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Item Efeitos da suplementação com creatina e cafeína sobre a força de fratura óssea em ratos submetidos a exercício de saltos verticais(Revista da Educação Física / UEM, 2012-01) Oliveira, Tânia Toledo; Gomes, Gilton de Jesus; Silva, Karina Ana da; Natali, Antônio José; Costa, Neuza Maria Brunoro; Franco, Frederico Souzalima CaldoncelliEste estudo investigou se a suplementação com creatina e cafeína, isoladamente ou combinadas, interfere na força de fratura óssea em ratos jovens exercitados. Os ratos foram divididos aleatoriamente em oito grupos: sedentários placebo, exercitado placebo, sedentário creatina, exercitado creatina, sedentário cafeína, exercitado cafeína, sedentário creatina + cafeína e treinado creatina + cafeína. Os grupos suplementados receberam creatina (carga: 0,430g/kg de peso corporal, por sete dias; e manutenção: 0,143g/kg, por 35 dias), cafeína (10mg/100g de peso corporal, por 35 dias) ou creatina+cafeína. Os grupos exercitados executaram saltos verticais na água (4x10 saltos com 1 minuto de intervalo entre séries, 5 dias/sem) por seis semanas. A ingestão de cafeína reduziu a espessura, o peso e a força de fratura do fêmur dos ratos, independentemente do exercício. A cafeína e a creatina+cafeína aumentaram a excreção urinária de cálcio. O exercício de saltos elevou a força de fratura, independentemente da suplementação, mas não alterou o peso e as dimensões do fêmur dos animais.Item Identification and expression levels of pig miRNAs in skeletal muscle(Livestock Science, 2013-06) Verardo, L. L.; Nascimento, C. S.; Silva, F. F.; Gasparino, E.; Toriyama, E.; Barbosa, A. R.; Costa, K. A.; Lopes, P. S.; Guimarães, S. E. F.; Périssé, I. V.MicroRNAs are a class of naturally occurring non-coding RNAs. Typically they are $22 nucleotides long and suppress translation of their targets genes. Several laboratories have attempted to identify miRNAs from pig muscle and the bioinformatics strategies using ESTs have proved to be successful for this aim. In this study we report an in silico identification of ncRNA in pig EST libraries focusing on novel pig miRNAs and further investigated the differential expression of pigs miRNAs (known and novel) by quantitative real-time PCR during preand postnatal stage from Commercial and local breed Piau pigs skeletal muscle tissue. We identified two miRNAs not yet described in pigs: hsa-miR-1207-5p and hsa-miR-665. Besides, we found 288 target genes for hsa-miR-1207-5p and 214 for hsa-miR-665; from them, four are muscle specific genes. Through expression analyses, differences were found between pre- and postnatal stages and genetics groups. The findings of miRNAs and their muscle-specific targets in pigs will be helpful for understanding the function and processing of this RNA class in the future. Besides, the miRNAs differentially expressed between Commercial and Piau breeds suggest that they can be used to uncover phenotypic differences across different genetic groups.Item Concentração de proteína solúvel por bradford revela diferenças no metabolismo de plantas de ora-pro-nobis em diferentes doses de nitrogênio(Revista Brasileira de Agropecuária Sustentável, 2013-07) Guimarães, Dimitrius Santiago P. Simões Fróes; Souza, Maria Regina de Miranda; Hirano, Rafael T.; Pereira, Paulo Roberto Gomes; Baracat-Pereira, Maria CristinaO ora-pro-nobis (Pereskia aculeata Mill.) é um alimento rico em proteínas, muito utilizado no meio rural e com importância crescente na indústria de alimentos e farmacológica. O objetivo deste trabalho foi determinar a concentração de proteínas pelos métodos de Bradford e Kjeldahl, em folhas de plantas de ora-pro-nobis submetidas a diferentes doses de adubação nitrogenada, comparando estes métodos. As folhas das plantas de ora-pro-nobis adubadas com diferentes doses de N (0, 50, 100, 200 e 400 kg de N/ha) foram coletadas aos 423 dias após o plantio (DAP). Para o método de Bradford, as folhas foram trituradas com nitrogênio líquido e maceradas em Tris-HCl 50 mM, pH 7,0, o homogenato centrifugado e a proteína solúvel determinada no sobrenadante. Para avaliar o perfil proteico, as amostras dos diferentes tratamentos foram separadas por SDS-Tricina-PAGE 14%. O método de Kjeldahl tradicional foi realizado usando-se o fator de correção 6,25. Os resultados por ambos os métodos indicaram que houve alterações nas concentrações e composição de proteínas presentes em função da disponibilidade de N no solo. A proteína total por Kjeldahl aumentou até a dose de 100 kg de N/ha, e a proteína solúvel por Bradford aumentou nas doses de N entre 50 e 200 kg/ha. Pelo SDS-Tricina-PAGE, verificou-se aumento da intensidade das bandas consonante com o método de Bradford. Estes resultados sugerem que a avaliação de proteínas solúveis pelo método de Bradford permite detectar diferenças no metabolismo das plantas de ora-pro-nobis, expressando informações biológicas relevantes para estudos fisiológicos e nutricionais.Item The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H + - ATPase in Saccharomyces cerevisiae cells(Cell Calcium, 2012-01) Bouillet, L. E. M.; Fietto, L. G.; Cardoso, A. S.; Perovano, E.; Pereira, R. R.; Ribeiro, E. M. C.; Trópia, M. J. M.; Tisi, R.; Martegani, E.; Castro, I. M.; Brandão, R. L.Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H+-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H+-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H+-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca2+-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H+-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H+-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP3. Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H+-ATPase activation.Item Ecto-nucleotidase activities of promastigotes from Leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome(PLOS Neglected Tropical Diseases, 2012-10-11) Fietto, Juliana L. R.; Leite, Pauline M.; Gomes, Rodrigo S.; Figueiredo, Amanda B.; Serafim, Tiago D.; Tafuri, Wagner L.; Souza, Carolina C. de; Moura, Sandra A. L.; Melo, Maria N.; Ribeiro-Dias, Fátima; Oliveira, Milton A. P.; Rabello, Ana; Afonso, Luı́s C. C.Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease.Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages.Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.Item High-yield secretion of multiple client proteins in Aspergillus(Enzyme and Microbial Technology, 2012-07-15) Gonçalves, Thiago A.; Segato, Fernando; Damásio, André R. L.; Lucas, Rosymar C. de; Squina, Fabio M.; Decker, Stephen R.; Prade, Rolf A.Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50–100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.Item Diversity of soil-dwelling Trichoderma in Colombia and their potential as biocontrol agents against the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary(Journal of General Plant Pathology, 2012-11-24) Smith, Alexander; Beltrán, Camilo A.; Kusunoki, Manabu; Cotes, Alba M.; Motohashi, Keiichi; Kondo, Takumasa; Deguchi, MichihitoTwenty-one isolates of Trichoderma spp. were collected from eight states in Colombia and characterized based on the 5′ end of the translation elongation factor-1α (EF1-α1) gene and RNA polymerase II gene encoding the second largest protein subunit (RPB2) by using mixed primers. Seven species of soil-dwelling Trichoderma were found: T. atroviride, T. koningiopsis, T. asperellum, T. spirale, T. harzianum, T. brevicompactum and T. longibrachiatum. Species identifications based on the EF1-α1 gene were consistent with those obtained from the RPB2 gene. Phylogenetic analyses with high bootstrap values supported the validity of the identification of all isolates. These results suggest that using the combination of the genes EF1-α1 and RPB2 is highly reliable for molecular characterization of Trichoderma species. Trichoderma asperellum Th034, T. atroviride Th002 and T. harzianum Th203 prevented germination of more than 70 % of sclerotia of Sclerotinia sclerotiorum in bioassay tests and are promising biological control agents. No relationship between mycelium growth rate and parasitism level was found.Item Thermostability improvement of Orpinomyces sp. xylanase by directed evolution(Journal of Molecular Catalysis B: Enzymatic, 2012-09) Trevizano, Larissa Mattos; Ventorim, Rafaela Zandonade; Rezende, Sebastião Tavares de; Silva Junior, Floriano Paes; Guimarães, Valéria MontezeThe methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching.Item Low linolenic soybeans for biodiesel: Characteristics, performance and advantages(Fuel, 2012-06-16) Santos, Eleonice Moreira; Piovesan, Newton Deniz; Barros, Everaldo Gonçalves de; Moreira, Maurilio AlvesSoybean is one of the main raw materials used for biodiesel production. However, the polyunsaturated fatty acids present in soybean seeds are not desirable for this purpose due to their low oxidative stability. Therefore, it is expected that the use of soybean cultivars with low linolenic acid content for biodiesel production will improve its oxidative stability and the cold filter plugging point (CFPP). This work presents the main characteristics, the advantages and performance of low linolenic acid soybean (LL) as compared to a conventional soybean variety (CO) for biodiesel production. The results showed that LL oil and protein contents were similar to those of CO. Phosphatide concentration was higher in LL oil, while total tocopherol content was lower in relation to CO. With respect to LL biodiesel performance, oxidative stability was much higher than that produced from CO, and the CFPP did not change even with the improved fatty acid profile of LL.Item Development of a method to quantify sucrose in soybean grains(Food Chemistry, 2012-02-15) Teixeira, Arlindo I.; Ribeiro, Lucas F.; Rezende, Sebastião T.; Barros, Everaldo G.; Moreira, Maurílio A.The sucrose content of soybean seeds affects the final flavor of soy-derived products. The aim of this work was to develop a simple, low-cost, spectrophotometric method for sucrose quantification in soybean seeds. To achieve this goal, we combined the action of invertase, an enzyme that hydrolyses sucrose into fructose and glucose, with glucose oxidase, an enzyme widely used for glucose quantification. This system was adapted to ELISA plates, making large-scale analyses possible at low cost, with potential application in routine analyses. To validate this method, sucrose content was determined in seeds of 14 soybean cultivars by this new method, as well as by HPLC and the enzymatic method of Stitt. The correlation coefficients were high and significant between the results obtained with the new method and the HPLC method (r = 0.9766) and the Stiff method (0.9461).