Muitas espécies de Gracilaria são de importância econômica devido à exploração do ágar e, em menor escala, por serem consumidas diretamente na alimentação. Recentemente pesquisas têm mostrado um bom potencial biológico dessas espécies, porém com pouca investigação relacionada a identificação das substâncias bioativas. Sob esta perspectiva, o objetivo geral deste estudo foi investigar os componentes químicos majoritários e avaliar o potencial biológico e nutricional de três espécies nativas obtidas no litoral do nordeste brasileiro: Gracilaria birdiae E. M. Plastino & E. C. Oliveira Gracilaria caudata J. Agardh e Gracilaria domingensis (Kützing) Sonder ex Dickie. A análise da composição química levou a identificação de cerca de 50 substâncias distribuídas em alcanos, ácidos graxos livres, monossacarídeos, ácidos sulfônicos, aminoácidos livres, esteróis, heterosídeos, lipídios polares, monoacilgliceróis, polissacarídeos sulfatados, aminoácidos do tipo micosporinas e sais halogenados. Ésteres de ácidos graxos, ácidos carboxílicos aromáticos, fitol e o derivado fitona, diidroactinidiolideo também foram detectados em pequenas quantidades. Gracilaria caudata e G. domingensis foram muito semelhantes quimicamente. Já G. birdiae foi menos diversa, compartilhando, porém, muitas das substâncias majoritárias. Os resultados desse trabalho sugerem grande diversidade química para espécies de Gracilaria, ampliando o conhecimento disponível para Rhodophyta. Atividades biológicas promissoras foram detectadas para diversos ensaios. O extrato hexânico de G. caudata foi ativo frente as artêmias, com o isolamento biomonitorado de duas possíveis substâncias bioativas. O isolamento biomonitorado guiado pela atividade bioestimulante in vitro de plântulas de Lactuca sativa L. (alface) resultou no ácido palmítico como sendo responsável pelo aumento em 83% do crescimento de raiz. Os polissacarídeos sulfatados presentes nos extratos aquosos de G. caudata e G. domingensis foram bioestimulantes de raiz e folha de alface. Além disso, esses polissacarídeos de G. domingensis inibiu a atividade da enzima transcriptase reversa do HIV-1. Para G. birdiae vários extratos se mostraram com potenciais citotóxico e fitotóxico. Os aminoácidos tipo micosporinas (asterina 330, chinorina, palitina, palitinol e porphyra 334), por serem substâncias comuns em algas vermelhas, foram isolados e avaliados quanto ao potencial biológico. A palitina inibiu a atividade da enzima transcriptase reversa do HIV-1, enquanto porphyra 334, chinorina e também palitina apresentaram boa atividade antioxidante com o ensaio do reagente Folin-Ciocalteu. Quanto ao aspecto nutricional, os resultados indicaram que as três algas avaliadas, principalmente G. birdiae, foram ricas em fibras dietéticas e sais minerais, formando um grupo alimentar único bem diferente dos alimentos tradicionais na mesa dos brasileiros. Em resumo, os resultados obtidos para as espécies de Gracilaria avaliadas reforçaram a ideia de ampla diversidade química para as rodófitas, mostraram resultados promissores em diferentes ensaios, além de apontarem um potencial uso como fonte suplementar nas dietas dos brasileiros / Many Gracilaria species are economically important due to the exploitation of agar and, to a lesser extent, because they are consumed directly in the food. Recent studies have shown a good biological potential of these species, but with few studies aimed at the identification of bioactive substances. From this perspective, the general objective of this study was to investigate the major chemical components and to evaluate the biological and nutritional potential of three native species obtained in the northeast Brazilian coast: Gracilaria birdiae E. M. Plastino & E. C. Oliveira Gracilaria caudate J. Agardh and Gracilaria domingensis (Kützing) Sonder ex Dickie. The chemical composition analysis resulted in the identification of about 50 metabolites distributed as alkanes, free fatty acids, monosaccharides, sulfonic acids, free amino acids, sterols, heterosides, polar lipids, monoacylglycerols, sulfated polysaccharides, mycosporine-like amino acids and halogenated salts. Esters of fatty acids, aromatic carboxylic acids, phytol and the phytone derivative, dihydroactinidiolide were also detected in small amounts. G. caudate and G. domingensis were chemically similar. On the other hand G. birdiae was less diverse, sharing, however, many of the major metabolites. The results of this work suggest a great chemical diversity for Gracilaria species, increasing the knowledge available for Rhodophyta. Promising biological activities were detected for several assays. The hexane extract of G. caudate was active in brine shrimp lethality assay, with the biomonitoring isolation of two possible bioactive substances. The biomonitoring isolation guided by in vitro biostimulant activity of young Lactuca sativa L. (lettuce) seedlings resulted in palmitic acid as responsible for 83% increase in root growth. The sulfated polysaccharides present in the aqueous extracts of G. caudate and G. domingensis were biostimulant of root and lettuce leaf. In addition, these polysaccharides from G. domingensis inhibited the activity of the HIV-1 reverse transcriptase enzyme. For G. birdiae several extracts showed cytotoxic and phytotoxic potentials. Mycosporine-like amino acids (asterina 330, shinorine, palythine, palythinol and porphyra 334), known as common red algae substances, were isolated and evaluated for biological potential. Palythine inhibited the activity of the HIV-1 reverse transcriptase enzyme, while porphyra 334, shinorine and palythine showed good antioxidant activity using the Folin-Ciocalteu reagent assay. Regarding the nutritional aspect, the results indicated that from the three algae evaluated, highlighting G. birdiae, all were rich in dietary fibers and minerals, forming a unique food that is very different from the traditional Brazilian food. In summary, the results obtained for the Gracilaria species studied reinforced the idea of a wide chemical diversity for red algae, showing promising results in different bioassays, besides indicating a potential use as a supplementary food source in Brazilian diets

Un criblage phytochimique à partir de quatre lichens saxicoles hydrophiles du genre Dermatocarpon a mis en évidence la présence inattendue de composés de type mycosporines dans ces chlorolichens. Leur présence a été confirmée dans des échantillons historiques d'herbiers couvrant une période de plus de 170 ans. Leur teneur était comprise entre 0,1 et 1,4 mg/g de lichen sec. Trois mycosporines ont été isolées de Dermatocarpon luridum collecté en Bretagne sur des rochers d'eau vive: deux mycosporines natives, la mycosporine glutaminol et la mycosporine glutamicol ainsi qu'un artéfact d'isolement, l'ester éthylique de la mycosporine glutamicol. Leurs propriétés physico-chimiques incluant le calcul de leur pKa ont été déterminées. L'étude phytochimique a également conduit à l'isolement de sept autres composés dont un acide aminé non protéique, un dipeptide et un céramide identifiés pour la première fois dans un lichen et le cérévistérol jusqu'ici non décrit dans D. luridum. Les activités cytotoxiques et photoprotectrives des composés disponibles en quantité suffisante ont été évaluées. Le cérévistérol a montré une cytotoxicité modérée sur huit lignées cellulaires contrairement aux cinq autres composés. Les propriétés photoprotectrices et activités antioxydantes de l'ester éthylique de la mycosporine glutamicol et d'un extrait aqueux semi-purifié de D. miniatum présentent un intérêt pour un possible développement comme actif solaire. / A screening of mycosporine-like compounds in four hydrophilic Dermatocarpon lichen species has highlighted the unexpected occurrence of such metabolites in chlorolichens. These compounds were confirmed and quantified in historical herbarium samples (15-50 mg) even more than 170 years old. Total mycosporine contents ranged from 0.1 to 1.4 mg/g of dry lichens. Two genuine mycosporines (mycosporine glutaminol and the more stable mycosporine glutamicol) with an artifact mycosporine glutamicol ethyl ester were isolated from Dermatocarpon luridum collected in a breton river. Their physico-chemical properties were also determined including pKa. The phytochemical studies led to the isolation of seven additional compounds in which a non-protein amino acid, a dipeptide and a ceramide were identified for the first time in lichens and cerevisterol for the first time in D. luridum. Compounds obtained in suitable quantity were evaluated for their cytotoxic and photoprotective properties. Except a moderate cytotoxicity of cerevisterol, the five tested compounds were not found toxic on the eight cell lines. Photoprotective properties of the mycosporine glutamicol ethyl ester and a semi-purified aqueous extract of Dermatocarpon miniatum along with some antioxidant activities are promising to develop new sunscreens.

Although mass coral bleaching events are generally triggered by high seawater temperatures, experiments have demonstrated that corals and reef-dwelling foraminifers bleach more readily when exposed to high energy, short wavelength solar radiation (blue, violet and ultraviolet [UVR]: Lambda ~ 280 - 490 nm). In seawater, colored dissolved organic matter (CDOM), also called gelbstoff, preferentially absorbs these shorter wavelengths, which consequently bleach and degrade the CDOM. Alteration of watersheds and destruction of coastal wetlands have reduced natural sources of CDOM to reefal waters. I tested the null hypothesis that CDOM does not differ between reefs that differ in coral health, and that water transparency to UVR is not a factor in reef health. I measured absorption of UVR and UV irradiance at various reefs in the Florida Keys that differ in distance from shore and degree of anthropogenic development of the adjacent shoreline. My results show that intact shoreline - associated reefs and inshore reefs tend to be exposed to lower intensities of UVR, and lower degrees of photic stress, than developed shoreline - associated reefs and offshore reefs. Absorption due to CDOM (ag320) was higher, and photic stress, as revealed by increased production of UV-absorbing compounds, Mycosporine - like Amino Acids (MAAs), was lower at the surface compared to the bottom. The following results support my conclusion: ag320 and UV attenuation coefficients (Kd 's) were higher at intact compared to developed shoreline - associated reefs, and at inshore compared to offshore reefs. Spectral slope, S, was higher at offshore compared to inshore reefs, indicating a higher degree of photobleaching of CDOM. Relative expression of MAAs was higher at developed compared to intact shoreline - associated reefs, at offshore reefs compared to inshore reefs, and at the surface compared to the bottom. Solar energy reaching the benthos at two inshore reefs of the same depth (6m) was approximately an order of magnitude higher at the reef near developed shoreline compared to the reef near intact shoreline, and may be due to greater degree of diffuseness of the underwater light field combined with lower ag at the developed shoreline-associated reef.

Aminoácidos tipo micosporinas (MAAs) são compostos responsáveis pela fotoproteção no ultravioleta de diversos organismos aquáticos. São sintetizados pela via do ácido chiquímico por algas, bactérias e fungos, de maneira similar à síntese de flavonóides em plantas superiores. Neste trabalho foram conduzidos estudos relacionados a estes compostos em algas. Protocolos de extração de diferentes algas foram testados alterando-se parâmetros como solventes, temperatura e condições de incubação. Os resultados mostraram que dependendo da alga estudada, diferentes condições podem mudar a concentração de MAAs extraída, ressaltando a importância de se testar diversos parâmetros na extração, evitando assim valores sub- ou superestimados de concentrações. O desenvolvimento de um método por HPLC permitiu a separação de 6 MAAs com boa resolução. A caracterização estrutural foi realizada majoritariamente por espectrometria de massas utilizando diferentes tipos de analisadores. Estas análises permitiram a proposição de mecanismos de fragmentação descritos pela primeira vez para esta classe de compostos e possibilitaram a identificação de diferentes MAAs em algumas micro e macroalgas. Ensaios in vitro foram realizados com o extrato obtido da macroalga Gracilaria domingensis no intuito de avaliar seu potencial uso em formulações cosméticas direcionadas à fotoproteção. Os testes de estabilidade quanto ao pH, temperatura e exposição à radiação ultravioleta bem como os ensaios de citotoxicidade, fototoxicidade e avaliação do fator de proteção solar sugeriram que este extrato pode ser promissor quando incorporado em formulações direcionadas para a fotoproteção. Este extrato não apresentou atividade antioxidante significativa. Os estudos com o dinoflagelado Prorocentrum minimum isolado de duas regiões diferentes, quando exposto às radiações ultravioleta, mostraram que houve uma indução das MAAs em ambas linhagens em todos os tratamentos realizados no período de 72 h. A indução foi mais rápida e pronunciada na linhagem oriunda de Lisboa, em Portugal do que na linhagem proveniente de Kattegat, na Dinamarca. Estes dados estão de acordo com as características do local de origem das linhagens, uma vez que os dinoflagelados originários de Portugal em seu meio natural estavam sujeitos a maiores irradiações. Medidas do rendimento quântico do fotossistema II indicaram que a síntese e acúmulo de MAAs em P. minimum isolada de Lisboa ofereceu vantagens na proteção do sistema fotossintético e na supressão de espécies reativas de oxigênio desta microalga quando comparada aos indivíduos da mesma espécie de regiões com menores irradiações. / Mycosporine-like amino acids (MAAs) are chemically related water soluble compounds responsible for UV photoprotection in many aquatic organisms. They are biosynthesized via the shikimate pathway by algae, bacteria and fungi in a similar manner to the biosynthesis of UV-screening flavonoids in terrestrial plants. In this work, studies related to this class of compounds were performed with algae. Extraction protocols of some algae were tested using diverse solvents at different temperature. The results showed that depending on the organism examined, both solvent concentration and temperature affected extraction efficiency and final MAA concentration. The improvement of a HPLC method separated a mix of 6 MAAs with good resolution. The structural characterization was made by mass spectrometry using different analyzers. The analysis by mass spectrometry allowed the proposition of new mechanisms of fragmentation and identification of different MAAs in some micro and macroalgae. In vitro preliminary assays were performed to evaluate the potential use of Gracilaria domingensis extract in suncare products. The extract showed no antioxidant activity, however the pH, temperature and UV exposure stability, as well the citotoxicity, phototoxicity and sun protection factor showed a potential commercial utilization for the extracts. The UV exposure experiments with the dinoflagellate Prorocentrum minimum isolated from Lisbon, Portugal and Kattegat, Denmark, showed that MAAs were induced in all treatments during 72 h. The induction was faster in the species from Lisbon than the ones from Kattegat. These data are in agreement with local isolation place, since the dinoflagellates from Portugal are submitted to high irradiance regime in their natural enviroment. The quantum yield of photosystem II suggested that the over production of MAAs by P. Minimum from Lisbon protects the photosynthetic apparatus, indicating that MAAs act as spectrally specific UV sunscreens in phytoplankton.

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No. of bitstreams: 1 saraiva_ainb_dr_rcla.pdf: 2224366 bytes, checksum: 14d1d03c22d3e56e61a484a95e72f7c4 (MD5) Previous issue date: 2017-06-23 / Nos últimos séculos a história do ser humano no planeta Terra tem sido marcada por intensos impactos ambientais antropogênicos negativos, dentre os quais destaca-se a maior exposição da radiação ultravioleta (UV) na superfície terrestre devido à destruição da Camada de Ozônio ocasionada pela liberação de substâncias tais como os CFCs (clorofluorcarbonetos). A radiação UV tem sido considerada com um dos principais fatores que afetam a distribuição dos organismos fotossintetizantes em ambientes aquáticos, tendo efeitos biológicos diversos como danos no DNA e em moléculas proteicas, alteração dos pigmentos fotossintéticos, modificações nos parâmetros fotossintéticos e crescimento desses organismos. Torna-se evidente, portanto, que as mudanças da incidência de radiação UV na superfície terrestre podem levar a alterações ecofisiológicas nas comunidades algais. Nesse contexto, este trabalho teve como objetivo central analisar as respostas fisiológicas (parâmetros fotossintéticos, concentração de pigmentos fotossintéticos e de substâncias fotoprotetoras) à radiação ultravioleta (UVA e UVAB) em macroalgas continentais (ambientes lóticos e aerofíticos). A idéia geral foi avaliar o nível de resposta (tendência de aumento ou diminuição em maior ou menor grau) na performance em função da exposição à radiação ultravioleta. Duas hipóteses gerais foram formuladas: 1) as respostas das macroalgas consistirão essencialmente de tendência de diminuição de sua performance fisiológica (parâmetros fotossintéticos e concentração de pigmentos) e aumento de substâncias fotoprotetoras sob exposição à radiação UV; 2) o nível de resposta será diferente entre os grupos de algas, sendo que para as algas verdes (tipicamente algas de sol) espera-se que sejam mais tolerantes à radiação UV, enquanto que as rodófitas (tipicamente algas de sombra) tenderão a ser mais sensíveis e as cianobactérias deverão apresentar respostas intermediárias. Hipóteses mais específicas foram formuladas para cada parte do trabalho (capítulos). Experimentos foram realizados em três condições: PAR apenas (400-700nm); PAR + UVA (320-700nm), UVA; PAR + UVA + UVB (280-700nm), UVAB. A tese foi dividida em três capítulos com seus respectivos objetivos e hipóteses: 1) “Respostas fotossintéticas dos estágios gametofítico e esporofítico da macroalga vermelha continental Kumanoa ambigua (Rhodophyta, Batrachospermales) sob radiação UV”. O objetivo foi analisar o desempenho fotossintético dos estágios (gametófito e esporófito, 'Chantransia') de K. ambigua em cultura sob radiação UV. Hipotetizou-se que os estágios do histórico de vida de K. ambigua exibiriam respostas fotossintéticas diferentes à exposição à UV. 2) “Efeitos da radiação UV na fotossíntese e fotoproteção em duas espécies de Cyanobacteria”. O objetivo foi analisar o desempenho fotossintético (parâmetros derivados da fluorescência da clorofila), as concentrações de pigmentos (clorofila a e carotenóides) e a presença de compostos absorventes de UV (aminoácidos tipo-micosporinas - MAAs e scitoneminas) em duas cianobactérias aerofíticas, Leptolyngbya cf. henningsii e Scytonema sp. em resposta à exposição à radiação UV em condições de cultura. Hipotetizou-se que ambas espécies teriam desempenho fotossintético semelhantes em resposta à exposição de radiação UV, mas elas difeririam em termos de concentrações de pigmento e formação de compostos absorvedores de UV. 3) Efeitos da radiação UV na fotossíntese de três espécies de macroalgas lóticas tropicais. O objetivo foi analisar o desempenho fotossintético (parâmetros derivados da fluorescência da clorofila), as concentrações de clorofila a e a presença de compostos absorventes de UV (MAAs) em Cladophora glomerata (Chlorophyta), Spirogyra sp. (Streptophyta) e Sirodotia delicatula (Rhodophyta) coletadas em condições naturais em resposta à exposição de radiação UV em condições laboratoriais. Hipotetizou-se que Spirogyra sp. and C. glomerata, típicas algas adaptadas ao sol, apresentariam performances fotossintéticas semelhantes, com uma leve resposta à exposição à radiação UV e que S. delicatula, caracterizada por ser uma alga adaptada à sombra, exibiria uma resposta distinta com uma maior sensibilidade à exposição à UV. Os principais resultados e conclusões encontrados foram: i) este é o primeiro trabalho que reportou não somente os efeitos da radiação UV sobre o desempenho fotossintético de macroalgas tropicais, bem como descreveu a presença de MAAs em duas espécies de algas vermelhas de água doce; ii) O gametófito de K. ambigua respondeu com um aumento da sua performance fotossintética, além da presença de shinorina (MAA) sob UVA, sugerindo que é menos sensível à radiação UV, particularmente UVA, em comparação com o esporófito em condições de cultura, além de indicar que a radiação UVA, quando moderada, tem efeitos benéficos em alguns organismos aquáticos, tais como o gametófito de K. ambigua; iii) Espécimes coletados em condições naturais de S. delicatula apresentaram dois tipos de MAAs (shinorina e palatina) e foram praticamente insensíveis aos tratamentos com radiação UV, sugerindo que não somente a presença de MAAs é um importante escudo de proteção contra radiação UV, mas também demonstra que apesar de ser uma rodófita tipicamente adaptada à sombra, pode tolerar altas irradiâncias, incluindo-se a radiação UV; iv) C. glomerata apresentou uma diminuição mais acentuada da fotossíntese em UVA em relação a UVAB, apesar de menor teor de clorofila a ter sido observado em UVAB; v) Spirogyra sp. apresentou uma diminuição da performance fotossintética em PAR, sugerindo que a radiação UVAB deve ser importante na indução de fotoreparo e/ou uma fonte energia para fotossíntese; vi) Apesar de serem algas verdes adaptadas ao sol, C. glomerata e Spirogyra sp., apresentaram respostas distintas no desempenho fotossintético e concentrações de clorofila a sob exposição da radiação UV, indicando que elas utilizam estratégias diferentes para lidar com a radiação UV; vii) A ausência de MAAs e de scitoneminas e a pouca variação do desempenho fotossintético indicam que L. cf. henningsii foi insensível à exposição a UV nas condições testadas, e que o aumento do conteúdo de carotenóides sob UVA e UVAB sugerem que estes pigmentos atuaram como escudo eficaz para a radiação UV; viii) Scytonema sp. apresentou um aumento da atividade fotossintética em UVA, sinalizando que também é capaz de usar UVA como fonte de energia para a fotossíntese, e/ou em mecanismos de reparo relacionados ao DNA. Em contrapartida, observou-se uma diminuição do desempenho fotossintético em UVAB, acompanhada de maiores concentrações de MAAs, sugerindo que estes pigmentos não foram suficientemente eficazes para evitar os efeitos negativos da radiação UV. Em síntese, as duas hipóteses iniciais foram refutadas: a primeira, pois nem sempre as algas responderam com tendência à diminuição de sua performance fotossintética ou de produção de substâncias fotoprotetoras à radiação ultravioleta. A segunda, pois as duas espécies de algas verdes, C. glomerata e Spirogyra sp., típicas algas adaptadas ao sol, não mostraram ser mais tolerantes à radiação UV em relação às algas vermelhas, consideradas adaptadas à sombra. Além disso, as respostas foram espécie-específicas, como exemplificado pelas duas espécies de cianobactérias (Leptolyngbya cf. henningsii e Scytonema sp.) e entre os dois estágios de vida de K. ambigua. Estes resultados são muito relevantes por representarem a primeira abordagem para compreensão da adaptação e aclimatação fisiológica de macroalgas tropicais às mudanças globais do clima, notadamente radiação UV. / The history of human on planet Earth has been marked by intense negative anthropogenic environmental impacts, which enhanced the exposure to ultraviolet (UV) radiation the Earth's surface due to depletion of the ozone layer caused by the release of substances, such as, CFCs (chlorofluorocarbons). UV radiation has been considered as one of the main factors that affect the distribution of photosynthetic organisms in aquatic environments, with varied biological effects, such as, damage to DNA and protein molecules, alteration of photosynthetic pigments, changes in photosynthetic parameters and growth of these organisms. Changes in the incidence of UV radiation on the Earth's surface can lead to ecophysiological alterations in algal communities. In this context, the objective of this investigation was to analyze the physiological responses (photosynthetic parameters, concentration of photosynthetic pigments and photoprotective substances) to ultraviolet radiation (UVA and UVAB) in inland macroalgae (lotic and aerophytic habitats). The general idea was to evaluate the level of response (trend of increase or decrease to greater or lesser degree) in photosynthetic performance to ultraviolet exposure. Two general hypotheses were formulated: 1) the responses of macroalgae will essentially consist of a trend to decrease in their physiological performance (photosynthetic parameters and pigment concentration) and increase photoprotective substances under UV exposure; 2) the response level will be different among algal groups, with green algae (typically sun-adapted algae) expected to be more tolerant to UV radiation, whereas red algae (typically shaded-adapted algae) to be more sensitive and the cyanobacteria having intermediate responses. Specific hypotheses were formulated for each part of the work (chapters). Experiments were performed under three conditions: PAR only (400-700nm); PAR + UVA (320-700nm), UVA; PAR + UVA + UVB (280-700nm), UVAB. The thesis was divided into three chapters with their respective objectives and hypotheses: 1) “UV-radiation effects on photosynthesis and photoprotection in gametophytic and sporophytic stages of the freshwater red alga Kumanoa ambigua (Rhodophyta, Batrachospermales)”. The aim was to analyze the photosynthetic performance of gametophytic and sporophytic (‘Chantransia’) stages of Kumanoa ambigua in culture under UV radiation. We hypothesized that the life history stages of K. ambigua would exhibit different photosynthetic responses to UV exposure; 2) “UV-radiation effects on photosynthesis and photoprotection of two species of Cyanobacteria”. The aim was to analyze the photosynthetic performance (parameters derived of chlorophyll fluorescence), pigment concentrations (chlorophyll a and carotenoids) and the presence of UV-absorbing compounds (mycosporine-like aminoacids - MAAs and scytonemin) in two terrestrial cyanobacteria, Leptolyngbya cf. henningsii and Scytonema sp. in response to UV radiation exposure under culture conditions. It was hypothesized that the responses to UV exposure would be similar in both species for photosynthetic performance but they would differ in terms of pigment concentrations and UV-absorbing compounds; 3) “UV-radiation effects on photosynthesis in three species of tropical lotic macroalgae”. The aim was to analyze the photosynthetic performance (parameters derived of chlorophyll fluorescence), chlorophyll a concentrations and the presence of UV-absorbing compounds (mycosporine-like aminoacids - MAAs) in three tropical lotic macroalgae, Cladophora glomerata (Chlorophyta), Spirogyra sp. (Streptophyta) and Sirodotia delicatula (Rhodophyta) collected in natural conditions in response to UV radiation exposure under laboratory conditions. We hypothesized that Spirogyra sp. and C. glomerata, typical sun-adapted algae, would present similar photosynthetic performance with slight responses to UV exposure, while S. delicatula, a typical shaded-adapted alga, would exhibit a distinct strategy with higher sensitivity to UV exposure. The main results and conclusions were: i) this is the first work that reported not only the effects of UV radiation on the photosynthetic performance of tropical macroalgae, but also described the presence of MAAs in two species of freshwater red algae; ii) the increase of photosynthetic performance and the presence of shinorine (MAA) under UVA, suggests that gametophyte of K. ambigua is less sensitive to UV radiation, particularly UV in comparison to the sporophyte under culture conditions. In addition, UVA radiation, in moderate levels, could have beneficial effects in some aquatic organisms, such as the gametophyte of K. ambigua; iii) specimens collected under natural conditions of S. delicatula had two types of MAAs (shinorine and palythine) and were practically insensitive to treatments with UV radiation. These results suggest that not only the presence of MAAs is an important protection shield against UV radiation, but also demonstrates that some Rhodophyta, typically shaded-adapted algae, can tolerate high irradiances, including UV radiation; iv) C. glomerata presented a more pronounced decrease of photosynthesis under UVA in relation to UVAB, although a lower chlorophyll a content has been observed in UVAB; v) Spirogyra sp. had a decrease in photosynthetic performance in PAR, suggesting that UVAB radiation should be important in the induction of photorepair and/or as energy source to photosynthesis; vi) C. glomerata and Spirogyra sp., although considered typical sun-adapted algae, presented distinct responses on photosynthetic performance and chlorophyll concentrations under UV exposure, indicating that they use different strategies to deal with UV radiation; vii) the lack of MAAs and scitonemins and the weak responses of photosynthetic performances indicate that L. cf. henningsii was insensitive to UV exposure under the conditions tested. In addition, the increased carotenoid content under UVA and UVAB suggests that these pigments act as effective shield for UV radiation; viii) Scytonema sp. showed an increase in photosynthetic activity under UVA, signaling that it is also capable to use UVA as a source of energy for photosynthesis, and/or in repair mechanisms of DNA. In contrast, a decrease in the photosynthetic performance in UVAB was observed, although associated to higher concentrations of MAAs, suggested that these pigments were not effective enough to avoid the negative effects of UV radiation. In summary, the two initial hypotheses were rejected: the first, because the algal species not always responded with a trend to decrease of their physiological performances or to produce photoprotective substances under UV radiation. The second, because the two green algae, C. glomerata and Spirogyra sp., typical sun-adapted, were not shown to be more tolerant to UV radiation in comparison to the red algae, considered as shade-adapted. In addition, the responses were species-specific as clearly demonstrated by the two cyanobacterial species (Leptolyngbya cf. henningsii e Scytonema sp.) and between the two stages of K. ambigua. These results are relevant to be a first approach to a understanding of the adaptation and physiological acclimation of tropical macroalgae to global climate changes, notably UV radiation.

Aminoácidos tipo micosporinas (MAAs) são compostos responsáveis pela fotoproteção no ultravioleta de diversos organismos aquáticos. São sintetizados pela via do ácido chiquímico por algas, bactérias e fungos, de maneira similar à síntese de flavonóides em plantas superiores. Neste trabalho foram conduzidos estudos relacionados a estes compostos em algas. Protocolos de extração de diferentes algas foram testados alterando-se parâmetros como solventes, temperatura e condições de incubação. Os resultados mostraram que dependendo da alga estudada, diferentes condições podem mudar a concentração de MAAs extraída, ressaltando a importância de se testar diversos parâmetros na extração, evitando assim valores sub- ou superestimados de concentrações. O desenvolvimento de um método por HPLC permitiu a separação de 6 MAAs com boa resolução. A caracterização estrutural foi realizada majoritariamente por espectrometria de massas utilizando diferentes tipos de analisadores. Estas análises permitiram a proposição de mecanismos de fragmentação descritos pela primeira vez para esta classe de compostos e possibilitaram a identificação de diferentes MAAs em algumas micro e macroalgas. Ensaios in vitro foram realizados com o extrato obtido da macroalga Gracilaria domingensis no intuito de avaliar seu potencial uso em formulações cosméticas direcionadas à fotoproteção. Os testes de estabilidade quanto ao pH, temperatura e exposição à radiação ultravioleta bem como os ensaios de citotoxicidade, fototoxicidade e avaliação do fator de proteção solar sugeriram que este extrato pode ser promissor quando incorporado em formulações direcionadas para a fotoproteção. Este extrato não apresentou atividade antioxidante significativa. Os estudos com o dinoflagelado Prorocentrum minimum isolado de duas regiões diferentes, quando exposto às radiações ultravioleta, mostraram que houve uma indução das MAAs em ambas linhagens em todos os tratamentos realizados no período de 72 h. A indução foi mais rápida e pronunciada na linhagem oriunda de Lisboa, em Portugal do que na linhagem proveniente de Kattegat, na Dinamarca. Estes dados estão de acordo com as características do local de origem das linhagens, uma vez que os dinoflagelados originários de Portugal em seu meio natural estavam sujeitos a maiores irradiações. Medidas do rendimento quântico do fotossistema II indicaram que a síntese e acúmulo de MAAs em P. minimum isolada de Lisboa ofereceu vantagens na proteção do sistema fotossintético e na supressão de espécies reativas de oxigênio desta microalga quando comparada aos indivíduos da mesma espécie de regiões com menores irradiações. / Mycosporine-like amino acids (MAAs) are chemically related water soluble compounds responsible for UV photoprotection in many aquatic organisms. They are biosynthesized via the shikimate pathway by algae, bacteria and fungi in a similar manner to the biosynthesis of UV-screening flavonoids in terrestrial plants. In this work, studies related to this class of compounds were performed with algae. Extraction protocols of some algae were tested using diverse solvents at different temperature. The results showed that depending on the organism examined, both solvent concentration and temperature affected extraction efficiency and final MAA concentration. The improvement of a HPLC method separated a mix of 6 MAAs with good resolution. The structural characterization was made by mass spectrometry using different analyzers. The analysis by mass spectrometry allowed the proposition of new mechanisms of fragmentation and identification of different MAAs in some micro and macroalgae. In vitro preliminary assays were performed to evaluate the potential use of Gracilaria domingensis extract in suncare products. The extract showed no antioxidant activity, however the pH, temperature and UV exposure stability, as well the citotoxicity, phototoxicity and sun protection factor showed a potential commercial utilization for the extracts. The UV exposure experiments with the dinoflagellate Prorocentrum minimum isolated from Lisbon, Portugal and Kattegat, Denmark, showed that MAAs were induced in all treatments during 72 h. The induction was faster in the species from Lisbon than the ones from Kattegat. These data are in agreement with local isolation place, since the dinoflagellates from Portugal are submitted to high irradiance regime in their natural enviroment. The quantum yield of photosystem II suggested that the over production of MAAs by P. Minimum from Lisbon protects the photosynthetic apparatus, indicating that MAAs act as spectrally specific UV sunscreens in phytoplankton.

Ultraviolet light is damaging but fish have evolved protective mechanisms, which allows them to live in shallow water reefs, high in UV radiation. This thesis details my investigation into the physiological ecology of solar ultraviolet (UV) absorbing compounds, known as mycosporine-like amino acids found in the external epithelial mucus, and examines the supporting role potentially played by a UV-induced DNA repair mechanism in coral reef fish of the Indo-Pacific. Using reverse phase chromatography and UV spectrophotometry, I examined whether the distribution of MAA compounds across different areas of the body is correlated with differential UV exposure. Comparisons were made between the MAA content and the absorbance spectra of mucus from the dorsal, ventral, caudal and head body surface areas in five species of Scaridae (Chlorurus sordidus, Scarus schlegeli, S. niger, S. psittacus and S. globiceps) from Ningaloo Reef, Coral Bay, Western Australia. All fish analysed had at least five MAAs present, and results showed that fish had increased UV absorbance in mucus over the dorsal area, which receives the brunt of UV radiation. Little UV protection was found in mucus from the ventral area, which receives the lower level of UV radiation mostly via reflection of the sand and reef surfaces. Furthermore, UV absorbance per mg dry mucus versus standard fish length showed that there is a positive relationship in C. sordidus with increasing size. I examined whether there is a difference in the quantity of UV screening compounds found in the mucus of fish along a longitudinal geographical gradient from inshore reefs (Lizard Island, Great Barrier Reef) to the outer edge reefs to oceanic reefs (Osprey Reef). MAA absorbance increased with longitudinal distance from the mainland landmass of Australia to more oligotrophic outer reefs, where UV attenuation is reduced and the ocean is more transparent to UV wavelength. I determined that fish living on inshore, more turbid reefs where UV attenuation in shallow waters is high have lower levels of MAA protection than fish from clear oceanic reefs. Furthermore, there seems to be a direct relationship between light attenuation and exposure with the quantity of protective sunscreening found in the mucus of reef fish. It is know that UV irradiation decreases with water depth and that mucus from fish with deep habitats absorbs less UV than that of fish from shallow habitats. It is unknown however, whether this UV protection is variable within the same individuals and if so, how fast changes 11 occur. To test this, I relocated 9 ambon damselfish from a deep reef (18 m) to a shallow reef (1.5 m) to expose fish to increased levels of UV and relocated another 7 fish from a shallow to a deep reef to expose fish to decreased levels of UV. One week after relocation, all fish were returned to their original reef site to determine whether MAA levels would return to their initial levels. Fish relocated to a shallower depth were recovered and had a 60% (SD+/-2%) increase in mucus UV absorbance. Conversely, the fish relocated to a deeper depth were recovered and had a 41% (SD+/-1%) decrease mucus UV absorbance. No difference was found between UV absorbance of relocated and original fish at both depth. Six days after fish were returned to their original reef, mucus UV absorbance levels had returned to 67% +/- 4% of the original level. These results show that mucus UV absorbance is variable in individual ambon damselfish and that the sunscreen protection typical for a certain depth is reached in relocated fish within just a few days of relocation. The rate of MAA loss is higher than the accumulation of MAAs suggesting that diet is not the sole determining factor involved in the sequestration of MAAs to mucus. The cleaner fish Labroides dimidiatus performs a mutualistic service by removing ectoparasites such as gnathiid isopods as well other dead infected tissue from its clients. Cleaner fish however are also known to feed on client mucus. The benefits of eating mucus until recently were unclear. In this study, we analysed the mucus of several cleaner fish clients to determine whether mucus feeding has a nutritional advantage over gnathiids and whether cleaner fish obtain their own MAA protection through this dietary mucus ingestion. Results show that host fish that are infected with gnathiids of poor nutritional value, in contrast to those that harbour gnathiids with higher nutritional value, continuously exude mucus that has both high nutritional value and high MAA content. These findings support the conclusion that in a competitive market for cleaners some host fish are forced to offer more than parasites to cleaners. Ultraviolet light that is not filtered by UV absorbing compounds such as MAA may still lead to DNA damage such as the formation of cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts (6-4 PPs). However, coral reef fish have alternative mechanisms to overcome UV induced damage via the photolyase DNA repair mechanisms. We experimentally demonstrated for the first time that a coral reef fish species, the moon wrasse Thalassoma lunare has the ability to repair DNA damage via photoreactivation. Fish both with and without MAA protection were irradiated with UVB wavelength to induce DNA lesions. Half of the experimental fish were then exposed to photoreactivating wavelength to induce DNA repair 12 while the other fish were blocked from the repair mechanisms. Fish which had undergone DNA repair had the lowest number of lesions regardless of mucus MAA protection. When fish were blocked from photoreactivation wavelengths MAA sunscreens clearly served a photoprotective role. The amount of damage was greatest in fish which both lacked MAAs and which were also blocked from photoreactivating wavelengths. Thus for the overall UV protection of fish both the MAA sunscreens as well as the DNA repair system play a significant role in counteracting UV damage. Ultraviolet protection by MAA sunscreens is ubiquitous in marine fish. To date the same 5 MAA compounds (palythine (λmax 320 nm), asterina (λmax 330 nm), palythinol (λmax 332 nm), usujirene (λmax 357 nm) and palythene (λmax 360nm) have been identified in the mucus of several different species of reef fish from Australia. Here we report the first evidence of the presence of additional UV absorbing compounds found in the mucus of fish from Indonesia. Using UV spectroscopy the mucus of four species of fish was compared between both geographical regions. The presence of an additional peak between 294-296 nm wavelengths suggests the presence of gadusol and/or deoxygadusol, which are photoprotective compounds, thought to be the precursors of MAAs. Thus, UV protecting compounds in the mucus of fish may not be as conserved between different regions as previously assumed. Our knowledge concerning the effect of UV radiation has advanced considerably in the past decade and my research findings contribute to the better understanding of protective mechanisms of marine fish. The correlations I have found between UV attenuation/exposure, depth, and longitude of sampled individuals lead me to believe that mucus UV absorbing MAA compounds are a highly efficient adaptive defence.