Identificação e avaliação de propriedades de polissacarídeos sulfatados de diferentes fontes naturais que possibilitem sua aplicabilidade biotecnológica

Sulfated polysaccharides (SP) are widely distributed in animals and seaweeds tissues. These polymers have been studied in light of their important pharmacological activities, such as anticoagulant, antioxidant, antitumoral, anti-inflammatory, and antiviral properties. On other hand, SP potential to synthesize biomaterials like as nanoparticules has not yet been explored. In addition, to date, SP have only been found in six plants and all inhabit saline environments. However, the SP pharmacological plant activities have not been carrying out. Furthermore, there are no reports of SP in freshwater plants. Thus, do SP from marine plants show pharmacological activity? Do freshwater plants actually synthesize SP? Is it possible to synthesize nanoparticles using SP from seaweed? In order to understand this question, this Thesis was divided into tree chapters. In the first chapter a sulfated polysaccharide (SPSG) was successfully isolated from marine plant Halodule wrightii. The data presented here showed that the SPSG is a 11 kDa sulfated heterogalactan contains glucose and xylose. Several assays suggested that the SPSG possessed remarkable antioxidant properties in different in vitro assays and an outstanding anticoagulant activity 2.5-fold higher than that of heparin Clexane® in the aPTT test; in the next chapter using different tools such as chemical and histological analyses, energy-dispersive X-ray analysis (EDXA), gel electrophoresis and infra-red spectroscopy we confirm the presence of sulfated polysaccharides in freshwater plants for the first time. Moreover, we also demonstrate that SP extracted from E. crassipes root has potential as an anticoagulant compound; and in last chapter a fucan, a sulfated polysaccharide, extracted from the brown seaweed was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution for hydrophobic chains of 1H NMR was approximately 93%. SNFfuc-TBa125 in aqueous media had a mean diameter of 123 nm and zeta potential of -38.3 ± 0.74 mV, measured bydynamic light scattering. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0 43.7% at SNFuc concentrations of 0.05 0.5 mg/ mL and RAEC non-tumor cell line proliferation displayed inhibition of 8.0 22.0%. On the other hand, nanogel improved CHO and RAW non-tumor cell line proliferation in the same concentration range. Flow cytometric analysis revealed that this fucan nanogel inhibited 786 cell proliferation through caspase and caspaseindependent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle

Aspectos biológicos do peixe-olhudo-dentinho, Synagrops bellus (Actinopterygii: Acropomatidae), da plataforma externa e talude superior do estado de São Paulo, Brasil.

Biological aspects of the blackmouth bass Synagrops bellus from the outer shelf and upper slope along the coast of São Paulo, southeastern Brazil, are presented. The species represented about 71.6% and 9.7% in number of the total catch performed by balloom trawl in the isobaths of 300m and 500m respectively. Body sizes of 266 individuals ranged between 130 and 265mm total length, with sex ratio of 55.9% males, and 44.1% females, where most individuals were in maturation stage. Twenty two food items were found, pointing out Myctophidae fishes, Penaeidea and Caridea shrimps, Brachyuran megalopae, Enoploteuthidae and Cranchiidae cephalopods, pteropods and tunicates. The intestinal coefficient increases as the body size increase, and the number of gill rakers ranged between 16 and 17. Length-weight relationship was WT = 6.0 x 10-6 x TL3.12, r2 = 0.9495. Synagrops bellus is an important link between zooplankton and micronekton, and demersal and pelagic predators in the outer shelf and upper slope in southwestern Brazilian coast.

Nenneguya garavelli n. sp and Myxobolus peculiaris n. sp (Myxozoa : Myxobolidae) in the gills of Cyphocharax nagelli (Osteichthyes : Curimatidae) from Rio do Peixe Reservoir, Sao Jose do Rio Pardo, São Paulo, Brazil

The present work describes myxozoans found in Cyphocharax nagelli (Characiformes: Curimatinae) commonly called saguiru collected from Rio do Peixe Reservoir, Sao Jose do Rio Pardo, São Paulo, Brazil. From a total of 38 examined fish, 24 were infected with Henneguya garavelli n. sp. (63% prevalence) and two with Myxobolus peculiaris n. sp. (5% prevalence) in the gills. Spores were studied by staining and fresh spores were observed by differential interference contrast optics. Henneguya garavelli n. sp. differs from Henneguya iheringi , Henneguya occulta, Henneguya cesarpintoi, Henneguya santae, Henneguya pisciforme, Henneguya amazonica, Henneguya striolata, Henneguya leporinicola and Henneguya chydadea in spore length and from Henneguya travassosi, Henneguya adherens, Henneguya malabarica, Henneguya piaractus and also Henneguya chydadea in polar capsule length and tail length. Myxobolus peculiaris n. sp. was very different when compared to other species of Myxobolus in its morphology and the biggest size of spore body. The authors present tables with comparative measurements of Brazilian myxozoan parasites. (c) 2006 Elsevier B.V. All rights reserved.

Substituição da farinha de peixe pelo farelo de soja em dietas para juvenis de curimba

Replacement of fishmeal by soybean meal in juvenile curimba (Prochilodus lineatus) diets was evaluated. Five isonitrogenous (26% crude protein) and isocaloric (4,000 kcal of gross energy kg(-1)) diets with 0, 25, 50, 75 and 100% of replacement were used. Fish (3.71 +/- 0.54 g) were distributed in 25 tanks (120 liters). The diets were provided for 84 days. Total replacement of fishmeal (100%) by soybean meal worsened weight gain, feed conversion and protein efficiency ratio. An increase in carcass fat was observed at the expense of reducing protein percentage, as well as an increase in hepatosomatic index. It was concluded that replacing 75.0% fish meal by soybean meal (33.2% of inclusion) did not affect the animal growth, although there been observed changes in corporal composition and hepatic hyperplasia.

Metacromasia para identificação de basófilos sanguíneos em surubim híbrido: contribuição metodológica

Padua S.B. & Ishikawa M.M. [Metachromasia for blood basophils identification in hybrid surubim catfish: methodological contribution]. Metacromasia para identificacao de basofilos sanguineos em surubim hibrido: contribuicao metodologica. Revista Brasileira de Medicina Veterinaria, 33(3):147-150, 2011. Centro de Aquicultura da Unesp/Jaboticabal Rod. Paulo Donato Castellane, s/n Bairro Rural, Jaboticabal, 14884-900, SP. Brasil. E-mail: santiagopadua@live.comDifferent protocols of fixation and hydrolysis of blood smears of hybrid surubim catfish for metachromasia to marking blood basophils were evaluated. For this reason, methanol, acid-alcohol and formalin vapor were tested as fixatives. For hydrolysis of blood smears, HCl, citric acid and 2-Mercaptoethanol + Urea + NaCl solutions was evaluated. After procedures, the blood smears was stained with toluidine blue (0.025%) diluted in McIlvaine buffer (pH 4). The different protocols for fixation and hydrolysis of blood smears for metachromasia influenced the quality of the reaction. Hydrolysis with 2-Mercaptoethanol + Urea + NaCl solutions provided negative results when using methanol and acid-alcohol as fixatives. The fixation with acid-alcohol associated with citric acid hydrolysis provides highest quality reason.

Identificação morfológica de organismos semelhantes à anaplasmataceae em monócitos de surubim híbrido (Pseudoplatystoma reticulatum x P. corruscans) – relato de caso

The aim of this study was to report the occurrence of Anaplasmataceae-like organisms in monocytes from the hybrid surubim catfish. During the hematological evaluation of fish infected by Pseudomonas sp. in a fish farm located in the State of Mato Grosso do Sul we observed into the monocytes the presence of numerous pleomorphic inclusions of various sizes, rough in appearance which presented basophilic staining. These inclusions were similar to elementary bodies, initial bodies and morule of the bacteria from Anaplasmataceae family, often diagnosed in domestic mammals. This is the first report of its occurrence possibly belonging to the family of Anaplasmataceae in cultured fish in Brazil. Additional studies are necessary for molecular characterization of this bacteria, pathogenic potential, life cycle and impact on the intensive production of fish.

Identificação e caracterização de genes vip e cry coleóptero-específicos em isolados de Bacillus thuringiensis

O objetivo deste trabalho foi identificar e caracterizar os genes cry3, vip1, vip2 e vip1/vip2 em uma coleção de 1.078 isolados de Bacillus thuringiensis potencialmente tóxicos para larvas de coleópteros. Foram utilizados pares de oligonucleotídeos iniciadores gerais obtidos a partir de regiões conservadas dos genes e do alinhamento de sequências consenso. Posteriormente, os isolados positivos foram caracterizados por meio da técnica de PCR‑RFLP, tendo-se utilizado enzimas de restrição específicas, para identificar novas subclasses de genes nos isolados. Cento e cinquenta e um isolados foram positivos para os genes avaliados, com maior frequência para o gene vip1/vip2 (139 isolados). Pela técnica de PCR‑RFLP, foram observados 14 perfis polimórficos, o que indica a presença de diferentes alelos e, consequentemente, de distintas subclasses desses genes.

Identificação dos princípios ativos presentes no extrato etanólico de cereus jamacaru e avaliação em ratos dos possíveis efeitos tóxicos e/ou comportamentais da exposição prolongada

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Identificação e fatores de virulência de Candida spp isoladas da cavidade bucal de transplantados renais do hospital universitário Onofre Lopes em Natal-RN

Despite Candida species are often human commensals isolated from various oral sites such as: tongue, cheek and palatal mucosa plus subgingival region, there are some properties linked to the organism commonly known as virulence factors which confer them the ability to produce disease. Oral candidiasis is one of the main oral manifestations reported in literature related to kidney transplant patients. The objectives of the present study were to identify and investigate virulence factors of yeasts isolated from the oral cavity of kidney transplant recipients admitted at the Hospital Universitário Onofre Lopes, in Natal RN. Seventy Candida species isolated from 111 kidney transplant recipients were investigated in this study. Identification of the isolates was performed by using the evidence of germ tube formation, hypertonic broth, tolerance to grow at 42°C, micromorphology and biochemical profiles. We observed a high rate of isolation of yeasts from the oral cavity of kidney transplant recipients (63.1%) being C. albicans was the most prevalent species. Oral candidiasis was diagnosed in 14.4% of transplant recipients. We evaluated virulence properties of the isolates regarding to: biofilm formation on polystyrene microplates as well as XTT reduction, adherence to acrylic resin and human buccal epithelial cells and proteinase activity. Most isolates were able to form biofilm by the method of adhesion to polystyrene. All isolates of Candida spp. remained viable during biofilm formation when analyzed by the method of XTT reduction. The number of CFU attached to the acrylic resin suggested high adherence for C. parapsilosis. C. albicans isolates showed higher median adherence to human buccal epithelial cells than non-C. albicans Candida isolates. Nevertheless, this difference was not statistically significant. C. dubliniensis showed low ability to adhere to plastic and epithelial cells and biofilm formation. Proteolytic activity was observed for all the isolates investigated, including the unique isolate of C. dubliniensis. There was a statistically significant association between proteinase production and the presence of oral candidiasis. Studies related to oral candidiasis in renal transplant recipients are limited to clinical and epidemiological data, but investigations concerning Candida spp. virulence factor for this group of individuals are still scarce. We emphasize the importance of studies related to virulence factors of yeasts isolated from this population to contribute to the knowledge of microbiological aspects of oral candidiasis

Análises espaciais na identificação das áreas de risco para a esquistossomose mansônica no município de Lauro de Freitas, Bahia, Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efetividade de métodos de identificação sexual em tilápias-do-nilo (oreochromis niloticus) revertidas sexualmente com hormônio em ração com diferentes granulometrias

Three methods of sexual identification were evaluated in Nile tilapia (Oreochromis niloticus) at ages of 30, 60 and 90 days that were submitted to sexual reversion under administration of androgen hormone 17 alpha-methyltestosterone added to crumble diets with different granules sizes (0.25, 0.35 and 0.50 mm) in dosage of 60mg/kg. The techniques of sexual identification employed were: a) Macroscopic examination of urogenital papilla; b) Microscopic examination of gonads through stain with carmine-acetate and c) Microscopic examination of gonads through histological routine. In the presence of three methods of sexual identification evaluated, histology of gonads has shown the most confident diagnosis. The grain diameters of rations did not interfere in morphological characteristics of gonads, neither in secondary sexual characters of fish.

Identificação de genes cry de Bacillus thuringiensis no controle de Sphenophorus levis, o bicudo da cana-de-açúcar

A bactéria B. thuringiensis caracteriza-se pela produção de proteínas tóxicas a representantes de diversas ordens de insetos, as quais são codificadas por genes cry. Este trabalho foi realizado com objetivo de selecionar isolados de B. thuringiensis, por meio da caracterização morfológica e molecular, identificando as diferentes subclasses dos genes cry3 e cry35 e determinar a patogenicidade contra Sphenophorus levis, uma das mais importantes pragas da cultura da cana-de-açúcar. Foram utilizados 1163 isolados de B. thuringiensis e com a observação em microscópio com contraste de fases foram confirmadas como pertencentes à espécie de B. thuringiensis. O material genético foi purificado pela matriz de troca iônica Instagene Matrix e submetido a PCR com iniciadores gerais cry3 e cry35 identificando-se 30 isolados contendo genes com potencial para o controle de coleópteros, os quais juntamente com as linhagens-padrão de B. thuringiensis var. tenebrionis, B. thuringiensis var. morrissone e B. thuringiensis var. tolworthi foram utilizados para a realização do bioensaio. Através de análise discriminante alocaram-se os isolados em quatro grupos quanto à toxicidade de B. thuringiensis. Os grupos ficaram assim definidos: um grupo que promovem até 10% de mortalidade contendo as testemunhas e duas linhagens;um grupo que causou 39% de mortalidade contendo três linhagens padrão e dez isolados; um grupo com 52% de mortalidade contendo treze isolados e um grupo com 70% de mortalidade contendo cinco isolados, os quais devem ser considerados promissores no controle biológico de S. levis.

Morfologia e quantificação da microbiota intestinal do curimbatá (Prochilodus lineatus) e do cascudo cinza (Pterygoplichthys anisitsi) cultivados em cativeiro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Variação periódica da triiodotironina (T3) plasmática e sua ação na reprodução induzida do matrinxã, Brycon cephalus (Günther, 1869) em cativeiro

O matrinxã, Brycon cephalus, espécie nativa oriunda da Bacia Amazônica, apresenta características adequadas para a piscicultura. Entretanto, trata-se de peixe reofílico, sendo necessário manejo adequado para induzir à reprodução. O objetivo deste trabalho foi caracterizar o perfil da triiodotironina (T3) plasmática no matrinxã, durante 16 meses (outubro/97 a janeiro/99), relacionando-o com a maturação sexual, além de testar a ação do T3 associado ao extrato pituitário de carpa na reprodução induzida da espécie. O experimento foi conduzido no Centro Nacional de Pesquisa de Peixes Tropicais - CEPTA, Pirassununga, SP, e no Departamento de Morfologia e Fisiologia Animal, da Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal - UNESP. Foram amostrados, mensalmente, 8 a 12 peixes, de ambos os sexos, totalizando 173 animais, de onde retirou-se sangue para dosagem do T3 plasmático. As gônadas foram removidas para análise histológica com identificação do sexo e determinação do estádio de maturação. Analisaram-se testículos e ovários de 161 peixes em corte transversal, tendo predominado machos (63,35%) e o estádio sexual imaturo entre ambos. A concentração plasmática de T3 foi maior de dezembro a janeiro, para machos e fêmeas, coincidindo com o período de maior atividade reprodutiva da espécie e maiores temperaturas da água. em janeiro/99, reprodutores de matrinxã foram induzidos com extrato de pituitária de carpa (EPC) associado à administração de T3 (20 mg/kg em 0,1 mL de suspensão oleosa). Os resultados sugeriram que o T3 atuou sinergicamente à gonadotropina do extrato hipofisário e que o tratamento agudo de triiodotironina com o EPC pode estimular o eixo hipotálamo-hipófise-ovário. O tempo de eclosão das larvas provenientes das fêmeas tratadas com o T3 foi menor e o crescimento inicial e a sobrevivência dessas larvas, maiores.

First record of Tuxophorus caligodes (Siphonostomatoida, Tuxophoridae) in sea-farmed cobia, Rachycentron canadum, in Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)