, celulares y moleculares, lo que permite la aparición ordenada y secuencial de los distintos tipos celulares (Livesey y Cepko, 2001b). Entre las diversas moléculas y factores tróficos que influencian el desarrollo de los bastones se encuentran el Ácido Retinoico (AR) y el Ácido Docosahexaenoico (ADH). El AR ejerce una amplia variedad de efectos durante el desarrollo de los vertebrados y la diferenciación celular. Juega un rol crucial en la determinación del patrón antero-posterior del cuerpo, en la espermatogénesis, y en la formación y crecimiento de los miembros y de la piel. Además, es crítico para el desarrollo temprano del ojo y diferenciación de los FRs (Stenkamp y col., 1993; Prabhudesai y col., 2005; Hyatt y col., 1996; Khanna y col., 2006). El AR ejerce sus efectos en las células uniéndose y activando a receptores nucleares que funcionan como factores de transcripción y regulan así la transcripción génica. Por otro lado, en nuestro laboratorio se ha establecido que el ADH promueve la supervivencia y diferenciación de los FRs de retina de rata en cultivo, y que sus efectos anti-apoptóticos ocurren a través de la estimulación de la vía de la ERK/MAPK y de la modulación de la expresión de proteínas anti y pro-apoptóticas.El objetivo general de este trabajo fue estudiar los efectos del AR en el desarrollo de neuronas amacrinas y FRs de retina in vitro. Para ello utilizamos cultivos neuronales de retinas de rata postnatal desarrollados en medio químicamente definido, los cuales fueron suplementados con AR y/o ADH. Dado que el AR es un factor de diferenciación celular nuestra hipótesis fue que, al igual que otros factores tróficos, esta molécula promovería además la supervivencia de los FRs. Sin embargo, cuando el AR se agregó al día 0 se incrementó el porcentaje de FRs apoptóticos, lo cual se correspondió con una pérdida de funcionalidad mitocondrial. Esta apoptosis pudo ser bloqueada completamente por el tratamiento con un pan-inhibidor de caspasas previo a la suplementación con AR. Estos resultados sugieren que el AR induciría la muerte de los FRs a través de un mecanismo apoptótico que involucra la pérdida de la actividad mitocondrial y activación de caspasas. Como el AR está ubicuamente presente en la retina y es esencial para su desarrollo, la preservación de FRs viables requeriría que su efecto pro-apoptótico fuera contrarrestado por la presencia simultánea de moléculas de supervivencia, como el ADH. Para poner a prueba esta hipótesis agregamos ADH a los cultivos previo al tratamiento con AR. Este agregado previno la muerte de los FRs inducida por el AR, respaldando la hipótesis de que durante el desarrollo se requeriría la presencia de otros factores de supervivencia para prevenir esta muerte. Notablemente, la inducción de apoptosis por AR afectó selectivamente a los FRs, resultando inalteradas las neuronas amacrinas.Dado que el AR es reconocido por sus efectos promotores de la diferenciación, su efecto inductor de la muerte de los FRs fue un hallazgo inesperado. Esta observación hizo necesario verificar si, en las condiciones experimentales ensayadas, el AR favorecía o no la diferenciación. Comprobamos que el AR promovió marcadamente la diferenciación, en paralelo al aumento en el porcentaje de células apoptóticas. Determinamos, por inmunocitoquímica y Western Blot, que el AR incrementó la cantidad de FRs que expresaron opsina y periferina, proteínas características de FRs maduros, y que desarrollaron procesos apicales, rudimentos de los segmentos externos propios de estas neuronas maduras. Además, el AR aumentó el número de FRs que desarrollaron neuritas y la extensión alcanzada por las mismas.Cabe destacar que a diferencia de los otros parámetros analizados, la estimulación del desarrollo de neuritas no fue selectiva para los FRs: el tratamiento con AR indujo el crecimiento de neuritas también en las neuronas amacrinas. Dado que el AR y el ADH tienen efectos similares sobre la diferenciación, y que se unen a receptores que forman heterodímeros (RAR y RXR respectivamente), decidimos estudiar sus posibles efectos aditivos o sinérgicos. El tratamiento simultáneo con ambos factores aumentó la expresión de opsina y periferina a valores semejan la suma de los dos por separado. Estos resultados implican que el AR y ADH contribuyen a la diferenciación de los FRs en forma aditiva, y sugieren que estimularían vías independientes para promover sus efectos. El hecho de que el AR indujera mayor expresión de proteínas y formación de estructuras de neuronas maduras, nos llevó a proponer que la funcionalidad de los FRs también podría estar estimulada. Sin embargo, observamos que el AR no estimuló la hidrólisis del GMPc, característica indicativa de una cascada de fototransducción activa y por consiguiente de capacidad de respuesta a la luz, ni la capacidad de incorporar neurotransmisores (como glutamato en los FRs y GABA en las neuronas amacrinas) del medio extracelular. Estos resultados indican que, aunque el AR promueve la diferenciación de los FRs y neuronas amacrinas, por sí solo no logra la maduración funcional de estas neuronas en cultivo, sugiriendo que se requeriría la presencia de otros factores. La observación de que el AR inducía simultáneamente la diferenciación y simultáneamente la apoptosis nos hizo suponer que podría tener efectos distintos sobre distintas sub-poblaciones de FRs o sobre sub-poblaciones celulares en distintos estadios de maduración. Para corroborar esta hipótesis, se suplementaron los cultivos con AR al día 0, cuando la proliferación aún era activa, y al día 2, momento en el cual ya no había progenitores en proliferación. Notablemente, al tratar los cultivos al día 2, el AR estimuló la diferenciación de los FRs, aunque ya no se observó un aumento en la apoptosis. Estos resultados indican que el AR actuaría en forma diferencial según el estadio de desarrollo de los FRs, induciendo la apoptosis en una sub-población de aquellos que aun son progenitores indiferenciados y acelerando la diferenciación en los que ya han abandonado el ciclo celular. Diversos trabajos han demostrado que el AR influye en la proliferación y la adquisición de un fenotipo particular en progenitores de retina embrionarios. Esto sugirió que el incremento en el número de células diferenciadas inducido por el AR podría ser resultado de un mayor número total de FRs debido a que el AR podría estar modificando la proliferación o redirigiendo el destino celular. Sin embargo, al analizar distintos parámetros relacionados con estos eventos, como la incorporación de BrdU, la expresión de p27, nestina, Crx y HPC-1 (marcadores de FRs y neuronas amacrinas, respectivamente), observamos que el AR no indujo una salida temprana del ciclo ni modificó la determinación de la identidad celular. Esto implica que al menos en las condiciones experimentales descritas, y en ese momento del desarrollo postnatal temprano, el AR no altera la salida del ciclo ni regula la identidad celular de estas neuronas in vitro. Para comprender mejor los mecanismos de acción del AR sobre los FRs, estudiamos la modulación de las vías de señalización intracelular implicadas en sus efectos. Se ha involucrado al AR en la activación de la quinasa p38, relacionada con la regulación de la apoptosis en varios tipos celulares. Cuando investigamos si el AR activaba la vía de p38 en los FRs, el análisis por Western Blot e inmunocitoquímica demostró que el AR promovió rápidamente la activación de esta vía de señalización, y que el bloqueo de dicha activación con un inhibidor específico de p38 evitó la apoptosis de los FRs. Paralelamente, la inhibición de esta vía redujo significativamente, aunque no por completo, la diferenciación de los FRs. Esto sugiere que la vía de señalización de p38 sería la preferencialmente activada por el AR para activar la apoptosis de los FRs y al menos una de las involucradas en inducir su diferenciación. Trabajos previos han mostrado que en la estimulación de la supervivencia de los FRs promovida por ADH interviene la activación de ERK/MAPK. Por ello, sería posible que el efecto deletéreo del AR implicara una modulación de esta vía. Sin embargo, no observamos cambios en la activación de dicha vía, indicando que no estaría afectada en el proceso de muerte inducido por AR. Por otro lado, teniendo en cuenta que la actividad de p38/MAPK podría ser regulada por interacción con la vía de PI3K/Akt, determinamos si el AR era capaz de modular esta vía en los FRs. El tratamiento con AR redujo la cantidad de P-Akt, respaldando la hipótesis de que el efecto estimulatorio del AR sobre la vía de p38 involucraría también una inhibición de la actividad de PI3K/Akt. En conjunto, estos resultados muestran que el AR es requerido para promover la diferenciación de los FRs y que este proceso de diferenciación no está necesariamente ligado a la supervivencia de estas neuronas. Su presencia prematura podría inducir la muerte de los progenitores al inducirlos a diferenciarse cuando aun están demasiado inmaduros, lo que resalta la importancia de la presencia simultánea de factores tróficos para prevenir dicha muerte. En conclusión, este trabajo remarca la importancia de una adecuada sincronización entre los niveles de diferentes señales moleculares esenciales para el desarrollo de los FRs. El AR podría así ser una de las moléculas cruciales que contribuyen a definir el número final de FRs en la retina. Las principales conclusiones de esta tesis son: a) El AR induce la muerte por apoptosis en los progenitores de FRs mientras se encuentran en el ciclo celular, por una vía que involucra la pérdida de funcionalidad mitocondrial y la activación de caspasas. b) El AR induce la diferenciación de los FRs, estimulando la expresión de opsina, periferina y el crecimiento de neuritas. c) El AR promueve el crecimiento de las neuritas en las neuronas amacrinas. d) La inducción de apoptosis por parte del AR es selectiva para los FRs. e) El AR no altera la proliferación ni modifica el destino de los progenitores. f) La inducción de de la diferenciación es independiente de que las células estén activas o no en el ciclo celular. g) Los procesos de apoptosis y diferenciación en los FRs inducidos por el AR dependen de la activación de la vía de p38/MAPK, que a su vez interacciona con la vía de PI3K/Akt. h) Un factor trófico lipídico, el ADH, protege a los FRs de la muerte inducida por AR. / The vertebrate retina has five neuronal types: photoreceptors (PHRs, rods and cones), bipolar, ganglion, horizontal and amacrine neurons, and non neuronal cells including the Müller glial cells. During development, these neurons are originated from progenitor cells that undergo a series of competence states, determined by genetic, cellular and environmental factors, thus allowing the sequential and organized appearance of the different cell types (Livesey y Cepko, 2001b). Retinoic Acid (RA) and Docosahexaenoic Acid (DHA) are among the different molecules and trophic factors that influence the development of rod PHRs. RA exerts a wide variety of effects during vertebrate development and cell differentiation. It plays a major role in the determination of the antero-posterior body axis, spermatogenesis, the formation and growth of body limbs and skin. Moreover, it is critical for the early development of the eye and PHR differentiation (Stenkamp y col., 1993; Prabhudesai y col., 2005; Hyatt y col., 1996; Khanna y col., 2006). RA binds to and activates nuclear receptors that function as transcription factors, thus regulating gene transcription. On the other hand, in our lab we have established that DHA promotes the survival and differentiation of rat PHRs in culture, and that these anti-apoptotic effects require the activation of the ERK/MAPK signaling pathway and the modulation of anti- and pro-apoptotic protein the expression. The general purpose of this work was to study the effects of RA on the development of amacrine neurons and PHRs in vitro. To that end, we used cultures obtained from postnatal rat retinas, developed in chemically defined media, which were supplemented with RA and/or DHA. Given that RA is a cell differentiation factor; our hypothesis was that, like other trophic factors, this molecule would also promote PHR survival. However, when RA was added at day 0, the percentage of apoptotic PHRs increased, in parallel with a loss of mitochondrial functionality. This apoptosis was completely blocked by incubating the cultures with a caspase inhibitor before RA addition. These results suggest that RA would induce PHR death through an apoptotic mechanism involving a loss of mitochondrial activity and caspase activation. Since RA is ubiquitously present in the retina and it is essential for development, the preservation of viable PHRs would require its pro-apoptotic effects to be counteracted by the simultaneous presence of survival molecules, such as DHA. To test this hypothesis, we added DHA to the cultures prior RA treatment; this addition prevented RA-induced PHR death, supporting the hypothesis of the necessity of other survival factors to prevent death during development. Noteworthy, RA-induced apoptosis was selective for PHRs, since amacrine neurons were not affected. Since RA is well known for its differentiation-promoting effects, the fact that it induced apoptosis was rather unexpected. This observation led us to test whether, under these experimental conditions, RA would promote or not PHR differentiation. RA indeed promoted differentiation, in parallel with an increase in the percentage of apoptotic PHRs. We determined, by immunocytochemistry and Western Blot, that RA increased the amount of PHRs that expressed opsin and peripherin, characteristic proteins of mature PHRs and of PHRs that developed apical processes, structures that resemble the initial steps of outer segment formation. Moreover, RA increased the percentage of PHRs that developed neurites and promoted neurite outgrowth. It is worth to note that, unlike other evaluated features, the stimulation of neurite outgrowth was not exclusive for PHRs; RA treatment also induced also neurite outgrowth in amacrine cells. Since RA and DHA have similar effects on differentiation, and they bind to receptors that form heterodimers (RAR y RXR respectively), we evaluated their possible additive or synergistic effects. The simultaneous treatment with both factors increased opsin and peripherin expression up to a value that resembled the sum of both metabolites alone. These results imply that RA and DHA contribute to PH differentiation in an additive fashion, and suggest that they stimulate independent pathways to that end. The fact that RA induced the expression of proteins and formation of structures of mature neurons, led us to propose that the functionality of these cells could also be stimulated. However, RA neither stimulated cGMP hydrolysis, a characteristic that would indicate an active phototransduction cascade and the ability to respond to light, nor the capacity to take up neurotransmitters (like glutamate in PHRs and GABA in amacrine neurons) from the extracellular medium. These results indicate that, although RA promotes PHR and amacrine cell differentiation, it is not enough of a stimulus to achieve functional maturity of these cells, suggesting that this functionality requires the presence of other factors. The finding that RA simultaneously induced differentiation and apoptosis led us to propose that it might have distinct effects on different PHR sub-populations or on populations at different developmental stages. To test this hypothesis, cultures were supplemented with RA at day 0, when proliferation is still active, and at day 2, when there are no longer proliferating progenitors. Noteworthy, when added at day 2, RA stimulated PHR differentiation, although no increase in apoptosis was evident. These results indicate that RA would act differentially depending on PHRs developmental stages, inducing apoptosis in a sub-population of undifferentiated progenitors and accelerating the differentiation in those which have already abandoned the cell cycle. Several studies have shown that RA influences proliferation and in the acquisition of a particular phenotype in embryonic retina progenitors. For that reason, the increase in the number of differentiated cells induced by RA could be due to a higher total number of PHRs, since RA might be redirecting cell fate or modifying proliferation. However, when we analyzed a number of parameters related to these events, such as BrdU incorporation and the expression of p27, nestin, CRX and HPC-1 (markers of PHRs and amacrine cells, respectively), we found RA neither induced cell cycle exit nor modified cell fate. This implies that, at least under the described experimental conditions, and at this particular time of development, RA would not alter the cell cycle exit or regulate cell identity. To better understand the mechanisms by which RA exerted its effects on PHRs, we studied the modulation of signaling pathways. RA has been involved in the activation of p38/MAPK, which related to the regulation of apoptosis in several cell types. When we evaluated whether RA activated the p38 pathway in PHRs, Western Blot and immunocytochemical analyses showed that it induced a rapid activation of this pathway, and the blockade of such activation with a specific inhibitor prevented PHR apoptosis. Moreover, the inhibition of this pathway led to a significant, though not complete, reduction of PHR differentiation. This suggests that the p38/MAPK would be the preferred signaling pathway activated by RA to induce apoptosis in PHRs, and at least one of the involved in the induction of their differentiation. Previous work has shown that DHA-stimulated survival in PHRs requires the activation of the ERK/MAPK pathway. Hence, the deleterious effect of RA might involve the modulation of this pathway. However, we found no changes in the activation of this pathway, indicating that it would not be related to RA-induced PHR death. On the other hand, given that p38/MAPK activity has been shown to be regulated by interaction with the PI3K/Akt pathway, we determined whether RA was capable of modulating this pathway in PHRs. Treatment with RA reduced the amount of P-Akt, supporting the hypothesis that the stimulatory effect of RA on the p38 pathway would involve the inhibition of PI3K/Akt activity. As a whole, these results show that RA is required for the induction of PHR differentiation, and that this process is not necessarily linked to the survival of these neurons. The premature presence of RA could elicit progenitor death as it might induce them to differentiate at a stage when they are still too immature, highlighting the need of the simultaneous presence of trophic factors to prevent this death. In summary, this work underscores the relevance of an adequate synchronization between the levels of different molecular cues essential for PHR development. RA might thus be one of the crucial molecules that contribute to define the final number of PHRs in the retina. The main conclusions of this thesis are: a) RA induces PHR progenitor apoptosis while they are active in the cell cycle, through a mechanism that involves the loss of mitochondrial activity and caspase activation. b) RA induces PHR differentiation, stimulating opsin and peripherin expression, and neurite outgrowth. c) RA promotes neurite outgrowth in amacrine neurons. d) RA-induced apoptosis is selective for PHRs. e) RA does not alter progenitor proliferation or the acquisition of cell fate. f) The induction of differentiation occurs regardless of the cells being active in the cell cycle or not. g) RA-induced differentiation and apoptosis processes in PHRs depend on the activation of p38/MAPK, which also interacts with PI3K/Akt. h) A lipid trophic factor, DHA, protects PHRs from RA-induced apoptosis.

Sensory neurons are key mediators of the transduction of external stimuli from the ear to the brain, essential for the sense of balance and hearing. Understanding when, where and how the sensory nervous system is assembled during development can provide insights on deafness and balance disorders. Here, I show in zebrafish that Her9 transcription factor is a key element in the regulation of the otic neurogenesis. Loss of Her9 function leads to the ectopic expression of neurogenic genes neurod and neurod4. Moreover, I show that Her9 acts downstream of Tbx1, and both genes are activated by retinoic acid signaling emanating from the paraxial mesoderm and negatively regulated by Hedgehog signaling. Altogether, the data demonstrates a role of retinoic acid in axial patterning and the establishment of a neurogenic domain through Tbx1 and Her9. At later stages, retinoic acid has an additional role by regulating neuronal differentiation in the statoacoustic ganglion. / Les neurones sensorials de l’oïda interna són mediadores claus en la transducció dels estímuls externs des de l’oïda interna al cervell. Entendre a on, quan i com el sistema nerviós sensorial s’organitza durant el desenvolupament embrionari pot ajudar en l’estudi de les malalties neurosensorials. En el present treball, mostro en peix zebra que el factor de transcripció Her9 és un element clau en el control de la neurogènesi òtica i que Her9 es troba sota el control directe del factor Tbx1. A més, ambdos factors estan regulats de manera positva per la via de senyalització de l’àcid retinoic i negativament per la vía de hedgehog. En resum, la tesis demostra un paper de l’àcid retinoic en la regionalització axial del primordi òtic en l’eix anteroposterior i l’establiment d’un domini neurogènic a través de Tbx1 i Her9. En estadis tardans, l’àcid retinoic regula la diferenciació neuronal en el gangli estato-acústic.

Submitted by GABRIELA RONCADA HADDAD null (gabriela.haddad@yahoo.com) on 2017-09-10T22:08:03Z No. of bitstreams: 1 Tese Doutorado 27 jul 2017 - Gabriela.pdf: 1172239 bytes, checksum: d6ae9b8e2d02ba5890bf385b02539433 (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-12T16:46:39Z (GMT) No. of bitstreams: 1 haddad_gr_dr_bot.pdf: 1172239 bytes, checksum: d6ae9b8e2d02ba5890bf385b02539433 (MD5) / Made available in DSpace on 2017-09-12T16:46:39Z (GMT). No. of bitstreams: 1 haddad_gr_dr_bot.pdf: 1172239 bytes, checksum: d6ae9b8e2d02ba5890bf385b02539433 (MD5) Previous issue date: 2017-08-21 / Introdução: A influência do ácido retinoico (AR) sobre o coração é bastante relevante, ocorrendo durante a embriogênese, diferenciação e desenvolvimento cardíaco. Estudos experimentais mostram que o AR pode induzir hipertrofia excêntrica com melhora da função cardíaca em coração de ratos sadios, e também reduzir a hipertrofia, modulando o sistema renina angiotensina aldosterona (SRAA), em animais hipertensos. Pouco se sabe sobre os efeitos do AR em coração de humanos. Pacientes portadores de acne fazem uso de um tipo de AR que é o 13- cis-AR, também chamado de isotretinoína e por isso possibilitam o estudo do papel do AR em humanos. Estudo prévio mostrou que com 2 meses de uso do 13-cis-AR houve remodelação cardíaca. Entretanto, não se sabe sobre os mecanismos ou se essas alterações são reversíveis. Objetivos: Portanto, os objetivos desse trabalho foram de comparar a avaliação morfofuncional cardíaca e variáveis do SRAA entre pacientes em uso de isotretinoína com um grupo controle. Adicionalmente, avaliar se as alterações são reversíveis em pacientes em uso de isotretinoína. Casuística e Métodos: Foram estudados 35 adolescentes e adultos jovens, com idade entre 14 e 23 anos, do sexo masculino, sendo 20 deles em uso de isotretinoína oral, na dose de 0,5 mg/kg/dia a 0,75 mg/kg/dia, acompanhados no ambulatório de dermatologia do Hospital das Clínicas da Faculdade de Medicina de Botucatu (FMBUNESP), aos 6 meses de tratamento. Os outros 15 pacientes foram convidados na comunidade ou apresentavam acne leve com indicação apenas de tratamento tópico. Foram realizados avaliação morfofuncional e doppler tissular por meio de ecocardiografia transtorácica, dosagens bioquímicas de rotina e dosagens de componentes do SRAA renina, angiotensina I, angiotensina II, aldosterona, angiotensina 1-7 e alamandina. Essas variáveis foram comparadas nos grupos controle e isotretinoína pelo teste t de student ou Mann Whitnney. Os pacientes que receberam isotretinoína foram estudados antes do início do tratamento, com 6 meses de tratamento e 2 meses após o término do tratamento. Esses momentos foram comparados por meio do teste de Anova de 1 via de medidas repetidas. O nível de significância adotado foi de 5%. Resultados: Os pacientes do grupo controle e isotretinoína apresentaram a mesma idade, índice de massa corcoral, pressão arterial e frequência cardíaca. Dosagens bioquímicas habitualmente solicitadas durante o tratamento como enzimas hepáticas, função renal e triglicérides também foram semelhantes entre os grupos. Os dados morfológicos mostraram aumento do diâmetro diastólico do ventrículo esquerdo (DDVE) acompanhado de aumento do débito cardíaco e do fluxo transmitral avaliado por E/E’. Houve aumento do volume do átrio esquerdo (AE), no limite da significância e tendência ao aumento da massa do ventrículo esquerdo (VE) e com espessura relativa da parede semelhante entre os grupos. Sobre o SRAA houve redução da angiotensina II e da renina. Na avaliação ecocardiográfica apenas dos pacientes em uso de isotretinoína observou-se que houve redução do AE e do índice de massa do VE (IMVE) após 2 meses do término do tratamento. Embora não significativo, o comportamento de E/E’ também foi de redução após o tratamento. Discussão: O 13-cis-AR promove remodelação cardíaca, provavelmente induzida por hipervolemia, levando a um padrão de hipertrofia excêntrica com melhora da função. Essas alterações provavelmente levaram a menor ativação do SRAA visto pela redução da renina e angiotensina II. Esse perfil de remodelação e de bloqueio do SRAA é semelhante ao que ocorre no exercício físico. Nesse estudo foi possível apenas avaliar o grupo isotretinoína, quanto às variáveis ecocardiográficas antes, durante e após o tratamento. Observa-se que o término do estimulo com 13-cis-AR reduz algumas variáveis como átrio esquerdo e massa do VE. Portanto, em corações normais de adultos jovens, o AR atenuou o efeito de SRAA e promoveu remodelação cardíaca do tipo excêntrica, com melhora da função, compatível com sobrecarga volêmica e com caráter transitório. / Background: The influence of retinoic acid (RA) in the heart is very relevant, occurring during the embryogenesis, differentiation and cardiac development. Experimental studies shown that RA induces excentric hypertrophy, with improvement of cardiac function in heart of healthy rats. In addition, it was observed that RA regulates renin angiotensin aldosterone system (RAAS), a regulatory system involved in blood pressure, volume homeostasis and cardiac hypertrophy. There is a lack of information about the role of RA on cardiac remodeling in adult humans. Otherwise there are patients with Acne that uses 13- cis-RA, also called isotretinoin, and this population allow the investigation of cardiac remodeling and RA treatment. In fact, previously study shown that the use of 13-cis-RA, for acne, for 2 months induced cardiac remodeling, however, no one knows if these changes are persistent and reversible. Objectives: the objectives of these study is to compare the cardiac morphofunctional evaluation and RASS variables in patients using isotretinoin and in control group. Additionally, evaluate if these changes are reversible in isotretinoin group. Casuistic and Methods: Study1: 35 young men, between 14 to 23 years, 20 in use of 13-cis-RA, in the dose of 0,5 mg/kg/day to 0,75 mg/kg/day, from dermatology clinic of São Paulo State University (FMB-UNESP), at 6 months of treatment. The others 15 patients had mild acne only with topic treatment. Morphofunctional evaluation, tissular Doppler, Biochemical evaluation, dosage of RAAS components were performed. Results were compared in isotretinoin and control group by t student or Mann Whitnney tests. Study 2: Only the isotretinoin group were evaluated before beginning of treatment (initial moment - M0), after 6 months of treatment (final moment - M1) and two months after finishing the medication (M2). This results were compared by One way pared Anova. The statistically significant level was set at 5%. Results: control and isotretinoin group presents similar age, body mass index, blood pressure and heart rate. Biochemical evaluation was also similar. The present study showed that young patients receiving 13-cis-RA for Acne treatment presented left ventricle and atrium chamber enlargement and increase in cardiac output and in mitral flows. There was a trend toward higher ventricular mass with preserved relative wall thickness. RAAS showed decreased in angiotensin II and renin. Considering only patients that received isotretinoin, it was observed that cardiac atrium size and flows returned to baseline 2 months after the end of treatment and cardiac structures such as ventricle mass and thickness reduced. Discussion: 13-cis-RA promotes cardiac remodeling, probably induced by hypervolemia, taking to a pattern of eccentric hypertrophy, with improvement of function. It is possible that hypervolemia or a direct effect of 13-cis-RA, reduces renin and angiotensin II. The remodeling phenotype described is compatible with cardiac remodeling induced by physical activity, marked by hypervolemia, excentric hypertrophy and increased cardiac output. In the isotretinoin group, the end of treatment reduces left atrium size and left ventricle mass. Therefore, in normal hearts of young adults, RA reduces the effect of RAAS and promotes eccentric cardiac remodeling, with improvement of function, compatible with volume overload and with transitory character.

Orientador: Francisco Eduardo Martinez / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T07:43:37Z (GMT). No. of bitstreams: 1 Fontanelli_BeatrizAparecidaFioruci_M.pdf: 2160007 bytes, checksum: 5e358e9b41de70f7fd7c801fae0e0378 (MD5) Previous issue date: 2011 / Resumo: A exposição ao etanol altera a concentração do retinol e do all-trans-ácido retinóico (atAR) em vários tecidos. Os retinóides, retinol e atAR, são importantes para a diferenciação e manutenção das células epiteliais da próstata. O atAR se liga aos receptores de ácido retinóico (RARa, ß e y) e a interação receptor/ligante com a sequência responsiva ao retinóide no DNA, levam à transcrição de genes alvos. Assim, o atAR exerce efeitos no crescimento celular, diferenciação e apoptose, sendo essencial no desenvolvimento e diferenciação de órgãos e tecidos. Nosso objetivo foi analisar o retinol, o ácido retinóico e seus receptores, bem como, o índice de proliferação celular e de apoptose no lobo dorsolateral da próstata de ratos adultos UCh. Os animais foram divididos em quatro grupos experimentais (n=10/grupo): UChA (ingestão voluntária de etanol a 10% (v/v); UChACo (controle - ausência de etanol); UChB (ingestão voluntária de etanol a 10% (v/v) e UChBCo (controle - ausência de etanol). Após 150 dias de experimentação, os animais foram eutanasiados por decapitação e o sangue do tronco e os lobos dorsolaterais das próstatas foram coletados e processados: (1) para análises da concentração do retinol e do atAR no plasma e na próstata por meio de HPLC; (2) e análises de microscopia de luz para a proliferação celular (Ki-67), apoptose (Tunel) e para os receptores de ácido retinóico, por meio dos anticorpos anti-RARa, -ß e -y. O consumo crônico de etanol diminuiu a concentração do retinol no plasma dos grupos UChB (consumo alto de etanol) e UChA (consumo baixo de etanol). A concentração do retinol foi ainda menor no plasma do grupo UChB comparado ao UChA. No entanto, a concentração do retinol no tecido prostático não teve diferença significativa entre os grupos. O atAR aumentou significativamente somente no plasma do grupo UChB. Na próstata, a concentração do atAR aumentou no grupo UChB, enquanto que no UChA não houve diferença estatística. O RAR? na próstata dorsal e lateral dos ratos UCh não foi alterada em função do consumo de etanol. Já os RARß e -? apresentaram aumento do sinal na próstata dorsal do grupo UChB. Não houve diferença no índice de proliferação celular e de apoptose nas próstatas dorsais e laterais dos grupos experimentais. Conclui-se que o etanol altera a concentração do retinol e do atAR no plasma. Essa alteração é diretamente proporcional à quantidade de etanol consumida. Já na próstata, o retinol não é alterado pelo etanol. O consumo alto de etanol altera a concentração do atAR na próstata dorsolateral e a expressão dos RAR ß e y na próstata dorsal. A alteração da expressão dos RAR pode aumentar a sensibilidade da próstata à ação do atAR. O etanol não altera a proliferação celular e a apoptose na próstata dorsal e lateral / Abstract: Ethanol exposure alters the concentration of retinol and all-trans retinoic acid (atAR) in several tissues. Retinoids (retinol and atAR) are essential for the differentiation and homeostasis of the prostate epithelial cells. atAR binds to retinoic acid receptors (RAR a, ß and ?) and the interaction receptor/ligand with the sequence responsive to retinoid into DNA lead to transactivation of target genes. Thus, atAR directly produces their effects on cell growth, differentiation and apoptosis. This study aimed to analyze the retinol and all-trans-retinoic acid concentrations and its atAR receptors as well as the cell proliferation and apoptosis index upon the dorsolateral prostate lobe of adult UCh rats. All animals were divided into four experimental groups (n = 10/group): UChA (10% ethanol (v / v) voluntary intake); UChACo (without ethanol consumption); UChB (10% ethanol (v / v) voluntary intake) and UChBCo (without ethanol consumption). After 150 days of experimentation, animals were sacrificed followed by decapitation and trunk blood and dorsolateral prostate lobes collected. Samples of plasma and prostate by concentration analysis of the retinol and atAR were processed for HPLC. The cell proliferation and apoptosis immunoreactivities were assessed by Ki-67 and Tunel, respectively, and nuclear receptors by anti-RAR a,-ß and-y. Chronic ethanol consumption reduced the concentration of plasma retinol in UChB (high ethanol intake) and UChA groups (low ethanol intake). The retinol concentration in plasma was even lower in UChB compared to UChA group. However, the retinol concentration in prostate tissue was not significantly different between the groups. Concentration of atAR increased in plasma of UChB group, and was 96% higher in the UChA group. The prostate, atAR increased in the UChB group, while in UChA group no statistical difference. There was no statistical difference in proliferation cell and apoptosis in the dorsal and lateral prostate lobes between the groups. The expression of RAR a in the dorsal and lateral prostate of UCh rats was not altered as a function of ethanol consumption. Already RAR ß and-y showed increased signal in the dorsal prostate UChB group. We conclude that ethanol alters the concentration of retinol and atAR in plasma. This change is directly proportional to the amount of ethanol consumed. In the prostate, retinol is not altered by ethanol. The high ethanol intake alters the concentration of atAR in dorsolateral prostate and the expression of RARß and RARy in the dorsal prostate. Alteration in expression of RAR can increase sensitivity to the action of the atAR in prostate. Ethanol does not alter cell proliferation and apoptosis in the dorsolateral prostate / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

O ácido retinoico (RA), metabolito ativo da vitamina A, exerce ampla atividade biológica sobretudo na modulação da resposta imunológica. A interação do RA com os seus receptores nucleares induz a transcrição de genes que atuam na homeostase de sítios imunológicos, principalmente no tecido linfóide associado à mucosa intestinal (GALT). O RA promove a diferenciação de células T reguladoras CD4&#43CD25&#43Foxp3&#43, a migração de células efetoras para a mucosa intestinal induzindo a expressão de CCR9 e 4&#9467, além de inibir a diferenciação de linfócitos T helper (Th) 17 no intestino, garantindo a homeostase intestinal. Entretanto, eventos mediados pelo RA durante o desenvolvimento do sistema imunológico neonatal ainda não são totalmente conhecidos, principalmente no contexto materno-fetal. Desta forma, o objetivo do trabalho foi avaliar o efeito da suplementação materna com RA, durante a amamentação, no sitema imunológico da prole. Para tanto, camundongos fêmeas C57BL6 Foxp3-GFP receberam 6 doses de RA (1mg/gavagem), durante o período de amamentação, e o grupo controle recebeu apenas óleo vegetal. Os resultados mostram que a suplementação materna com RA foi capaz modular o sistema imunológico da prole aumentando o percentual de linfócitos T reguladores (Treg) esplênicos nas proles com 6 semanas de idade. Além disso, houve aumento percentual de linfócitos Treg, TCD4&#43 e TCD8&#43 que expressam CCR9, tanto no baço quanto nos linfonodos mesentéricos da mães e de suas prole, o que pode proporcionar a migração de células para o intestino. Este efeito foi duradouro nas proles até 6 semanas de idade. A suplementação materna com RA elevou o percentual de linfócitos Treg e linfócitos B IgA&#43 no intestino das proles, e a concentração de imunoglobulina (Ig) A fecal, mas não alterou a composição da microbiota intestinal. Nas mães suplementadas houve redução das concentrações séricas de IgA e IgG. Em contraste com o efeito tolerogênico do RA na lâmina própria do intestino, observamos o aumento sérico de interferon (IFN)- nas proles de mães suplementadas e aumento na secreção de IFN- por esplenócitos induzida por CL097 (agonista de Toll-like receptor 7/8), sugerindo que o RA pode ter um impacto importante na deficiente resposta de perfil Th1 nos neonatos. Para averiguar o efeito modulatório in vivo da suplementação materna de RA, foi avaliada a indução de colite por sulfato de sódio dextrano (DSS) nas proles. Não houve perda de peso acentuado nas proles de mães suplementadas com RA quando comprado às proles de mães controles, além de apresentarem a permeabilidade intestinal conservada e aumento do fator de transformação do crescimento (TGF)- &#946 no homogenato intestinal, indicando menor dano no tecido epitelial do intestino. Apesar disto, o RA não foi capaz de inibir totalmente o processo inflamatório na colite. No conjunto, os achados evidenciam que a suplementação materna com RA foi importante no desenvolvimento da imunidade de mucosa e na manutenção da homeostase intestinal, sendo um importante metabólito para atenuar respostas inflamatórias. A indução sérica de IFN- e após estímulo com CL097 pode indicar o uso de RA como estratégia para potencializar respostas Th1, crucial contra infecções virais e bacterianas no período neonatal. / Retinoic acid (RA), the active metabolite of vitamin A, exerts extensive biological activity mainly in the modulation of the immune response. The interaction of RA with its nuclear receptors induces the transcription of genes that acts on the homeostasis of immunological sites, especially in gut associated lymphoid tissue (GALT). RA promotes the differentiation of CD4&#43CD25&#43Foxp3&#43 regulatory T cells, migration of effector cells to the intestinal mucosa through gut-homing receptors CCR9 and 4&#9467, besides inhibiting the differentiation of T helper (Th) 17 cells in the gut, guaranteeing intestinal homeostasis. However, events mediated by RA during the development of the neonatal immune system are not totally known, especially in the maternal-fetal context. Thus, the aim of the study was to evaluate the effect of maternal RA supplementation during breastfeeding on the immune system of offspring. For this, C57BL/6 Foxp3-GFP female mice with 8-10 weeks-old received 6 doses of RA (1mg / gavage) during the breastfeeding period, and the control group received only vegetable oil. The results show that maternal RA supplementation was able to modulate the offspring immune system by increasing the percentage of splenic regulatory T (Treg) cells in offspring at 6 weeks of age. In addition, there was an enhancement in the CCR9 expression on regulatory T cells and CD4&#43 and CD8&#43 T cells, in the spleen and in the mesenteric lymph nodes of mothers and their offspring, which can provide migration of cells into the gut. This effect was long-term in offspring up to 6 weeks of age. Maternal RA supplementation also increasing the percentage of regulatory T cells and B IgA&#43 cells in the offspring´s gut, beside increasing the fecal immunoglobulin (Ig) A concentration, but did not alter the composition of the intestinal microbiota. In the supplemented mothers, serum concentrations of IgA and IgG were reduced. In contrast to the tolerogenic effect of RA on intestinal lamina propria, we observed a serum increase of interferon (IFN)- in offspring and increasing in CL097(Toll-like receptor 7/8 agonist)-induced IFN- secretion by splenocytes, suggesting that RA may have a significant impact on the deficient Th1 profile response in neonates. To investigate the modulatory effect in of RA maternal supplementation, was evaluated the induction of colitis by dextran sodium sulfate (DSS) in the offspring. There was no significant weight loss in the offspring from mothers supplemented with RA in comparison with the offspring from control mothers, as well as having preserved intestinal permeability and increased transforming growth factor (TGF)- &#946 in the intestinal homogenate, indicating less damage to intestinal epithelial tissue. Despite this, RA was not able to totally inhibit the inflammatory process in colitis. Taken together, the findings show that maternal RA supplementation was important in the development of mucosal immunity and maintenance of intestinal homeostasis, being an important metabolite to attenuate inflammatory responses. Induction of serum IFN- after TLR7/8 (CL097) stimulation may indicate the use of RA as a strategy to potentiate Th1 responses, crucial against viral and bacterial infections in the neonatal period.

Existeix certa controvèrsia sobre els efectes del b-carotè com a promotor o protector del càncer de pulmó. A més, el b-carotè, com a precursor de l'àcid retinoic, podria estimular la termogènesi i regular l'adipositat corporal. La fura representa un bon model per estudiar els efectes de la ingesta de b-carotè ja que l'absorbeix de manera pràcticament intacta (semblant als humans). Hem demostrat que el b-carotè ingerit amb altres antioxidants no sembla tenir efectes inductors del càncer ja que no augmenta la proliferació cel·lular al pulmó i a més prevé els efectes del carcinogen benzo[a]pirè. D'altra banda, dosis farmacològiques de b-carotè fan a la fura efectes contraris als de l'àcid retinoic, ja que augmenten l'adipositat i resulten en una menor capacitat termogènica, principalment al teixit adipós retroperitoneal que, a la fura, presenta certes característiques de teixit adipós marró, ja que té un percentatge considerable d'adipòcits multiloculars i expressa quantitats significatives d'UCP1. / Existe controversia sobre los efectos del b-caroteno como promotor o protector del cáncer de pulmón. Además, el b-caroteno, como precursor del ácido retinoico, podría estimular la termogénesis y regular la adiposidad corporal. El hurón representa un buen modelo para estudiar los efectos de la ingesta de b-caroteno pues lo absorbe de manera prácticamente intacta (similar a los humanos). Hemos demostrado que el b-caroteno ingerido con otros antioxidantes no parece tener efectos inductores del cáncer puesto que no aumenta la proliferación celular del pulmón y además previene los efectos del carcinógeno benzo[a]pireno. Por otra parte, dosis farmacológicas de b-caroteno producen en el hurón efectos contrarios a los del ácido retinoico, ya que aumentan la adiposidad y reducen la capacidad termogénica, principalmente del tejido adiposo retroperitoneal que, en el hurón, presenta ciertas características de tejido adiposo marrón, al contener un porcentaje considerable de adipocitos multiloculares y expresar cantidades significativas de UCP1. / The effects of b-carotene promoting or protecting against lung cancer are unclear. Furthermore, b-carotene, as retinoic acid precursor, could induce thermogenesis and regulate body adiposity. The ferret represents a good model to study the effects of oral administration of b-carotene because it absorbs it almost intact (similarly to humans). We have shown that the intake of b-carotene together with other antioxidants does not seem to inducecancer as it does not increase cellular proliferation in lung and prevents the effects of the carcinogen benzo[a]pyrene. In addition, pharmacological b-carotene doses in ferrets have different effects from retinoic acid, increasing adiposity and decreasing thermogenic capacity, especially in the retroperitoneal adipose tissue which, in ferrets, resembles brown adipose tissue, since it has an important percentage of multilocular adipocytes and expresses significant amount of UCP1.

En un intento de optimizar el sistema de fecundación in vitro en la especie porcina se estudió la influencia de distintas condiciones de maduración y de cocultivo de los gametos. Se evaluó la reducción del tiempo de coincubación de los gametos a 10 min observándose un claro efecto del ratio espermatozoides:ovocito. El estudio de las necesidades de los espermatozoides en términos de aditivos del medio de fecundación y tiempo de coincubación reveló variaciones entre verracos. La utilización de un tiempo de coincubación tan corto como 2 min fue suficiente para obtener unas tasas de penetración y monospermia similares a las alcanzadas por los sistemas de FIV tradicionales. El sistema de FIV en pajuela con 10 min de coincubación aumentó la penetración monoespérmica y mejoró la calidad de los blastocistos. La adición de 5 nM de 9- cis ácido retinoico al medio de maduración aumentó significativamente la formación de blastocistos. / The present study was conducted in an attempt to optimize porcine in vitro fertilization system. For this purpose, the influence of maturation and gamete coculture conditions were studied. The coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocytes ratio. The needs of boar spermatozoa for IVF, in terms of additives to IVF medium and coincubation times vary among boars. The use of coincubation time as brief as 2 min is long enough to obtain good fertilization rates similar to those achieved from current long term exposure times in IVF. A straw IVF system in combination with a 10 min coincubation increased monospermic penetration and the quality of blastocysts compared with the microdrop-IVF system. The addition of 5 nM of 9- cis retinoic acid in the IVM medium increased blastocyst formation rate, suggesting that RA may play an important role during IVM.