Le nombre de cancers et notamment de cancers photoinduits étant en augmentation, il est indispensable d’identifier de nouvelles molécules protectrices de type filtres UV, réparatrices vis-à-vis des altérations de l’ADN mais également favorisant la réponse pigmentaire. Les lichens sont des organismes originaux qui possèdent des qualités de résistance remarquables aux rayonnements solaires dues notamment à la production de métabolites photoprotecteurs tels que les mycosporines. A partir de ce motif structural original, nous avons synthétisé divers analogues par catalyse à l’or en utilisant un outil de la chimie théorique (TD-DFT) pour orienter les synthèses. Les premières évaluations de leurs propriétés physico-chimiques des molécules ont montré des activités prometteuses. / For several years, melanoma is the cancer with 84,000 new cases per year in Europe, including over 7000 cases in France, in 2010. One of the major causes of this cancer is the excessive exposure to UV radiations and it has been suggested that UV-A may be the primary cause of sunlight-induced melanoma. Hence, sunscreens with a good absorption in the UV-A spectral range need to be developed with a good efficacy and photostability. Lichens are original sources for the development of new UV filters because they possess ability to absorb UV due to the presence of metabolites such as mycosporines or MAAs (mycosporines like aminoacid). Based on the general structure of mycosporines, we have synthesized various analogues of mycosporines using a gold-catalyzed strategy. To orientate the synthesis, we used the method of the quantum chemistry: TD-DFT (Time-dependent density functional theory). These products were analyzed for their UV profiles and some of them showed good absorbing properties in UVA range. The cytotoxicity assay showed no toxicity of all products evaluated.

No description available.

Yes / The photoprotection and isolation of marinomycin A using sporopollenin exine capsules (SpECs) derived from the spores of the plant Lycopodium clavatum is described. The marinomycins have a particularly short half-life in natural light, which severely impacts their potential biological utility given that they display potent antibiotic and anticancer activity. The SpEC encapsulation of the marinomycin A dramatically increases the half-life of the polyene macrodiolide to the direct exposure to UV radiation by several orders of magnitude, thereby making this a potentially useful strategy for other light sensitive bioactive agents. In addition, we report that the SpECs can also be used to selectively extract culture broths that contain the marinomycins, which provides a significantly higher recovery than with conventional XAD resins and provides concomitant photoprotection.

Un excès d’énergie lumineuse peut être délétère pour les organismesphotosynthétiques ; en effet, il en résulte la formation d’espèces réactives de l’oxygène ausein des centres réactionnels. Les cyanobactéries ont adopté divers mécanismes dephotoprotection afin de contrer ce phénomène. L’un d’eux repose sur l’activité de l’OrangeCarotenoid Protein (OCP), protéine soluble qui attache un kéto-caroténoïde (hydroxyechinenone).Subissant de fortes intensités de lumière bleu-verte, l’OCP se convertit d’uneforme inactive/orange vers sa forme active/rouge. L’OCP ainsi photoactivée possède la facultéd’interagir avec les phycobilisomes - principales antennes collectrices de lumière - induisantla dissipation de l’énergie collectée par ces gigantesques complexes sous forme de chaleur. Lapression d’excitation au niveau des centres réactionnels ainsi que la fluorescence du systèmedécroissent alors.L’OCP photoactivée se fixe au coeur des phycobilisomes qui sont majoritairementconstitués de protéines chromophorylées de la famille des allophycocyanines (APC). J’aiconstruit différentes souches mutantes de Synechocystis PCC 6803 en modifiant ousupprimant les sous-unités mineures d’APC (ApcD, ApcF et ApcE). Ces sous-unités jouent lerôle essentiel d’émetteurs terminaux des phycobilisomes, véhiculant l’énergie qu’ellesreçoivent à la Chlorophylle a. J’ai aussi démontré que le mécanisme photoprotectif associé àl’OCP chez ces mutants restait inchangé, aussi bien in vivo que in vitro. Ces résultatssuggèrent qu’aucun émetteur terminal n’est nécessairement requis pour l’attachement del’OCP aux phycobilisomes et sous-entendent que l’OCP interagit probablement avec unesous-unité majeure d’APC.Divers phycobilisomes, contenant 2, 3 ou 5 cylindres d’APC dans leur coeur, ont étéisolés à partir de cyanobactéries variées. Les OCPs de Synechocytis et d’Arthrospira ont étépurifiées à partir de souches mutantes de Synechocystis. J’ai alors mené une étude in vitro desinteractions entre ces OCPs et les phycobilisomes. Le nombre de cylindres d’APC présents ausein des phycobilisomes n’affecte en rien la diminution de fluorescence. De plus, j’ai constatéque l’OCP de Synechocystis est spécifique pour ses propres phycobilisomes alors que l’OCPd’Arthrospira interagit avec tous les phycobilisomes employés ici. Des hypothèses, fondéessur les structures disponibles, ont été formulées pour élucider ces différences.Les domaines N- et C-terminaux de l’OCP d’Arthrospira ont été dissociés parprotéolyse. Le domaine N-terminal isolé conserve le caroténoïde attaché, ayant uneconformation similaire à celle observée lorsque l’OCP est photoactivée. Ce domaine Nterminalest aussi capable d’induire une importante diminution de la fluorescence desphycobilisomes. A l’inverse, le domaine C-terminal isolé est incolore et n’a aucun effet sur lafluorescence des phycobilisomes. Ces résultats suggèrent que seul le domaine N-terminal del’OCP est impliqué dans l’interaction avec les phycobilisomes. Le domaine C-terminal quantà lui module son activité. / Too much light can be lethal for photosynthetic organisms. Under such conditionsharmful reactive oxygen species are generated at the reaction center level. Cyanobacteria havedeveloped photoprotective mechanisms to avoid this. One of them relies on the solubleOrange Carotenoid Protein (OCP) that binds a ketocarotenoid (hydroxyechinenone, hECN).Under strong blue-green illumination, OCP gets photoconverted from an orange inactive form(OCPo) to a red active one (OCPr). OCPr interacts with phycobilisomes, the majorcyanobacterial light harvesting antennae, and triggers heat dissipation of the excess lightenergy collected by these gigantic pigment-protein complexes. Consequently, excitationpressure on reaction centers and fluorescence emission decrease.OCPr binds to phycobilisome cores, containing mainly chromophorylated proteins ofthe allophycocyanin (APC) family. I constructed Synechocystis PCC 6803 mutants affected insome minor APC forms (ApcD, ApcF and ApcE). These special APCs play the role ofterminal emitters, i.e. funnel light energy to Chlorophyll a. Strong-blue green illuminationtriggered normal OCP-related fluorescence quenching in all mutant cells. The fluorescencedecrease induced by Synechocystis OCP in vitro was similar when using phycobilisomesisolated from wild-type or mutant cells. These results demonstrated that the terminal emittersare not needed for interaction with the OCP and they strongly suggested that OCPr interactswith one of the major APC forms of the phycobilisome core.Phycobilisomes containing 2, 3 or 5 APC cylinders per core were isolated fromdifferent cyanobacterial strains. Synechocystis and Arthrospira OCPs were purified from overexpressingSynechocystis mutant strains. I then performed in vitro OCP/phycobilisomeinteraction studies. The number of APC cylinders per core had no clear influence on theamount of fluorescence quenching. Both OCPs behaved very differently, one appearing muchmore species-specific than the other. Structure-based hypotheses were emitted to explain suchdissimilarity.Arthrospira OCP N-terminal and C-terminal domains were separated throughproteolysis. The isolated N-terminal domain retained a bound carotenoid, which displayedsimilar conformation than in OCPr. This isolated N-terminal domain triggered importantphycobilisome fluorescence quenching even under dark conditions. In contrast, the isolated Cterminaldomain attached no pigment and had no visible effect on phycobilisome emission. Itwas then proposed that only the N-terminal domain of OCP is implied in interactions withphycobilisomes. The C-terminal domain modulates its activity.

Les caroténoïdes jouent un rôle essentiel dans les premiers événements photosynthétiques. Ils absorbent la lumière et transfèrent l'énergie résultante en excitation aux molécules voisines, assurant le respect de la succession des étapes photosynthétiques. En plus de l'absorption de la lumière, les caroténoïdes protègent l'appareil photosynthétique du stress photo-oxydatif survenant en condition de lumière intense, évitant les éventuels dommages. Les propriétés électroniques des caroténoïdes sont à la base de leurs mécanismes d'action et dans ce travail de recherche une combinaison de techniques biochimiques et spectroscopiques est utilisée pour examiner plus loin ces mécanismes avec un accent mis sur le rôle photoprotecteur joué par des caroténoïdes. Les d'échantillons analysés représentent différent niveaux d'organisation des protéines collectrices de lumière contenants ces pigments. Dans cette thèse quatre études principaux ont étés réalisés pour comprendre comment: les propriétés d'absorption des caroténoïdes lutéine et -carotène près les plantes peuvent être réglées in vivo par le site de liaison à leur protéines, le majeur complexe de capture de la lumière (LHCII) et le photosystème II (PSII) respectivement; l'altération des gènes de la voie biosynthétique des caroténoïdes peut indirectement provoquer une altération du transport d'électrons dans l'organisme photosynthétique; des molécules artificielles sont capable d'imiter le mécanisme photoprotecteur de transfert d'énergie entre les états de triplet des chromophores en mimant les protéines de l'apparat photosynthétique; la flexibilité structurelle de l'LHCII peut être explorée en modifiant son environnement. / Carotenoids play an essential role in the first steps of photosynthesis. They absorb light and they transfer the resulting excitation energy to the neighboring molecules, guaranteeing the correct order of the photosynthetic events. Additionally, carotenoids are able to protect the photosynthetic apparatus from the oxidative stress occurring in high light condition. Biological functions of carotenoids involving interaction with light, such as photosynthesis, are determined by the electronic properties of the conjugated polyene chain that is characteristic of carotenoid molecules. Understanding how these properties are tuned, is essential for understanding the mechanisms underlying carotenoid functions. Here we show that, by using a combination of different spectroscopic and biochemical approaches, these characteristics can be assessed in different kind of samples having the carotenoid molecules as common denominator. In this thesis four major studied have been performed in order to study how: the absorption properties of the two -carotenes molecules in PSII-RC and those of the two luteins in LHCII are tuned in vivo by their protein binding site, the alteration of the genes involved in the biosynthetic pathway of carotenoids has a pleiotropic effect on the photosynthetic organisms, artificial constructs are able to reproduce the photoprotective mechanism of triplet-triplet energy transfer between chromophores by mimicking the naturally occurring photosynthetic proteins, the structural flexibility of the major light harvesting complex can be probed by modifying its surrounding environment.

Les cyanobactéries ont développé des mécanismes de photo-protection pour se prémunir des dommages causés par un excès de lumière. L’un d’eux repose sur l’activité de l’Orange Caroténoïde Protéine (OCP), protéine soluble qui attache un kéto-caroténoïde, l’hydroxy-echinenone. Sous illumination, l’OCP se photo-convertit en forme active et interagit avec les phycobilisomes pour dissiper l’énergie collectée sous forme de chaleur. En conséquence, l’énergie d’excitation reçue par les centres réactionnels et la fluorescence du complexe photosynthétique diminuent. L’OCP a aussi la faculté de neutraliser l’oxygène singulet pour lutter contre la photo-oxydation. J’ai développé un système d’expression hétérologue pour reconstituer la voie de biosynthèse de cette protéine dans E.coli. Ce système permet l’obtention d’une grande quantité d’OCP liant son caroténoïde in vivo. Grâce à ce système robuste et rapide, les OCPs de trois cyanobactéries : Synechocystis, Arthrospira et Anabaena ont été produites, liant différents caroténoïdes. Toutes les OCPs recombinantes sont photo-actives et capables de quencher la fluorescence des phycobilisomes in vitro. Elles possèdent toutes la faculté de neutraliser l'oxygène singulet quel que soit le caroténoïde lié. Ce système d'expression hétérologue nous a permis d’élucider les déterminants structurels impliqués dans la photo-activation et la structure de la forme active de l’OCP. Il constitue une avancée fondamentale dans l'étude des protéines à caroténoïde et dans la production d'antioxydants solubles qui présentent un grand intérêt pour l’industrie de la santé. / Cyanobacteria have developed some photo-protective mechanisms to protect themselves from stress caused by excess light. One of them relies on the activity of the soluble Orange Carotenoid Protein (OCP) that binds a keto-carotenoid, the hydroxyechinenone. Under illumination, the OCP gets photo-converted to an active form and can interact with phycobilisomes to dissipate the collected energy as heat. Consequently, the excitation energy arriving at the photosynthetic reaction centers and the phycobilisome fluorescence emission decrease. The OCP can also quench the singlet oxygen to fight against photo-oxidation. I developed a heterologous expression system in which the biosynthetic pathway of the OCP is built in E.coli. The expression system allows the production of a large amount of OCP binding its carotenoid in vivo. Thanks to this robust and fast expression system, OCPs from three different cyanobacteria: Synechocystis, Anabaena and Arthrospira were produced, binding different carotenoids. All recombinant OCPs are photoactive and able to induce a large phycobilisome fluorescence quenching. Moreover, they all have the ability to quench the singlet oxygen, whatever the bound carotenoid. This heterologous expression system allowed us to elucidate the structural determinants involved in the photo-activation and structure of the active form of the OCP. This work represents a fundamental advance in the study of caroteno-proteins and in the production of others soluble antioxidants that are of great interest to the health industry.

Les cyanobactéries sont des organismes photosynthétiques capables de convertir le CO₂ en composés organiques et de produire de l’oxygène en utilisant l’énergie lumineuse. Néanmoins, de fortes intensités lumineuses saturent l'appareil photosynthétique, ce qui conduit à la production d'espèces réactives de l'oxygène, dangereuses pour la cellule. Pour y faire face, la photoactive orange carotenoid protein (OCP) induit une dissipation thermique de l’énergie excédentaire récoltée par le complexe d’antennes, le phycobilisome (PBS), afin de diminuer l’énergie arrivant aux centres photochimiques. L'OCP est composé de deux domaines), le domaine C-terminal (CTD) et le domaine N-terminal (NTD), reliés par un domaine de liason flexible (linker). Pendant la photoactivation, le caroténoïde est transféré vers le NTD, les domaines se séparent et le NTD peut interagir avec le PBS. Trois familles d'OCP coexistent (OCPX, OCP1 et OCP2) dans les cyanobactéries modernes. Outre l'OCP, de nombreuses cyanobactéries contiennent également des homologues des domaines OCP, le CTDH et HCP. Les HCP sont une famille de protéines caroténoïdes présentant différents traits photoprotecteurs. La plupart d'entre eux sont de très bons quenchers d'oxygène singulet, et un subclade est capable d'interagir avec le PBS et d'induire une dissipation de l'énergie thermique comme l'OCP. Le rôle de CTDH était inconnu. La présence de ces homologues parallèlement à l'OCP a conforté l'idée générale que l'OCP a une origine évolutive modulaire et que la CTDH et HCP pourraient interagir pour former un complexe OCP-like ayant des caractéristiques et une fonction similaires à celles de l'OCP. Dans cette thèse, je présente la première caractérisation des protéines CTDH. Les CTDH sont des dimères se liant à une molécule de caroténoïde. Le rôle principal de la CTDH est de transférer son caroténoïde au HCP. De plus, les CTDH sont capables de récupérer les caroténoïdes des membranes contrairement aux HCP. Ces résultats suggèrent fortement que les CTDH sont des transporteurs de caroténoïde qui assurent le chargement en caroténoïde sur les HCP. Ce nouveau mécanisme de translocation des caroténoïdes pourrait être multidirectionnel. La résolution de deux structures tridimensionnelles de l'ApoCTDH d'Anabaena a montré que la queue C-terminale du CTDH (CTT) peut adopter différentes conformations. De plus, l'analyse de mutation a démontré que le CTT joue un rôle essentiel dans la translocation des caroténoïdes. Enfin, je rapporte une caractérisation moléculaire du linker reliant les domaines de différents OCP modernes et son rôle au cours de l'évolution de l'OCP. Tout d’abord, j’ai caractérisé les OCP des trois subclades, y compris l’OCPX non caractérisé. OCPX et OCP2 présentent une désactivation rapide par rapport à OCP1. Alors que OCP1 et OCPX peuvent dimériser, OCP2 est stable en tant que monomère. Enfin, j'ai constaté que le linker est essentiel pour la désactivation de l'OCP et qu'il régule la photoactivation. Dans OCP1 et OCPX, le linker ralentit la photoactivation, tandis que dans OCP2, il augmente le taux de photoactivation. L'analyse bioinformatique complète cette caractérisation et fournit une image claire de l'évolution de l'OCP pour répondre efficacement aux conditions de stress. / Cyanobacteria are photosynthetic organisms capable of CO₂ conversion into organic compounds and production of O2 by using light energy. Nevertheless, high light intensities saturate the photosynthetic apparatus leading to production of reactive oxygen species, which are dangerous for the cell. To cope with this, the photoactive Orange Carotenoid Protein (OCP) induces thermal dissipation of the excess energy harvested by the antenna complex, the phycobilisome (PBS) to decrease the energy arriving at the photochemical centers. The OCP is composed of two domains connected by a flexible linker, the C-terminal domain (CTD) and the N-terminal domain (NTD). During photoactivation, the carotenoid is translocated to the NTD, the domains separate and the NTD is able to interact with the PBS. Three OCP families co-exist (OCPX, OCP1 and OCP2) in modern cyanobacteria. In addition to the OCP, many cyanobacteria also contain homologs of OCP domains, the CTDH and HCP. The HCPs are a family of carotenoid proteins with different photoprotective traits. Most of them are very good singlet oxygen quenchers, and one sub-clade is able to interact with the PBS and to induce thermal energy dissipation like OCP. The role of CTDH was unknown. The presence of these homologs in parallel to the OCP supported the general idea that the OCP has a modular evolutionary origin and that the CTDH and HCP can interact forming an OCP-like complex with similar characteristics and function than the OCP.In this thesis, I present the first characterization of the CTDH proteins. CTDHs are dimers binding a carotenoid molecule. The main role of the CTDH is to transfer its carotenoid to the HCP. In addition, CTDHs are able to uptake carotenoids from membranes but not HCPs. These results strongly suggested that the CTDHs are carotenoid carriers that ensure the proper carotenoid loading into HCPs. This novel carotenoid translocation mechanism could be multidirectional. The resolution of two tridimensional structures of the ApoCTDH from Anabaena showed that the C-terminal tail of the CTDH (CTT) can populate different conformations. Moreover, mutational analysis demonstrated that the CTT has an essential role in carotenoid translocation. Finally, I report a molecular characterization of the flexible linker connecting the domains of different modern OCPs and its role during the evolution of the OCP. First, I characterized OCPs from the three subclades, including the uncharacterized OCPX. OCPX and OCP2 present a fast deactivation compared with OCP1. While OCP1 and OCPX can dimerize, OCP2 is stable as monomer. Finally, I found that the linker is essential for the OCP deactivation and it regulates the photoactivation. In OCP1 and OCPX the linker slows down the photoactivation, while in OCP2 it increases the photoactivation rate. Bioinformatic analysis complements this characterization and provides a clear picture of the evolution of the OCP to respond efficiently to stress conditions.

Foram estudadas trinta e uma cepas fúngicas não identificadas, as quais foram denominadasX1 a X31. O potencial fotoprotetor foi avaliado pela medida espectrofotométrica da absorçãodos extratos na região do UV (280-400 nm). Os extratos com os melhores perfis de absorção em cultura estacionária foram X1, X2, X6, X12, X13, X18, X19, X22, X24 e X31 e, em cultura agitada X4 e X17. A reprodutibilidade do processo foi avaliada e as cepas fúngicas que apresentaram coeficiente de variação menor que 15% foram selecionadas para o estudo de fotoestabilidade. A fotoestabilidade dos extratos foi avaliada pela medida da viabilidade celular de fibroblastos L929 tratados com extratos previamente irradiados sob radiação UVA (11,2 J/cm2) e UVB (3,43 J/cm2) e extratos não irradiados, bem como, pela comparação das áreas sob as curvas de absorção na região do UV dos extratos irradiados e não irradiados. Os extratos selecionados para o estudo de fotoestabilidade foram X4, X12, X19, X22, X24 e X31. Os extratos não irradiados apresentaram os seguintes valores deIC50 para viabilidade celular (citotoxidade): X4-130µg/ml, X19-20µg/ml, X22-10 µg/ml e X24-60µg/ml. Após a radiação UVA e UVB, os extratos apresentaram redução significativa da viabilidade celular em relação ao IC50 dos extratos não irradiados. Sob luz UVB, os extratos X12 (IC50 35µg/ml) e X31 (IC50 70µg/ml) mantiveram a mesma porcentagem de redução da viabilidade celular quando comparado ao IC50 dos extratos não irradiados. No entanto após exposição à luz UVA, o extrato X12 aumentou a viabilidade celular de 50% (quando não irradiado) para 75% (irradiado). Enquanto que o extrato X31, mesmo após a radiação UVA, manteve a mesma redução de 50% da viabilidade celular. Nessa etapa os extratos selecionados foram os X12 e X31. O espectro de absorção na região do UV obtido para o extrato X12 mostrou uma redução da absorbância de 28,3% sob radiação UVB e de 60% sob radiação UVA em relação ao extrato não irradiado. O extrato X31 apresentou uma redução da absorbância de 17,6% e30% sob radiação UVB e UVA respectivamente, em relação ao extrato não irradiado. Os fungos selecionados foram identificados por PCR, sugerindo que o fungo X12 seja o Aspergillus terreus e o X31 seja o Talaromyces pinophilus. Por fim, foi feita a identificação da substância ativa do extrato X12 empregando a técnica de desreplicação, a qual fez o uso da instrumentação analítica acoplada UHPLC-DAD-(ESI)-HRMS associada ao banco de dados Chapman& Hall\'s Dictionary of Natural Products (DNP). No extrato X12 o composto majoritário foi identificado como sendo a citreoviridina. Assim, os resultados do presente trabalho permitiu estabelecer um procedimento para a seleção de fungos produtores de compostos absorvedores de radiação UV, que poderia ser aplicado na obtenção de novos filtros orgânicos naturais para protetores solares. / It were studied thirty-one fungal strains not identified, which were named X1 to X31. The photoprotective potential was assessed spectrophotometrically by measuring absorption of the extract in the UV region (280-400 nm). The extracts that presented the best absorption profiles in stationary culture were X1, X2, X6, X12, X13, X18, X19, X22, X24 and X31, and X4 and X17 in stirred culture. The reproducibility of the process was evaluated and fungal strains that showed a coefficient of variation lower than 15% were selected for the study of photostability. The photostability of the extracts was assessed by measuring cell viability of L929 fibroblasts treated with extracts previously irradiated under UVA light (11,2 J/cm2) and UVB (3,43 J/cm2) and not irradiated extracts, as well as by comparison of the areas under the curves of absorption in the UV region of the irradiated and non-irradiated extracts. The extracts selected for the study of photostability were X4, X12, X19, X22, X24 and X31. The non-irradiated extracts showed the following IC50 values for cell viability (cytotoxicity): X4- 130?g/ml X19-20?g/ml, X22-10/ml and X24-60?g/ml. After UVA and UVB radiation, the extracts showed significant reduction in cell viability compared to the IC50 of the unirradiated extracts. Under UVB light, the X12 extracts (IC50 35?g/ml) and X31 (IC50 70mg/ml) maintained the same percentage of cell viability reduction when compared to the IC50 of the unirradiated extracts. However after exposure to UVA light, X12 extract increased the cell viability from 50% (when not irradiated) to 75% (irradiated). While X31 extract even after the UVA irradiation, remained the same 50% of reduction in cell viability. At this stage the selected extracts were X12 and X31. The absorption spectrum in the UV region obtained for X12 extract showed a decrease in absorbance of 28.3% under UVB and 60% under UVA radiation relative to non-irradiated extract. The X31extract showed a reduction in absorbance of 17.6% and 30% in UVA and UVB radiation, respectively, compared to non-irradiated extract. The selected fungi were identified by PCR, suggesting that X12 fungus is Aspergillus terreus and X31 is the Talaromyces pinophilus. Finally it was identified the active substance of X12 extract employing dereplication technique which makes use of coupled analytical instrumentation UHPLC-DAD- (ESI) HRMS associated to the Chapman and Hall\'s Dictionary of Natural Products (DNP) database. The majority compound of X12 extract was identified as the citreoviridin. Thus, the results of this study allowed us to establish a procedure for the selection of producers of UV absorbing compounds from fungi, which could be applied in obtaining new natural organic filters for sunscreens.

Le rôle du mélanocyte dans la pigmentation de la peau n'est plus a démontré, par contre son rôle photoprotectif est controversé. Le rôle du mélanocyte a été étudié en comparant des épidermes reconstruits avec 100 % de kératinocytes et des épidermes reconstruits avec 95 % de kératinocytes et 5 % de mélanocytes. Dans un premier temps, l'effet d'une irradiation UVB aiguë a été étudié sur ces deux types de reconstructions, puis l'étude a été élargie aux effets des UVA et des UVA+B. Ces études ont permis de montrer, qu'après irradiation, la présence de mélanocytes au sein de l'épiderme reconstruit prévient l'apoptose sans pour autant protéger de façon significative de la formation des lésions directes de l'ADN (CPD et 6-4PP) et permet le maintien du rapport SOD/Catalase (principales enzymes antioxydantes). Par contre, la présence de mélanocytes au sein de l'épiderme amplifie les oxydations lipidiques et protéiques UV-induites mais semble prévenir l'oxydation de l'ADN. Les mélanocytes possèdent en plus de la mélanine, une plus grande concentration en acides gras polyinsaturés membranaires que les kératinocytes. Afin d'estimer quelle est la part de la mélanine et quelle est celle des acides gras polyinsaturés mélanocytaires dans les réponses UV-induites, des épidermes reconstruits avec des kératinocytes ont été suppléméntés avec des acides gras polyinsaturés. Cette étude a permis de mettre en évidence que ce sont les acides gras qui induisent l'amplification des oxydations lipidiques et protéiques alors que la mélanine protège l'ADN de l'oxydation induite par la lipoperoxydation. Ces différentes études ont également permis de montrer que face aux UV, l'épiderme reconstruit se comporte de façon similaire à l'épiderme normal in-vivo. Dans un dernier temps, des crèmes solaires et des antioxydants systémiques ont été testés et ont permis de confirmer que le modèle d'épiderme reconstruit est tout à fait adapté au test de molécules photoprotectrices.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A radiação ultravioleta (UV) induz diversos efeitos nocivos nos organismos e a quantidade desta radiação que atinge a biosfera é afetada pela concentração de ozônio, latitude, altitude, clima e reflexão especular. As respostas de briófitas em relação aos efeitos da radiação UV e a presença de compostos que absorvem esta radiação têm sido estudadas. Sanionia uncinata, Holomitriopsis laevifolia e Leucobryum laevifolium são espécies de musgos encontrados em locais expostos a alta incidência de radiação UV e com habitats distintos. Considerando que as respostas de musgos contra os efeitos da radiação UV e seus mecanismos de proteção ainda são pouco caracterizados, o objetivo deste estudo foi investigar o potencial fotoprotetor e possíveis riscos toxicológicos associados aos extratos dos musgos S. uncinata, proveniente da Antártica e H. laevifolia e L. laevifolium, proveniente do Amazonas. Seus extratos metanólico (EM), aquoso (EA), hidroalcoólico (EH) e etanólico (EE) foram estudados com a caracterização química por absorção ao UV e visível e pela cromatografia líquida de alta eficiência; quantificação do índice total de compostos fenólicos; determinação da capacidade captadora do radical 2,2-difenil-1-picril-hidrazila a fim de avaliar as atividades antioxidantes; avaliação do potencial de fotoproteção cutânea pela determinação do fator de proteção solar; avaliações do potencial mutagênico e citototóxico, através do ensaio de Salmonella/microssoma, utilizando as cepas TA97, TA98, TA100, TA102 e TA104; do potencial fotomutagênico através do ensaio de fotomutagenicidade, usando as cepas TA102 e TA104; e investigação dos efeitos genotóxicos e fotogenotóxicos, pelo ensaio de micronúcleo e fotomicronúcleo, respectivamente, usando diferentes linhagens celulares estabelecidas. Foram encontradas atividades fotoprotetoras e antioxidantes e observou-se que os extratos se apresentaram singulares devido a sua composição química. Os resultados fotoprotetores, além dos mutagênicos/fotomutagênicos, genotóxicos/fotogenotóxicos e suas respectivas avaliações citotóxicas também permitiram selecionar extratos e suas concentrações, como promissores candidatos em fotoproteção Assim, os EA e EH de H. laevifolia e L. laevifolium apresentam, no geral, os resultados mais significativos, tornando-se potenciais para avaliações refinadas em fotoproteção e na separação de componentes que possam levar a futuras aplicações como antioxidantes e protetores solares ou como adjuvantes. / The ultraviolet radiation (UV) induces many harmful effects in all living organisms and the amount of this radiation that reaching the ground is affected by many factors including ozone concentration, latitude, altitude, climate and specular reflection. The responses of bryophytes against the effects of UV radiation and the presence of compounds that absorb the UV region have been studied. Mosses Sanionia uncinata, Holomitriopsis laevifolia and Leucobryum laevifolium are found in locations exposed to UV at high levels of radiation and in different habitats. Whereas that the responses of mosses against the effects of UV radiation and their protection systems are poorly characterized yet, the aim of this study was to investigate photoprotective potential and possible toxicological risks associated with extracts of mosses S. uncinata (from Antarctica) and H. laevifolia and L. laevifolium (from Amazônia). Methanol (ME), aqueous (AE), hydroalcoholic (HE) and ethanolic (EE) were studied by: chemical characterization by UV/visible spectrophotometry and by High performance liquid chromatography; phenolic content estimation; 2,2-diphenyl-1-picrylhydrazyl scavenging activity; potential of skin photoprotection by in vitro determination of sun protection factor; the mutagenic potential, and cytotoxic by Salmonella/microsome assay, using the TA97, TA98, TA100, TA102 and TA104 strains; photomutagenic potential by photomutagenicity test, using TA102 and TA104 strains and; investigation of genotoxic and photogenotoxic effects by micronucleus test and photo-micronucleous assay, respectively, using different established cell lines. Photoprotective and antioxidant activities were found and it was observed that the extracts showed strong uniqueness due to its chemical composition. From the photoprotective, mutagenic/photomutagenic and genotoxic/photogenotoxic results and their cytotoxic evaluations it was possible to select extracts and their concentrations as promising candidates for photoprotection. Thus, the EA and EH of H. laevifolia and L. laevifolium demonstrated the most significant results, becoming potential for refined evaluations in photoprotection and separating components that can lead to future applications such as sunscreens and antioxidants or as adjuvants.