This thesis describes structural studies on the interactions between the fucosylated chondroitin sulfate (fCS) oligosaccharides and human proteins known as selectins. fCS is a carbohydrate obtained from sea cucumbers, that can be classified as a branched glycosaminoglycan (GAG). It has attracted much attention due to its anti-coagulant, anti-inflammatory, antimetastatic and anti-HIV properties and its structure was previously determined by NMR. Selectins constitute a family of proteins involved in cell adhesion processes, such as inflammation, attachment of viral particles and migration of tumour cells. fCS oligosaccharides have been shown to bind to selectins, which is likely a reason behind their biological activity. However, the mechanism of this interaction is currently unknown. The initial part of the thesis describes the experimental work on expression and purification of the recombinant L- and P-selectin constructs in Pichia pastoris, Escherichia coli and HEK 293 cells. The aim of these experiments was to produce two constructs for each selectin, a single domain construct, consisting of the C-type lectin domain only, and a double domain construct, consisting of both the C-type lectin and the EGF-like domains. The intention was that the recombinant proteins would be labelled with 13C and 15N to allow for the in-depth structural NMR studies on the fCS-selectin interaction. Various experimental approaches have been explored, including the use of different cell lines, modifications to construct design, as well as alterations to expression and purification conditions. Although it was not possible to produce soluble selectin constructs in either bacterial or yeast cells, protein expression tests in HEK293 cells, performed in collaboration with the Oxford Protein Production facility (OPPF), led to production of a soluble L-selectin construct, consisting of the L-selectin C-type lectin domain. The produced L-selectin construct, as well as two commercially available constructs of the Land P-selectin extracellular domains, were used in the Saturation Transfer Difference (STD) NMR experiments to provide new information about the nature of the fCS-selectin binding. The STD experiments allowed to identify the regions within the fCS oligosaccharides that are in direct contact with the protein and likely play an important role in this interaction. Experiments on different protein constructs allowed the comparison of fCS binding to P-selectin and to two different recombinant constructs of L-selectin. Results of these studies suggest that the binding occurs via a similar mechanism for both L- and P-selectins and that the fCS oligosaccharides bind to one-domain L-selectin construct with similar affinity as to a larger construct, consisting of the entire extracellular region of the protein. Alongside the experimental work, theoretical in silico studies on the fCS-selectin binding were undertaken as part of this project. The existing X-ray structures of selectin complexes were subjected to Molecular Dynamics (MD) simulations, which allowed to explore the dynamic behaviour of E-selectin upon binding to sialyl Lewis x (sLex). It was found that sLex forms a more favourable interaction with the extended conformation of E-selectin and that the protein in this conformation is characterised by a high degree of interdomain flexibility, with a new type of interdomain movement observed in the MD studies on this complex. In further in silico studies, the fCS oligosaccharides were docked to the existing P-selectin structures. The docking tests were performed on the computationally produced fCS trisaccharides with fucose branches either 2,4 or 3,4-sulfated. Results were evaluated with MD simulations and analysed in the light of current knowledge of selectin-ligand binding and the STD NMR experimental results. The in silico studies allowed to identify a subset of P-selectin residues that are likely involved in the interaction with fCS oligosaccharides in vivo. The conformational behaviour of P-selectin upon binding to fCS was also explored and it was found that the interdomain hinge is flexible during this interaction and allows transition from bent to extended conformational state. Finally, a new NMR method was developed to facilitate the studies of complex carbohydrates, incorporating the concepts of G-matrix Fourier Transform (GFT) NMR into 2D HSQC and 2D HSQC-TOCSY experiments. The method allows to separate peaks in the regions of high spectral overlap, providing information that can simplify the assignment process. The new experiments facilitated the structural evaluation of a sample containing a mixture of oligosaccharides resulting from the depolymerisation of fCS polysaccharide.

Orientador: Marcos Jun Watanabe / Coorientador: Letícia Andreza Yonezawa / Banca: Carlos Alberto Hussni / Banca: Alexandre Augusto de Oliveira Gobesso / Resumo: O objetivo deste estudo foi avaliar o efeito da administração de maltodextrina em dose única sobre as respostas fisiológicas e metabólicas de cavalos submetidos a testes padrão de exercício progressivo (TPEP) e a sessões sequenciais de exercício de alta intensidade e rápida aceleração (Teste de Exercícios Sequenciais - TES) em esteira ergométrica. Para tanto, o estudo foi dividido em 2 etapas: o Estudo 1 foi realizado visando determinar o efeito da maltodextrina administrada por via oral em dose única em resposta a três quantidades (0,2g/kg; 0,4g/kg; 0,6g/kg) sobre as curvas glicêmica e insulinêmica de equinos em repouso, com o objetivo de determinar a dose de maltodextrina que apresentasse maior persistência das concentrações sanguíneas de glicose associada com menor estímulo insulinêmico, e o Estudo 2 que avaliou o efeito da maltodextrina em resposta a dois tipos de exercícios: TPEP e TES. Foram utilizados oito equinos da raça puro sangue Árabe, machos castrados, com idade de 2 a 8 anos divididos aleatoriamente em dois grupos de quatro animais que passaram por dois tratamentos, suplementados com maltodextrina (GM, n=8) e controle (GC, n=8). Animais submetidos ao GM receberam 0,4g/kg de maltodextrina enquanto os submetidos ao GC receberam carboximetilcelulose pela via oral 30min antes dos exercícios. O TPEP foi realizado em esteira com inclinação de 6% e consistiu em fases sequenciais de velocidades progressivas de 1,8 m/s por um período de 5min, seguindo a 4,0 m/s por 3min, 6,0 m/s por 2min, e fases subsequentes a 7,0; 8,0; 9,0 e 10,0m/s por 1min em cada velocidade, ou até quando os cavalos não conseguiram manter o galope mesmo sendo estimulados. O TES seguiu o protocolo com um período de aquecimento ativo de velocidade de 1,7m/s por 1min (sem inclinação), trote a 3,5m/s por 5min (sem inclinação) e passo a 1,5m/s por 5min (6% de inclinação), ao término deste período, a velocidade... / Abstract: The aim of this study was to evaluate the effect of acute administration of maltodextrin on the physiological and metabolic responses of horses submitted to standard incremental exercise test (TPEP) and sequential sessions high intensity exercise and rapid acceleration (Sequential Test Exercises - TES ) on a treadmill. Therefore, the study was divided into two steps: Study 1 was carried out to determine the effect of maltodextrin orally administered acutely in response to three amounts (0.2 g / kg, 0.4 g / kg; 0.6g / kg) on glucose and insulin curves of horses at rest, allowing determine the amount of maltodextrin to present greater persistence of blood glucose concentrations associated with lower insulinemic stimulus, and the second study that evaluated the effect of maltodextrin in response to two types exercises: TPEP and TES. Eight were used adult horses of the Arab horses, gelding were randomly divided into two groups of four animals that underwent two treatments, supplemented with maltodextrin (GM n = 8) and control (CG n = 8). Animals was used to GM received 0.4 g / kg of maltodextrin while undergoing GC received carboxymethylcellulose orally 30 minutes before exercise. The TPEP was performed on a treadmill with incline of 6% and consisted of sequential stages of progressive speeds of 1.8 m / s for a period of 5 minutes, followed at 4.0 m / s for 3 minutes, 6.0 m / s per 2min, and subsequent phases 7,0, 8.0; 9.0 and 10,0m / s for 1 min at each speed, or even when the horses could not keep galloping even being stimulated. The TES followed the protocol with an active heating period of speed of 1.7 m / s for 1min (0% incline), trot to 3.5 m / s for 5 minutes (0% incline) and step-by-1.5m / s for 5 minutes (6% incline), at the end of this period, the speed of the treadmill was quickly increased to speed corresponding to 105% of HRmax maintained for 50s. After this period, called sprint, the speed was immediately reduced to ... / Mestre

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-02-17T14:00:34Z No. of bitstreams: 2 Dissertação - Gerson Francisco Duarte Júnior - 2015.pdf: 3229718 bytes, checksum: 81bfec5942eb1f9b2ce2a8218a18cdba (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-02-18T11:48:18Z (GMT) No. of bitstreams: 2 Dissertação - Gerson Francisco Duarte Júnior - 2015.pdf: 3229718 bytes, checksum: 81bfec5942eb1f9b2ce2a8218a18cdba (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-02-18T11:48:18Z (GMT). No. of bitstreams: 2 Dissertação - Gerson Francisco Duarte Júnior - 2015.pdf: 3229718 bytes, checksum: 81bfec5942eb1f9b2ce2a8218a18cdba (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-07-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The current work describes some instrumental advances on contactless conductivity detection (C4D) as well as the development of ionic electrodes to be coupled on microchips electrophoresis (MSE). Firstly, was constructed a high-resolution C4D detector based on openC4D electronic. Frequency and amplitude was optimized resulting in 400 kHz and 4 Vpp, respectively, for optimal values. These conditions was able to be used in different MSE devices including polymethylmethacrylate, polydimethysiloxane and polyester-toner. For liquid electrodes fabrication, channels were engraved on a polymeric plate using a CO2 laser system and sealed with adhesive membrane. After that, PDMS microchip was reversibly sealed against the polymer membrane. The electrode channels were filled with different ionc solutions (KCl, NaCl and LiCl). The KCl solution with 2 mol/L provide the best peak intensity. Then analytical performance of the ionic electrode was test in a separation of K+, Na+ e Li+ (300 μmo/L each). A good separation was observed and limit of detection achieved were 28, 40 and 58 μmol/L for K+, Na+ e Li+, respectively. The biggest advantage the use of ionic electrode are related of easiness fabrication process, simplicity and lower cost per device. Additionally, a commercial system of MSE-C4D was propose in a separation of sugars. The analytical separation was realized under the alkaline condition (pH>12), in order to provide the negative charge for sugars. After optimization of buffer and experimental conditions, a mixture containing fructose, galactose, glucose, lactose and sucrose was separated in less 3 min. / O presente trabalho descreve alguns avanços instrumentais relacionados à construção de um detector condutométrico de alta resolução e também ao uso de eletrodos iônicos para detecção condutométrica sem contato (C4D) acoplada aos microssistemas eletroforéticos (MSE). Além disso, o trabalho também descreve o desenvolvimento de metodologia para a análise de açúcares voltadas à aplicações na produção do etanol de 2ª geração. Inicialmente, foi construído e otimizado um detector condutométrico para MSE baseado na eletrônica OpenC4D. Os valores de frequência e amplitude obtidos com a otimização foram de 400 kHz e 4 Vpp, respectivamente. Com os valores otimizados, o detector se mostrou versátil ao utilizá-lo em MSE fabricados em diferentes substratos. Para a construção dos eletrodos iônicos, primeiramente foram gravados canais em placas poliméricas e selados com uma membrana adesiva. Um microchip de PDMS foi selado reversivelmente contra este suporte. Em comparação aos eletrodos metálicos, os eletrodos iônicos oferecem algumas vantagens, tais como, simplicidade, facilidade de fabricação, e baixo custo por dispositivo. Diferentes soluções foram avaliadas como eletrodos, dentre as quais a solução de KCl 2 mol/L forneceu os melhores resultados em termos de intensidade de sinal. O desempenho analítico dos novos eletrodos foi avaliado para a separação de uma mistura padrão de K+, Na+ e Li+ (300 μmol/L cada). Neste estudo, foram obtidos limites de detecção entre 28 e 58 μmol/L. Estes valores são comparáveis aos obtidos com os convencionais eletrodos metálicos. Uma metodologia para separação de frutose, glicose e sacarose utilizando um sistema comercial de MSE-C4D foi proposta. As análises foram baseadas na utilização de um pH alcalino (>12) que garante a ionização destas espécies para a forma aniônica. Após a otimização das condições experimentais onde um tampão composto por NaOH/Na2HPO4 75/10 mmol/\L foi o que forneceu melhores resultados em temos de resolução entre par frutose e glicose. com estas condições uma mistura contendo frutose, galactose, glicose, lactose e sacarose foi separada em um tempo de 3 min.

A tuberculose (TB) é uma doença infectocontagiosa causada principalmente pelo Mycobacterium tuberculosis (Mtb). A TB era uma doença considerada em vias de extinção, porém sua incidência voltou a aumentar devido a diversos fatores, como o fluxo migratório dos chamados \"Países Reservatórios\" e a pandemia da AIDS. Em 2013, a OMS estimou 9 milhões de novos casos de TB e 1,5 milhões de mortes. A multirresistência aos quimioterápicos disponíveis justifica a procura por novos tuberculostáticos. Face ao exposto, o trabalho objetivou a síntese de potenciais inibidores da maltosiltransferase (GlgE), um novo e promissor alvo contra o Mtb, utilizando Planejamento com base na Estrutura do Alvo Molecular -- Structure-Based Drug Design (SBDD). A comparação das simulações de dinâmica molecular do sistema apo da GlgE e em complexo com a maltose-1-fosfato (M1P) mostrou as mudanças estruturais, em nível atômico, decorrentes da ligação com seu substrato. Levando em consideração estes dados, as simulações de docking dos potenciais inibidores revelaram que análogos glicosídicos e maltosídicos triazólicos ligados a grupos aromáticos substituídos possuem melhores perfis de interação com o sítio ativo da GlgE e, por essa razão, foram escolhidos para síntese. Assim, sintetizaram-se análogos glicosídicos e maltosídicos através da click chemistry, obtendo-se rendimentos variados. Posteriormente, realizaram-se ensaios enzimáticos e microbiológicos no Institute for Tuberculosis Research (ITR), University of Illinois at Chicago, USA. Apesar de os ensaios enzimáticos estarem em andamento, os compostos ensaiados apresentaram atividades que variaram de moderada a fraca, frente à cepa H37Rv. Dois compostos glicosídicos exibiram valores de Concentração Inibitória Mínima (CIM) inferiores a 60 µM, sem efeito citotóxico em células VERO em concentrações superiores a 250 µM. Tais resultados indicam que o análogo glicosídico triazólico aromático, como o NDS 112, pode ser utilizado como protótipo interessante para o planejamento de novas séries de compostos capazes de atuar como tuberculostáticos / Tuberculosis (TB) is an contagious infectious disease mainly caused by Mycobacterium tuberculosis (Mtb). TB was a disease considered in extinction, but its incidence has risen again due to several factors, such as the migration from \"Reservatoir Countries\" and AIDS pandemic. In 2013, WHO estimated 9 million new TB cases and 1.5 million deaths. Multidrug resistance to drugs available in chemotherapy validates the search for new TB drugs. In this context, this work aimed to the design and synthesis of potential inhibitors of maltosyltransferase (GlgE), a new and promissor target against Mtb using Structure-Based Drug Design. Comparison between molecular dynamic simulations of GlgE apo system and in complex with maltose-1-phosphate (M1P) showed important structural changes at molecular level due to substrate binding. Considering these data, docking simulations of potential inhibitors revealed that glucoside and maltoside triazoles analogues bound to substituted aromatic groups have better interaction profiles with the GlgE active site and for this reason they were chosen for synthesis. Thus, glucoside and maltoside triazole analogues were synthetized through click chemistry with different yields. Then, enzymatic and microbiological assays were performed at Institute for Tuberculosis Research (ITR), University of Illinois at Chicago, USA. Although the enzymatic assays are ongoing, componds were tested against H37Rv strains and they have shown activity ranging from moderate to weak. Two compounds presented Minimal Inhibitory Concentration (MIC) values below 60 µM, without cytotoxic effect in VERO cells in concentrations higher than 250 µM. These results indicate that glucosidic aromatic triazoles analogues, such as NDS 112, can be used as a prototype for design new series of compounds capable of acting as antituberculosis agents

A vegetação ao longo do rio Amazonas é submetida a dois períodos muito bem marcados ao longo do ano: estação de cheia e estação seca. Características morfológicas e fisiológicas das espécies de várzea foram amplamente descritas, porém, uma das principais características é o metabolismo de carboidratos que tem sido muito pouco explorada. Com o objetivo de estudar a resiliência dos carboidratos não estruturais da árvore amazônica Senna reticulata (Leguminosae), a mesma foi cultivada sob diferentes estresses abióticos, como luz baixa combinada com elevada concentração de CO2 atmosférico, bem como disponibilidade hídricas distintas (alagamento das raízes e seca). Os resultados encontrados reforçam o papel do amido nas folhas como principal órgão armazenador desta espécie e também a alta resiliência desta reserva, que foi mantida mesmo em condições de baixa irradiância, em alagamento e em condições de seca. Estes resultados também destacam a forte ligação entre as reservas de amido e a manutenção do crescimento. Além disso, esta espécie mostrou estratégias ecofisiológicas distintas em relação a diferentes estresses hídricos (alagamento x seca) e uma alta capacidade de se recuperar depois de um período de seca, padrão este que pode estar diretamente relacionado à manutenção do crescimento durante o estabelecimento de plantas jovens no primeiro período de fase terrestre / The vegetation along the Amazon River is subjected to two markedly periods: flooded and drought seasons. Morphological and physiological traits for floodplain species have been largely described; however, one of the main features is the carbohydrate metabolism, which has been poorly explored. In order to study the resilience of non-structural carbohydrates of the Amazonian tree Senna reticulate (Leguminosae), this species was grown under different abiotic stresses such as low light combined with elevated CO2 and distinct water availabilities (waterlogging and drought). Results reinforce the role of starch in leaves as a main storage organ of this species and also the high resilience of this reserve under low light, waterlogging and drought conditions. These findings also highlight the tight connection between starch reserves and growth maintenance. Additionally, this species has shown very distinct eco physiological strategies to cope with different water availabilities and a high ability to recover after drought that might be strictly related to growth maintenance during seedling establishment in the first period of terrestrial phase

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-09-09T11:45:13Z No. of bitstreams: 1 2011 - Vanessa de Almeida Raia.pdf: 1415435 bytes, checksum: 6553cdee2bd5a5f2a363cb3cd11f812d (MD5) / Made available in DSpace on 2016-09-09T11:45:13Z (GMT). No. of bitstreams: 1 2011 - Vanessa de Almeida Raia.pdf: 1415435 bytes, checksum: 6553cdee2bd5a5f2a363cb3cd11f812d (MD5) Previous issue date: 2011-01-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / To fill some gaps about the intrinsic mechanisms of the biology of oviposition of R. (B.) microplus, we evaluated the concentration of lipids and carbohydrates, as well as the variability of lipid in eggs per day of posture. For this, engorged females weighing between 151 and 360 mg were placed in controlled environment (27 ? 1 ? C, 80 ? 5% RH, darkness), and after beginning of laying, three samples of the eggs pool were daily collected, packed and preserved at -20 ? C. Samples 1 and 2, weighing 50 mg each, were used to quantitate lipids and glucose, and to evaluate the lipid profile, respectively; and the third sample, containing 1 g of eggs, was used to quantitate glycogen. The determination of total lipids was performed using phospho-vanillin reagent, while the concentrations of cholesterol, triglycerides and glucose were obtained by colorimetric determination using endpoint enzymatic kit. The level of glycogen was performed using the acid 3,5 dinitrosalicylic reactive . The results were analyzed by nonparametric Kruskal-Wallis and the relationship between the concentrations of substrates and the days of laying was assessed by polynomial regression. There was no significant difference in the concentrations of these substrates in eggs from different days of oviposition, as there was no relationship of these concentrations with the days of laying. After lipid extraction, the samples were performed in thin layer chromatography, high-performance (HPTLC) for neutral lipids and phospholipids. Then, densitometry was performed using the Image Master Total Lab and the daily percentage of each lipid in the samples was calculated. Among the neutral lipids were identified the hydrocarbons, esterified cholesterol, triglycerides, fatty acids, free cholesterol and diacylglycerol. Of these, the cholesterol ester showed the highest variation, it was not detected in eggs at the fifth day of oviposition. Among the phospholipids were detected the phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine and sphingomyelin fosfatidilinusitol. This latter was absent in eggs of the seventh day onwards. Thus, the different rates of hatchability of eggs from different laying days cannot be attributed to the concentrations of total lipids, although some specific lipids such as cholesterol ester and sphingomyelin might influence the difference in larval hatchability, more researches are needed for clarify the role of these substrates in embryogenesis / Objetivando preencher algumas lacunas sobre os mecanismos intr?nsecos da biologia da oviposi??o de R. (B.) microplus, foram avaliadas as concentra??es de lip?dios e carboidratos, assim como a variabilidade lip?dica dos ovos postos em diferentes dias de postura. Para isso, f?meas ingurgitadas pesando entre 151 e 360 mg foram colocadas em estufa biol?gica sob condi??es controladas (27 ? 1oC, 80 ? 5% UR, escotofase), e ap?s in?cio da postura, tr?s amostras di?rias do ?pool? de ovos foram coletadas, acondicionadas e preservadas a ?20?C. As amostras 1 e 2, pesando 50 mg cada, foram utilizadas para dosar lip?dios e glicose, e avaliar o perfil de lip?dios, respectivamente; a amostra 3, contendo 1 g de ovos foi utilizada para dosar glicog?nio. A dosagem de lip?dios totais foi realizada atrav?s do reagente de fosfo-vanilina, enquanto as concentra??es de colesterol, triglicer?dios e glicose foram obtidas atrav?s de determina??o colorim?trica com a utiliza??o de kit enzim?tico de ponto final. A dosagem de glicog?nio foi realizada atrav?s do reativo ?cido 3,5 dinitrosalic?lico. Os resultados das dosagens foram analisados pelo teste n?o param?trico de Kruskal-Wallis e a rela??o entre as concentra??es dos substratos e os dias de postura foi avaliada atrav?s de Regress?o polinomial. N?o foram constatadas diferen?as significativas entre as concentra??es destes substratos nos ovos de diferentes dias de oviposi??o, assim como n?o houve rela??o dessas concentra??es com os dias de postura. Ap?s extra??o dos lip?dios das amostras, foram realizadas cromatografias em camada delgada de alto desempenho (HPTLC) para lip?dios neutros e fosfolip?dios. Em seguida, a densitometria foi realizada atrav?s do programa Image Master Total Lab e o percentual di?rio de cada lip?dio nas amostras foi calculado. Dentre os lip?dios neutros foram identificados hidrocarbonetos, colesterol esterificado, triglicer?dios, ?cidos graxos, colesterol livre e diacilglicerol. Destes, o colesterol esterificado foi o que apresentou maior varia??o, n?o sendo detectado nos ovos do quinto dia de oviposi??o. Dentre os fosfolip?dios foram detectados o ?cido fosfat?dico, fosfatidiletanolamina, fosfatidilcolina, fosfatidilinusitol e esfingomielina. Sendo este ?ltimo ausente nos ovos do s?timo dia de postura em diante. Assim, as diferentes taxas de eclodibilidade por dia de postura n?o podem ser atribu?das ?s concentra??es de lip?dios totais, embora alguns lip?dios espec?ficos como o colesterol esterificado e a esfingomielina possam ter influ?ncia na diferen?a de eclodibilidade larval, sendo necess?rios mais estudos para eclarecer o papel desses substratos na embriog?nese

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-03-21T17:26:15Z No. of bitstreams: 1 2016 - Monique de Moura Gurgel.pdf: 5504454 bytes, checksum: 56c93476cae66a9ba7cfc9cad8b26016 (MD5) / Made available in DSpace on 2017-03-21T17:26:15Z (GMT). No. of bitstreams: 1 2016 - Monique de Moura Gurgel.pdf: 5504454 bytes, checksum: 56c93476cae66a9ba7cfc9cad8b26016 (MD5) Previous issue date: 2016-04-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The chemical analysis and structural analyzes of tropical biomass products has become a potential interest for the production of biofuels. Residues from coconut is promising because it doesn`t compete with the food industry and it can be transformed into a higher product added value due the lignocellulosic origin. Green coconuts had been separated between fiber and parenchyma following by anatomical characterization which it could better be understood and visualized the effect of pretreatment delignification; ozonolysis, with the chemical and compositional changes in the cell wall. To access the carbohydrates, lignin must be removed, thus it had been explored the changes induced by ozone under pH conditions; with addition of acetic acid and sodium hydroxide, and treatment time in milli-Q water. The ozonolysis was carried out at pH 3, 5 and 8 for 2; 4 and 6 hours at 25 ? C. Analyses were performed before and after the ozonolysis reaction to investigate the change of polysaccharides and lignin with the following techniques: SEM, Py-GC/MS, HPLC, FT-IR and TGA. With Scanning electron microscope and infrared spectroscopy, it was possible to observe the degradation of structural components of the fibers, in the form of "spring", from 4 hours of oxidation and changes in the intensities of the bands. According to the Py-GC/MS results, compounds resulting from lignin had been identified and stablished S/G ratio as 0,73. With a lignin Klason content of 26.28% from green coconut fiber and 25.45% from parenchyma. The best conditions of ozone treatment were determined to be the fiber with no addition of reagents for 4 hours under oxidation as determined by the increase of glucose content in the hydrolysate to 45.96 % after acid hydrolysis of ozone treated fibers. / An?lises qu?micas e estruturais dos produtos de biomassa tropical tem se tornado um potencial interesse para produ??o de novos produtos que agreguem valores sustent?veis, dentre eles, os biocombust?veis. Res?duos de coco se tornam promissores pois n?o competem com a ind?stria aliment?cia e podem ser transformados em um produto de maior valor agregado visto serem de origem lignocelul?sica. Nesse contexto, os cocos verdes foram separados entre fibra e par?nquima e por conseguinte, caracterizados a n?vel anat?mico para que melhor pudesse ser compreendido a rela??o do tratamento utilizado para deslignifica??o; no caso, a ozon?lise, assim como as altera??es qu?micas e composicionais da parede celular. Para acessar os carboidratos, a lignina precisa ser removida, portanto foi explorado as mudan?as induzidas pelo oz?nio sob condi??es de pH; atrav?s adi??o de ?cido ac?tico e hidr?xido de s?dio, e tempo. O tratamento com oz?nio em solu??o de ?gua biodeionizada foi conduzido em pH 3, 5 e 8 por 2, 4 e 6 horas a temperatura de 25?C. As an?lises foram realizadas com amostras anteriores e posteriores ? rea??o de ozon?lise, investigando as altera??es dos polissacar?deos e da lignina com as seguintes t?cnicas: MEV, Pi-GC/MS, CLAE, FT-IR e TGA. De acordo com a Microscospia eletr?nica de varredura e Espectroscopia de infravermelho, foi poss?vel observar a degrada??o dos componentes estruturais das fibras, em forma de ?mola?, a partir de 4 horas de oxida??o e altera??es nas intensidades das bandas respecivamente. De acordo com a Pi-GC/MS, os derivados da lignina foram identificados e estabelecido uma rela??o S/G de 0,73 para fibra. O teor de lignina de Klason foi de 26,28% para a fibra e 25,45% para o par?nquima. Em visto disso, chegou-se a conclus?o que a melhor condi??o de tratamento com oz?nio foi obtido com as fibras sem adi??o de reagente sob 4 horas de exposi??o ao agente oxidante, determinado pelo aumento do teor de glicose a 45,96%.

A tuberculose (TB) é uma doença infectocontagiosa causada principalmente pelo Mycobacterium tuberculosis (Mtb). A TB era uma doença considerada em vias de extinção, porém sua incidência voltou a aumentar devido a diversos fatores, como o fluxo migratório dos chamados \"Países Reservatórios\" e a pandemia da AIDS. Em 2013, a OMS estimou 9 milhões de novos casos de TB e 1,5 milhões de mortes. A multirresistência aos quimioterápicos disponíveis justifica a procura por novos tuberculostáticos. Face ao exposto, o trabalho objetivou a síntese de potenciais inibidores da maltosiltransferase (GlgE), um novo e promissor alvo contra o Mtb, utilizando Planejamento com base na Estrutura do Alvo Molecular -- Structure-Based Drug Design (SBDD). A comparação das simulações de dinâmica molecular do sistema apo da GlgE e em complexo com a maltose-1-fosfato (M1P) mostrou as mudanças estruturais, em nível atômico, decorrentes da ligação com seu substrato. Levando em consideração estes dados, as simulações de docking dos potenciais inibidores revelaram que análogos glicosídicos e maltosídicos triazólicos ligados a grupos aromáticos substituídos possuem melhores perfis de interação com o sítio ativo da GlgE e, por essa razão, foram escolhidos para síntese. Assim, sintetizaram-se análogos glicosídicos e maltosídicos através da click chemistry, obtendo-se rendimentos variados. Posteriormente, realizaram-se ensaios enzimáticos e microbiológicos no Institute for Tuberculosis Research (ITR), University of Illinois at Chicago, USA. Apesar de os ensaios enzimáticos estarem em andamento, os compostos ensaiados apresentaram atividades que variaram de moderada a fraca, frente à cepa H37Rv. Dois compostos glicosídicos exibiram valores de Concentração Inibitória Mínima (CIM) inferiores a 60 µM, sem efeito citotóxico em células VERO em concentrações superiores a 250 µM. Tais resultados indicam que o análogo glicosídico triazólico aromático, como o NDS 112, pode ser utilizado como protótipo interessante para o planejamento de novas séries de compostos capazes de atuar como tuberculostáticos / Tuberculosis (TB) is an contagious infectious disease mainly caused by Mycobacterium tuberculosis (Mtb). TB was a disease considered in extinction, but its incidence has risen again due to several factors, such as the migration from \"Reservatoir Countries\" and AIDS pandemic. In 2013, WHO estimated 9 million new TB cases and 1.5 million deaths. Multidrug resistance to drugs available in chemotherapy validates the search for new TB drugs. In this context, this work aimed to the design and synthesis of potential inhibitors of maltosyltransferase (GlgE), a new and promissor target against Mtb using Structure-Based Drug Design. Comparison between molecular dynamic simulations of GlgE apo system and in complex with maltose-1-phosphate (M1P) showed important structural changes at molecular level due to substrate binding. Considering these data, docking simulations of potential inhibitors revealed that glucoside and maltoside triazoles analogues bound to substituted aromatic groups have better interaction profiles with the GlgE active site and for this reason they were chosen for synthesis. Thus, glucoside and maltoside triazole analogues were synthetized through click chemistry with different yields. Then, enzymatic and microbiological assays were performed at Institute for Tuberculosis Research (ITR), University of Illinois at Chicago, USA. Although the enzymatic assays are ongoing, componds were tested against H37Rv strains and they have shown activity ranging from moderate to weak. Two compounds presented Minimal Inhibitory Concentration (MIC) values below 60 µM, without cytotoxic effect in VERO cells in concentrations higher than 250 µM. These results indicate that glucosidic aromatic triazoles analogues, such as NDS 112, can be used as a prototype for design new series of compounds capable of acting as antituberculosis agents

O objetivo deste estudo foi determinar os efeitos da suplementação com maltodextrina, na substituição do amido na dieta de eqüinos avaliando a digestibilidade aparente total da matéria seca e nutriente e as diferenças nas respostas glicêmicas e insulinêmicas das dietas determinadas. Foram utilizados quatro eqüinos fêmeas com idade aproximada de 30 meses, peso médio de 400 kg, pertencentes ao rebanho da USP. Os animais foram imunizados contra tétano, vermifugados e pulverizados contra ectoparasitos. A dieta foi composta por 50% de concentrado e 50% de volumoso, formulada para atender as exigências de crescimento e mantença da categoria eqüina utilizada. As dietas continham 50% de amido no concentrado, substituídas em 33, 66% e 100% de maltodextrina. A colheita total de fezes foi feita em um período de 24 horas durante 03 (três) dias, com animais mantidos em baias, com piso de concreto, sem cama. No primeiro dia de colheita de fezes foram realizadas as coletas de sangue em tubos previamente preparados com estabilizantes, a partir da veia jugular, 30 minutos antes, 30 minutos, 90 minutos, 150 minutos, 210 minutos após o fornecimento do concentrado para dosagem de glicose e insulina. Não foi observado efeito linear ou quadrático para os coeficientes de digestibilidade aparente total da matéria seca e de nutriente, no entanto foi observado uma maior coeficiente de digestibilidade aparente total da ração com 100% de substituição do amido pela maltodextrina no concentrado, para a matéria seca, matéria orgânica, proteína bruta, fibra em detergente neutro e ácido. Em relação as respostas glicêmicas e insulinêmicas foi observado efeito quadrático para a glicose quando se analisou a área abaixo da curva desta variável. Da mesma forma foi observado um comportamento linear para a insulina. Quando se analisou a resposta das variáveis em função do tempo se observou um pico de glicose e insulina entre os tempo de 1,5 a 2 horas pós-alimentação, em relação a glicose a ração com 66% de substituição apresentou maior pico e a ração com 100% de substituição apresentou maior pico de insulina. O resultado do uso da maltodextrina sobre a fisiologia digestiva e o comportamento pós-brandial em eqüinos, permite sua inclusão na dieta dos animais. / The objective of this study was to determine the effects of supplementation with maltodextrin, the replacement of starch in the diet of horses by evaluating the apparent digestibility of dry matter and nutrient and differences in glycemic responses and insulinemic certain diets. There were four female horses aged approximately 30 months, average weight of 400 kg, belonging to the flock of USP. The animals were immunized against tetanus, wormed and pulverized against ectoparasites. The diet was composed by 50% concentrate and 50% forage diet formulated to meet the demands of growth and maintenance of category equine use. The diets contained 50% starch in the concentrate, replaced in 33, 66 and 100% of maltodextrin. The total collection of feces was made in a period of 24 hours over 03 (three) days, with animals kept in stalls with concrete floor, without bedding. On the first day of faeces were carried out blood samples in tubes previously prepared with stabilizers from the jugular vein 30 minutes before 30 minutes, 90 minutes, 150 minutes, 210 minutes after delivery of the concentrate to measure glucose and insulin. Were not observed effects linear or quadratic for the coefficients of apparent digestibility of dry matter and nutrient, however it was observed a higher coefficient of apparent digestibility of the ration with 100% replacement of starch by maltodextrin in concentrated, dry matter, organic matter, crude protein, neutral detergent fiber and acid. For the glycemic responses and insulin levels were observed quadratic effect for glucose when we analyzed the area under the curve of this variable. Similarly, it was observed a linear behavior for insulin. When analyzing their response as a function of time was observed a peak of glucose and insulin between the time of 1.5 to 2 hours post-feeding in relation to glucose diet with 66% replacement had a higher peak and cooperation with 100% replacement had a higher peak insulin. The results of maltodextrin use on digestive physiology and its post prandial behavior in equines allow its inclusion in animal diets.

Acteur clé de la symbiose hôte-microbiote, les IgA sécrétoires modulent le microbiote et participe à l'homéostasie intestinale. Chez la souris, les IgA sont polyréactives, en reconnaissent diverses bactéries, elles régulent la composition du microbiote et réduisent l'inflammation intestinale. Ces observations ouvrent des perspectives thérapeutiques intéressantes. Cependant, les caractéristiques des IgA et leurs spécificités restent mal connues chez l'homme. L'objectif de ce travail a donc été d'étudier la spécificité de la réponse IgA, en distinguant IgA1 et IgA2. La mise au point d'une technique de production in vitro d'IgA monoclonales 100% humaines issues de l'intestin nous a permis de montrer le profil de polyréactivité des IgA. Chaque IgA interagit avec un spectre large mais défini de bactéries commensales. Par ailleurs, les IgA1 et les IgA2 ciblent la même fraction du microbiote. Les réponses IgA1 et IgA2 convergent aussi au niveau des épitopes polysaccharidiques reconnus. Si les IgA sécrétoires contribuent largement à l’homéostasie intestinale, des IgG sériques anti-microbiote jouent un rôle dans la relation symbiotique hôte-microbiote. Leur présence n’ayant pas encore été démontrée chez l’homme en condition physiologique, ce travail a eu pour objectif secondaire de les explorer. Le développement d’une technique de cytométrie bactérienne nous a permis de détecter des IgG ciblant les bactéries commensales dans le sérum des individus sains. Ces IgG anti-microbiote sont dirigées vers les bactéries déjà reconnues par les IgA sécrétoires, elles présentent des spécificités propres à chaque individu. / IgA, the dominant immunoglobulin produced in the gut, plays diverse roles ranging from toxin neutralization, immune functions regulation, intestinal homeostasis maintenance. It is now well established that polyreactive IgA, which target multiple bacteria, can modulate gut microbiota composition and have promising therapeutic effects. However, IgA features remain elusive in humans. We therefore determined the reactivity profile of native monoclonal antibodies from human colon and compared IgA1 and IgA2. We found that IgA are polyreactive and bind a diverse but restricted subset of gut commensals. Most commensals were dually coated by IgA1 and IgA2, yet IgA2 alone coated a distinct fraction of colonic bacteria. Besides their common microbial targets, IgA1 and IgA2 exhibited overlapping anti-carbohydrate repertoires. An essential link between IgA and IgG responses against microbiota has been recently demonstrated in mice. Nevertheless, it remains unclear whether symbiotic bacteria could induce systemic IgG under homeostatic conditions in humans. Hence, we characterized anti-microbiota IgG in serum of healthy donors by bacterial flow cytometry. We found that each individual harbored a diverse and private panel of anti-commensals IgG that converge with secretory IgA to cover a restricted fraction of the gut microbiota. Patients with IgA deficiency or common variable immunodeficiency (CVID) exhibited a distinct set of anti-commensals IgG suggesting that IgA replacement in addition to polyvalent IgG might be beneficial to treat gastro-intestinal symptoms in these patients.