Ce travail porte sur les propriétés des nanocristaux de cellulose, leur modification de surface et le développement de matériaux avancés. Diverses approches sont utilisées sur ces substrats nanométriques visant à modifier leurs propriétés de surface et étendre leur utilisation dans des applications très sophistiquées, telles que la postsulfation et la désulfatation, le greffage et l'adsorption de polymères, l’oxydation sélective, le greffage moléculaire et l'inclusion "hôte-invité". Sur la base de modifications de surface, l'analyse des propriétés (pour différents taux de groupements sulfates) et divers nanomatériaux dérivés des nanocristaux de cellulose sont étudiés et préparés, notamment des nanocristaux sulfatés à différents taux, des nanocomposites extrudés, des éponges biocomposites et des hydrogels supramoléculaires. L'effet d’un gradient de groupements sulfates sur la chimie de surface, la morphologie et les propriétés physiques des nanocristaux de cellulose est discuté et notamment quatre modèles de section transversale sont comparés pour la détermination de la mesure du degré de substitution surfacique des nanocristaux de cellulose. Une stratégie nouvelle de protection impliquant une double couche polymère et la compatibilisation physique et/ou chimique des nanocristaux de cellulose est proposée afin de promouvoir à la fois la stabilité thermique des nanoparticules et la compatibilité des nanocristaux avec des matrices polymères non polaires au cours de la mise en forme par extrusion. En participant àla réticulation pour la construction de matériaux avancés, des nanocristaux de cellulose sélectivement oxydés (et de la cellulose microfibrillée oxydée pour comparaison) sont introduits dans de l'alginate pour développer des éponges biocomposites présentant une meilleure stabilité mécanique et une meilleure stabilité structurelle. Grâce à la conception intelligente par inclusion 'hôte-invité' in situ entre des nanocristaux de cellulose chimiquement modifiés et la cyclodextrine,deux polysaccharides hydrophiles sont combinés dans des hydrogels supramoléculaires pour l'administration de médicaments. En un mot, cette thèse contribue à l’avancée des nanocristaux de cellulose dans les domaines de l'analyse des propriétés et le développement des applications. / The present work focuses on the properties of cellulose nanocrystals, their surface modification and development of advanced materials. Diverse approaches are employed on these nanoscaled substrates aiming to modify their surface properties and extend their use in highly sophisticated applications, such as postsulfation and desulfation, polymer grafting and adsorption, selective oxidation, molecular grafting, and ‘host-guest' inclusion. On the basis of surface modifications, properties analysis (for different sulfate group contents) and various nanomaterials derived from cellulose nanocrystals are investigated and prepared, including gradient sulfated nanocrystals, extruded nanocomposites, biocomposite sponges, and supramolecular hydrogels. The effect of gradient degrees of sulfate groups on cellulose nanocrystals to surface chemistry, morphology and physical properties are discussed, particularly four cross-section models are compared for the determination of the surface degree of substitution on cellulose nanocrystals. A novel strategy involving a double-polymer-layer shield and physical and/or chemical compatibilization of cellulose nanocrystals is proposed, in order to realize both improvement of thermal stability and promotion of compatibility for nanocrystals with non-polar polymeric matrices during processing by melt-extrusion. With the idea of participating as crosslinking aid for the construction of advanced materials, selectively oxidized cellulose nanocrystals (with oxidized microfibrillated cellulose as comparison) are introduced in alginate for the development of biocomposite sponges with improved mechanical stability or structural stability. Through the smart design of in situ ‘host−guest' inclusion between chemically modified cellulose nanocrystals and cyclodextrin, two hydrophilic polysaccharides are combined in supramolecular hydrogels for use as drug delivery. In a word, this dissertation contributes to the advances of cellulose nanocrystals in the topics of property analysis and application development.

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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos de comunidades de esponjas marinhas são escassos no Brasil, sendo este trabalho pioneiro nessa abordagem para a Ilha Grande - RJ, um local de alta diversidade biológica. A estrutura das assembleias de esponjas marinhas e da comunidade bentônica marinha séssil foi avaliada, a partir de índices descritores de diversidade, em seis pontos da Ilha Grande e ilhas próximas, sendo três do lado continental e três do lado oceânico. As assembleias foram comparadas entre os diferentes lado e profundidade, através da contagem do número de indivíduos e área de cobertura por foto-quadrados. Paralelamente, as esponjas foram coletadas, fixadas e posteriormente identificadas através de metodologia e literatura especializada. Foi encontrado um total de 5.457 indivíduos, representando as Classes Demospongiae e Calcarea, distribuídos em 41 espécies e nove morfotipos, indicando maior diversidade para Lagoa Azul e menor para Parnaióca, sendo o local com maior riqueza a Ilha do Abraão. Dentre as espécies identificadas, quatro dominaram mais de 50% do total e 26 não alcançaram nem 5% da abundância absoluta. Análises de variância por GLM só evidenciaram diferença significativa para profundidade com substratos diferentes (F= 2,79; p<0,04), enquanto o fator lado (F= 2,23; p>0,16) e a interação entre os fatores (F= 1,17; p>0,38) não tiveram diferença estatística. As análises multivariadas de ordenação Cluster e MDS indicaram a formação de quatro assembleias de esponjas: 1) quatro locais com substrato não consolidado; 2) Lagoa Azul com substrato não consolidado; 3) locais oceânicos de substrato rochoso; e 4) locais continentais de substrato consolidado. Já para a comunidade geral, 49 espécies foram encontradas, sendo o Filo Porifera o de maior representatividade específica, apesar das macroalgas terem formado o grupo mais abundante. A comunidade bentônica foi dominada por quatro espécies, que juntas alcançaram média de 50% da cobertura bentônica: alga calcária incrustante, algas formadoras de tapete de turf, a esponja Iotrochota arenosa e o zoantídeo Palythoacaribaeorum. Estatisticamente, o lado continental se mostrou diferente do oceânico, o qual possui maior riqueza, diversidade e uniformidade de espécies, muito provavelmente pelo menor número de espécies dominantes e aliado a isso maior heterogeneidade de habitats, o que promove o aumento da diversidade. Quinze novas espécies estão sendo registradas para a Baía da Ilha Grande, sendo três novas espécies, as quais estão sendo descritas por especialistas, e 12 são novos registros de espécies ou gêneros para a região, evidenciando que a diversidade de esponjas marinhas na BIG é alta e ainda pouco conhecida e que a formação de assembleias pode ser devida a singularidade de cada local, implicando na necessidade de conservação dos costões rochosos da Ilha Grande e cercanias, a qual pode ser manejada através da realização de rápidos levantamentos sobre a riqueza e o número de indivíduos da espécie na região / Marine sponge community studies are scarce in Brazil and this is a pioneering study in this approach on Ilha Grande RJ, a high diversity region. The structure of marine sponges assemblages and sessile marine benthic organisms was evaluated from diversity descriptors index in six sites of Ilha Grande and nearby islands, three in continental side and three on oceanic side. Assemblages were compared between sides and depths counting number of individuals and its coverage area by photo-quadrats method. At the same time, the sponges were collected, fixed and, in lab, identified through specialized methodology and literature. There was a total of 5.457, representing Class Demospongiae and Calcarea, distributed in 41 species and nine morphotypes (50 possible species) with greater diversity to Lagoa Azul on the inside and lower for Parnaióca, outside, while Ilha do Abraão had the greatest richness. Among the identified species, four dominated more than 50% of the total and 26 did not reach even 5% of total abundance. General linear model (GLM) on ANOVA highlighted significant difference only between depths with different substrates (F=2.79; p<0.04), while side (F=2.23; p>0.16) and the interaction between factors did not have differences (F=117; p>0.38). Ordination multivariate analyses, Cluster and MDS, distinguished four sponge assemblages: 1) four sites with unconsolidated substrate; 2) Lagoa Azul with unconsolidated substrate; 3) oceanic sites with hard substratum; 4) continental sites with hard substrate. For the community, 49 species were found and Phylum Porifera was the highest specific representation, despite macroalgae have formed the most abundant group. Encrusting calcareous algae, turf algae, sponge Iotrochotaarenosa, exclusive of the continental side and zoanthid Palythoacaribaeorum added approximately 50% of the benthic cover in all sites. The sides were significantly different with highest richness, diversity and evenness for oceanic side while continental had more abundance of species, most probably due to fewer dominant species. Moreover, greater heterogeneity of habitats may promote increased diversity. The 15 new species now recorded which three are new for science and 12 represent new species or genus records for Ilha Grande Bay revealing this area as one of the highest diverse area in marine sponges from Brazil, despite the low knowlegment. Furthermore, this result implies the need to increase the preservation of the rocky shores of the region and shows that a quick survey of the number of species and their abundance is sufficient to manage a conservation area

A descoberta de fármacos a partir de produtos isolados de organismos marinhos tem apresentado um grande crescimento nos últimos anos, principalmente devido aos avanços tecnológicos analíticos, síntese química e biotecnologia. Dentre estes, as esponjas representam uma das principais fontes de metabólitos protótipos para diversas atividades, destacando-se os efeitos antitumorais. Neste contexto, este trabalho teve como objetivo a investigação química e biológica de três esponjas coletadas na costa sul-brasileira: Haliclona tubifera, Polymastia janeirensis e Scopalina ruetzleri. Considerando a correlação entre câncer, distúrbios da coagulação e desbalanço de espécies reativas de oxigênio (EROs), foram realizados ensaios visando a aquisição destas atividades e a identificação de substâncias bioativas. Para a esponja H. tubifera foram observados interessantes efeitos antitumorais em células de glioma e neuroblastoma humano (IC50 < 20 μg/ml), além das atividades antioxidante e anticoagulante para a fração acetato de etila. O composto majoritário desta fração foi isolado como um derivado N-Boc e sua configuração foi estabelecida utilizando um novo protocolo de dicroísmo circular e semissíntese de derivados. Assim, este esfingosídeo de cadeia longa isolado (2R,3R,6R,7Z)-2-amino-7-octadecene-1,3,6-triol, foi denominado halisphingosine A. Um novo composto minoritário, halisphingosine B foi obtido usando técnicas de isolamento em escala nanomolar. Sua configuração absoluta foi estabelecida por comparação com o composto A. Da mesma forma, para a esponja S. ruetzleri, a fração acetato de etila demonstrou os resultados mais promissores. Um potencial efeito anticâncer e de inibição dos radicais peroxila foi observado. Além disso, um efeito modulador da peroxidação lipídica foi evidenciado em ensaio ex vivo de dienos conjugados. Através da análise por RMN de 1H, verificou-se que a fração era majoritariamente constituída por ácidos graxos, os quais foram derivatizados para caracterização por Cromatografia Gasosa (GC/FID). Foram identificados 32 ácidos graxos principalmente poli-insaturados (53%). Ácidos graxos minoritários não usuais para o ambiente marinho também foram caracterizados. A esponja P. janeirensis apresentou os efeitos citotóxicos mais promissores em células de glioma e neuroblastoma humano, com um IC50 < 1,0 μg/ml para o extrato aquoso (pH 6,8), sendo este efeito pH-dependente, uma vez que o extrato (pH 5,8) não alterou a viabilidade celular. Para P. janeirensis, foi também investigado seu potencial de defesa química em modelo de Zebrafish. Foi observado que o extrato aquoso desencadeia um efeito de fuga, alterando significativamente o comportamento espaço-temporal de peixes Danio rerio. Analisando em conjunto, os dados do presente trabalho representam uma nova contribuição para o estudo químico e biológico de espécies de esponjas marinhas da costa sul-brasileira e apontam as potencialidades destas esponjas na busca de moléculas protótipos para fármacos, especialmente relacionados à terapia do câncer. / Drug discovery from marine natural products has increased in the past few years, mainly due to technological advances in spectroscopy, chemical synthesis and biotechnology. Among all marine animals, sponges represent one of the major sources of prototype metabolites for several biological activities, highlighting the antitumor effects. In this context, this study carried out chemical and biological investigation of three sponges collected on the South Brazilian coastline: Haliclona tubifera, Polymastia janeirensis and Scopalina ruetzleri. Considering the correlation between cancer, clotting disorders and imbalance of reactive oxygen species (ROS), experiments were conducted for acquisition of these activities and identification of bioactive compounds. H. tubifera showed an interesting cytotoxic effect in human neuroblastoma and glioma cell lines (IC50 <20 μg/ml), antioxidant and anticoagulant effect for ethyl acetate (EtOAc) fraction. The major compound of EtOAc fraction was isolated as an N-Boc derivative and its configuration was established using a new circular dichroism protocol with the production of semi-synthetic derivatives. This long chain sphingoid base (2R,3R,6R,Z)-2-aminooctadec-7-ene-1,3,6-triol was named as halisphingosine A. A new minor compound, halisphingosine B was obtained using nanomol scale techniques and their absolute configuration was established by comparison with compound A. Likewise, for the sponge S. ruetzleri, the EtOAc fraction showed the most promising results. A potential anticancer effect, inhibition of peroxyl radicals and modulation effect of lipid peroxidation was observed. Fingerprint 1H NMR analysis showed that this fraction is mainly constituted of fatty acids. Through Fatty Acid Methyl Ester (FAMEs) analysis by GC/FID, it was possible to identify 32 fatty acids, of which around 50% were Polyunsaturated Fatty Acids (PUFAs). In addition, some minor unusual fatty acids for the marine biosphere were identified. It was observed for P. janeirensis the most promising cytotoxic effects on human glioma and neuroblastoma cells, with an IC50 <1.0 μg/ml to aqueous extract (pH 6.8), being this effect pH-dependent, since the extract (pH 5.8) did not affect the cell viability. Moreover, P. janeirensis was investigated along their potential chemical defense in Zebrafish model. Aqueous extract trigged an escape effect, significantly altering the spatio-temporal swimming activity of animals. Taken together, the data presented from this study represent a new contribution to chemical and biological research of marine sponge species from South Brazilian coastline, and point the potentialities of sponges to search chemical prototypes for drugs, especially related to cancer therapy.

Bioerosion is a major process that affects the carbonate balance on coral reefs, and excavating sponges from the genus Cliona are some of the most important bioeroders on Caribbean reefs. The orange boring sponge, Cliona delitrix, is an abundant excavating sponge offshore southeast Florida that frequently colonizes dead portions of live stony corals, killing live coral tissue as it grows. With the recent decline in coral cover attributed to combined environmental and anthropogenic stressors, the increasing abundance of excavating sponges poses yet another threat to the persistence of Caribbean coral reefs. In the first part of this study, I explored distributional patterns of C. delitrix offshore southeast Florida and compared yearly sponge growth/corresponding coral tissue loss rates across habitats of different depths. C. delitrix densities and growth rates were significantly higher on the outer reef, where coral colonies also showed some of the fastest tissue retreat rates. More sponge individuals were found on sites with higher coral densities, likely resulting from the higher availability of preferred coral skeleton substrate. C. delitrix showed a clear preference for boulder stony coral species, which could alter the coral community composition in the future and allow an increase in branching and foliose species. The growth rates of C. delitrix offshore southeast Florida are slower compared to rates from other locations, likely a result of intense fouling of the coral-sponge interface by other spatial reef competitors. These results suggest that outer reef sites with high boulder coral density offshore southeast Florida are most vulnerable to C. delitrix colonization and may continue to suffer the greatest impacts of coral bioerosion. Excavating sponges are also strong competitors for space on coral reefs; able to colonize, excavate, and kill entire live stony corals. Despite the known negative effects of excavating sponges on stony corals very few studies have experimentally tested the competitive nature of this interaction. In the second part of this study, I examined the effect of manual removal of the excavating sponge, Cliona delitrix (Pang 1973), on tissue loss of the stony coral Montastrea cavernosa (Linnaeus 1767), and its possibility as a restoration technique. A total of 33 M. cavernosa colonies colonized by small C. delitrix sponges (up to 10 cm in diameter) were examined. Sponges were removed using a hammer and chisel from 22 of the affected colonies, and 11 colonies were left alone as controls. After sponge removal, the resultant cavities in the coral skeletons were filled to minimize future colonization by other bioeroders and promote coral tissue growth over the excavation. Cement was used as fill material on 11 of the colonies, and the remaining 11 cavities were filled with epoxy. Standardized photos of each colony were taken immediately, at 6 months and 12 months after sponge removal. Results show a significant reduction in coral tissue loss in colonies where sponge was removed, and both fill materials performed similarly reducing coral tissue loss. I also found that a majority of experimental corals showed no return of C. delitrix to the colony surface a year after removal. This study demonstrates that eliminating the bioeroding sponge competitor may promote recovery of the affected stony coral. Additionally, the sponge removal technique can be applied to any stony coral colonized by C. delitrix to preserve, or at least slow the loss of, remaining live tissue.

Les matériaux carbonés présentent de nombreux avantages pour les domaines des nanotechnologies et de l'environnement.La mixité de chiralité des nanotubes de carbone limite leur application dans les appareils électroniques et le nano-confinement. Dans une première partie, ce travail de thèse s'est concentré sur la séparation en chiralité de nanotubes de carbone de type CoMoCAT, afin d'élaborer de nouveaux nano-conteneurs.Après sélection en chiralité, nous avons évalué les propriétés sous hautes pressions des fagots de nanotubes sélectionnés, et leur interaction avec l'eau. Les résultats ont montré que les fagots supportent des pressions jusqu'à 17 GPa, avant de subir un effondrement radial réversible, permettant de les utiliser en tant que nano-enclumes.L'élaboration d'une éponge de carbone (super)hydrophobe pour le traitement des eaux après pollution aux hydrocarbures a été décrite dans une seconde partie. La pyrolyse de mousses polymères a permis de conserver la très grande porosité de la mousse (> 99%), tout en lui conférant des propriétés proches de la superhydrophobie et de grandes capacités d'absorption de pétrole et solvants organiques (85-200 g/g). L'élasticité du matériau permet sa régénération par simple compression mécanique : récupération du polluant et réutilisation de l'absorbant. De plus, cette caractéristique reste valable même après une centaine de cycles de compression-décompression, en conservant 81% de sa capacité d'absorption dans le cas du pétrole brut / Carbon materials present many advantages for the nanotechnology and environment fields. The chirality mixity of carbon nanotubes limits their application in electronic devices and the nano-confinement. In the first part, this thesis has focused on the chirality separation of CoMoCAT carbon nanotubes, in order to elaborate new nano-containers.After the chirality selection, the properties of selected nanotubes bundles under high pressure were evaluated, as well as their interaction with water. The results show that the bundles support pressures until 17 GPa, before to undergo a reversible collapse, allowing their application as nano-anvils.The (super)hydrophobic carbon sponge elaboration for the clean-up of water polluted by oils was described in the second part. The polymeric foams pyrolysis allows to keep the very high foam porosity (> 99%), to give properties next to the superhydrophobicity and large absorption capacities in oils and organic solvents (85-200 g/g). The material elasticity allows its regeneration by simple mechanical compression : recovery of pollutant and re-use of the absorbant. Moreover, this feature remains valid after a hundred compression-decompression cycles, with 81% of the initial crude oil absorption capacity

The hybrid nanostructures exhibit excellent performances in various fields such as catalysis, sensing, and energy conversion as compared to their individual ones. The thesis deals with the new methods for the synthesis of different type of hybrids with doped/pristine carbon nanostructures in the form of graphene, multiwall carbon nanotubes (MWCNTs) as one component and metals nanostructures (Ag, Pd, Pt and Au), carbide (Fe3C), metal chalcogenides (Ni3S2 and Co9S8) and oxide (CoO) as the other components. Various synthesis techniques such as modified galvanic replacement reaction at room temperature, hydrothermal, microwave and pyrolysis have been explored for the synthesis of different hybrid nanostructures. Furthermore, various hybrid nanostructures have been explored for various catalytic activities such as oxygen reduction reaction (ORR), oxygen evolution reaction (OER) and 4-nitrophenol (4-NP) reduction. It may be noted that the ORR and OER which are undoubtedly vital for their applications in fuel cells, metal-air batteries and water oxidation reaction. Interestingly, the catalytic activities of these hybrid nanostructures are comparable or better as compared to the commercial benchmark precious catalysts.

Orientador: Fabiana Fantinatti-Garboggini / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:39:46Z (GMT). No. of bitstreams: 1 Mantovani_CristinaKampus_M.pdf: 1137189 bytes, checksum: 2db8a522e2d39a0ae80a5e302d4d79e8 (MD5) Previous issue date: 2011 / Resumo: Nas últimas décadas um grande número de compostos de interesse biotecnológico, como por exemplo, citotoxinas, agentes antifúngicos, antimicrobianos, antivirais e anticancerígenos têm sido isolados de esponjas marinhas. Entretanto, estudos comprovam que, em muitos casos, os compostos ativos desses animais são oriundos de micro-organismos associados, que podem compor até 60% do volume tecidual das esponjas. A presente proposta teve por objetivo a caracterização taxonômica da diversidade de bactérias cultiváveis associadas às esponjas coletadas no litoral norte do estado de São Paulo, Brasil, e a avaliação da atividade antimicrobiana a partir de extratos orgânicos brutos dessas bactérias. Um total de 86 bactérias foi recuperado das esponjas Axinella corrugata, Dragmacidon reticulata, Chelonaplysilla erecta e Petromica citrina utilizando diferentes meios de cultivo. A diversidade das bactérias foi caracterizada utilizando dados de morfologia, ARDRA (Amplified Ribossomal Restriction Analysis) e sequenciamento do gene RNA ribossomal 16S, cuja análise permitiu a identificação de membros pertencentes aos filos Proteobacteria, Actinobacteria, Bacteroidetes e Firmicutes num total de 15 gêneros distintos. O gênero Pseudovibrio foi o único presente em todas as esponjas amostradas, e os gêneros Bacillus, Ruegeria, Vibrio, Staphylococcus e Erythrobacter estavam presentes em mais de uma esponja. A esponja Dragmacidon reticulata apresentou a maior diversidade bacteriana, englobando oito diferentes gêneros, dentre eles, um representate do gênero Cyclobacterium, o qual até onde se sabe, foi isolado pela primeira vez de uma esponja marinha. O gênero Bacillus esteve presente em três esponjas, mas na Petromica citrina, endêmica do Brasil, o gênero ficou representado em 74% dos isolados obtidos. Este estudo foi o primerio relato sobre a diversidade de bactérias cultiváveis da esponja Petromica critrina. Todos os isolados foram avaliados quanto à presença ou ausência dos fragmentos dos genes PKS (Polyketide Synthases) e NRPS (Non Ribossomal Peptide Synthetases), visando à investigação do potencial biotecnológico das bactérias, e mais da metade delas apresentaram pelo menos um dos genes estudados. Uma triagem da atividade antimicrobiana utilizando o método da difusão em bloco de ágar demonstrou que 21 isolados foram promissores para produção de antimicrobianos. Destes isolados foram obtidos os extratos orgâncios brutos, os quais foram testados quanto à determinação da concentração inibitória mínima contra oito micro-organismos indicadores. Um total de 13 extratos orgânicos brutos, em sua maioria respresentantes do gênero Bacillus, demonstraram ação contra o micro-organismo Bacillus subtilis ATCC 6051 e um deles demonstrou ação contra o micro-organismo Escherichia coli ATCC 11775. A numerosa inibição de estirpes de Bacillus por outros Bacillus sugere que a atividade possa ser gerada por bacteriocinas, polipeptídeos produzidos pela via ribossomal que atuam na inibição de crescimento de grupos próximos de micro-organismos. Sua possível função no meio ambiente é prover vantagem seletiva através da eliminação de um competidor relativamente próximo. Ainda, um representante do gênero Exiguobacterium apresentou atividade antimicrobiana contra B. subtilis, resultado este não descrito até o presente na literatura / Abstract: In recent decades a large number of compounds of biotechnological interest, such as cytotoxins, antifungal, antimicrobial, antiviral and anticancer substances have been isolated from marine sponges, however, studies show that, in many cases, the active compounds are actually produced by associated microorganisms, which can comprise up to 60% of the volume of sponge tissue. This proposal aimed to characterize the taxonomic diversity of culturable bacteria associated with sponges collected in the northern coast of São Paulo, Brazil, and to evaluate the antimicrobial activity from crude organic extracts of these bacteria. A total of 86 bacteria were recovered from sponges the Axinella corrugata, Dragmacidon reticulata, Petromica citrina and Chelonaplysilla erecta using different culture media. The diversity of bacteria was characterized using data from morphology, ARDRA (Amplified Ribossomal Restriction Analysis) and sequencing of 16S ribosomal RNA gene, whose analysis allowed the identification of members belonging to the phyla Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, in a total of 15 distinct genera. The genus Pseudovibrio was the only one present in all sponges sampled, and the genera Bacillus, Ruegeria, Vibrio, Staphylococcus and Erythrobacter were present in more than one sponge sampled. The sponge Dragmacidon reticulata showed the highest bacterial diversity, encompassing eight different genera, among which the genus Cyclobacterium, which, as far as is known, was first isolated from a marine sponge. The genus Bacillus was present in three sponges, but in Petromica citrina, endemic to Brazil, the genus accounted for 74% of the isolates. This study was the first report on the diversity of culturable bacteria from the sponge Petromica critrina. All isolates were evaluated for the presence or absence of NRPS (non ribossomal peptide synthetases) and PKS (polyketide synthase) genes in order to investigate the biotechnological potential of bacteria, and over half of the isolates had at least one of these genes. A screening of antimicrobial activity using the diffusion agar disk method showed that 21 isolates were promising for the production of antibiotics. Crude organic extracts from these isolates were produced and tested against eight indicator microorganisms to determine the minimum inhibitory concentration (MIC). A total of 13 crude organic extracts, most of the genus Bacillus, showed inhibitory activity against the microorganism Bacillus subtilis ATCC 6051, and one of them showed activity against the microorganism Escherichia coli ATCC 11775. The large inhibition of Bacillus strains to other Bacillus strains suggests that the activity can be generated by bacteriocins produced through ribosomal polypeptides that inhibit close groups of microorganisms. Its possible role in the environment is to provide a selective advantage by eliminating a relatively close competitor. Still, a representative of the genus Exiguobacterium showed antimicrobial activity against B. subtilis, which was not described in the literature up to date / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Zusammenfassung Ulrike Jörn Untersuchung des Glukose-, Harnstoff- und Kreatiningehalts in der Tränenflüssigkeit bei Katzen mit normalen und erhöhten Blutplasmawerten Klinik für Kleintiere der Veterinärmedizinischen Fakultät, Universität Leipzig Eingereicht im Februar 2018, verteidigt im Oktober 2018 73 Seiten, 13 Abbildungen, 20 Tabellen, 208 Literaturangaben Die Entnahme von Tränenflüssigkeit und Untersuchung von klinisch relevanten Metaboliten ist derzeit ein hochinteressantes Forschungsthema in der Diabetesforschung beim Menschen. Auch für die Tiermedizin ist dieses schnelle und einfache Verfahren relevant und könnte Blutuntersuchungen ergänzen, reduzieren oder sogar ersetzen. Aufgrund der Minimalinvasivität eignet sich die Tränenanalyse insbesondere bei der stressempfindlichen Katze und trägt so zum Tierwohl bei. Zum gegenwärtigen Zeitpunkt gibt es keine Untersuchungen der Glukosekonzentration bei der Katze. Die derzeit vorliegenden Untersuchungen beinhalten die semiquantitative Untersuchung der Harnstoffkonzentration bei Hunden und Katzen und die quantitative Untersuchung der Harnstoff- und Kreatininkonzentration beim Pferd. Ziele dieser Arbeit waren (1) die Bestimmung eines geeignetes Material für eine praktikable Tränenentnahme und (2) die quantitative Analyse der Metaboliten Glukose, Harnstoff und Kreatinin in der Tränenflüssigkeit von Katzen mit normalen und erhöhten Blutplasmakonzentrationen. Dazu wurden zunächst an einer kleineren Anzahl an Tieren (n=13) die Materialien Kapillare, Filterpapierstreifen und Polyurethanschwamm auf die Kriterien Handhabung, Verletzungsgefahr, Flüssigkeitsaufnahme und -abgabe und Entnahmezeit getestet. Im Anschluss wurde aufgrund der Praktikabilität der Polyurethanschwämme die quantitative Analyse bei 60, 72 und 48 Katzen durchgeführt. In der statistischen Untersuchung wurde kein signifikanter Unterschied für Glukose und Harnstoff zwischen beiden Augen bestimmt. Die lakrimale Glukosekonzentration konnte bei 60 Katzen erfasst werden und lag mit 16 % deutlich unter der Glukosekonzentration des Blutplasmas. Es wurde eine moderate Korrelation zwischen Tränenflüssigkeit und Blutplasma gefunden (r=0,743; p<0,001). Bei einem Cut-Off-Wert von 1,6 mmol/l waren Sensitivität und Spezifität 90 % und 81%. Harnstoff konnte bei 72 Katzen in der Tränenflüssigkeit bestimmt werden und lag mit 7 % etwas über der Glukosekonzentration des Blutplasmas. Es wurde eine starke Korrelation zwischen Tränenflüssigkeit und Blutplasma nachgewiesen (r=0,971; p<0,0001). Bei einem Cut-Off-Wert von 14,35 mmol/l lagen Sensitivität und Spezifität bei 100 %. Die Ergebnisse der lakrimalen Kreatininbestimmung (n=48) konnten nur deskriptiv beschrieben werden, da viele Tiere Konzentrationen unter der gerätespezifischen Nachweisgrenze von 18 µmol hatten. Die Konzentrationen in der Tränenflüssigkeit bei Katzen mit normalen und erhöhten Blutplasmawerten lagen dabei deutlich unter der Konzentration des Blutplasmas. Diese Arbeit zeigt, dass die Entnahme, Verarbeitung und Analyse von den Metaboliten Glukose, Harnstoff und Kreatinin in der Tränenflüssigkeit möglich ist. Die vorliegenden Ergebnisse der quantitativen Studie sind trotz Limitationen ein wichtiger Beitrag für die Etablierung neuer minimalinvasiver Verfahren durch Nutzung der Tränenflüssigkeit zur Diagnostik bei der Katze.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Aufbau und Funktion des Tränenfilmes 4 2.2 Übertritt von Metaboliten in die Tränenflüssigkeit 5 2.2.1 Permeabilität von Kornea und Konjunktiva 5 2.2.1.1 Aufbau und Funktion der Kornea 6 2.2.1.2 Aufbau und Funktion der Konjunktiva 6 2.2.1.3 Zusätzliche Permeabilitätsfaktoren 7 2.2.2 Übertritt von Glukose in die Tränenflüssigkeit 8 2.2.2.1 Untersuchung der lakrimalen Glukosekonzentration 8 2.2.2.2 Transportsysteme für Glukose 10 2.2.2.3 Veränderungen von Kornea und Konjunktiva bei Diabetes mellitus 11 2.2.2.4 Anwendung der lakrimalen Glukosemessung in Human- und Tiermedizin 13 2.2.3 Übertritt von Harnstoff in die Tränenflüssigkeit 14 2.2.3.1 Untersuchung der lakrimalen Harnstoffkonzentration 14 2.2.3.2 Transportsysteme für Harnstoff 15 2.2.4 Übertritt von Kreatinin in die Tränenflüssigkeit 17 2.2.4.1 Untersuchung der lakrimalen Kreatininkonzentration 17 2.2.4.2 Metabolismus des Kreatinins 18 2.2.4.3 Sekretion von Kreatinin durch Transportsysteme (OCTS, MATE, MDR1) 19 2.2.5 Veränderungen der Kornea und Konjunktiva bei Niereninsuffizienz 20 2.2.6 Vergleich der Metabolitkonzentrationen in beiden Augen 21 2.3 Methodische Verfahren zur Entnahme von Tränenflüssigkeit 21 2.3.1 Tränenentnahme mit Kapillaren 21 2.3.2 Tränenentnahme mit Filterpapierstreifen 22 2.3.3 Tränenentnahme mit Polyurethantupfer, Zelluloseacetat und Polyesterkegel 23 2.3.4 Experimentelle Materialuntersuchungen zum Aufnahme- und Abgabeverhalten 24 3 Material und Methoden 26 3.1 Testmaterialien zur Tränenentnahme 26 3.2 In-vitro-Vorversuche 26 3.2.1 Untersuchung der Flüssigkeitsaufnahme der Materialien 26 3.2.2 Untersuchung der Flüssigkeitsabgabe der Materialien 27 3.3 In-vivo-Studien 29 3.3.1 Untersuchte Katzen 29 3.3.2 Durchführung der In-vivo-Versuche 30 3.4 Statistische Analysen 35 4 Ergebnisse 37 4.1 In-vitro-Versuche 37 4.1.1 Untersuchung der Flüssigkeitsaufnahme der Materialien 37 4.1.2 Untersuchung der Flüssigkeitsabgabe der Materialien 37 4.2 In-vivo-Studien 39 4.2.1 Vergleich der verschiedenen Materialien an der Katze 39 4.2.2 Quantitative Bestimmung der Glukosekonzentration 40 4.2.2.1 Vergleich der Tränenkonzentrationen untereinander 41 4.2.2.2 Vergleich der Tränenkonzentration mit dem Blutplasma 44 4.2.3 Quantitative Bestimmung der Harnstoffkonzentration 47 4.2.3.1 Vergleich der Tränenkonzentrationen untereinander 48 4.2.3.2 Vergleich der Tränenkonzentrationen mit dem Blutplasma 50 4.2.4 Quantitative Bestimmung der Kreatininkonzentration 53 5 Diskussion 56 5.1 In-vitro-Versuche 56 5.2 In-vivo-Versuche 56 5.2.1 Anwendung der verschiedenen Testmaterialien an der Katze 56 5.2.1.1 Handhabung der Probenentnahme 57 5.2.1.2 Entnahmezeit und Volumen 58 5.2.2 Quantitative Bestimmung der Glukosekonzentration 59 5.2.3 Quantitative Bestimmung der Harnstoffkonzentration 63 5.2.4 Quantitative Bestimmung der Kreatininkonzentration 65 5.3 Klinische Relevanz dieser Untersuchungen 67 6 Zusammenfassung 70 7 Summary 72 8 Literaturverzeichnis 74

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.