O extrato metanólico da esponja Darwinella sp. coletada na costa do Rio do Janeiro, exibiu atividade leishmanicida. O extrato bruto foi particionado em três frações: fração hexânica, fração de AcOEt e fração XAD (extração da fração orgânica do extrato aquoso mediante mistura de resinas XAD-2, XAD-4 e XAD-7). Cada uma das frações foi avaliada em bioensaio atividade leishmanicida, mostrando que a fração AcOEt apresentou 100% da morte dos parasitas. A fração de AcOEt foi fracionada usando cromatografia por exclusão de tamanho (Sephadex&reg LH-20) e extrações em fase sólida (SPE) com diferentes colunas pré-empacotadas. Os compostos foram isolados e purificados por cromatografia de alta eficiência acoplada a detector ultravioleta (CLAE-UV). Sete compostos foram isolados, dos quais quatro deles foram identificados espectroscopicamente. O membranolídeo, o éster metílico da oxeatamida A, a oxeatamida H e a oxeatina são diterpenos espongianos que possuem um esqueleto aplysulfurano nitrogenado, excetuando o membranolídeo. Tanto o éster metílico da oxeatamida A quanto a oxeatamida H, pertencem a uma série de compostos denominados de oxeatamidas. O membranolídeo, o éster metílico A e a oxeatina foram avaliados em teste de atividade antiparasitária contra Leishmania infantum, porém nenhum deles apresentou atividade leishmanicida. / The sponge Darwinella sp. was collected in Rio de Janeiro’s coast and its methanolic extract showed leishmanicidal activity. The crude extract was partitioned in three fractions: hexane, AcOEt and XAD fractions. This last one was obtained by resin adsorption (mixture of XAD-2, XAD-4 and XAD-7 resins) of organic constituents from the aqueous fraction and subsequent recovery by extraction from the resins mixture. All of three fractions were also tested in leishmanicidal bioassay. The AcOEt fraction promoted 100% of parasite death. This fraction was separated into less complex fractions by size exclusion chromatography (Sephadex&reg LH-20) and solid phase extractions (SPE). The compounds were isolated and purified by high-performance liquid chromatography (HPLC) combined with on-line UV detector. Seven substances were isolated, which four of them were spectroscopically identified. The membranolide, the oxeatamide A methyl ester, the oxeatamide H and the oxeatin are spongian diterpenes with nitrogenous aplysulphurane skeleton, except for the membranolide. The oxeatamides are a series of nitrogenous aplysulphurane metabolites that includes the oxeatamide A methyl ester and the oxeatamide H. All of these compounds, except the oxeatamide H, were evaluated for antiprotozoal activity against Leishmania infantum. None of these compounds displayed leishmanicidal activity.

The development of a new technique capable of analyzing compounds crystallographically without first needing to crystallize them has been recently described. The present thesis aims to demonstrate the potential of such a technique, which utilizes crystalline sponges, in order to regularly order guest compounds in a porous media. The structural stability of the molecular sponges, which are highly porous metal-organic frameworks (MOFs), is first investigated, revealing that the Co-based MOF, 1, undergoes two remarkable transformations. This thesis also demonstrates how the technique can be employed to visualize the motion and occupancy of gaseous guests in a MOF. The Zn-based MOF, 4, was found to physisorb and chemisorb molecular iodine, leading to the formation of a variety of polyiodide species. The flexible nature of the host was determined to be an essential component in the exceptionally large iodine uptake capacity of the MOF. These results illustrate that the crystalline sponge method can be an effective strategy for directly visualizing guest molecules and obtaining vital information on the interactions formed between the host and guest.

The giant barrel sponge, Xestospongia muta is an iconic and essential species of the coral reefs in South Florida. The sponge has primary roles providing ecosystem services and creating unique habitats for diverse microbial communities. On April 27, 2012 an outbreak of Sponge Orange Band Disease (SOB) was detected off the coast of South Florida. The disease begins with sponge bleaching, followed by mesohyl or “mesohyl” necrosis and often total mesohyl disintegration. Sampling from two diseased populations at Boynton Beach and Fort Lauderdale, FL took place on May 11th and May 29th, 2012. Each of the nine diseased sponges from Boynton Beach and the five diseased sponges from Fort Lauderdale had three separate mesophyl samples collected to examine the effects of disease progression on the microbial community. These included healthy mesohyl from a diseased sponge (HoD), the boundary layer which captured the advancing line of diseased mesohyl (BL) and diseased mesohyl from a diseased sponge (D). Mesohyl from three sponges with no visible signs of SOB disease were also collected from each sampling location to use for healthy controls (HC). Sequencing of the V4 region of the 16S rRNA gene was performed on all of these samples via the “454” pyrosequencing on a Titanium GS FLX platform. The microbial communities associated with the diseased samples revealed a microbiome shift that followed the progression of Sponge Orange Band Disease (SOB) and was dominated by Bacteroidetes, Protebacteria and Chloroflexi. No singular or group of microbes were solely found within the infected mesohyl of Xestospongia muta from both sampling site populations; therefore there is no unequivocal candidate as a definite microbial causative SOB agent. But there were bacteria associated with disease progression that included Armatimonadetes, Caldithrix, Chlorobi, Fibrobacteres, Fusobacteria, GN02, KSB3, OP1, OP2, OP8, Planctomycetes, SR1, TM6, Tenericutes, Verrucomicrobia, WPS-2 and ZB3. Verrucomicrobia and Plantomycetes increased significantly within the D and the BL populations, which was consistent within all the diseased sponges. This study provides a deep sequencing profile of microbial communities within Xestospongia muta affected with SOB Disease and provides a new insight into the sponge healthy microbiome.

Marine micro-organisms are important components of various biogeochemical cycles, complex food webs and ecological niches. Metagenomic sequencing can provide rapid profile of metabolic activities within the sponge and resident microbes. However, the study of metatranscriptomes from sponges using high throughput sequencing technology has only recently begun. Through this study we isolated, characterized and compared metatranscriptome profiles of Axinella corrugata host and sponge-specific microbial communities using 454 pyrosequencing technology. Four cDNA libraries (two eukaryotic and two prokaryotic) were generated from Axinella corrugata sponge samples collected in December 2009 and May 2010, and were characterized to a) reveal which metabolic genes were actively expressed and b) reveal possible interactions between the sponge and its microbial symbionts. The techniques used for isolation of mRNA and cDNA normalization also helped in optimization of whole-transcriptome amplification. More than 130,000 ESTs were generated for the two seasonal sponge samples and the metagenomic data sets were analyzed using bioinformatics tool, MG-RAST. Several stress-related transcripts were found which can increase our understanding of sensitivity of the sponge to changes in physical parameters in nature. The involvement of the sponge and its microbial consortia is depicted through actively expressed nitrogen and sulfur metabolism genes. Novel genes involved in several functional pathways may be discovered upon further studying hypothetical genes found across all four metagenomic data sets. Metatranscriptomic data sheds light on the functional role of microbes within the sponges and the extent of their involvement in sponge metabolism. 16S rRNA analysis was also carried out using genomic DNA of the same samples, to better elucidate the bacterial taxa abundance in the sponge. This study provides a profile of active mRNA trancripts in Axinella corrugata which include eukaryotic as well as prokaryotic sequences. The data analysis of this research provides new information at the cross-disciplinary interface between molecular biology and computational science.

Previous studies have shown that bacteria associated with coral diseases are not found in the surrounding water column at detectable levels, yet at the same time, coral diseases are becoming more prominent. Sponges are coral reef residents, which expel filtered seawater that is practically sterile of microbes. Therefore sponges harbor very diverse and abundant microbial communities. This leads to the possibility that coral disease associated bacteria (CDAB) may be present within reef sponge microcosms. In order to identify internal microbes, nonculturable techniques including fluorescent in situ hybridization (FISH), electron microscopy (EM) and 16S small subunit (SSU) rRNA gene cloning and sequencing were applied to local Florida reef sponges Agelas tubulata, Amphimedon compressa and Aplysina fistularis. This study targeted potential coral bacterial pathogens with FISH including Aurantimonas coralicida, Cytophaga sp., Desulfvibrio spp., Firmicutes, Serrattia marcescans, and Vibrio shiloni AK-1. All of the targeted coral disease associated bacteria were found within A. compressa and A. tubulata with FISH, but not in every individual. Differences in the spatial arrangement of targeted microbes were also seen within these sponge hosts. For instance, the two anaerobic bacteria Desulfovibrio spp. and S. marcescans were found in aggragates. In addition, electron microscopy revealed a higher abundance of bacteria in Applysina fistularis choanosome compared to the ectosome.

Sponges are major macrofaunal components of coral reef ecosystems, often outnumbering corals in richness, abundance and coverage. Although reefs along southeastern Florida have exhibited substantial deterioration over the last few decades and are currently under intensive scrutiny, most investigations have omitted sponges. This study focuses on reef sponge assemblages along Broward County, Florida, from 2002 through 2006 and examines changes in sponge assemblage composition, areal coverage and abundance over time. The photographic database derives from an ongoing study of stony and soft coral coverage carried out by the Nova Southeastern University (Nova Southeastern University) and includes twenty-five 20 x 2-m transects. Forty images covering 1 m2 each were taken per transect (two rows of 20) via scuba. Sponges were identified and their areal coverage measured in a total of 5,000 images using Coral Point Count with Excel extensions (CPCe) software. Data includes species counts, minimum and maximum coverage by species, transect and year, as well as total and average areal coverage by transect and year. Dominant species include Spirastrella coccinea, Niphates erecta, Amphimedon compressa and encrusting species. Comparisons among transects and years were carried out using Primer software. Stations on the Middle and Outer Reef tracts cluster together separately from those of the inshore Ridge Complex stations in all factors analyzed (areal coverage, composition, abundance). Values for the Ridge Complex stations (plus a single Inner Reef site) are much more variable than those for the Middle and Outer Reefs. Univariate analyses (ANOVA, Linear regressions) of areal coverage,richness and abundance over time reveal no temporal trends with one exception; sponge abundance on Outer Reef stations showed a significant decline over the study period. During 2005, multiple transects were affected by hurricanes Katrina, Wilma and Rita, which damaged or removed some sponges and buried others. The more common species, such as branching species appear to have recovered more rapidly, but species such as Xestospongia muta have survived the storms and maintained both numbers and coverage. Tracking of selected individual sponges in five quadrats over the study period revealed that some species (e.g., Xestospongia muta, Cliona delitrix) remained for the entire period, while some others (e.g., Niphates erecta, N. digitalis, Amphimedon compressa, Iotrochota birotulata) either appeared or disappeared between yearly images, or exhibited substantial growth (Ircinia strobilina).

The giant barrel sponge, Xestospongia muta is a prominent component in southeast Florida reef communities and is often injured from anthropogenic or natural disturbances. The resulting complete or partial shearing of X. muta barrels frequently leads to mortality of loose fragments that do not typically reattach. This study examines the degree to which sponge size and injury severity affects recovery and growth, and explores artificial reattachment success and growth of fragments. In June 2008, 65 sponges were subjected to injury treatments of approximately 50% or 90% barrel removal. A new, simple method was developed to secure these removed barrels (fragments) to the substrate. For 15 months, all donor, fragment and control sponges were monitored for survival, recovery and growth (change in height), and fragments were additionally monitored for reattachment. All injured sponges showed signs of recovery within 1 month. Within 7 months the reattachment method had already proved successful with more than 80% of all fragments becoming attached; final reattachment success after 15 months was 87%. Survival remained high for donors (97%), fragments (94%) and controls (97%). Size class and injury treatment did not significantly affect survival for donors. However, larger fragments had significantly lower survival and reduced reattachment success. Fragment growth rates were significantly lower than donor and control sponges. All sponges showed significant increase in height from post-cutting to the final monitoring. No injured donors reached pre-cut heights, however those from the smaller size class are projected to reach pre-injury heights faster than the larger size class.

Low light intensity habitats harbor unique sciophilous benthic communities and are a source of novel and unique sponge fauna. However, the community structure of these habitats is poorly studied to date. Thus, this study attempts to understand the composition and structure of sciophilous sponge populations in southeast Florida. Fifty limestone plates were placed on a shallow reef in Fort Lauderdale for two years (2010-2012). To identify the sponge community and their patterns over time, all plates were photographed at the end of each year. Then, samples were taken from each of the live sponge specimens observed on the plates and processed in the laboratory for taxonomical identification. A total of 45 different sponge species were found, the majority corresponding to the Poecilosclerida Order. Eighteen were identified to species level, twenty-two to genus, and five were undetermined. Eight sponges constitute new records to Florida, and four are potentially new species. The most dominant species include Oscarella sp.1, Dysidea etheria, Mycale sp.1, Halisarca caerula and Tedania ignis. Species richness significant varied among years, and species cover among sectors (inner and outer reef). However, sponge assemblages were similar between years with slightly variation between sectors. This study found a diverse and complex composition and structure of sponges that is quite distinct from the sponge assemblages on the open reefs. A guide of the biodiversity of cryptic sponge species was created to facilitate further studies in low light intensity habitats.

Dans cette thèse, on a étudié les propriétés rhéologiques et moussantes de phases de bicouches, de type lamellaire et éponge, en utilisant le système expérimental SDS/hexanol/saumure. On a également préparé des mousses avec ces deux phases de bicouches, dont on a étudié la stabilité et les mécanismes d’évolution dans le temps. En premier lieu, les propriétés rhéologiques de la phase éponge ont été examinées. Les échantillons ayant une fraction volumique de membrane phi ≥ 0.08 se comportent comme des fluides Newtoniens. Avec les moins concentrés, phi = 0.05 et 0.024, un changement de viscosité est observé à environ 1000 et 100 s⁻¹, respectivement. Ce changement pourrait être lié à une transition de la phase éponge vers une phase lamellaire induite par cisaillement. Les changements de viscosité qui suggèrent une transition éponge-lamellaire induite par l’écoulement ont été plus facilement détectés pour l'échantillon de fraction volumique phi = 0.024. Pour cet échantillon, il a été constaté qu’à des températures plus basses ou lors de l’ajout d’un polymère hydrosoluble (PEG), la variation de la viscosité liée à la transition de phase éponge-lamellaire se produit à des valeurs inférieures de la vitesse de cisaillement. L’étude des propriétés rhéologiques de la phase lamellaire a ensuite été menée. Les expériences ont révélé le comportement de gel faible des échantillons. A une fréquence de 10 rad/s et dans un intervalle de température compris entre 5 à 50 °C, il n’y a aucun changement perceptible des modules G’ et G’’, donc pas de transition de phase, sauf pour les échantillons les moins concentrés : dans ce cas, la température a un effet plus marqué, et la structure lamellaire évolue probablement. Lorsque le taux de cisaillement augmente, il a été observé que les échantillons avec phi ≥ 0.10 présentent une forte augmentation de la viscosité à partir d’une valeur seuil du taux de cisaillement. Cette observation, combinée avec des résultats de Rhéo-SAXS, a permis de conclure qu’il y a une transition vers une phase de vésicules (ou oignons) induite par l’écoulement. Les expériences de Rhéo-SAXS ont montré que, après l’arrêt du cisaillement, la phase de vésicules relaxe vers la phase lamellaire initiale au bout d’un temps typique d’une demi-heure. Enfin, des mousses ont été obtenues par incorporation de bulles d’air dans les phases lamellaire et éponge précédemment étudiées. Les mousses faites avec les phases lamellaires présentent une grande stabilité, probablement en raison de la viscosité élevée de ces gels. De plus, ces gels présentent des contraintes seuils d’écoulement faibles. Lorsque les bulles sont petites, la contrainte seuil est supérieure à la contrainte de pesanteur sur les bulles de la mousse (force d’Archimède). Mais ces bulles grossissent avec le temps (mûrissement dû aux différences de pression de Laplace entre bulles) et lorsque la contrainte de pesanteur dépasse la contrainte seuil du gel, le drainage de la mousse commence. On a constaté que la taille des bulles évoluait avec le temps en suivant une loi de puissance. Cependant, l’exposant (≈ 0.25) est plus petit que ceux trouvés pour le mûrissement d’Ostwald (0.333, valable pour des bulles isolées) ou le mûrissement des mousses (0.5). Le fait que l’exposant trouvé soit plus petit pourrait être lié à la réorientation des domaines lamellaires lors du mûrissement. Enfin, les mousses préparées avec des phases éponge présentent une faible stabilité probablement due aux passages internes entre bicouches qui forment la structure éponge, passages qui pourraient faciliter la coalescence des bulles. Deux scénarii ont été détectés lors de l’évolution de ces mousses: à basse fraction volumique de membrane, l’effondrement de la mousse commence pendant son drainage, alors que les mousses faites avec les échantillons les plus concentrés ont le temps de mûrir avant de s’effondrer. / In this thesis a study of the rheological and foaming properties of the lamellar and sponge phases of the SDS/hexanol/brine system was performed. Shear rate and temperature sweeps were done to analyze the rheology of the lamellar and sponge phases, adding frequency sweeps for the lamellar phase samples. Also, foamability and foam stability tests of foams made with these two phases were done. Sponge phase samples with membrane volume fractions phi ≥ 0.08 showed a Newtonian behavior, whereas in the less concentrated ones, phi 0.05 and 0.024, a change in viscosity was seen at shear rate values near to 1000 and 100 s ⁻¹, respectively. This change can be linked to a shear induced transition sponge to lamellar phase. Results indicate that temperature and the addition of the hydrosoluble polymer PEG affect the shear rate value at which this transition occurs. Lamellar phase samples with phi ≥ 0.10 presented a sharp increase in viscosity at intermediate shear rate values. The standard rheology technique together with Rheo-SAXS experiments allowed us to infer that there is a shear induced phase transition to vesicle phase. Rheo-SAXS data confirmed that the average interlamellar vesicle phase decreases by the effect of shear rate, and, when shear is stopped, the vesicle structure is relaxed and goes back to the lamellar phase after certain time. Foams made with lamellar phase samples showed a high stability and it was found that the time evolution of the bubble diameter follows a power law equation with exponents smaller than those reported for coarsening and Ostwald ripening. The small values of these exponents seem to be related with orientation defects of the liquid crystalline phase. On the opposite, the foams made with the sponge phase samples presented poor stability, as a possible consequence of the passages that form their structure that could facilitate coalescence of bubbles.

Les microARN (miARN) sont de petits ARN non-codants dont la principale fonction est de réprimer l’expression génique en s’hybridant par complémentarité de séquence à leurs cibles ARN. L’activité des miARN est également régulée par leurs cibles qui entrent en compétition pour leur liaison. Certains de ces ARN compétiteurs endogènes (ARNce) résistent à la répression induite par le miARN et vont alors les séquestrer. Ils sont appelés éponges à miARN. La dérégulation des réseaux d’ARNce et des éponges à miARN est impliquée dans des processus pathologiques tels que le cancer. Au cours de ma thèse, nous nous sommes intéressés à la séquestration des miARN dans le mélanome cutané. Le mélanome provient de la transformation maligne du mélanocyte, une cellule spécialisée dans la production de pigment. S’il n’est pas pris en charge à temps, des métastases apparaissent et se disséminent rapidement dans l’organisme (ganglions, foie, poumons, cerveau, etc.). Des solutions thérapeutiques existent mais une faible proportion de patients y répondent de manière efficace nécessitant de nouvelles stratégies de traitement. Nous avons mis en évidence que l’ARN messager (ARNm) de TYRP1, gène spécifiquement exprimé dans le mélanocyte et donc le mélanome, porte le rôle d’éponge à miARN dans le mélanome métastatique. Ce rôle est indépendant de la fonction protéique de TYRP1. Nous avons déterminé que l’ARNm de TYRP1 séquestre le suppresseur de tumeurs miR-16 via des sites de liaison (MRE-16) non-canoniques. Les MRE-16 non-canoniques permettent à l’ARNm de TYRP1 de ne pas être dégradé par le miR-16 et le rendent donc plus stable dans la cellule de mélanome. La majorité du pool de miR-16 est ainsi séquestrée et ne peut donc plus réprimer ses cibles intervenant dans la prolifération cellulaire et la croissance tumorale in vivo. Afin de remettre en activité le miR-16 au sein de la cellule de mélanome, nous avons utilisé la technologie du « target site blocker » (TSB), un oligonucléotide antisens modifié ayant une forte stabilité et affinité pour sa cible. Le TSB, spécifique du MRE-16 de l’ARNm de TYRP1, entre en compétition pour la liaison à l’ARNm de TYRP1 avec le miR-16 pour permettre sa libération et son action sur ses cibles effectrices. Nous avons montré in vitro et in vivo via un modèle murin de xénogreffe de tumeur dérivée de patient que la stratégie du TSB est efficace contre le mélanome métastatique. Ces travaux ont permis l’identification d’un nouveau mécanisme oncogénique basé sur la séquestration de miARN et proposent une nouvelle stratégie de thérapie ciblée contre le mélanome métastatique. / MicroRNAs (miRNAs) are small non-coding RNAs. They fine tune gene-expression through specific complementary interaction with their RNA targets. The miRNA repressive function towards a given RNA is highly regulated and in part dependent on the abundance of its other targets competing for miRNA’s binding. Some of these competing endogenous RNAs (ceRNAs) can resist to miRNA-mediated RNA decay thereby sequestering miRNAs. They are named miRNA sponges. Deregulation of ceRNAs and miRNA sponges networks are implicated in many pathologic processes including cancer. My PhD work focused on miRNA sequestration in cutaneous melanoma. Melanomas arise from the malignant transformation of melanocytes; the skin-cell specialized in pigment production. Most melanoma undergoes metastatic evolution, with metastatic cells spreading rapidly in the entire organism (lymph node, liver, lungs, brain, etc.). Early and complete resection of primary in situ melanoma is thus determinant for patient outcome. Since 2010, potent therapeutic options have been developed. Unfortunately, patients ultimately develop resistance while some are non-responders. There is thus an urgent need to develop new therapeutic strategies to treat metastatic melanoma. We have identified that the Tyrosinase Related Protein 1 (TYRP1) mRNA function as a miRNA sponge. TYRP1 is specifically expressed in the melanocytic lineage. TYRP1 mRNA governs melanoma growth endorsing thereby a non-coding function. We demonstrated that TYRP1 mRNA sequesters the tumor suppressor miR-16 via non-canonical miRNA binding sites (MREs-16). Non-canonical miR-16 binding lacks mRNA decay function favoring TYRP1 mRNA stability and miRNA sequestration. Sequestered miR-16 can no more repress its canonical targets involved in cell proliferation and tumor growth. To reset miR-16’s activity and block melanoma growth, we used “Target Site Blocker” (TSB). TSBs are modified antisense oligonucleotides with enhanced stability and affinity to its target. We designed a TSB, named TSB-T3, overlapping specially TYRP1 non-canonical MRE-16. We first showed that TSB-T3 binds to TYRP1 mRNA and competes with miR-16. Freed miR-16 binds to its canonical targets inducing their decay. TSB-T3 blocks melanoma cell growth in vitro and in vivo, using patient-derived tumor xenograft. We thus showed for the first time that TSB’s strategy redirecting a tumor suppressor miRNA is a potent tool to monitor metastatic melanoma growth. Together my PhD work brings out a new oncogenic mechanism based on miRNA sequestration and proposes an original strategy of targeted therapy against metastatic melanoma.