Made available in DSpace on 2015-05-14T12:59:39Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 5219644 bytes, checksum: 21dfe60a9b7bfb0358d99227ad19922d (MD5) Previous issue date: 2012-08-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Algae and marine sponges have been the targets of important scientific researches in the last 50 years, with a large number of secondary metabolites isolated and identified, showing a wide structural variety where certain compounds are useful in characterizing classes of algae and sponges fact of great importance for chemists, taxonomists and ecologists. These substances exhibit many biological activities, such as defense against consumers and competitors, and colonial and pathogen organisms; or they display reproductive function. This thesis describes the results from the chemical study of Sargassum polyceratium Montagne, a seaweed from the Sargassaceae family, and Aplysina fistularis Pallas, a marine sponge from the Aplysinidae family. Both species had their chemical constituents identified by spectroscopic methods such as IR, mass and 1H and 13C-1D and -2D NMR, and by comparison with literature. The following porphirinic derivatives were isolated from S. polyceratium: 132-hydroxy-(132-R)-phaephytin a, 132-hydroxy-(132-S)-phaeophytin a,phaeophytin a and the steroid fucosterol. The chemical study of A. fistularis led us to the reisolation of the alkaloid and acetamide: 2-(3,5-dibromo 4methoxypheny,N,N,Ntrimethylethanamonium and (3,5-dibromo-4-ethoxy-1-hydroxy-4-methoxycyclohexa-2,5-dienyl) acetamide, and to the isolation of alkaloid 3,5-dibromo-4-[3 dimethylamonium]propoxyphenyl]-N,N,N trimethylethanamonium (aplysfistularine),reported for the first time. The ethanolic extract from S. polyceratium was evaluated for psicopharmacological effects, showing behavioral changes of CNS depressant type. The ethanolic, hexanic and dichloromethanic extracts as well as the mixture (132-hydroxy-(132-R)-phaephytin a and 132-hydroxy-(132-S)-phaeophytin a) were not capable of inhibiting the growth of the tested lineages, showing negative antimicrobial activity. However they were capable of modifying the antibiotic activity, modulating the bacterial resistance between 2x to 4x. The isolated compounds from A. fistularis show inhibition of the DNA enzyme Topoisomerase II-α, with further biological studies needed for determining the compounds potency. Therefore, the present results contribute for the chemical study of marine organisms from species S. polyceratium and A. fistularis. / As algas e as esponjas marinhas têm sido alvos de pesquisas científicas importantes nos últimos 50 anos, de onde foram isolados e caracterizados um grande número de metabólitos secundários, com ampla variedade estrutural, determinadas substâncias podem caracterizar as classes de algas e esponjas, fato de grande importância para os químicos, taxonomistas e ecologistas. Estas substâncias exercem inúmeras atividades biológicas, como: defesa contra consumidores, competidores, organismos coloniais e patógenos ou apresentam função reprodutiva. Este trabalho descreve os resultados do estudo químico dos organismos marinhos Sargassum polyceratium Montagne e Aplysina fistularis Pallas, uma alga marinha da família Sargassaceae e uma esponja marinha da família Aplysinidae, respectivamente. Os constituintes químicos foram identificados através da análise de dados obtidos por métodos espectroscópicos como infravermelho, massas e ressonância magnética nuclear de 1H e 13Cuni e bidimensionais, além de comparação com valores da literatura. Da espécie S. polyceratium foram isolados os derivados porfirínicos: 132-hidroxi-(132-R)-feofitina a, 132- hidroxi-(132-S)-feofitina a, feofitina a e o esteróide fucosterol. O estudo químico da A.fistularis resultou no reisolamento do alcaloide e da acetamida: 2-(3,5-dibromo-4- metoxifenil)-N,N,N-trimetiletanamônio e (3,5-dibromo-4-etoxi-1-hidroxi-4-metoxicicloexa-2,5-dienila) acetamida, e no isolamento do alcaloide 3,5-dibromo-4-[3 dimetilamônio]propoxifenil]-N,N,N-trimetiletanamônio (aplysfistularina), aqui descrito pela primeira vez. A avaliação dos efeitos psicofarmacológicos do extrato etanólico do S. polyceratium induziu alterações comportamentais, do tipo depressora do SNC. Os extratos etanólico, hexânico, diclorometânico e a mistura (132-hidroxi-(132-R)-feofitina a e 132-hidroxi-(132-S)-feofitina a) não foram capazes de inibir o crescimento das linhagens testadas, apresentando atividade antimicrobiana negativa. Porém foram capazes de modificar a atividade antibiótica, diminuindo entre 2x à 4x a resistência bacteriana. As substâncias isoladas da A. fistularis mostraram inibição da enzima DNA Topoisomerase II-α, sendo necessárias outras avaliações biológicas para determinar a potência dos compostos. Assim, os resultados obtidos neste trabalho contribuíram para o estudo químico dos organismos marinhos das espécies S. polyceratium e A. fistularis.

Made available in DSpace on 2015-04-01T14:16:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1615747 bytes, checksum: 2bbe77aa39b7f33d5f7b0781f235bb2d (MD5) Previous issue date: 2014-08-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A new lectin was purified and characterized from marine sponge Aplysina fulva (AFL). The crude extract was obtained with Tris HCl 0.1 M pH 7.4 containing NaCl 0.15 M buffer. Haemagglutinating activity detection and soluble protein measurement were performed with the crude extract and after each purification step. Isolation of AFL was performed by affinity chromatography in Sepharose CL 4B column, and the retained peak was applied in a DEAE Sephacel ion exchange chromatography column. Analysis of DEAE Sephacel retained peak on polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of a single band with approximately 27.5 kDa. Under non-denaturating conditions it shows the presence of a single 57 kDa band. The novel lectin showed large amount of hydrophobic amino acids, high temperatures resistance and better hemagglutination activity in the range of neutral to alkaline pHs. AFL was able to inhibit the growth of strains of P. aeruginosa, C. albicans and C. tropicalis. The lectin did not show toxic effects on cells of breast cancer, but had agglutination effect on Leishmania brasiliensis promastigotes by recognizing galactose residues on its surface. / Uma nova lectina foi purificada e caracterizada a partir da esponja marinha Aplysina fulva (AFL). O extrato bruto foi produzido a partir da solubilização de proteínas em solução tampão Tris HCl 0,1 M pH 7,4 NaCl 0,15 M, sendo este utilizado na realização dos ensaios de atividade hemaglutinante e dosagem de proteínas pelo método de Bradford. O primeiro passo de purificação de AFL foi por meio de cromatografia de afinidade em coluna de Sepharose CL 4B. O pico não retido foi eluido com o mesmo tampão de extração e o pico retido foi eluido com tampão Glicina 0,1 M pH 2,6 NaCl 0,15 M. O mesmo foi dialisado, liofilizado e ressuspendido em Tris HCl 0,025 M pH 7,6 e submetido a cromatografia de troca iônica em DEAE Sephacel. A análise do pico retido da DEAE Sephacel por eletroforese nativa em gel de poliacrilamida (PAGE) mostrou a presença de uma única banda com aproximadamente 57 kDa, como também uma única banda com aproximadamente em 27,5 kDa em presença de SDS (PAGE-SDS). A nova lectina apresenta uma grande quantidade de aminoácidos hidrofóbicos, mostrando-se resistente a altas temperaturas e com melhor atividade hemaglutinante na faixa de pH neutro a alcalino. AFL foi capaz de inibir o crescimento de cepas de P. aeruginosa, C. albicans e C. tropicalis. A nova lectina não apresentou efeitos tóxicos para células de câncer de mama, entretanto mostrou-se hábil em aglutinar formas promastigotas de Leishmania brasiliensis através do reconhecimento de galactose em sua superfície.

A common injury type associated with vessel groundings on coral reefs is generation of coral rubble. As a substrate rubble is dynamic and can be mobilized by normal wave agitation, causing secondary damage to surviving corals and preventing coral larvae and invertebrates from surviving. Typical rubble management options include offsite removal and onsite stabilization using artificial materials. Sponges show promise as a restoration tool for rubble stabilization, especially sponges that are known to have rapid regeneration rates, low mortality, and the capability to quickly form attachment points to natural and artificial substrates. As an effective restoration tool a sustainable source of sponge is required in order to minimize impact on natural populations. The purpose of this project is to evaluate the feasibility of sponge propagation for use as a restoration material following groundings on coral reefs in Biscayne National Park, FL. If small sponge fragments could be collected from donor sponges and propagated on artificial growout structures (i.e. nurseries) a sustainable source of sponge fragments could be maintained for future restoration activities. If feasible, this technique would create a continually growing sponge source that would require a single harvest, reducing pressure on the local sponge populations. Fragments of four species of native sponges, Aplysina fulva¸ Amphimedon compressa, Aplysina cauliformis, and Iotrochota birotulata, were harvested and attached to artificial growout structures composed of PVC rods and cement paver tiles. Fragment necrosis, attachment, sponge retention (persistence throughout the study), complexity, and growth rates for all four species were recorded monthly for one year. Two different PVC types were utilized to compare attachment material effects on growth. Donor sponges were monitored to assess recovery and growth after harvesting, and intact sponges in the natural population were also monitored as a control. Transplanted fragments had the highest retention of any treatment (96-100% of fragments remained), growth rates ranging from 105% to 424%, low necrosis rates, and all but one species increased in complexity. There was no significant difference in sponge growth on different PVC types or between sites. Within one month all donor wounds inflicted by harvest were completely healed, and by the end of the study donors had significantly higher growth rates than the control treatment, ranging from 400% to 781% of initial sponge volume. Donor sponges also had low necrosis rates, but were less complex than controls. This is a promising, practical and low cost method, and based on these results, is a feasible way of producing sponges for us in restoration, with little negative impact on the natural populations.

Die Schwammskelette der Ordnung Verongida zeichnen sich durch das Fehlen mineralischer Komponenten aus. Stattdessen bestehen sie aus Spongin, einem kollagenartigen Protein, und Chitin. Im Rahmen der vorliegenden Arbeit wurden die aus solch einem Chitin-Protein-Komplex bestehenden Skelette der Schwammspezies Aplysina cavernicola und Ianthella basta aus der Ordnung Verongida untersucht. Aufgrund ihrer morphologischen Unterschiede wurde für jede Schwammart eine eigene Methode zur Isolierung der Schwammskelette entwickelt. Die isolierten Skelette konnten anschließend mit verschiedenen Methoden wie REM, ATR-FTIR-Spektroskopie und NMR-Spektroskopie charakterisiert werden. Weiterhin wurde eine Methode zur Extraktion und Analyse der in den Skeletten befindlichen Aminosäuren mittels GC-MS entwickelt. Die Untersuchungen zeigten deutlich, dass es sich bei Spongin um ein kollagenartiges und halogeniertes Protein handelt, welches je nach Schwammart Unterschiede in der Aminosäurezusammensetzung aufweist. Darüber hinaus gelang es zum ersten Mal das Chitin aus dem Chitin-Protein-Komplex mittels Phosphorsäure zu entfernen. Aus den gewonnenen Erkenntnissen konnte abschließend ein Modell zum Aufbau des Chitin-Protein-Komplexes in der Schwammspezies Aplysina cavernicola entwickelt werden.

Die Schwammskelette der Ordnung Verongida zeichnen sich durch das Fehlen mineralischer Komponenten aus. Stattdessen bestehen sie aus Spongin, einem kollagenartigen Protein, und Chitin. Im Rahmen der vorliegenden Arbeit wurden die aus solch einem Chitin-Protein-Komplex bestehenden Skelette der Schwammspezies Aplysina cavernicola und Ianthella basta aus der Ordnung Verongida untersucht. Aufgrund ihrer morphologischen Unterschiede wurde für jede Schwammart eine eigene Methode zur Isolierung der Schwammskelette entwickelt. Die isolierten Skelette konnten anschließend mit verschiedenen Methoden wie REM, ATR-FTIR-Spektroskopie und NMR-Spektroskopie charakterisiert werden. Weiterhin wurde eine Methode zur Extraktion und Analyse der in den Skeletten befindlichen Aminosäuren mittels GC-MS entwickelt. Die Untersuchungen zeigten deutlich, dass es sich bei Spongin um ein kollagenartiges und halogeniertes Protein handelt, welches je nach Schwammart Unterschiede in der Aminosäurezusammensetzung aufweist. Darüber hinaus gelang es zum ersten Mal das Chitin aus dem Chitin-Protein-Komplex mittels Phosphorsäure zu entfernen. Aus den gewonnenen Erkenntnissen konnte abschließend ein Modell zum Aufbau des Chitin-Protein-Komplexes in der Schwammspezies Aplysina cavernicola entwickelt werden.