L’impact biologique exact des acides gras trans (TFA) sur la santé globale n’est toujours pas clair. Actuellement, il y a deux sources principales de TFA : 1- ceux qui se produisent naturellement dans les produits laitiers et de viande à la suite de la biohydrogénation chez les ruminants (R-TFA) et 2-ceux formés par des procédés industriels (I-TFA). Les I-TFA ont des effets néfastes sur le risque cardiovasculaire (CV), la résistance à l’insuline et le diabète de type 2. Cependant, les résultats actuels suggèrent que les R-TFA peuvent avoir des effets bénéfiques sur les facteurs de risque du diabète. À ce jour, peu d’études animales ont été menées à l’aide de R-TFA. Ces études suggèrent des mécanismes d’action potentiels qui peuvent expliquer les effets de la R-TFA sur le diabète de type 2 : diminution de l’inflammation, modification des niveaux d’expression génique et diminution de la graisse hépatique. L’objectif de cette étude était de développer une formulation qui pourrait encapsuler des R-TFA de manière isolée sans les autres constituants du lait. Pour ce faire, une stratégie de formulation visant à obtenir des solutions de TFA stables, solubles dans l’eau et biodisponibles ont été développées. Dans un premier temps, nous avons développé des formulations encapsulant les TFA dans des nanovésicules de 100 nm en moyenne. Nos résultats démontrent que ces formulations sont stables physiquement et chimiquement pendant une semaine. Dans un deuxième temps, nous avons administré ces formulations contenant les TFA aux animaux, par gavage. Les résultats obtenus démontrent que nos formulations assurent la biodisponibilité des TFA, sans moduler les concentrations plasmatiques d’autres acides gras. Enfin, nous avons démontré la versatilité de cette technique en coencapsulant des TFA avec d’autres molécules hydrophiles et hydrophobes telles que la vitamine D3 et la L-leucine. Ces résultats démontrent l’intérêt de l’utilisation de nanovésicules en sciences des aliments pour l’encapsulation de composés non solubles dans l’eau. In fine, ces formulations représentent une base solide pour mener à bien les études in vivo futures et comprendre les effets réels des R-TFA sur le DT2 et ses facteurs de risques.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2010-2011 / Notre groupe de recherche a récemment démontré que des apports très élevés en acides gras trans naturels (AGTn) et industriels (AGTi) avaient des effets comparables sur les lipides et lipoprotéines plasmatiques. L'impact des différents AGT sur les processus impliqués dans l'homéostasie du cholestérol demeure toutefois inconnu et il nous est apparu important d'investiguer dans cette voie. Ce mémoire présente les résultats d'une étude dont l'objectif était de comparer l'impact des AGTi et des AGTn sur les marqueurs de la synthèse endogène et de l'absorption intestinale du cholestérol. Les résultats démontrent que le niveau d'absorption du cholestérol est modifié de façon plus importante suite à la consommation d'AGTn que d'AGTi, mais cette différence ne se reflète pas dans les concentrations de LDL-C entre les deux sources d'AGT. Il semble également que la consommation d'AGT n'affecte pas la synthèse endogène de cholestérol, peu importe leur origine.

Si la découverte récente de la souche virale JFH1, capable de réaliser un cycle viral complet en culture cellulaire, a permis de mieux caractériser le déroulement de l’infection du virus de l’hépatite C VHC, de nombreux aspects de la biologie du virus demeurent méconnus. Parmi ceux-ci, les mécanismes gouvernant l’encapsidation de l’ARN génomique viral au sein des particules restent à élucider. Cette thèse décrit la mise point d’un système de mobilisation hétérologue permettant l’analyse de l’interaction de la protéine de capside Core avec l’ARN viral. Ce système nous a permis d’identifier les régions de la protéine impliquées dans la liaison au génome viral, tout en autorisant son transfert dans des cellules naïves. Cette approche de mobilisation dépendante de la Core est complétée d’une étude de recrutement hétérologue basée sur la reconnaissance de l’ARN du phage MS2 avec la protéine de manteau Coat. Cette stratégie novatrice permet le recrutement efficace des ARNs marqués tout en autorisant leur localisation cellulaire. / The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. To study packaging in vivo, we developed a novel heterologous system to evaluate the interactions of HCV Core capside with viral RNA. This system allowed us to pinpoint Core domains involved in RNA binding, and package and transfer HCV RNA into a lentiviral vector. Aside from this Core dependent recruitment, we engineered retoviral vectors to trans-package MS2-tagged RNAs using MS2 Coat protein interaction. This system allowed us to efficiently recruit MS2-tagged replicons into naive cells and offers the opportunity to visualize HCV RNAs in Huh7.5 cells.

Le travail de recherche proposé dans cette thèse porte sur la question de la transmission psychique inconsciente, l’objet de recherche s’attachant plus précisément à définir les différentes modalités de transit de la réalité psychique. C’est à partir d’une clinique auprès d’enfants suivis en hôpital de jour ou en centre médico-psychologique que sont interrogées les formes, les voies et les fonctions des transits, des échanges qui s’organisent entre les différentes scènes subjectives, qu’il s’agisse de la scène du rêve, du jeu, du corps, mais encore celle de l’autre de la relation. La scène psychique au regard de sa croissance, de son développement, ne peut se penser sans l’objet, aussi le modèle proposé en appui sur la théorie psychanalytique mais explorant aussi d’autres épistémologies comme la phénoménologie, les neurosciences, l’embryologie, est un modèle intersubjectif. A la suite des auteurs kleiniens mais aussi des travaux des groupalistes, il a pour objet de construire une représentation de l’espace intermédiaire ou espace « entre », situé entre les scènes et à partir duquel s’organisent les transactions et la transmission psychique. Il est possible de caractériser ce lieu de la connexion, d’en proposer une métaphore, une topologie, de montrer qu’il a une concrétude et des processus spécifiques. Penser l’entre-deux et l’alternance entre les scènes subjectives sur lesquelles se montre la réalité psychique amène aussi à interroger les modes de représentation de la conflictualité, à penser en termes de scénalité, d’inter-scénalité, de trans-scénalité, à rechercher également qu’elle est l’instance intrapsychique organisatrice de sa mise en jeu et de sa mise en scène. Parce que le moi est un être frontière et assure une fonction d’articulation, on peut décrire, en usant de la métaphore théâtrale, un moi dramaturge et scénographe et montrer comment les scènes subjectives interconnectées permettent alors de retrouver un lien associatif et une continuité narrative. Lorsque la souffrance psychique est trop grande et ne peut être psychisée, la scène éloignée (par exemple la scène phobique), se révèle être un puissant attracteur de la réalité psychique et donne une forme au conflit. Du fait d’une coïncidence, d’une mêmeté ou autre, elle vient en relais, elle est à penser comme un lieu où se déposent et se déchargent les tensions à l’instar d’une zone de transits ou parfois d’une zone de quarantaine maintenant à distance le surcroit de douleur et évitant une contamination de l’ensemble. Certains mécanismes identificatoires jouent un rôle fondamental dans la transmission psychique, notamment l’identification projective qui permet que se véhicule la souffrance et notamment qu’elle soit communiquée au thérapeute. De fait, la scène thérapeutique parce qu’elle attire à elle des contenus psychiques toxiques, non-élaborés, ceux-ci pouvant être liés à une transmission transgénérationnelle, opère aussi comme une scène à distance où s’organise un partage des expériences affectives et émotionnelles, cela étant à penser dans le cadre d’une relation transféro-contre-transférentielle. La prise en compte des jeux d’alternance impliquant souvent plusieurs scènes interconnectées entre elles comme les scènes thérapeutique, transférentielle mais aussi corporelle, onirique, fantasmatique, qui sont autant de lieux possibles d’expression du conflit psychique, amène en dernier lieu et en guise d’ouverture à un prochain travail de recherche à envisager une autre modalité d’associativité que nous nommons associativité inter-scénale. / The research work proposed within the framework of this thesis focuses upon the unconscious psychic transmission, with a particular emphasis on the various forms of transits of psychological reality. Based on The forms, channels and the roles of the transits, the interaction between the various subjective scenes, be they the dream scene, the game scene, the body scene or the Other in the relationship, have been studied in a child clinic with children in a day hospital or in a medical psychological centre. The psychological scene in view of its growth, its development, is unthinkable without the object, that is why the model proposed on the psychoanalytical theory, but also investigating other epistemologies such as phenomenology, neurosciences, embryology, is an intersubjective model. Following the kleinian authors, but also the groupalists’ work, its purpose is to build a representation of the intermediary space, or space in between, located between the scenes and from which psychological transactions and transmissions organize themselves. It is possible to characterize this place of connection, to propose a metaphor for it, a topology, to show it is real, with specific processes. Considering the between-two and the alternating between the subjective scenes on which the psychological reality shows, also leads us to ask questions about the modes of representation of conflicts, to think in terms of scenality, inter-scenality, trans-scenality, also to investigate the intrapsychic occurrence which articulates its operation and staging. Because the self is a border being and it has a function of articulation, one could evoke, with a theatrical metaphor, a dramatist and scenographer self, and show how the interconnected subjective scenes then allow to get back to an associative link and a narrative continuity.When mental suffering is too acute and it cannot be psychologized, the remote scene (the phobic scene for instance) proves to be a powerful attraction of psychic reality, and it gives a shape to the conflict. Because of a coincidence, a sameness, or other, it is a relay, one should think about it as a place where tensions lay and discharge as in a transit area or sometimes a quarantine zone keeping the excess pain away and avoiding a contamination of the whole.Some identifiable mechanisms play a key role in the psychologicaltransfer, particularlythe projective identification which ensures pain is carried out and, especially, communicated to the therapist. Indeed, because the therapeutic scene attracts undeveloped toxic psychic contents which may be linked to a transgenerational transmission, it also operates like a distant scene in which a sharing of the affective and emotional experiences takes place. The coverage of the alternation games often involving several scenes interconnected together such as the therapeutic scenes, transferential as well as corporeal, oniric, fantasmal, which represent as many potential places of expression of psychological conflict, finally leads us, thus opening up to a future research work, to consider another method of associativity which we name inter-scenal associativity.

A doença de Chagas, também conhecida como tripanossomíase americana, é uma das doenças tropicais mais devastadoras e é causada pelo protozoário Trypanosoma cruzi. O parasita expressa uma enzima de superfície, Trypanosoma cruzi trans-sialidase (TcTS), responsável pela transferência de ácidos siálicos de células do hospedeiro para moléculas de -galactose terminais presentes em glicoproteínas de sua superfície. As moléculas de glicoproteína sialiladas estão envolvidas na adesão e subsequente penetração do parasita em células hospedeiras. Logo, TcTS desempenha papel fundamental no reconhecimento e na invasão de células do hospedeiro. Além disso, a ausência de trans-sialidase em seres humanos faz de TcTS um alvo potencial a ser explorado, no entanto nenhum inibidor desta enzima (em concentração nanomolar) é conhecido até o momento. Considerando a importância da unidade de galactose e da função carboxila do ácido siálico para interações no sítio activo de TcTS, focamos na síntese de derivados de galactose com ácido succínico em diferentes posições do anel de açúcar e sua avaliação biológica em TcTS. Além disso, foi proposta uma busca por novos inibidores de TcTS através de técnicas de modelagem molecular, como triagem virtual baseada no sítio ativo da enzima. -D-galactose e -Dgalactopiranosídeo de metila, disponíveis comercialmente, foram tratados com reagentes adequados para fornecer alguns intermediários com apenas uma hidroxila livre em poucas etapas. O tratamento destes compostos com anidrido succínico em piridina levou à formação dos respectivos derivados com ácido succínico em todas as posições do anel galactosídico. Após etapa de desproteção foi obtido apenas o derivado com ácido succínico na posição 4 (85), que foi testado em TcTS por ensaio fluorimétrico in vitro para avaliação de sua atividade inibitória. Adicionalmente, 85 foi testado em ensaios in vitro para avaliação de sua atividade tripanocida e citotóxica. De acordo com os ensaios biológicos, o composto 85 apresentou atividade inibitória promissora (56%) na concentração de 1,0 mM. Esse resultado preliminar foi importante para mostrar que esse tipo de derivado pode atuar como inibidor de TcTS e para orientar a síntese de novos derivados de galactose. O valor da atividade tripanocida foi inferior a 40% (0,5 mM) e não foi observada citotoxicidade na concentração de 0,5 mM. Os estudos de triagem virtual realizados neste trabalho através de simulações de \"docking\" resultaram na seleção das 50 melhores moléculas, baseada na orientação de maior pontuação, dentre 50.000 encontradas na base de dados diverset. O próximo passo envolve novos estudos para filtrar as moléculas mais promissoras para serem testadas em TcTS. / Chagas\' disease, also known as American trypanosomiasis, is one of the most devastating tropical diseases and it is caused by the protozoan Trypanosoma cruzi. The parasite expresses a cell surface enzyme, Trypanosoma cruzi trans-sialidase (TcTS), responsible for the transference of sialic acids from host cells to terminal -galactose molecules present on its glycoprotein surface. The sialylated glycoprotein molecules are involved in the attachment and subsequent penetration of the parasite into host cells. As a result, TcTS plays a key role in the recognition and invasion of host cells. Moreover, the lack of trans-sialidase in humans makes TcTS a potential drug target to be explored, however no strong inhibitors (at nanomolar range) of this enzyme are known to date. Considering the importance of galactose unit and the carboxyl function in sialic acid for interactions in the active site of TcTS, we have focused on the synthesis of galactose derivatives containing succinic acid in different positions of the sugar ring and on their biological evaluation against TcTS. In addition, we have proposed the search for new TcTS inhibitors applying molecular modeling techniques, like virtual screening based on the enzyme´s active site. Commercially available ,-D-galactose and methyl--D-galactopyranoside were treated with suitable reagents to afford some intermediates with just one free hydroxyl group in few steps. Treatment of these compounds with succinic anhydride in pyridine afforded derivatives thereof with succinic acid in all galactosidic ring positions. After deprotection step it was only obtained the derivative containing succinic acid at position 4 (85), which was tested in in vitro TcTS fluorimetric assay for evaluation of its inhibitory activity. In Addition, 85 was tested in in vitro assays for assessment of their trypanocidal activity and cytotoxic. According to the biological assays, compound 85 showed promising inhibitory activity (56%) at 1.0 mM concentration. This preliminary result was important both to show that this type of derivative can act as an inhibitor of TcTS and to guide the synthesis of new derivatives of galactose. The trypanocidal activity value was lower than 40% (0.5 mM) and there was no cytotoxicity at 0.5 mM concentration. The virtual screening studies performed in this work through docking simulations resulted in the selection of the 50 top-ranked molecules, based in the highest score orientation, among 50.000 found at diverset data base. The next step involves new studies to filter the most promising molecules to be tested against TcTS.

As mulheres trans são consideradas e tratadas como figuras abjetas, por não se enquadrarem no sistema de normas de gênero e sexualidade vigentes na sociedade. Com isso, há os processos de estigmatização e discriminação à sua identidade de gênero, produzindo a negação de seus direitos enquanto cidadãs, inclusive do direito à saúde integral. Por outro lado, recentemente foram produzidas políticas públicas voltadas a essa população, com o intuito de garantir os seus direitos humanos. Diante desse cenário, buscou-se descrever e compreender como se dá o acesso das mulheres trans aos serviços de saúde e à saúde integral no município de Curitiba, à luz das práticas sociais, individuais e programáticas envolvidas em tal processo. Para tanto, foram realizadas entrevistas semiestruturadas, com a utilização do recurso das cenas, com sete mulheres trans residentes em Curitiba. Para a análise do material obtido, foram eleitas as cenas que mais expressavam o seu (não)acesso à saúde integral, que compuseram, posteriormente, uma narrativa sobre o percurso e vivência delas na área da saúde, tendo como pano de fundo o quadro dos direitos humanos e da vulnerabilidade, e as normas regulatórias de gênero. O exame desse material possibilitou observar que as mulheres trans têm uma trajetória na área da saúde marcada pela violação de seus direitos humanos e à saúde, demonstrando que, por mais que existam documentos que garantam esses direitos, a efetividade destes na vida cotidiana das mulheres trans esbarra em barreiras individuais, sociais e programáticas (que são permeadas e produzidas primordialmente pelas normas regulatórias de gênero), que aumentam a sua vulnerabilidade ao adoecimento. À luz das cenas e narrativas trazidas pelas mulheres trans entrevistadas e considerações apontadas, ao longo deste texto, com base no quadro teórico adotado são oferecidas na conclusão algumas recomendações para o aumento da qualidade das políticas públicas e legislação vigentes, para que assim haja um aumento na efetividade das ações pautadas nesses documentos / The trans women are considered and treated as abject beings, because they dont frame in the system of gender and sexuality norms effective in the society. With that, there are the stigmatization and discrimination processes to their gender identity, producing the denial of their rights while citizens, including the right to the integral health. On the other hand, recently, public politics turned to that population were produced, with the intention of guaranteeing their human rights. Before that, it was looked for to describe and to understand how the trans womens access to the services of health and the integral health in the municipal district of Curitiba happens, to the light of the social, individual and programmatic practices involved in this process. For so much, semi-structured interviews were accomplished, with the use of the scenes resource, with seven trans women residents in Curitiba. For the analysis of the obtained material, they were chosen the scenes that expressed more their (not) access to the integral health, which composed, later, a narrative on the course and existence of them in the health field, having as backdrop the picture of the human rights and of the vulnerability, and the regulatory norms of gender. The exam of that material made possible to observe that the trans women have a path in the health field marked by the violation of their human rights and their right to the health, demonstrating that, no matter how much documents that guarantee those rights exist, their effectiveness in the trans women\'s daily life bumps into individual, social and programmatic barriers (that are permeated and produced firstly by the regulatory norms of gender), that increase their vulnerability to the illness. To the light of the scenes and narratives brought by the trans women interviewees and of the considerations pointed along this text, with base in the adopted theoretical picture, they are offered in the conclusion some recommendations for the increase of the quality of the current public politics and legislation, so that there is an increase in the effectiveness of the actions ruled in these documents

O SL trans-splicing (do inglês, spliced leader trans-splicing) catalisado pelo spliceossomo em Trypanosoma brucei é responsável pelo processamento dos pré-mRNAs policistrônicos em mRNAs maduros. Esta maquinaria é associada a partir de pequenas partículas ribonucleoproteicas nucleares (snRNPs) U1, U2, U4/U6 e U5 constituídas de pequenos RNAs nucleares (snRNAs), um complexo canônico de sete proteínas Sm (SmB, SmD3, SmD1, SmD2, SmE, SmF e SmG) e fatores proteicos específicos. O núcleo de proteínas Sm de T. brucei apresenta variações com funções desconhecidas, como a substituição do heterodímero SmD3/SmB por Sm16,5K/Sm15K na snRNP U2, e de SmD3 por SSm4 na snRNP U4. Na primeira parte deste trabalho, investigou-se a interação destes diferentes complexos Sm recombinantes com os snRNAs U2, U4 e U5 obtidos por transcrição in vitro. Todos os complexos apresentaram alta afinidade pelo snRNA cognato. Observou-se, ainda, que apenas o núcleo Sm que contém Sm16,5K/Sm15K associado ao snRNA U2 interage com alta afinidade com U2A/U2B. Adicionalmente, foi obtida a estrutura cristalográfica de U2A/U2B de T. brucei, que revela uma organização similar àquela já descrita para ortólogas de Homo sapiens. Entretanto, há um desvio de pelo menos 6 Å no ponto médio da alça carregada positivamente no domínio RRM de U2B para a acomodação do snRNA U2. Além disso, observou-se uma longa hélice-α adicional na extremidade C-terminal de U2A. A análise dos três núcleos Sm de T. brucei a partir da combinação de modelagem molecular e espalhamento de raios-X a baixo ângulo revela estruturas de barril-β altamente torcido com interior carregado positivamente para interação com snRNAs. A principal diferença entre as estruturas encontra-se nas extremidades C- e N-terminal dos variantes de proteínas Sm, possivelmente para interação com U2A no braço 1 do spliceossomo, no caso de Sm15K/Sm16,K, e com U5-220K e U5-200K no corpo do spliceossomo, no caso de SSm4. Na segunda parte deste trabalho, a expressão homóloga da proteína U5-200K de T. brucei completa e do produto truncado no seu cassete helicase/ATPase/Sec63 N-terminal levou à copurificação de um subcomplexo de snRNP U5 composto por U5-220K, U5-116K, U5-40K e U5-Cwc21, sendo que a proteína recombinante completa ainda copurificou as proteínas Sm. Experimentos de imunolocalização mostraram que a proteína U5-200K truncada não é direcionada ao núcleo, como é o caso da proteína completa. As células que expressam a proteína truncada apresentaram um defeito de crescimento significativo, e os processamentos de pré-mRNA por cis- e SL trans-splicing foram ligeiramente afetados, já que a proteína truncada não entra no núcleo, onde deveria exercer sua atividade. Os resultados apresentados indicam a formação de um subcomplexo de snRNP U5 ainda no citoplasma, sendo que as proteínas Sm devem ser um sinal para o seu transporte nuclear mediado por importina-β. Em leveduras, a proteína Aar2 substitui U5-200K no citoplasma, regulando assim a biogênese de snRNP U5, porém esta proteína não foi identificada em T. brucei. Os resultados apresentados neste trabalho contribuem como o primeiro estudo estrutural de proteínas spliceossomais de um parasita do homem e também com novas informações sobre a biogênese das partículas ribonucleoproteicas U2 e U5 de T. brucei. / The spliced-leader (SL) trans-splicing catalyzed by the spliceosome in Trypanosoma brucei is responsible for processing polycistronic pre-mRNAs into mature mRNAs. The spliceosome machinery is assembled by small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 that are composed by small nuclear RNAs (snRNAs), a canonical complex of Sm proteins (SmB, SmD3, SmD1, SmD2, SmE, SmF, SmG) and specific factors. The Sm core peculiarly varies in T. brucei, where SmD3/SmB are replaced by Sm16.5K/Sm15K in U2 snRNP and SmD3 is substituted by SSm4 in U4 snRNP. In the first part of this thesis, we investigated the interaction of the different recombinant Sm cores with U2, U4 and U5 snRNAs obtained by in vitro transcription. All the protein complexes bind the cognate snRNA with high affinity. Only the Sm core that contains Sm16.5K/Sm15K associated with U2 snRNA interacts with the recombinant U2A/U2B subcomplex. Additionally, the crystallographic structure of T. brucei U2A/U2B was obtained, showing an overall organization similar to the one observed in the human counterpart. However, we observed a 6 Å deviation in the medium point of a positively charged turn in the RRM motif of U2B to accommodate U2 snRNA. Besides, a long α -helix was observed in the C-terminal region of U2A. Structural analysis of Sm core variations in T. brucei was proceeded using molecular modelling techniques associated with small angle X-ray scattering. The quaternary structure models show seven Sm proteins as β-barrels with positively charged interior for cognate snRNA interaction. The main difference among these Sm core structures resides in the C- and N-terminal regions of the variant proteins, probably enabling the interaction of Sm15K/Sm16,5K with U2A in the spliceosomes arm 1, and the association of SSm4 with U5-220K and U5-200K in the spliceosomes body. In the second part of this thesis, homologous expression of full-length and N-terminally truncated U5-200K from T. brucei led to the copurification of a U5 snRNP subcomplex containing U5-220K, U5-116K, U5-40K and U5-Cwc21. The full-length U5-200K construct also copurified Sm proteins. Immunolocalization experiments showed that the truncated U5-200K protein is not directed to the nucleus as is the case for the full-length protein. Cells that expressed the truncated protein showed a significant growth defect and the pre-mRNA processing by cis- and SL trans-splicing was negatively affected since the truncated protein did not enter the nucleus where it should be active. The results suggest that a subcomplex of U5 snRNP begins to be assembled in the cytoplasm and the Sm proteins may be the signal for the nuclear transport mediated by β-importin. In yeast, Aar2 replaces U5-200K in the cytoplasm in another regulation step. However, Aar2 has not been identified in T. brucei. The results presented here contribute with the first structural study of spliceosomal proteins of a human parasite and give new insights into the biogenesis of U2 and U5 snRNPs in T. brucei.

Em virtude da necessidade de proteger a pele contra as espécies reativas de oxigênio, geradas excessivamente após exposição ao raios UV, substâncias antioxidantes têm sido adicionadas aos fotoprotetores. Entretanto associações fotoinstáveis podem levar à formação de intermediários reativos, que são prejudiciais ao organismo, e à redução da atividade fotoprotetorados filtros solares e antioxidantes. Além disso, as características de penetração dos filtros solares e antioxidantes influenciam diretamente a segurança e a eficácia dos fotoprotetores. Dessa forma, o objetivo deste trabalho foi desenvolver e avaliar a fotoestabilidade e a penetração cutânea de formulações fotoprotetoras contendo diferentes associações de filtros solares acrescidas ou não de trans-resveratrol e beta-caroteno, por meio do uso de CLAE e espectrofotometria e de células de Franz, respectivamente. Para tal, foram desenvolvidas formulações contendo diferentes associações de filtros solares (associações 1, 2 e 3), que apresentavam em comum avobenzona, metoxicinamato de etil-hexila e octocrileno, acrescidas de trans-resveratrol e beta-caroteno, isoladamente ou em combinação. Para avaliar a fotoestabilidade, amostras das formulações foram aplicadas em lâminas de vidro e expostas à radiação UVA e, a seguir, submetidas a análises por CLAE, para determinar o teor dos filtros solares e antioxidantes em estudo, e por espectrofotometria, para determinação da razão UVA/UVB. Para avaliar a penetração cutânea, as formulações contendo a associação de filtros solares mais fotoestável foram aplicadas sobre a pele de orelha de porco, e foi realizada a quantificação das substâncias em estudo, presentes no estrato córneo (EC), na epiderme/derme (E+D) e na solução receptora, por CLAE. O estudo de fotoestabilidade por CLAE dos filtros solares e antioxidantes presentes nas formulações avaliadas mostrou que, apesar de terem apresentado boa fotoestabilidade, todas as substâncias foram fotoinstáveis, com exceção do bemotrizinol, octiltriazona e octocrileno. Além disso, foi observado que o uso dos antioxidantes em associação foi melhor do que utilizá-los separadamente em fotoprotetores. As análises espectrofotométricas mostraram que, após irradiação, houve redução significativa da razão UVA/UVB de todas as formulações e que as formulações contendo bemotrizinol (associação 2) e octiltriazona (associação 3) apresentaram, respectivamente, aumento e redução significativos da razão UVA/UVB, com relação às formulações que não apresentavam esses filtros. No estudo de penetração cutânea, foi observado que, após aplicação das formulações contendo bemotrizinol (associação 2), os filtros solares e antioxidantes avaliados penetraram a pele, mas não permearam até a solução receptora, ou seja, ficaram retidos no EC, majoritariamente, e na E+D. Além disso, foi observada redução significativa da retenção no EC dos filtros solares e antioxidantes avaliados na presença do beta-caroteno. Os resultados desse estudo contribuíram para mostrar os benefícios da utilização de uma combinação de antioxidantes em fotoprotetores, uma vez que a combinação do trans-resveratrol e do beta-caroteno, resultou em melhor fotoestabilidade das formulações e também apresentou vantagens no estudo de penetração cutânea, pois reduziu a penetração dos filtros solares. / Due to the need to protect the skin against reactive oxygen species, which are excessively generated after exposure to UV rays, antioxidants compounds have been added to sunscreens. However, photounstable combinations can lead to generation of reactive intermediates, that are harmful to the body, and decrease the photoprotective activity of UV-filters and antioxidants. Furthermore, the penetration characteristics of UV-filters and antioxidants influence directly sunscreens safety and efficacy. Thus, the aim of this study was to develop and to evaluate the photostability and cutaneous penetration of sunscreen formulations containing different combinations of UV-filters supplemented or not with trans-resveratrol and beta-carotene, by using HPLC and spectrophotometry and Franz cells, respectively. For this, formulations containing different UV-filters combinations (combinations 1, 2 and 3), which had in common avobenzone, ethyl-hexyl methoxycinnamate and octocrylene, supplemented with trans-resveratrol and beta-carotene, alone or in combination, were prepared. For photostability evaluation, samples of the formulations were spread onto glass plates, exposed to UVA radiation and then analyzed by HPLC to determine the concentration of UV-filters and antioxidants under study, and by spectrophotometry to determine the UVA/UVB ratio. To assess cutaneous penetration, formulations containing the most photostable combination of UV-filters were applied on pig ear skin, and the quantification of substances under study present into stratum corneum (SC), viable epidermis plus dermis (E+D) and receptor fluid was performed by HPLC. HPLC photostability study, which analyzed the UV-filters and antioxidants present in formulations, showed that all substances, despite having good photostability, were photounstable, except bemotrizinole, octyltriazone and octocrylene. Furthermore, it was observed that the use of antioxidants in combination was better than using them separately in sunscreens. The spectrophotometric analysis showed that after irradiation, all formulations had a significant decrease of UVA/UVB ratio, and that formulations containing bemotrizinole (combination 2) and octyltriazone (combination 3) presented, respectively, significant increase and decrease of the UVA/UVB ratio, in comparison with formulations that did not contained these UV-filters. In the cutaneous penetration study it was observed that after application of formulations containing bemotrizinole (combination 2), the UV-filters and antioxidants under study penetrated the skin, but did not reach the receptor fluid, which means that they were retained in SC, predominantly, and in E+D. Furthermore, a significant decrease on SC retention of UV-filters and antioxidants under study in the presence of beta-carotene was observed. The results of this study contributed to demonstrate the benefits of using a combination of antioxidants in sunscreens, since the combination of trans-resveratrol with beta-carotene, resulted in improved photostability of the formulations and also showed advantages in the skin penetration study, because it reduced UV-filters penetration.

Diese Arbeit beschreibt die Realisierung mehrerer theoretischer Ansätze zur Synthese von Oligonucleotiden, die an gezielter Position mit der trans-{PtII(NH3)2}2+ - Spezies modifiziert sind. Es wurde ein Synthesebaustein "Pt-T" synthetisiert, der die direkte Einführung eines N3-platinierten Thymidins während der Oligonucleotidsynthese ermöglicht. Unter den Bedingungen der Standard - H-Phosphonat - Synthese werden bei Verwendung des synthetisierten platinierten Synthesebaustein "Pt-T" platinierte Oligonucleotide erhalten, die eine trans-[PtII(NH3)2Py(N3-T)]+ - Modifizierung enthalten. Mit Hilfe der Sanger - Sequenzierung konnte gezeigt werden, daß die an T angebundene trans-{PtII(NH3)2Py}2+ - Platinspezies T7 DNA Polymerase blockiert. Weiterhin konnte gezeigt werden, daß die Spaltung der 5'-Phosphorsäurediesterbindung durch Schlangengift - Phosphodiesterase infolge dieser Platinmodifizierung deutlich langsamer abläuft. Durch Ersatz von Pyridin durch 1,6-Lutidin bleibt die Reaktionsfähigkeit der trans-Position am Platin erhalten. Durch geeignete Reaktionsbedingungen wurde das nach erfolgter Oligonucleotidsynthese an einem 4-mer gebundene Platin bifunktional an den selben Strang gebunden und so trans-[PtII(NH3)2{d(TTTG)-N3-T(2),N7-G(4)}]+, ein kurzes Oligonucleotid mit intrastrand-crosslink, synthetisiert. Die durchgeführte enzymatische Hydrolyse zeigt eine hohe Beständigkeit gegenüber dem Abbau mit Schlangengift - Phosphodiesterase und alkalischer Phosphatase. / This work describes the synthesis of oligodeoxynucleotides, which are modified at a specific position with the trans-{PtII(NH3)2}2+ - species. A platinated monomer building block "Pt-T" has been synthesized separately prior to automated synthesis. Platin modified oligonucleotides were elongated by use of standard H-phosphonate chemistry. The use of the synthesized platinated building block "Pt-T" leads to platinated oligonucleotides with a trans-[PtII(NH3)2Py(N3-T)]+ - modification. The synthesized oligonucleotides have been subjected to sequencing by the Sanger - method and it could be shown, that this modification blocks T7 DNA polymerase. Furthermore it could be shown, that the cleavage of the 5'-phosphodiester bond by snake venom phosphodiesterase due to these modification runs down clearly more slowly. In consequence of substitution of pyridine by 2,6-lutidine the reactivity of the trans - position of the platinum remains received. After oligonucleotide synthesis the platinum became crosslinked, thus trans-[PtII(NH3)2{d(TTTG)-N3-T(2),N7-G(4)}]+, a short oligonucleotide with intrastrand-crosslink, was synthesized. The enzymatic hydrolysis showed a high constancy facing the degradation with snake venom phosphodiesterase and alkaline phosphatase.

trans-Sialidase de Trypanosoma cruzi (TcTS) pertence à família de glicoproteínas de superfície do parasita e constitui um dos poucos exemplos naturais de glicosiltransferases superficiais encontradas em eucariotes. T. cruzi é incapaz de sintetizar ácido siálico e utiliza esta enzima para retirar este monossacarídeo de glicoconjugados do hospedeiro para sialilar moléculas aceptoras, como mucina-GPI, presentes na sua membrana plasmática. Esta enzima é específica em catalisar, preferencialmente, a transferência de ácido siálico para moléculas de mucina, originando ligações -2,3 com moléculas de galactose aceptoras na superfície do parasita. As moléculas de mucina sialiladas estão envolvidas no processo de aderência e subseqüente penetração do parasita nas células do hospedeiro. Considerando a heterogeneidade das moléculas de mucina de T. cruzi, não existem compostos disponíveis que atuem como substratos glicopeptídicos sintéticos. Desta forma, este trabalho teve como objetivo o desenvolvimento de métodos químicos e químico-enzimáticos de síntese de glicopeptídeos de mucina de T.cruzi para investigação da natureza das interações moleculares críticas envolvidas no reconhecimento e processamento de glicosídeos contendo ácido siálico, na presença de trans-sialidase. O melhor entendimento destas interações conduziu ao planejamento racional de potenciais inibidores seletivos de trans-sialidase de T. cruzi. Alguns dos principais glicopeptídeos, obtidos por metodologias de síntese em fase sólida e síntese químico-enzimática com a enzima 1,4-galactosiltransferase (1,4-GalT) foram: NH2(Thr)2-(LacNAc)-(Thr)3-GlyOH 2, NH2(Thr)2-(LacNAc)-(Thr)3-GlyOH 4 e NH2(Thr)2-(LacNAc)-(Thr)3-(LacNAc)-(Thr)3-GlyOH 5. Como precursores destes glicopeptídeos os blocos de construção GlcNAc-FmocThrOH 24, GlcNAc-FmocThrOH 25, Gal-FmocThrOH 27 e LacNAc-FmocThrOH 75 foram sintetizados por meio de reações de glicosilação do aminoácido treonina 18, contendo os grupos protetores N-Fmoc e O-Bn, com os correspondentes açúcares GlcNAcCl 12, GalBr 13 e LacN3Cl 33, seguidas de reações de hidrogenólise (10% Pd-C/ H2) para desproteção do grupo O-Bn e posterior acoplamento em cadeia peptídica rica em seqüência de treonina. Os aminoácidos glicosilados GlcNAc-ThrOH 22 e GlcNAc-ThrOH 23, totalmente desprotegidos, foram empregados em reações enzimáticas com a enzima 1,4-GalT, sendo obtidos os dissacarídeos glicosídicos LacNAc-ThrOH 53 e LacNAc-ThrOH 41 (também sintetizado quimicamente). Os dissacarídeos glicosídicos 41 e 53, os glicopeptídeos 2 e 4 e o aminoácido glicosilado Gal- ThrOH 28 foram submetidos aos ensaios enzimáticos com a enzima TcTS, sendo verificada a sialilação de todos os aceptores testados em rendimentos elevados, o que confirmou que estes compostos podem atuar como potenciais substratos da enzima TcTS. / Trypanosoma cruzi trans-sialidase (TcTS) belongs to the family of glycoproteins expressed on the surface of the parasite and constitutes one of the few examples of natural surface glycosyltransferases found in eucariotes. T. cruzi can not synthesize sialic acid itself and uses a trans-sialidase enzyme to scavenge this monosaccharide from host glycoconjugates to sialylate acceptors molecules, such as GPI (glycosylphosphatidylinositol)-anchored mucins, that are present in parasite plasma membrane. This enzyme is specific to catalise, preferentially, the transference of sialic acid to mucin glycoproteins, originating -2,3- linkages with acceptor galactose molecules in the parasite surface. The sialylated mucin molecules are involved in the attachment and subsequent penetration of the parasite into host cells. Given the heterogeneity of T. cruzi mucin molecules, there are no suitable synthetic occurring sources of TcTS glycopeptide substrates. Thus, the objective of this work was the development of chemical and chemoenzymatic methods of synthesis of T. cruzi mucin glycopeptides in order to investigate the nature of the molecular interactions involved in recognition and processing of containing sialic acid glycosides, in the presence of trans-sialidase. A better understanding of these interactions led to drug design of selective potential inhibitors for T. cruzi trans-sialidase. Some of the main glycopeptides obtained by methods of solid phase and chemoenzymatic synthesis with 1,4-galactosyltransferase (1,4-GalT) enzyme were: NH2(Thr)2-(LacNAc)- (Thr)3-GlyOH 2, NH2(Thr)2-(LacNAc)-(Thr)3-GlyOH 4 and NH2(Thr)2-(LacNAc)-(Thr)3- (LacNAc)-(Thr)3-GlyOH 5. As precursors of these glycopeptides, the building blocks GlcNAc-FmocThrOH 24, GlcNAc-FmocThrOH 25, Gal-FmocThrOH 27 and LacNAc- FmocThrOH 75 were synthesized by glycosylation reactions of the threonine amino acid 18, containing the protecting groups N-Fmoc and O-Bn, with the correspondent sugars GlcNAcCl 12, GalBr 13 and LacN3Cl 33, followed by hydrogenolysis reactions (10% Pd- C/ H2) for deprotection of O-Bn group and later coupling into the peptide chain rich in threonine sequence. The glycosylated amino acids GlcNAc-ThrOH 22 and GlcNAc-ThrOH 23, totally deprotected, were employed in enzymatic reactions with 1,4-GalT, being obtained the disaccharide glycosides LacNAc-ThrOH 53 and LacNAc-ThrOH 41 (also chemically synthesized). The disaccharide glycosides 41 and 53, the glycopeptides 2 and 4 and the glycosylated amino acid Gal-ThrOH 28 were submitted to enzymatic assays with TcTS enzyme, being verified the sialylation of all tested acceptors in high yields, which confirmed that these compounds can act as potential acceptors substrates for TcTS enzyme.