A análise química do extrato bruto de esponja marinha é um método clássico de descoberta de novas drogas. Metabólitos secundários e peptídeos são geralmente os resultados neste tipo de pesquisa, utilizando diferentes e consecutivos protocolos de purificação. Neste estudo de uma amostra de extrato aquoso esponja separada numa coluna Sephadex®-G25 foi purificado por HPLC, numa tentativa de isolar uma única molécula. Com uma base de dados de RNA e a análise in silico espera-se que o RNAm pode ser relacionado com a molécula precursora do composto bioativo ou mesmo o seu gene. A dissociação de células a partir da matriz da esponja é um método importante para a extração de RNA optimizado em quantidade e qualidade. Frações extrato bruto do organismo e dados de RNAm a partir de células isoladas da esponja podem ser comparados por uma abordagem de comparação dos dados. Ensaios de MTS foram utilizados para analisar a bioatividade e microscopia confocal foi a principal ferramenta para examinar os danos em células tumorais (T47D). / The chemical analysis of sea sponge crude extract is a classic method in drug discovery. Secondary metabolites and peptides are usually the results in this type of research using different and consecutives purification protocols. In this study a sample of sponge aqueous extract separated in a sephadex-G25 column was purified by HPLC in an attempt to isolate a single molecule. With a RNA databank and in silico analysis is expected that the mRNA may be related with the precursor molecule of the bioactive compound or even its gene. The dissociation of cells from the sponge matrix was an important method to the optimized RNA extraction in quantity and quality. Crude extract fractions of the organism and mRNA data from sponge-isolated cells were obtained to a next step cross-data approach. MTS assays were used to analyze the bioactivity and confocal microscopy was the main tool to scan the damage in tumor cells (T47D).

A análise química do extrato bruto de esponja marinha é um método clássico de descoberta de novas drogas. Metabólitos secundários e peptídeos são geralmente os resultados neste tipo de pesquisa, utilizando diferentes e consecutivos protocolos de purificação. Neste estudo de uma amostra de extrato aquoso esponja separada numa coluna Sephadex®-G25 foi purificado por HPLC, numa tentativa de isolar uma única molécula. Com uma base de dados de RNA e a análise in silico espera-se que o RNAm pode ser relacionado com a molécula precursora do composto bioativo ou mesmo o seu gene. A dissociação de células a partir da matriz da esponja é um método importante para a extração de RNA optimizado em quantidade e qualidade. Frações extrato bruto do organismo e dados de RNAm a partir de células isoladas da esponja podem ser comparados por uma abordagem de comparação dos dados. Ensaios de MTS foram utilizados para analisar a bioatividade e microscopia confocal foi a principal ferramenta para examinar os danos em células tumorais (T47D). / The chemical analysis of sea sponge crude extract is a classic method in drug discovery. Secondary metabolites and peptides are usually the results in this type of research using different and consecutives purification protocols. In this study a sample of sponge aqueous extract separated in a sephadex-G25 column was purified by HPLC in an attempt to isolate a single molecule. With a RNA databank and in silico analysis is expected that the mRNA may be related with the precursor molecule of the bioactive compound or even its gene. The dissociation of cells from the sponge matrix was an important method to the optimized RNA extraction in quantity and quality. Crude extract fractions of the organism and mRNA data from sponge-isolated cells were obtained to a next step cross-data approach. MTS assays were used to analyze the bioactivity and confocal microscopy was the main tool to scan the damage in tumor cells (T47D).

Submitted by Alberto Vieira (martins_vieira@ibest.com.br) on 2017-10-18T22:14:47Z No. of bitstreams: 1 618387.pdf: 5097477 bytes, checksum: b8518d78d9bd177997c0fd650714809f (MD5) / Made available in DSpace on 2017-10-18T22:14:47Z (GMT). No. of bitstreams: 1 618387.pdf: 5097477 bytes, checksum: b8518d78d9bd177997c0fd650714809f (MD5) Previous issue date: 2004 / PETROBRAS / A taxonomia de Amphimedon é complicada devido à sua ampla distribuição e abundância de morfotipos. O objetivo deste trabalho foi caracterizar as espécies de Amphimedon da costa brasileira pelo estudo combinado de técnicas morfológicas e de polimorfismos do DNA. Foram utilizadas amostras provenientes da coleção do Museu Nacional (MN-UFRJ) de Amphimedon viridis, A. compressa e A. aff. compressa. O DNA genômico foi extraído e as regiões dos espaçadores internos transcritos (ITS-1 e ITS-2) dos genes que codificam para o RNA ribossomal (RNAr) nuclear foram amplificados. Após a amplificação, as amostras foram submetidas à técnica do polimorfismo de Conformação de Fita Simples (SSCP). Os resultados apresentados apoiam a ideia de que A. aff. compressa inclui espécies distintas quando utilizamos a regiões de ITS1 como caracter molecular. Do mesmo modo, estes resultados indicam a possibilidade das Amphimedon viridis da região Nordeste e Sudeste serem coespecíficas, ocorrendo homogeneamente ao longo da costa brasileira. Nossos resultados permitiram a diferenciação das espécies de Amphimedon pela técnica conjugada de PCR-SSCP, demonstrando a viabilidade deste método na distinção de espécies de esponjas marinhas. / The taxonomy of Amphimedon is complicated due to its wide distribution and abundance of morphotypes. The objective of this work was to characterize the species of Amphimedon from the Brazilian Coast by the combined study of morphology and DNA polymorphisms. The samples utilized were derived from the collections of Museu Nacional (MNRJ, and comprised Amphimedon viridis, A. compressa e A. aff. compressa. The genomic DNA was extracted and the regions of the internal transcribed spacers (ITS-1 and ITS-2) coding for nuclear ribossomal RNA were amplified. After amplifications, samples were subjected to the Single Strand Conformation Polimorphism (SSCP) technique. The individuals of Amphimedon viridis show a similar ITS 1 migration pattern, even when coming from distant collecting localities on the Brazilian Coast. The results presented support the idea that A. aff. compressa comprises distinct species when we utilize ITS 1 regions as a molecular character. In the same way, these results indicate the possibility that A. viridis from the northeastern and southeastern Brazilian regions are conspecific, occurring homogeneously along the Brazilian coast. Our results enabled the differentiation of Amphimedon species through the combined PCR-SSCP technique, showing the potential use of this method for the distinction of species of marine sponges.

Les travaux de thèse exposés dans ce manuscrit portent sur l'étude chimique de quatre éponges marines collectées à Madagascar : Monanchora unguiculata, Amphimedon sp., Aulospongus gardineri et Biemna laboutei. Pour les trois premières, l'extraction, l'isolement et l'identification des métabolites secondaires par différentes techniques chromatographiques (CLMP, CLHP…) et spectroscopiques (UV-visible, SMHR, RMN 1D et 2D…) ont été envisagés. Quinze métabolites secondaires de type alcaloïdes guanidiniques, dérivés de la bromotyrosine, N-acyléthanolamines et polyynes, ont été isolés de ces éponges. Huit sont de structures nouvelles. La valorisation des molécules isolées a ensuite été envisagée via l'évaluation de leurs activités biologiques. Deux d'entres elles, la fromiamycaline (M70) et la ptilomycaline F (MU7) ont montré une excellente activité antipaludique et quatre, la fromiamycaline (M70) et les ptilomycalines E (MU6), G (MU8) et H (MU9) une cytotoxicité sur cellules KB prometteuse. L'étude de la dernière éponge, Biemna laboutei, portait sur des molécules précédemment isolées au sein du LCSNSA. Les travaux entrepris comprenaient une vérification des configurations relatives de neuf alcaloïdes guanidiniques déterminées par RMN grâce à la probabilité DP4+. Les configurations relatives déterminées par RMN de sept des molécules ont ainsi pu être confirmées et trois d'entre elles, les nétamines G (E8), P (E17) et R (E19) ont fait l'objet d'une étude plus approfondie par modélisation moléculaire afin de déterminer leur configuration absolue par comparaison de leur spectre de Dichroïsme Circulaire Électronique expérimental avec un spectre calculé. / The work described in this manuscript concerns the chemical study of four sponges from Madagascar: Monanchora unguiculata, Amphimedon sp., Aulospongus gardineri and Biemna laboutei. For the first three sponges, the study was devoted to the extraction, isolation and identification of secondary metabolites by various chromatographic (MPLC, HPLC…) and spectroscopic (UV, HRMS, 1D and 2D NMR…) techniques. Fitfteen secondary metabolites including guanidine alkaloids, bromotyrosin derivated, N-acylethanolamines and polyynes were isolated from these sponges. Eight are new molecules. The biological activities of the isolated compounds were then evaluated. Two of them, fromiamycalin (M70) and ptilomycalin F (MU7) showed a very good antimalarial activity and four of them, fromiamycalin (M70) and ptilomycalins E (MU6), G (MU8) and H (MU9) a promising cytotoxicity against KB cells. The study of the last sponge, Biemna laboutei, was performed on molecules already isolated in the LCSNSA. This work included the determination of the relative configuration of nine guanidine alkaloids by using the DP4+ probability. The relative configuration of seven molecules was confirmed. Three of them, netamines G (E8), P (E17) and R (E19) were selected for a study by molecular modeling to determine their absolute configuration by comparison of their experimental Electronic Circular Dichroism spectrum with a calculated spectrum.

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.