Le dogme relatif à la radiorésistance du Système Nerveux Central (SNC) a vécu. Les progrès en neurosciences permettent aujourd'hui de reconsidérer les dysfonctionnements cognitifs radio-induits observés au décours des radiothérapies ou après un accident d'irradiation, et d'envisager des moyens diagnostiques et thérapeutiques adaptés. Nous avons développé un modèle Rat afin d'étudier les effets d'une irradiation gamma corps entier à dose sublétale (4,5 Gy). Celle-ci induit des troubles de l'apprentissage et de la mémorisation d'une tâche en cours d'acquisition durant le premier mois – lesquels sont prévenus par l'administration d'une molécule radioprotectrice de référence (amifostine) – tandis qu'elle ne semble pas perturber la mémoire rétrograde. Précocement, une vague apoptotique survient 5 à 9 heures après exposition dans la zone sous-ventriculaire avec, en parallèle, une neurogenèse anéantie. Deux jours après irradiation, l'étude métabolique ex vivo réalisée par RMN HRMAS (High Resolution Magic Angle Spinning) suggère la présence d'un œdème cérébral tandis que l'étude des lipides cérébraux en RMN liquide confirme l'atteinte membranaire (élévation du cholestérol et des phospholipides). Le profil lipidique se normalise ensuite tandis qu'une réaction gliale apparait. Enfin, 1 mois post-irradiation, l'élévation du GABA, neurotransmetteur inhibiteur du SNC, dans 2 structures cérébrales distinctes, s'accompagne d'une diminution de la taurine dans l'hippocampe qui persiste 6 mois. Notre modèle intégré permet ainsi de valider des biomarqueurs quantifiables en spectroscopie RMN in vivo – prochaine étape expérimentale – et de tester de nouvelles thérapeutiques radioprotectrices.

Durant el període de formació del Sistema Nerviós Central (SNC) una multitud de factors participen en el desenvolupament de les diferents estructures cerebrals. Aquests factors controlen tant la migració cel·lular com el correcte posicionament de les diferents cèl·lules dins el SNC. La Reelina és un proteïna soluble de matriu extracel·lular expressada al llarg del desenvolupament i de l'edat adulta, encara que la seva funció principal s'hipotetitza durant el desenvolupment. La present tesi abarca tres funcions diferents de la Reelina en el control de la migració cel·lular durant el desenvolupament i l'edat adulta.En el primer apartat d'aquesta Tesi, hem demostrat que la Reelina pot controlar el nivell de fosforilació en mode I de la proteïna MAP1B durant la migració de les neurones que formen l'escorça cerebral. Aquesta fosforilació en mode I s'ha associat al control de l'estabilitat dels microtúbuls durant la migració cel·lular. L'estudi morfològic que hem desenvolupat en l'animal deficient per la proteïna MAP1B ens ha corraborat que existeix una relació entre la cascada de senyalització desencadenada per la Reelina i l'activitat de la proteïna MAP1B. Finalment també hem descrit que en resposta a la Reelina la quinasa GSK3-beta fosforila principalment MAP1B en mode I, encara que la participació, en menor grau, de la quinasa Cdk5/p35 no pot ser descartada.En el següent punt del nostre estudi, hem relacionat la via de senyalització MAPK/ERK amb la cascada activada per la Reelina. Hem comprovat que la Reelina pot induir la fosforilació d'mDab1 i la subseqüent activació de la proteïna PI3K, que a la seva vegada pot fosforilar a la proteïna Akt1/PKB. A partir d'aquest resultats, hem demostrat que l'activació de PI3K en reposta a la Reelina indueix l'activació de MEK1/2 i que aquesta activació desenvoca en la fosforilació de les proteïnes Erk1 i Erk2. També hem descrit que les formes fosforilades d'Erk1/2 en resposta a Reelina es transloquen a nucli en neurones corticals on indueixen l'expressió del factor de transcripció Egr-1. Un punt important d'aquest estudi es basa en el fet que hem pogut relacionar la cascada Reelin-MAPK/ERK en el procés de desadhesió de les cèl·lules de la zona subventricular (o SVZ) que participen en la via de la migració rostral (o RMS). A partir del fet que en presència de la Reelina, les cèl·lules que surten d'explants de SVZ no migren homofílicament sino que pateixen un proces de desadhesió migrant de forma individualitzada, hem descrit que si bloquegem farmacològicament o genèticament la via d'activació MAPK/ERK l'efecte de desadhesió induït per la Reelina queda inhibit. Amb aquest resultat podem concloure que en resposta a la Reelina les proteïnes Erk1/2 es fosforilen i que aquesta fosforilació és necessària per que la Reelina desenvolupi els seu paper en desadhesió dels neuroblasts que participen en la RMS.El tercer i darrer punt d'aquesta Tesi es basa en el control per part de la Reelina de la migració radial de les cèl·lules granulars del cerebel. En resposta a Reelina les cèl·lules granulars del cerebel inhibeixen la seva migració i aquesta resposta es induïda mitjançant l'activació de la via de les MAPK/ERK. El bloqueig farmacològic d'aquesta via inhibeix l'efecte de la Reelina. Amb aquestes dades podem concloure que l'activació, induïda per la Reelina, de la via de les MAPK/ERK no només és important en el control de la migració en el cerbell anterior sino que també és important en el cerebel, y que aquesta pot ser una cascada de senyalització que participa àmplimant en les funcions on s'hipotetitza la funcionalitat de la Reelina. / "Role of Reelin signalling during neuronal migration in the Central Nervous System"During the development of the Central Nervous System (CNS) a multitude of factors work together to generate the different brain structures. Some of these factors control both cellular migration and cellular positioning inside the CNS. Reelin is a soluble protein of the extracellular matrix expressed during both development and adulthood, however its principal function is hypothesized to occur during development. This current Thesis addresses three different functions of Reelin on neuronal migration during embryonic and postnatal development.In the first chapter, we have shown that Reelin controls mode I phosphorylation of MAP1B protein during neuronal migration in the neocortex. Mode I phosphorylation has been related to the microtubule stability during cell migration. We developed morphological studies on map1b-deficient mice that corroborated the relationship between Reelin signalling cascade and MAP1B activity. Furthermore, we also described that this relation is done through the Reelin-dependent GSK3 activation, which phosphorylates MAP1B in mode I.In the second chapter, we addressed the relationship of Reelin signalling and the MAPK/ERK pathway. We showed that Reelin induces the phosphorylation of Dab1 and subsequent PI3K/Akt1 activation. Based on these results, we first described that Reelin-dependent PI3K activation induces MEK activation, which triggers Erk1 and Erk2 (Erk1/2) phosphorylation. We also showed that these active phospho-Erk1/2 proteins translocate into the nucleus inducing the expression of the Egr-1 transcription factor. Furthermore, we have demonstrated that this Reelin-induced pathway is activated during rostral migratory stream migration when neuroblasts face Reelin expressed in the olfactory bulb. The activation of ERK pathway in response to Reelin is crucial to change neuroblast migration from tangential chain migration to radial individual migration into the olfactory bulb.In the last chapter, we studied the role of Reelin in the control of radial migration of granule cells during the postnatal development of the mouse cerebellum. We shared that cerebellar granule cell migration is blocked by Reelin stimulation through MAPK/ERK activation. Furthermore, when ERK activation was pharmacologically block the Reelin effect on granule cells migration was inhibit.

Dorsoventral patterning of the vertebrate nervous system is achieved by the combined activity of morphogenetic signals secreted from dorsal and ventral signalling centres. The Shh/Gli pathway plays a major role in patterning the ventral neural tube; however, the molecular mechanisms that limit target gene responses to specific progenitor domains remain unclear. Here, we show that Wnt1 and Wnt3a, by signalling through the canonical β-catenin/Tcf pathway, control expression of dorsal genes and suppression of the ventral programme, and thatthis role in DV patterning depends on Gli activity. Additionally, we show that Gli3 expression is controlled by Wnt activity. Identification and characterization of highly conserved non-coding DNA regions around the human Gli3 gene revealed the presence of transcriptionally active Tcf-binding sequences. These indicated that dorsal Gli3 expression might be directly regulated by canonical Wnt activity. In turn, Gli3, by acting as a transcriptional repressor, restricted graded Shh/Gli ventral activity to properly pattern the spinal cord.Additionally, the Wnt canonical pathway and Hedgehog signalling have been linked to cell proliferation in a variety of systems, however interaction of these pathways to control cell cycle progression have not been studied. In the developing vertebrate nervous system, although Shh and Wnt ligands are expressed at the opposite ventral and dorsal signalling centres, reports demonstrate that proliferation of neural progenitors require both activities throughout the dorsoventral axis. Here we demonstrate the integration of both pathways to control the length of G1 phase, and the absolute requirement of an upstream Hedgehog activity for the Wnt-mediated regulation of the key cell cycle activator CyclinD1 expression and for G1 progression. Although Wnt canonical activity appeared restricted to the control of G1 phase, Hedgehog activity additionally regulates the length of G2 phase through the regulation of ate cell cycle activators such as CyclinA2 and CyclinB2/3. These findings support a key role for Hedgehog in growth control, as a regulator of G1 and G2 phases of cell cycle and importantly as an upstream regulator of the canonical Wnt activity.KEY WORDS: SHH, WNT, Neural Tube, Patterning, CNS, Cell Cycle. / "La integración de las vías de señalización de Shh y Wnt controla la morfogénesis del SNC"TEXTO: La formación del patrón dorsoventral del sistema nervioso central en vertebrados está controlada por la acción de señales morfogenéticas. Estasseñales son secretadas por centros de señalización situados en el extremo dorsal, ectodermo y placa del techo, y ventral, notocorda y placa del suelo, del tubo neural. La vía de señalización de Shh/Gli juega un papel principal en el establecimiento temprano de la región ventral del tubo neural. Sin embargo, losmecanismos moleculares que restringen la expresión de los genes responsables de establecer este patrón a un dominio concreto no son del todo conocidos. En este trabajo mostramos que las señales morphogenéticas Wnt1y Wnt3a, activando la vía de señalización canónica mediada por β-catenina/Tcf, regulan la expresión de genes dorsales y reprimen el programa ventral, mediante un mecanismo que depende de la actividad Gli. Además, mostramosque la expresión de Gli3 está controlada por la vía de Wnt. La identificación y caracterización de regiones no codificantes altamente conservadas alrededor del locus de Gli3 humano revela que contienen sitios de unión consenso para los factores Tcf/Lef-1 activos. Esto indica que la expresión dorsal de Gli3 está controlada directamente por la actividad de la vía canónica de Wnt. A su vez, Gli3, actuando como un represor transcripcional, restringe la actividad del gradiente ventral Shh/Gli para establecer el patrón dorsoventral del tubo neural correctamente.Por otro lado, las vías canónicas de señalización celular de Wnt y Hedgehog regulan la proliferación celular en varios contextos de forma conjunta, Sin embargo, posiblesm interacciones de estas vías de señalización en el control del ciclo celular no han siso estudiadas. Durante el desarrollo del sistema nervioso de vertebrados, aunque las proteínas Shh y Wnts se expresan en extremos dorso-ventralmente opuestos del tubo neural, varios trabajos demuestran que la proliferación de los progenitores neuronales requiere ambas actividades a lo largo de todo el eje dorsoventral. En este trabajo, demostramos que esnecesaria la integración de ambas vías de señalización para controlar la duración de la fase G1 del ciclo celular. Además, mostramos el requerimiento de la actividad Hedgehog para la regulación mediada por Wnt de la expresión del activador de ciclo celular ciclina D1, componente clave para la progresión através de G1. Aunque la actividad de la vía canónica de Wnt se limita al control de la transición G1/S, adicionalmente, la actividad Hedgehog regula la duración de la fase G2 mediante la regulación de las ciclinas de fase G2 ciclina A2 y Ciclinas B2/B3. En conjunto, estos resultados proponen un papel fundamentalen el control del crecimiento para la actividad Shh/Gli como reguladora de las fases G1 y G2 del ciclo celular y además como reguladora por encima de la actividad canónica de Wnt.

El linfoma de células del manto (LCM) es una entidad heterogénea tanto en sus aspectos biológicos como en su comportamiento clínico. La existencia de pacientes con mala respuesta al tratamiento sugiere que, de manera análoga a otros síndromes linfoproliferativos crónicos, existen alteraciones en los mecanismos de citotoxicidad inducida por fármacos. La heterogeneidad clínica del LCM queda reflejada, entre otros, por la variabilidad en la presentación de expresión hemoperiférica e infiltración del sistema nervioso central (SNC), dos complicaciones controvertidas y no suficientemente estudiadas. La hipótesis planteada en la presente tesis fue que la heterogeneidad observada en el LCM podría ser debida a la existencia de alteraciones en los mecanismos de apoptosis en un subgrupo de pacientes y se relacionaría con el pronóstico de éstos. Por otro lado, el análisis exhaustivo de los pacientes con expresión hemoperiférica o infiltración del SNC permitiría establecer cuáles son las características clínicas y biológicas que determinan la aparición de estas complicaciones en determinados enfermos. Para confirmar esta hipótesis nos planteamos los siguientes objetivos: 1) estudiar las bases moleculares de la regulación de la apoptosis en el LCM, tanto en líneas celulares como en células primarias de pacientes afectos; 2) analizar la incidencia y el impacto pronóstico de la expresión hemoperiférica en los enfermos con LCM mediante citología y citometría de flujo y 3) estudiar la incidencia y los factores determinantes de la infiltración del SNC en pacientes afectos de esta hemopatía.Para el estudio de las bases moleculares de la apoptosis en el LCM se analizaron cuatro líneas celulares portadoras de la t(11;14)(q13;q32), característica de esta entidad, y células primarias de 10 pacientes con LCM. El análisis de la viabilidad celular mediante marcaje con anexina V y yoduro de propidio, la detección de la pérdida de potencial de membrana y la producción de ROS, la determinación de caspasa 3 activa y de los cambios de conformación de BAX y BAK se llevaron a cabo mediante citometría de flujo (CMF). El análisis de diversas proteínas de la familia de BCL-2 se realizó mediante "Western blot". El estudio del ciclo celular se efectuó asimismo mediante CMF. El análisis de las alteraciones cromosómicas existentes en los LCM leucemizados se realizó mediante hibridación genómica comparada (CGH). Las diferencias entre subgrupos de pacientes se analizaron mediante la prueba exacta de Fisher y la t de Student. El análisis de supervivencia se llevó a cabo mediante el método de Kaplan y Meier y las diferencias observadas en términos de supervivencia se analizaron con el "log-rank test". El análisis de variables dependientes del tiempo se efectuó mediante el método de Mantel y Byar. Los resultados obtenidos en el primer trabajo demuestran que la citotoxicidad inducida por mitoxantrona en las células del LCM fue debida a la activación de la vía mitocondrial de apoptosis, y tiene lugar, probablemente, de manera dependiente de la integridad de los sensores de daño al ADN. Los resultados del segundo trabajo permiten concluir que la expresión hemoperiférica detectada mediante CMF se observa en la mayoría de los pacientes con LCM, incluso en aquellos con recuentos linfocitarios normales. Aunque la leucemización morfológica no se asoció en nuestro estudio con ninguna alteración citogenética específica detectable mediante CGH, los casos con linfocitosis >/5 x 10(9)/L presentaron anomalías citogenéticas diferenciales y un peor pronóstico. Los resultados del tercer trabajo demuestran que la infiltración del SNC se presenta fundamentalmente en pacientes con LCM blastoide, índice proliferativo elevado, niveles de LDH sérica elevados e IPI de riesgo intermedio/alto o alto, en general en el contexto de recidivas o progresiones sistémicas. / "BIOLOGICAL AND CLINICAL PROGNOSTIC FACTORS IN MANTLE CELL LYMPHOMA"TEXT:Mantle cell lymphoma (MCL) is characterized by the presence of translocation t(11;14)(q13;q32), an aggressive clinical course and poor response to chemotherapy. Few data concerning drug-induced apoptosis in MCL have been reported. The aim of the first study that constitutes the present thesis was to analyze the mechanisms of drug-induced apoptosis in MCL. Four cell lines carrying the t(11;14) and primary cells of 10 patients with MCL were incubated in vitro with several drugs currently used in the treatment of lymphoproliferative disorders and drug-induced cell death was characterized. Our results support that MCL cells have functional apoptotic machinery but require the integrity of functional DNA-damage response genes for its activation. From a clinical standpoint, extranodal involvement is a well-known feature in patients with MCL. Relatively few studies to date have focused on the peripheral blood (PB) involvement and the incidence of leukemic expression in MCL varies highly in different studies. The objective of our second study was to analyze the incidence, and the biological and clinical significance of leukemic involvement in patients with MCL. We investigated the incidence of PB involvement by both morphologic and flow cytometry (FC) analyses. Clinical features, genetic abnormalities detected by comparative genomic hybridization (CGH) and patient outcome were also determined. Leukemic expression at diagnosis detected by FC was a highly common feature, even in patients with a normal lymphocyte count. Although morphologically apparent leukemic expression was not associated with specific chromosomal alterations detected by CGH, a lymphocyte count >/ 5 x 10(9)/L was correlated with particular genetic abnormalities and a poor outcome.The incidence of central nervous system (CNS) involvement in patients with MCL is also highly variable in different studies, and predicting factors and outcome of CNS infiltration in these patients have not been thoroughly investigated. The aim of our study was to assess the incidence and factors for CNS involvement in MCL. In addition, we analyzed the clinical features, therapy and outcome of patients with MCL once CNS infiltration was detected. Our results suggest that, in most cases, CNS involvement occur late in the course of the disease, as part of a generalized relapse or progression. Blastoid histology, high proliferative index, high serum LDH and high-risk IPI are the variables associated with a higher risk to develop this complication.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2016. / Made available in DSpace on 2017-05-23T04:12:02Z (GMT). No. of bitstreams: 1 345773.pdf: 2975746 bytes, checksum: 75cb9b7e973aa13e7c87f1908fea3eb7 (MD5) Previous issue date: 2016 / A neurotoxicidade do metilmercúrio (MeHg) em indivíduos adultos é relativamente conhecida, contudo são escassos os trabalhos que analisam os estudos sobre a ação desse metal em duas estruturas do SNC durante o desenvolvimento. Este trabalho teve como objetivo relacionar os efeitos do MeHg sobre a organização estrutural e ultraestrutural do mesencéfalo e cerebelo, bem como identificar sua ação nos processos de proliferação e diferenciação celular, utilizando como modelo experimental embriões de Gallus domesticus. Os embriões foram tratados in ovo no 3° dia de incubação (E3) com uma única dose de metilmercúrio (MeHg) (0,1 µg diluído em 50 µL de salina) e analisados no 10° dia de incubação (E10). Os embriões do grupo controle foram tratados somente com 50 µL de solução salina.As análises morfológicas e morfométricas do mesencéfalo e cerebelo foram realizadas a partir de secções histológicas coradas com HE. A quantificação e imunolocalização de proteínas relacionados ao ciclo celular (ciclina E; p21 e p53), diferenciação celular (NeuN; TubulinaIII e GFAP), proliferação celular (fosfohistona H3) e dano ao DNA(?PH2Ax), bem como relacionados a dinâmica mitocondrial (Drp-1 eMfn-1) foram obtidos a partir de técnicas de imuno-histoquímica e citometria de fluxo. Foram realizados ensaios de detecção do MeHg através do espectrofotômetro de absorção atômica e análises por microscopia eletrônica de transmissão, para investigar alterações ultraestruturais. Os resultados mostraram deposição de Hg no mesencéfalo e cerebelo. A exposição ao MeHg provocou aumento da expressão da p21 no mesencéfalo e p53 no cerebelo, e significativa redução da expressão da ciclina E e de fosfohistona H3, sugerindo parada no ciclo celular e da proliferação celular. No grupo tratado foram observadas alterações ultraestruturais, sendo as mais evidentes nas mitocôndrias das células do mesencéfalo e principalmente do cerebelo. Os insultos ao ciclo e a estrutura celular alteraram os processos de diferenciação, provocando uma diminuição no número das células das linhagens neural e glial. Este estudo revelou que os insultos do MeHg no cerebelo foram mais severos do que no mesencéfalo, demonstrando que a toxicodinâmica do MeHg é dependente do período do desenvolvimento, podendo comprometer funções importantes para a sobrevivência do embrião.<br> / Abstract : Neurotoxicity of Methylmercury (MeHg) has been studied for years, but their relationship with the development of two CNS structures (midbrain and cerebellum) is highly unusual. This study aimed to analyze the effects of MeHg in the structural and ultrastructural organization and its influence on the processes of proliferation, differentiation and cell cycle of the midbrain and cerebellum of Gallus domesticus embryos. The embryos were treated in ovo on the 3rd day of incubation (E3) with a single dose of methylmercury (MeHg) (0.1ug in 50 uL of diluted saline) and analyzed on the 10th day of incubation (E10). The control group embryos were treated only with 50 uL saline solution. The morphological and morphometric analysis of the midbrain and cerebellum sections were taken from histological sections stained with hematoxylin-eosin. The quantification and immunolocalization of proteins related to cell cycle (Cyclin E, p21and p53), cell differentiation (NeuN; tubulin III and GFAP), cell proliferation (phosphohistone H3) and DNA damage (yPH2Ax) and related mitochondrial dynamics (Drp-1 and Mfn-1) were obtained from immunohistochemistry and flow cytometry techniques. Metal detection assays were conducted using the atomic absorption spectrophotometer and analysis by transmission electron microscopy to investigate ultrastructural changes. The results showed deposition of Hg in the midbrain and cerebellum. Exposure to MeHg caused increased p21 expression in the midbrain and p53 in the cerebellum, and significant reduction in the expression of cyclin E and phosphohistone H3, suggesting cell cycle arrest and cell proliferation. Ultrastructural changes were observed, being more evident in the mitochondria of cells of the midbrain and especially of cerebellum. The insults to the cycle and the cellular structure altered the processes of differentiation, causing a decrease in the number of cells of neural and glial lineages. This study revealed that MeHg insults in the cerebellum were more severe than in the midbrain, demonstrating that the toxicology of MeHg is dependent on the developmental period and could compromise functions important for the survival of the embryo.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-07-17T13:05:37Z No. of bitstreams: 1 YwllianeDaSilvaRodriguesMeurer_TESE.pdf: 1373910 bytes, checksum: d491e059868659b49cc5906532ddf376 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-07-18T14:01:06Z (GMT) No. of bitstreams: 1 YwllianeDaSilvaRodriguesMeurer_TESE.pdf: 1373910 bytes, checksum: d491e059868659b49cc5906532ddf376 (MD5) / Made available in DSpace on 2017-07-18T14:01:06Z (GMT). No. of bitstreams: 1 YwllianeDaSilvaRodriguesMeurer_TESE.pdf: 1373910 bytes, checksum: d491e059868659b49cc5906532ddf376 (MD5) Previous issue date: 2017-03-13 / Inibidores seletivos de recapta??o de serotonina (ISRS) s?o amplamente utilizados no tratamento de depress?o e ansiedade em v?rios est?gios da vida do indiv?duo, inclusive durante a gravidez ou lacta??o. Nessa circunst?ncia, com o feto in utero ou em amamenta??o, o mesmo ser? exposto a influ?ncia da hiperestimula??o seroton?rgica capaz de desorganizar o desenvolvimento morfofuncional do SNC. A modula??o do circuito seroton?rgico no c?rebro em desenvolvimento pode alterar a organiza??o e forma??o de diferentes redes neurais espec?ficas e repercutir na express?o comportamental do indiv?duo. Neste sentido, utilizamos o ISRS ? fluoxetina (dose: 10mg/kg)? no per?odo de desenvolvimento PND7PND21, para investigar poss?veis altera??es persistentes na neuroqu?mica e no comportamento de ratos machos e f?meas durante as idades p?snascimento de 45 (PND45) e 90 dias (PND90). Outros tr?s grupos experimentais foram utilizados como controle da administra??o farmacol?gica, s?o eles: Naive (animais n?omanipulados), Sham (animais residentes nas gaiolas de tratamento sem manipula??o) e Veh (animais residentes nas gaiolas de tratamento que recebiam inje??o de ?gua destilada). Nosso trabalho re?ne um conjunto de dados comportamentais complementares e tamb?m in?ditos relacionados a avalia??o do comportamento mnem?nico, da ansiedade e tipodepressivo nos animais nessas diferentes idades e em ambos os sexos. Aqui observamos aumento na ansiedade (avaliada no teste de campo aberto), altera??es de mem?ria de curto prazo (atrav?s dos paradigmas de reconhecimento de objetos e alterna??o espont?nea), bem como manifesta??o do comportamento tipodepressivo (usando os testes de prefer?ncia de sacarose e nata??o for?ada) na prole em idade de 45 dias. Enquanto que na idade adulta (PND90) foi observada (atrav?s dos paradigmas comportamentais usados na avalia??o da prole juvenil) redu??o nos n?veis de ansiedade, manuten??o das altera??es de mem?ria de curto prazo, bem como atenua??o do perfil tipodepressivo. Em ambas idades foram observadas redu??o no n?mero de neur?nios parvalbuminapositivos, contudo somente animais em idade PND45 tratados com fluoxetina apresentaram redu??o significativa e ainda quando comparados com os grupos Naive e Sham, mas n?o ao grupo Veh. Por isso, mais estudos s?o necess?rios para investigar os efeitos persistentes da serotonina sobre a matura??o, prolifera??o e migra??o de interneur?nios para as regi?es corticais. Os animais expostos ao ISRS apresentam maior tra?o de altera??es nos comportamentos de ansiedade e tipodepressivo. Nosso trabalho exp?e altera??es mnem?nicas decorrentes da exposi??o previa a fluoxetina, sugerindo altera??es persistentes no circuito corticol?mbico. Estes achados sugerem que a modula??o do circuito seroton?rgico durante per?odos cr?ticos do desenvolvimento do sistema nervoso central pode alterar a organiza??o dos diferentes circuitos neuroqu?micos e induzir altera??es comportamentais, as quais podem repercutir para o surgimento de dist?rbios neuropsiqui?tricos. / Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed to treat depression, anxiety and other disorders. The developmental exposure to selective serotonin reuptake inhibitors (SSRIs) results in persistent behavioural impairment into adulthood. In this way, serotonergic overexpression in early life may lead to structural and functional changes in brain circuits that control cognitive and emotional behaviour. Here, we addressed the question of how postnatal (PND7PND21) exposure to fluoxetine affects memory, anxietyand depressionlike behaviours, as well as neurochemical markers of interneurons and serotonergic cells in brain areas related to these behaviours in juvenile (PND45) and adult (PND90) female and male rats. In a first stage, we analysed both female and male rat?s performances in several behavioural tasks and investigated the expression of serotonin (5HT) in the dorsal and median nucleus of raphe, and parvalbumin (PV) in PFCm and hippocampus at PND45. We found that earlylife exposure to fluoxetine increased anxietyand depressionlike behaviours (more in female compared to male animals), and induced a working memory impairment only in the juvenile male. Afterwards, we performed behavioural and neurochemical analysis of male and female adult rats (PND90), where we found that fluoxetine affects only anxietyrelated male behaviour. Also, the memory impairment (more in male than female) and depressivelike profile (both sexes) remained in adult age. Moreover, the exposure to fluoxetine affect PV immunoreactivity in the hippocampus in any sex at PND45 and PND90; however, adult animals appear to recover neurochemical deficits observed at the juvenile age. The results revealed developmental fluoxetine effects on juvenile behaviour that can have implications for affective disorders and mnemonic processes. These results revealed persistent changes a sex and agemanner related to developmental exposure to fluoxetine, where serotonergic modulation induce differential profile of anxietyand depressionlike behaviour and mnemonic impairment on female and male rats at juvenile and adult age. Also, suggest a sexdependent compensatory mechanism, which it is possibly related to serotonergic sinalisation. Circuits may involve subcortical and cortical information processing, including subcortical limbic and possible (pre)frontal areas. Thus, our findings suggest that serotonergic modulation during critical periods of SNC development may alter the organisation of excitatoryinhibitory circuit and induce behavioural changes, which may have repercussions for the onset of neuropsychiatric disorders.

Os receptores para mineralocorticoides (MR) e glicocorticoides (GR) representam importantes sítios de ligação para os glicocorticoides. Enquanto a ativação crônica e excessiva de GR leva à atrofia do hipocampo, a ativação do MR é neuroprotetora. Considerando as alterações no giro denteado (GD), decorrentes do envelhecimento, e a possível participação do MR e GR neste processo, o objetivo deste trabalho foi correlacionar o desbalanço de tais receptores com a degeneração neuronal nesta região do hipocampo de cães idosos. Para isso, utilizaram-se cortes histológicos do hipocampo de 6 cadáveres caninos jovens/adultos (até 8 anos de idade) e 12 idosos (idade igual ou superior a 10 anos), de ambos os sexos e de qualquer raça, os quais foram submetidos à: coloração de Nissl, para a identificação de células nervosas; imuno-histoquímica, para o estudo da expressão do MR e GR; e marcação fluorescente (fluoro-jade B), para detecção de neurônios em degeneração. A camada polimórfica do GD de cães idosos apresentou redução (em 16%) na contagem de neurônios e maior número de neurônios em degeneração. Em conjunto com estas alterações celulares, a menor marcação/expressão do MR e a menor relação MR:GR foram correlacionadas com a degeneração neuronal na camada polimórfica do GD de cães idosos. Além disso, cadelas idosas apresentaram menor densidade celular na camada granular, quando comparadas aos machos idosos, sugerindo uma ação diferencial dos esteroides sexuais nas alterações do GD. Os resultados indicam que o desbalanço na relação MR:GR pode interferir na sobrevivência neuronal no GD de cães idosos. / The mineralocorticoid (MR) and glucocorticoid (GR) receptors bind the glucocorticoid hormones. The chronic and excessive GR activation leads to hippocampus atrophy. By contrast, MR activation is neuroprotective. Considering the aging changes in the dentate gyrus (DG) and the possible participation of MR and GR in this process, the objective of this study was to correlate the unbalance of these receptors with the neuronal degeneration in this hippocampal region of aged dogs. For that purpose, cadaveric histologic sections of hippocampus of 6 young/adult dogs (until 8 years old) and 12 aged dogs (more than 10 years old), of both sex and any breed, were included. The Nissl staining and imunochemistry were performed to identify nerve cells and to study the MR and GR expression, respectively. Moreover, fluorescent labeling (fluoro- Jade B) was used to detect degenerating neurons. The polimorfic layer of the DG of aged dogs showed reduction (of 16%) on the neurons counting and more degenerating neurons. Together with this cells changes, the less MR expression and MR:GR relation were correlated with the neuronal degeneration in the polimorfic layer of DG of aged dogs. Besides that, aged females presented lower cell density in the granular layer, when compared to aged males, suggesting a differential sex steroid action on changes in the GD. Our results indicate that unbalance on the MR:GR relation may interfere with neuronal survival in the DG of aged dogs.

Une étape critique au cours du développement de médicaments est la mesure ou la prédiction des concentrations du médicament dans un tissu cible, qui peuvent ensuite être liées à des mesures de leur efficacité ou leur toxicité. Les concentrations de médicaments ne pouvant être mesurées dans le cerveau humain, ils doivent être simulés ou prédits en utilisant des approches alternatives de modélisation. L'objectif de cette thèse est de développer in silico des approches de prédiction combinant à la fois des données précliniques in vitro et in vivo dans un modèle physiologique structuré, avec une stratégie translationnelle afin de permettre la simulation de concentrations totales et libres des médicaments dans le cerveau humain. Des modèles pharmacocinétiques physiologiques (PBPK) ont été développés dans cette thèse et évalués pour des médicaments de référence déjà sur le marché, et pour un médicament en cours de développement clinique dans l'industrie pharmaceutique. Ces modèles ont été développés à partir de stratégies de type « Bottom-up » sur la base de données in vitro pour la prédiction de la distribution des médicaments dans le cerveau et comparées à des méthodes de type «top-down » en utilisant des données in vivo. Cette thèse est une thèse sur article construite à partir de 5 articles scientifiques qui sont soit publiés soit en cours de soumission. Le premier article est une revue de la littérature publiée dans le Journal de l'AAPS. Cette revue discute les modèles PBPK disponibles actuellement et a permis d’élaborer des hypothèses de travail dans cette thèse afin de proposer des améliorations de ces modèles. Le deuxième article un article de recherche original publié dans Molecular Pharmaceutics. Cet article vise à démontrer l'élaboration d'une approche cohérente de modélisation pharmacocinétique chez le rat qui peut s’adapter en fonction de la quantité et de la qualité des données obtenues in vivo au cours du développement des médicaments. Un arbre de décision a été construit pour faciliter le paramétrage et la structure appropriée du modèle en fonction des données disponibles. Le troisième article est un article de recherche original publié dans Journal of Pharmaceutical Sciences. Cette étude porte sur le développement d'un modèle PBPK pour la prédiction de la pénétration cérébrale des médicaments, dans lequel son transfert à travers la BHE a été traité de façon mécanistique en séparant les paramètres régissant la quantité (perméabilité) à travers la BHE de sa liaison dans le tissu cérébral. Une stratégie de type vitro - vivo en fonction de la perméabilité des médicaments à travers les monocouches cellulaires in vitro a été proposé afin d'extrapoler la composante de transport actif du composé à l’aide de facteurs d’extrapolation (RAF). Deux autres articles sont en cours d’écriture ou soumis. Ces articles viennent compléter les approches de PBPK pour les médicaments du SNC décrites dans les deux autres articles originaux. Une dernière partie de la thèse constitue la discussion qui met très clairement en évidence l'importance du choix d’une approche de modélisation appropriée ou mieux encore la combinaison des approches fondées sur les connaissances physiologiques, les données expérimentales et les applications prévues dans le développement du médicament. L'avantage du paramétrage mécanistique dans ces modèles PBPK est qu’il améliore leur prédictivité et la simulation de différences inter-espèces. Cette thèse a considérablement contribué à démontrer la nécessité d’associer des données in vitro à des données in vivo dans la structuration des modèles PBPK qui se révèlent alors comme des outils précieux pour la prédiction de la pharmacocinétique cérébrale chez l'homme. / A critical step during drug development is the measurement or the prediction of drug concentrations in the target tissue, which can then be linked to measures of drug efficacy or toxicity. Drug concentrations cannot be directly measured in the human brain, and must be simulated or predicted using alternative modeling approaches. The objective of this thesis is to develop in silico approaches to predict BBB penetration of drugs, combining in vitro and in vivo preclinical data in a physiologically structured model, with a translational strategy to allow the simulation of total and free drug concentrations in the human brain. Physiologically based pharmacokinetic (PBPK) models were developed and evaluated for reference molecules already on the market, as well as for a drug currently under clinical development within the pharmaceutical industry. These models were developed based on both ‘bottom-up’ (model parameter values predicted from in vitro data) and ‘top-down’ (model parameters estimated from in vivo data) strategies. This thesis is comprised of 5 scientific papers which are either published or submitted to peer-reviewed journals. The first article is a review of the literature, published in the AAPS journal. This review discusses the currently published PBPK models available for the mechanistic prediction of BBB penetration of drugs, and proposes a strategy for in vitro-in vivo (IVIVE) extrapolation. The second article is an original research article published in Molecular Pharmaceutics. This article aims to show the development of a coherent pharmacokinetic modeling approach in the rat which can be adapted based on the quantity and quality of data obtained in vivo during the development of new drugs. A decision tree was constructed to enable the appropriate parameterization and model structure based on the available data. The third article is an original research article published in Journal of Pharmaceutical Sciences. This article was based on the development of a PBPK model for the mechanistic prediction of BBB penetration of drugs, in which the active and passive components of permeability were considered separately, as well as the intra-brain tissue binding parameters. An in vitro-in vivo strategy was proposed to extrapolate the active transport component using a relative activity factor (RAF) to account for in vitro-in vivo differences in transporter activity and/or abundance. Two additional articles are either submitted or under preparation. These articles extend the PBPK approaches described in the previous two published original research articles. The final part of this thesis consists of a discussion which emphasizes clearly the importance of the appropriate choice of modeling approach, or even better, a combination of approaches based on physiological knowledge, experimental data and knowledge gathered during the course of drug development. The advantage of mechanistic parameterization of PBPK models is the improved ability for inter-species extrapolation for the subsequent simulation of free or total drug concentrations within the human brain. This thesis has considerably contributed to this rapidly evolving field of CNS drug research and development, showing the importance of combining in vitro and in vivo data within a physiologically based model structure, thus providing a valuable tool for the quantitative prediction of the penetration of drugs in the human brain.

Desenvolveram-se métodos bioanalíticos para determinação de anfotericina B e fluconazol em apenas 200 L de plasma e líquor (LCR) através da cromatografia líquida de alta eficiência (CLAE UV-VIS). A anfotericina B foi determinada através de CLAE-VIS utilizando p-nitrofenol como padrão interno, após purificação das matrizes biológicas com acetonitrila, seguida da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por tampão acetato 0,1M pH 5,0 e acetonitrila (50:50,v/v) 0,5mL/min em 385nm; o tempo de corrida foi 15 min. Através da validação o método mostrou-se robusto com 0,2-25,0 &#181;g/mL(linearidade, r2 0,9999), LD 0,1 &#181;g/mL, precisão (5,4% e 6,9%), exatidão expressa através do erro sistemático (3,3% e 2,2%): intra e interdias). Os estudos de estabilidade evidenciaram 1,0% para o erro sistemático e 3% de precisão na bandeja (tempo e condição de análise por 24 h), e os ciclos de congelamento evidenciaram boa estabilidade uma vez que todos os ensaios foram realizados em Laboratório de luz amarela. O fluconazol foi determinado através de CLAE-UV utilizando carbamazepina como padrão interno, após purificação das matrizes biológicas pela extração líquido-líquido com diclorometano em meio alcalino, seguido da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por água UP e acetonitrila (70:30,v/v) 0,5mL/min em 210nm; o tempo de corrida foi 15 min. O método mostrou-se robusto com 0,2-250 &#181;g/mL(linearidade, r2 0,9998), LD 0,1&#181;g/mL, com boa recuperação absoluta (98%) e relativa (100%), precisão 0,5%/1,3%, exatidão expressa através do erro sistemático (1,2%). Evidenciou-se ótima estabilidade para os extratos em bandeja (tempo e condição de análise por 24 h), na longa duração (20&#176; C, 9 meses) e através dos ciclos de congelamento. Investigaram-se 21 pacientes adultos de ambos os sexos portadores de meningite criptocócica com AIDS após internação emergencial em terapia de alta dose com anfotericina B (1mg/Kg) e fluonazol (400 mg, 12/12 horas) durante 12 semanas. O monitoramento das concentrações de anfotericina B e fluconazol no plasma e no LCR forneceram as razões que permitiram estimar a penetração dos antifúngicos no SNC. Obtiveram-se concentrações de anfotericina B, médias (IC95%): 2,30 (0,02-5,08) &#181;g/mL no plasma e 0,30 (0,19-0,36) &#181;g/mL no LCR. As concentrações do fluconazol, médias (IC95%) foram: 31,7 (20,1-43,3) &#181;g/mL no plasma e 19,4 (11,1-27,7) &#181;g/mL no LCR. Com base nos resultados obtidos conclui-se que a penetração da anfotericina B foi insuficiente (10-27%), enquanto que a do fluconazol mostrou-se adequada com valores médios (IC95%) de 67 (47-87) %. / Analytical methods were developed to determine amphotericin B and fluconazole in only 200 L of plasma and in cerebrospinal fluid (CSF) by liquid chromatography (HPLC UVVIS). Amphotericin B was determined by HPLC - VIS using p-nitrophenol as internal standard, after the purification of biological matrices using acetonitrile, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of acetate buffer 0.1M pH 5.0 plus acetonitrile (50:50,v/v) 0.5mL/min at 385nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-25,0&#181;g/mL (linearity, r2 0.9999), DL 0.1&#181;g/mL, precision (5.4%/6.0%), accuracy expressed as systematic error (3.3%/2.2%). The stability was investigated, error systematic was 1% for the vials on the rack (time and conditions of drug analysis, 24h). Thawing cycles showed good stability after three freezing-thawing cycles. All procedures were performed under yellow light at room temperature. Fluconazole was determined by HPLC - UV using carbamazepine as internal standard, after the purification of biological matrices using liquid-liquid extraction in alkaline medium, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of purified water plus acetonitrile (70:30,v/v) 0.5mL/min at 210nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-250 &#181;g/mL(linearity, r2 0.9998), DL 0.1&#181;g/mL. Absolute recovery was 98% and relative recovery was 100%, intra/interday precision were 0,5/-1,3%; accuracy expressed as systematic error were 1.2%/1.2%.and relative recovery was 100%. Good stability for the vials on the rack (time and conditions of drug analysis, 24h) and long term stability (at 20o C for 9 months) were demonstrated. Also thawing cycles showed good stability after three freezing-thawing cycles. Twenty one adult patients of both sex were investigated. Inpatients with meningitis by Cryptococcus neoformans with AIDS were under high dose therapy with amphotericin B 1mg/Kg plus fluonazole 400 mg, every 12h during 12 weeks. Therapeutic monitoring of amphotericin B and fluconazole in plasma and in CSF showed ratios that indicate the penetration of antifungal drugs into CNS. Mean (CI95%) data were for amphotericin B 2.30 (0.02-5.08 ) &#181;g/mL in plasma and 0.30 (0.19-0.36) &#181;g/mL in CSF. Fluconazole showed 31.7 (20.1-43.3) &#181;g/mL in plasma and 19.4 (11.1-27.7) &#181;g/mL in CSF. Based on data obtained we conclude that the penetration of amphotericin B was poor (10-27%) while fluconazole was adequate 67% (47-87%), mean (CI95%).

Desenvolveram-se métodos bioanalíticos para determinação de anfotericina B e fluconazol em apenas 200 L de plasma e líquor (LCR) através da cromatografia líquida de alta eficiência (CLAE UV-VIS). A anfotericina B foi determinada através de CLAE-VIS utilizando p-nitrofenol como padrão interno, após purificação das matrizes biológicas com acetonitrila, seguida da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por tampão acetato 0,1M pH 5,0 e acetonitrila (50:50,v/v) 0,5mL/min em 385nm; o tempo de corrida foi 15 min. Através da validação o método mostrou-se robusto com 0,2-25,0 &#181;g/mL(linearidade, r2 0,9999), LD 0,1 &#181;g/mL, precisão (5,4% e 6,9%), exatidão expressa através do erro sistemático (3,3% e 2,2%): intra e interdias). Os estudos de estabilidade evidenciaram 1,0% para o erro sistemático e 3% de precisão na bandeja (tempo e condição de análise por 24 h), e os ciclos de congelamento evidenciaram boa estabilidade uma vez que todos os ensaios foram realizados em Laboratório de luz amarela. O fluconazol foi determinado através de CLAE-UV utilizando carbamazepina como padrão interno, após purificação das matrizes biológicas pela extração líquido-líquido com diclorometano em meio alcalino, seguido da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por água UP e acetonitrila (70:30,v/v) 0,5mL/min em 210nm; o tempo de corrida foi 15 min. O método mostrou-se robusto com 0,2-250 &#181;g/mL(linearidade, r2 0,9998), LD 0,1&#181;g/mL, com boa recuperação absoluta (98%) e relativa (100%), precisão 0,5%/1,3%, exatidão expressa através do erro sistemático (1,2%). Evidenciou-se ótima estabilidade para os extratos em bandeja (tempo e condição de análise por 24 h), na longa duração (20&#176; C, 9 meses) e através dos ciclos de congelamento. Investigaram-se 21 pacientes adultos de ambos os sexos portadores de meningite criptocócica com AIDS após internação emergencial em terapia de alta dose com anfotericina B (1mg/Kg) e fluonazol (400 mg, 12/12 horas) durante 12 semanas. O monitoramento das concentrações de anfotericina B e fluconazol no plasma e no LCR forneceram as razões que permitiram estimar a penetração dos antifúngicos no SNC. Obtiveram-se concentrações de anfotericina B, médias (IC95%): 2,30 (0,02-5,08) &#181;g/mL no plasma e 0,30 (0,19-0,36) &#181;g/mL no LCR. As concentrações do fluconazol, médias (IC95%) foram: 31,7 (20,1-43,3) &#181;g/mL no plasma e 19,4 (11,1-27,7) &#181;g/mL no LCR. Com base nos resultados obtidos conclui-se que a penetração da anfotericina B foi insuficiente (10-27%), enquanto que a do fluconazol mostrou-se adequada com valores médios (IC95%) de 67 (47-87) %. / Analytical methods were developed to determine amphotericin B and fluconazole in only 200 L of plasma and in cerebrospinal fluid (CSF) by liquid chromatography (HPLC UVVIS). Amphotericin B was determined by HPLC - VIS using p-nitrophenol as internal standard, after the purification of biological matrices using acetonitrile, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of acetate buffer 0.1M pH 5.0 plus acetonitrile (50:50,v/v) 0.5mL/min at 385nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-25,0&#181;g/mL (linearity, r2 0.9999), DL 0.1&#181;g/mL, precision (5.4%/6.0%), accuracy expressed as systematic error (3.3%/2.2%). The stability was investigated, error systematic was 1% for the vials on the rack (time and conditions of drug analysis, 24h). Thawing cycles showed good stability after three freezing-thawing cycles. All procedures were performed under yellow light at room temperature. Fluconazole was determined by HPLC - UV using carbamazepine as internal standard, after the purification of biological matrices using liquid-liquid extraction in alkaline medium, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of purified water plus acetonitrile (70:30,v/v) 0.5mL/min at 210nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-250 &#181;g/mL(linearity, r2 0.9998), DL 0.1&#181;g/mL. Absolute recovery was 98% and relative recovery was 100%, intra/interday precision were 0,5/-1,3%; accuracy expressed as systematic error were 1.2%/1.2%.and relative recovery was 100%. Good stability for the vials on the rack (time and conditions of drug analysis, 24h) and long term stability (at 20o C for 9 months) were demonstrated. Also thawing cycles showed good stability after three freezing-thawing cycles. Twenty one adult patients of both sex were investigated. Inpatients with meningitis by Cryptococcus neoformans with AIDS were under high dose therapy with amphotericin B 1mg/Kg plus fluonazole 400 mg, every 12h during 12 weeks. Therapeutic monitoring of amphotericin B and fluconazole in plasma and in CSF showed ratios that indicate the penetration of antifungal drugs into CNS. Mean (CI95%) data were for amphotericin B 2.30 (0.02-5.08 ) &#181;g/mL in plasma and 0.30 (0.19-0.36) &#181;g/mL in CSF. Fluconazole showed 31.7 (20.1-43.3) &#181;g/mL in plasma and 19.4 (11.1-27.7) &#181;g/mL in CSF. Based on data obtained we conclude that the penetration of amphotericin B was poor (10-27%) while fluconazole was adequate 67% (47-87%), mean (CI95%).