CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Nesta Tese sÃo estudados por meio da tÃcnica de espectroscopia Raman dois cristais de aminoÃcidos submetidos a condiÃÃes de altas pressÃes. O primeiro deles à uma amostra de L-Valina deuterada. Inicialmente, foi feita a tentativa de identificaÃÃo de todas as bandas Raman que aparecem no espectro da L-Valina deuterada, comparando-se com os resultados da L-Valina hidrogenada existentes na literatura. A seguir foi feito o estudo da amostra comprimida com argÃnio numa cÃlula de pressÃo a extremos de diamantes no intervalo entre 0,0 GPa e 12 GPa. Deste estudo pode-se concluir que entre 0,0 GPa e 1,3 GPa a L-Valina deuterada sofre uma mudanÃa estrutural, conforme informaÃÃes fornecidas pelos espectros Raman do material na regiÃo dos modos externos. Em pressÃes mais elevadas, entre 5 GPa e 7 GPa, ocorrem modificaÃÃes nos espectros Raman em praticamente todas as regiÃes espectrais, o que aponta tambÃm para uma possÃvel mudanÃa conformacional das molÃculas na cÃlula unitÃria. Forneceu-se tambÃm os valores (∂ωj/∂P)T para os diversos modos de vibraÃÃo do cristal nas vÃrias fases apresentadas pelo mesmo. O segundo aminoÃcido investigado foi a L-Isoleucina, que foi estudada atravÃs de espectroscopia Raman sob condiÃÃes de altas pressÃes atà cerca de 7,5 GPa tendo como lÃquido compressor o Ãleo mineral. Da anÃlise na regiÃo espectral 50 1/cm - 3200 1/cm , pode-se inferir que acima de 2,5 GPa e de 5,0 GPa ocorrem grandes mudanÃas conformacionais das molÃculas de L-Isoleucina na cÃlula unitÃria. Quando se faz o grÃfico do nÃmero de onda de dois modos da rede do cristal de L-Isoleucina investigados em funÃÃo da pressÃo, observam-se descontinuidades claras em aproximadamente 2,3 GPa e 5,0 GPa. Estas mudanÃas foram associadas a modificaÃÃes nas ligaÃÃes de hidrogÃnio, uma vez que as principais variaÃÃes de frequÃncias foram observadas em bandas associadas ao rocking do NH3+ e ao rocking do CO2 . / In this thesis, are studied by Raman spectroscopy of two amino acid crystals subjected to conditions of high pressures. The first is a sample of deuterated L-Valine. Initially, an attempt was made to identify all the bands that appear in the Raman spectrum of deuterated L-Valine, comparing with the results of L-Valine hydrogenated in the litera- ture. The following study was conducted on the sample compressed in a cell with argon pressure to extremes of diamonds in the range between 0.0 GPa and 12 GPa this study can conclude that between 0.0 and 1.3 GPa, the L-Valine suffers a deuterated structural change, according to information provided by the Raman spectra of the material in the region of external modes. At higher pressures, between 5 GPa and 7 GPa, changes occur in the Raman spectra in nearly all spectral regions, which also points to a possible con- formational change of molecules in the unit cell. It also provided the values (∂ωj/∂P)T for the various modes of vibration of the crystal at various stages presented by the same. The second investigation was the amino acid L-Isoleucine, which was studied by Raman spectroscopy under high pressures up to about 7.5 GPa with the compressor as a liquid mineral oil. Analysis in the spectral region 50 1/cm - 3200 1/cm , one can infer that above 2.5 GPa and 5.0 GPa large conformational changes occur in the molecule of L-Isoleucine in the unit cell. When you make a graph of the wave number of two-mode network of the crystal of L-Isoleucine investigated as a function of pressure, there are clear discontinuities at approximately 2.3 GPa and 5.0 GPa These changes were associated with changes in the connections hydrogen, since the major changes were observed in the frequency bands associated with the NH3+ rocking and the rocking of CO2-.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Objetivo: Investigar o perfil dos aminoÃcidos salivares em crianÃas saudÃveis e com diferentes graus de desnutriÃÃo, portadoras e livres de cÃrie da primeira infÃncia (CPI), correlacionando esses achados com experiÃncia de cÃrie e nÃveis de estreptococos do grupo mutans (EGM) em saliva. MÃtodos: Cento e vinte e duas crianÃas, de 12 â 71 meses de idade, com e sem CPI foram selecionadas para participar do estudo. Termos de consentimento foram assinados e as crianÃas foram divididas nos grupos saudÃveis (GS, n = 47), levemente (GI, n = 22), e moderadamente (GII, n = 53) desnutridas. NÃveis de desnutriÃÃo foram classificados segundo os padrÃes de crescimento da OMS 2006. Amostras de saliva nÃo estimulada foram coletadas de todos os participantes e subseqÃentemente centrifugadas. Sobrenadantes foram extraÃdos, liofilizados, armazenados a â 20o C e utilizadas para anÃlise de aminoÃcidos em um analisador de aminoÃcidos Biochem 20 plus. Saliva estimulada foi tambÃm coletada e usada para detecÃÃo de EGM. Estas amostras foram cultivadas em meio de cultura MSB (ufc/mL). Os resultados das concentraÃÃes de aminoÃcidos foram expressos em ÂM/mL. O exame dentÃrio foi realizado para cÃlculo do Ãndice ceo-s. Os dados foram analisados por meio do teste do Qui-quadrado de Pearson e de um modelo LogÃstico BinÃrio. Resultados foram considerados significantes quando p-valor < 0.02. Resultados: Quarenta aminoÃcidos foram identificados, com grande variabilidade em suas concentraÃÃes. AnÃlise da presenÃa/ausÃncia de cada aminoÃcido, e presenÃa/ausÃncia de cÃrie demonstrou uma associaÃÃo entre asparagina e CPI em GII (p=0.003). RegressÃo logÃstica mostrou que o risco à experiÃncia de cÃrie aumenta com a idade (p = 0.003). A presenÃa de alanina (p = 0.014) e carnitina (p = 0.008) reduziram as chances de experiÃncia de CPI. A presenÃa de histidina aumentou significativamente o risco à cÃrie (p = 0.012). Entretanto, EGM nÃo aumentou o risco à CPI (p = 0.065) nesse modelo. ConclusÃo: A presenÃa de aminoÃcidos especÃficos na saliva de crianÃas com DEP predispÃem a um maior ou menor risco à experiÃncia de cÃrie. / Aim: The aim of the present study was to identify the free amino acid content in whole saliva of children with protein-energy malnutrition (PEM), with and without early childhood caries (ECC), correlating these findings with caries experience and mutans streptococci (MS) levels in saliva. Methods: One hundred and twenty two, 12 â 70 months-old children, with or without ECC were selected to participate in the study. Consent forms were signed and children were divided into healthy (GH, n = 47), and mildly (GI,n = 22) or moderately (GII,n = 53) malnourished groups. Malnourishment levels were classified according to WHO 2006 growth standards. Unstimulated whole saliva was collected from all participants and centrifuged. Supernatants were extracted, lyophilized, stored at â 20oC and used for amino acid analysis, on a Biochem 20 plus amino acid analyzer. Stimulated whole saliva was also collected from all subjects, and used for MS detection on MSB agar medium (cfu/mL). Amino acid concentrations were expressed in ÂM/mL. Dental examination was performed for calculation of dmfs scores. Pearson Chi-Square test and a Logistic Binary Model were used for statistical analysis. Results were considered significant when p-value < 0.02. Results: Forty amino acids were identified, with great variability in their concentrations. Analysis of presence/absence of each amino acid and presence/absence of caries demonstrated an association between asparagine and ECC in GII (p = 0.003). Logistic regression showed that caries experience increased with an increase in age (p = 0.003). The presence of alanine (p = 0.014) and carnithine (p = 0.008) reduced the chances of experiencing ECC. The presence of histidine significantly increased caries risk (p = 0.012). However, MS counts did not significantly increase the risk of experiencing ECC (p = 0.065) in this model. Conclusion: Presence of specific amino acids in saliva of children with PEM predisposes to a higher or lower risk of caries experience.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-05-22T13:34:00Z No. of bitstreams: 1 2016 - Ronner Joaquim Mendon?a Brasil.pdf: 1892406 bytes, checksum: 2af27f8c4405579c52f8e3c5a83f0bce (MD5) / Made available in DSpace on 2017-05-22T13:34:00Z (GMT). No. of bitstreams: 1 2016 - Ronner Joaquim Mendon?a Brasil.pdf: 1892406 bytes, checksum: 2af27f8c4405579c52f8e3c5a83f0bce (MD5) Previous issue date: 2016-04-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Lysine is an essential amino acid used as a reference in diets based on the ideal protein concept. Determining the actual requirement for lysine is of great importance to formulate efficient feed without amino acids limitation or excesses. Three experiments were conducted in the Poultry Complex of Integrated Production Centre at Federal Rural University of Rio de Janeiro, objecting to estimate the digestible lysine requirements to broilers of low genetic potential for growth through the assessment of performance, carcass characteristics and breast meat quality. The first experiment corresponds to growth phase I (29-49 days), the second growth phase II (50-69 days) and the third final phase (70-84 days), where during each experiment was used different chickens. All experiments were conducted in a completely randomized design, with five treatments, four replications and twenty experimental units. The treatments were set with increasing lysine amounts obtained from the addition of L-Lysine HCl to the basal diet, replacing the ingredient corn starch as follows: 0.871, 1.011, 1.151, 1.291 and 1.431% in the growth phase I; 0.803, 0.943, 1.083, 1.223 and 1.363% in the growth phase II; and 0.766, 0.906, 1.046, 1.186% and 1.326% in the final phase. At the three stages studied, the lysine values nfluenced quadratically feed intake, weight gain and feed conversion. Lysine intake increased with enhanced amounts of lysine, however, it was detected a decrease in its usage efficiency. It was possible to measure the nutritional requirements for feed intake (1.298, 1.109, 1.150%), weight gain (1.183, 1.199, 1.162%) and feed conversion (1.203, 1.162, 1.126%), respectively for the growth phases I, II and final. The absolute weight as well as carcass and cuts yield were influenced by digestible lysine values of rations, where the highest carcass yield for growth phase I (73.62%) was estimated in the value of 1.162%, growth phase II (74.24%) in the value of 1.068%, and final phase (69.34%) in the value of 1.065% for digestible lysine. For chemical composition of breast meat, birds slaughtered at 70 days of age (growth phase II) was observed linear increase in moisture percentage and crude protein, and reduction the ether extract percentage. Nevertheless, it was not observed significant effect of broilers slaughtered at 85 days of age (final phase). For birds slaughtered at 70 and 85 days of age, the weight loss was significant by thawing and cooking the breast meat, increasing as improved lysine values. Therefore, optimizing feed conversion of Redbro Plum? broilers might be recommended, respectively, 1.203, 1.162, and 1.126% of lysine in the diet at phases 29 to 49, 50 to 69, and 70 to 84 days of age, corresponding to a proportion of digestible lysine:crude protein 6.09, 6.34, and 6.36%. / A lisina ? um amino?cido essencial utilizado como refer?ncia em ra??es formuladas com base no conceito de prote?na ideal. A determina??o da real exig?ncia de lisina ? de grande import?ncia na formula??o de ra??es eficientes, sem limita??es ou excessos de amino?cidos. Tr?s experimentos foram conduzidos no Setor de Avicultura do Centro de Produ??o Integrada da Universidade Federal Rural do Rio de Janeiro, com os objetivos de estimar as exig?ncias de lisina digest?vel para frangos de corte de menor potencial gen?tico para crescimento, atrav?s da avalia??o do desempenho, caracter?sticas de carca?a e de qualidade da carne do peito. O primeiro experimento corresponde a fase de crescimento I (29-49 dias), o segundo a fase de crescimento II (50-69 dias) e o terceiro a fase final (70-84 dias), sendo que em cada experimento foram utilizados frangos distintos. Todos os experimentos foram conduzidos em delineamento experimental inteiramente casualizado, com cinco tratamentos, quatro repeti??es e 20 unidades experimentais. Os tratamentos foram constitu?dos com valores crescentes de lisina digest?vel obtidos a partir da adi??o de L-Lisina HCl ? dieta basal, em substitui??o ao ingrediente amido de milho., sendo: 0,871; 1,011; 1,151; 1,291 e 1,431% na fase de crescimento I; 0,803; 0,943; 1,083; 1,223 e 1,363% na fase de crescimento II; e 0,766, 0,906; 1,046; 1,186% e 1,326% na fase final. Nas tr?s fases estudadas os valores de lisina digest?vel influenciaram de forma quadr?tica o consumo de ra??o, ganho de peso e convers?o alimentar. O consumo de lisina aumentou com os valores crescentes de lisina digest?vel, no entanto foi verificado uma redu??o na efici?ncia de utiliza??o de lisina. Foi poss?vel a estimativa das exig?ncias nutricionais para o consumo de ra??o (1,298; 1,109; 1,150%), ganho de peso (1,183; 1,199; 1,162%) e convers?o alimentar (1,203; 1,162; 1,126%), respectivamente para as fases de crescimento I, II e final. Os pesos absolutos e rendimentos de carca?a e cortes foram influenciados pelos valores de lisina digest?vel das ra??es, sendo que na fase de crescimento I o maior rendimento de carca?a (73,62%) foi estimado no valor de 1,162%, na fase de crescimento II (74,24%) no valor de 1,068%, e na fase final (69,34%) no valor de 1,065% de lisina digest?vel. Para composi??o qu?mica da carne do peito, nos frangos abatidos aos 70 dias de idade (fase de crescimento II) foi verificado efeito linear crescente no porcentual de umidade e prote?na bruta, e decrescente no porcentual de extrato et?reo. J? nos frangos abatidos aos 85 dias de idade (fase final), n?o foi constatado efeito significativo. Nos frangos abatidos aos 70 e 85 dias de idade, as perdas de peso por descongelamento e cozimento na carne do peito foram significativas, aumentando a medida que aumentou os valores de lisina digest?vel. Para otimiza??o da convers?o alimentar de frangos de corte Redbro Plum? podem ser recomendados, respectivamente, 1,203, 1,162 e 1,126% de lisina digest?vel na dieta nas fases de 29 a 49, 50 a 69 e 70 a 84 dias de idade, correspondendo a uma rela??o lisina digest?vel:prote?na bruta de 6,09; 6,34 e 6,36%.

O comportamento do óxido nítrico (NO) em sistemas biológicos vem sendo explorado desde a descoberta de sua importância em processos regulatórios, fisiológicos e em patologias. Atualmente há o grande interesse no desenvolvimento de compostos que possam liberar o NO de forma controlada em locais específicos nas células. Neste contexto, os complexos rutênio-nitrosilos têm se mostrado bastante promissores, visto que podem liberar NO no interior celular por processos redutimétricos, viabilizando sua utilização como agentes antitumorais e/ou tripanocida. Este estudo se baseia na premissa de que a liberação de NO pode ser direcionada para o interior celular através da modulação dos ligantes de forma a obter complexos biocompatíveis. Assim, o presente trabalho visou o delineamento de complexos rutênio-nitrosilos inéditos contendo ligantes polipiridínicos e/ou derivados de aminoácidos e, condução de experimentos a nível biológico. Os novos complexos rutênio-nitrosilos contendo os ligantes 3-etinilpiridina (3-etpy), derivado triazol de lisina e derivado imínico de triptofano foram sintetizados e purificados com êxito. Os ligantes foram caracterizados por RMN de 1H e 13C RMN, HSQC e 1H-1H COSY, espectrometria de massas e espectroscopia no infravermelho. Os complexos foram caracterizados por espectroscopia na região do UV-visível e FTIR, análise elementar, voltametria cíclica e espectrometria de massas. Os complexos foram testados frente a linhagens celulares B16-F10, MCF-7, MDA-MB231 e comparados com os testes em células de mama sadia MCF-10A. Além disso, os complexos foram aplicados a formas amastigotas de Trypanosoma cruzi. Os resultados de viabilidade celular mostraram que os complexos contendo derivado triazol de lisina ocasionam citotoxicidade às células MCF-7, MB-231 e B16-F10. O complexo cis-[Ru(NO)(bpy)2(3-etpy)](PF6)3.2H2O (onde bpy = 2,2\'-bipiridina) apresentou excelente resultado nos testes in vitro e in vivo frente a cepa Tulahuén LacZ e modelo murino, quando da sua associação ao benzonidazol. Os resultados obtidos sugerem possíveis relações existentes entre estrutura-atividade, abrindo novos caminhos para a pesquisa na busca pelo entendimento deste sistema, assim como o desenvolvimento de uma nova classe de compostos. / The behavior of nitric oxide (NO) in biological systems has been explored since the discovery of its importance in regulatory, physiological, and pathological processes. Currently, there is a great interest in the development of compounds that can release NO at specific sites inside cells in a controlled manner. In this context, the ruthenium-nitrosyl complexes have shown to be very promising, since they can release NO inside cells by reduction processes, turning possible its aplications as antitumor and/or trypanocides agents. This study is based on the premise that the release of NO can be directed to inside cells by modulating the ligands in order to obtain biocompatible complexes. In this way, the present work aimed at the design of new ruthenium-nitrosyl complexes containing polypyridine ligands and/or amino acid derivatives, and tests of biological activity. The new ruthenium-nitrosyl complexes containing 3-ethynylpyridine, triazole lysine derivative, and tryptophan imino derivative were synthesized and purified successfully. Ligands were characterized by 1H and 13C NMR, HSQC, and COSY, mass spectrometry and infrared spectroscopy. The complexes were characterized by spectroscopy in the UV-visible region and FTIR, elemental analysis, cyclic voltammetry, and mass spectrometry. The complexes were tested against cell lines B16-F10, MCF-7, MDA-MB231 and the normal breast cell model MCF-10A. In addition, the complexes were tested against amastigote forms of Trypanosoma cruzi. Cell viability results showed that triazole derivative-lysine complexes cause cytotoxicity to MCF-7, MB-231, and B16-F10 cells. The cis-[Ru(NO)(bpy)2(3-etpy)](PF6)3.2H2O (where bpy = 2,2\'-bipyridine) complex presented excellent results in vitro and in vivo tests against the Tulahuén LacZ strain and the murine model, respectively, when it was associated with benzonidazole. The results suggest possible relationships between structure and activity, opening new prospectives in the search for understanding this system, as well as the development of a new class of compounds.

Poucos são os trabalhos relacionados com a determinação das exigências nutricionais por aminoácidos de espécies nativas brasileiras, dentre as quais se destaca o dourado, Salminus brasiliensis (Cuvier, 1816), o maior peixe de escamas da Bacia do Prata, espécie reofílica e ictiófaga que chama atenção pelo excelente sabor da carne e pela beleza de seu tegumento. Este trabalho teve por objetivo estudar a exigência em aminoácidos e o uso de fonte de proteína de origem vegetal - farelo de soja (FS) - na nutrição do dourado. A exigência em lisina foi determinada em ensaio dose-resposta e os dados coletados foram analisados por meio de regressão polinomial e segmentada. Foi utilizada a relação A/E=[(aminoácido essencial÷total de aminoácidos essenciais+cistina+tirosina)x1.000], para estimar as exigências nutricionais dos demais aminoácidos essenciais. As unidades experimentais (UE) foram constituídas por lotes de 12 juvenis de dourado (11,4±0,2g; 9,4±0,9cm) condicionados a aceitar ração seca (43%PB e 4.600kcal EB), alojados em caixas de polipropileno com capacidade de 300L, com troca parcial de água num sistema fechado de recirculação e aeração. Os tratamentos correspondiam aos níveis crescentes de lisina: 1,0; 1,5; 2,0; 2,5; 3,0 e 3,5% na dieta num delineamento inteiramente aleatorizado - DIA (n=4). A exigência dietética em lisina para peso final (PF), ganho de peso (GP) e taxa de crescimento específico (TCE) foi de 2,15% da dieta ou 5% da proteína dietética; 2,5% de lisina 5,8% da proteína dietética, resultou no melhor índice de conversão alimentar (CA). A exigência por arginina foi determinada em ensaio dose-resposta utilizando a relação A/E e perfil de aminoácidos corporais de dourado. As UE foram constituídas por lotes de 12 juvenis de dourado (27,0±0,8g; 12,6±0,7cm) e os tratamentos correspondiam aos níveis crescentes de arginina: 1,0; 1,5; 2,0; 2,5 e 3,0% na dieta (DIA; n=4). A exigência dietética em arginina para PF, GP, consumo de ração e TCE foi de 1,48% da dieta ou 3,43% da proteína dietética. O valor 1,40% de arginina dietética ou 3,25% de arginina na proteína acarretou a melhor CA. O uso da regressão segmentada foi o mais apropriado para determinação da exigência dietética de lisina e arginina. A relação A/E foi uma ferramenta confiável para estimar níveis de exigência em aminoácidos essenciais. Para determinar efeitos do uso do farelo de soja em dietas para a espécie, grupos de 12 juvenis de dourado (27,0±0,8g; 12,6±0,7cm) foram submetidos a um ensaio de desempenho alimentados com dietas formuladas com base nos seguintes ingredientes: farinha de peixe+aminoácidos sintéticos (FP), farelo de soja+aminoácidos sintéticos (FS) e mistura farinha de peixe+farelo de soja+aminoácidos sintéticos (MIX) (DIA; n=4). A excreção indireta de nitrogênio e fósforo dos animais foi avaliada por meio do monitoramento dos parâmetros de qualidade de água nos aquários utilizando lotes de 20 juvenis (74,3±10,6g; 18,7±0,8cm) nas mesmas condições experimentais e com o uso das dietas da primeira parte do ensaio (n=3). Foram coletadas periodicamente (0, 2, 4 e 6 h pós-alimentação) amostras de água para determinação de amônio (NH4 +), amônia total (NH3) e fósforo total (P). O uso do farelo de soja e a suplementação de aminoácidossintéticos revelaram-se alternativas eficazes para substituir ou minimizar o uso da farinha de peixe na dieta desta espécie. Devido os resultados pouco conclusivos da excreção de metabólitos nitrogenados e de fósforo, maiores esforços precisam ser realizados para esclarecer este tópico de extrema importância para a consolidação de uma aquicultura sustentável. / Only a few research report address dietary amino acids requirement of Brazilian fish; this is true especially in regard to dourado, Salminus brasiliensis (Cuvier, 1816), a migratory, carnivorous Characin, which is not only the largest scale fish of the Prata Basin but also a prized aquaculture and sport fishing species. This study address dietary amino acids requirements and the use of a plant protein source soybean meal in the nutrition and feeding of dourado. Dietary lysine requirements were determined in doseresponse assay and collected data analyzed by polynomial and broken-line regression. The A/E relationship [AE = (essential amino acid÷total essential amino acids+cystine+tyrosine) x 1.000] was used to estimate the nutritional requirements of other essential amino acids. Groups of 12 juvenile dourado (11.4±0.2g; 9.4±0.9cm) were stocked in polypropylene tanks (300L) with partial water exchange in a closed recirculation, aerated system, conditioned to accept dry feed (43%CP and 4,600kcal CE), and then fed experimental diets containing increasing levels of lysine: 1.0; 1.5; 2.0; 2.5; 3.0 and 3.5% in a completely randomized design (n=4). Dietary lysine requirement for optimal final weight (WF), weight gain (WG) and specific growth rate (SGR) was 2.15% of diet or 5% of the dietary protein; 2.5% dietary lysine or 5.8% of lysine in dietary protein yielded optimum feed conversion rate (FCR). Dietary arginine requirements were also determined in dose-response assay, also based on A/E relationship and carcass amino acid profile of dourado. Groups of 12 juvenile dourado (27.0±0.8g; 12.6±0.7cm) were fed with experimental diets containing increasing levels of arginine: 1.0; 1.5; 2.0; 2.5 and 3.0% completely randomized design (n=4). Dietary arginine requirement for WF, WG, feed consumption and SGR was 1.48% of diet or 3.43% of the dietary protein; 1.40% dietary arginine or 3.25% of arginine in the protein caused the best FCR. The use of the broken-line regression was the most appropriate technique for determination of the dietary lysine and arginine requirement. A/E relationship was a reliable tool to estimate levels of essential amino acids requirements. To study the inclusion of soybean meal in diets for the species, the growth performance of groups of 12 juvenile dourado (27.0±0.8g; 12.6±0.7cm) fed with diets containing fish meal+ synthetic amino acids (FM), soybean meal+synthetic amino acids (SM) and mix of fish meal+soybean meal+synthetic amino acids (MIX) was evaluated. The indirect excretion of nitrogen and phosphorus was also evaluated by monitoring water quality parameters in the tanks stocked with groups of 20 juvenile dourado (74.3±10.6g; 18.7±0.8cm) in the same experimental conditions (n=3). Water samples were collected periodically (0, 2, 4 and 6 h after-feeding) for determination of ammonium (NH4 +), total ammonia (NH3) and total phosphorus (P). The use of soybean meal and synthetic amino acids are an efficient alternative to substitute or to minimize the use of the fish meal in the diets for the species, however, results regarding excretion of nitrogen andphosphorus were inconclusive, so additional efforts are needed to clarify this issue of importance for the consolidation of a sustainable aquiculture.

As teorias neurobiológicas defendem que a esquizofrenia é essencialmente causada por alterações bioquímicas e estruturais do cérebro. A importância que os aminoácidos tem com a fisiopatologia da esquizofrenia e, por este envolvimento encontrar-se pouco esclarecido é que esta pesquisa teve como propósito desenvolver e validar uma metodologia analítica para a análise simultânea dos aminoácidos: glutamato, aspartato, serina, glicina, arginina e lisina em plasma de pacientes com diagnóstico de esquizofrenia, utilizando para isto a cromatografia líquida de alta eficiência com detecção por fluorescência. Os analitos foram inicialmente extraídos através do processo de precipitação protéica, com o solvente orgânico acetonitrila. Para que pudessem ser detectados por fluorescência os aminoácidos foram derivatizados empregando orto-ftalaldeído na presença de B-mercaptoetanol. O tempo total de separação foi de 18 minutos em coluna analítica LiChroCART® RP-18 (Merck, 125mm, 4mm d.i., 5m) no modo de eluição gradiente com tampão acetato de sódio 0,1 mol/L (pH 7,0, A) e acetonitrila e água (B), na proporção 70:30, respectivamente, para posterior detecção por fluorescência (excitação 340 nm, emissão 450 nm). O método foi validado segundo os critérios estabelecidos pela Agência Nacional de Vigilância Sanitária (ANVISA). A resposta do detector encontrou-se linear na faixa de 9,6 a 192 pmol/mL para todos os aminoácidos. O limite de detecção estabelecido foi de 7,68 pmol/mL e o limite de quantificação foi de 9,6 pmol/mL. A recuperação foi superior a 70%. A precisão e exatidão intra-ensaio variou de 3,6% a 5,3% e 3,1% a 8,3%, respectivamente. A precisão e exatidão interensaio variou de 3,6% a 7,1% e 3,1% a 11,4%, respectivamente. A especificidade foi determinada para os seguintes interferentes: lorazepam, diazepam, clonazepam, alprazolam, haloperidol, levomepromazina, propranolol, ranitidina, fluoxetina, olanzapina, carbamazepina, diclofenaco, amiodarona, sulfametoxazol e, ainda, plasma hemolisado, normal e lipêmico. O método desenvolvido e validado mostrou ser eficiente na determinação simultânea de aminoácidos podendo ser aplicado em amostras de pacientes esquizofrênicos a fim de investigar seu envolvimento com esta desordem psiquiátrica tão intrigante. / Schizophrenia is primarily caused by structural and biochemical changes in the brain according to the main neurobiological theory, including the amino acids concentrations in the human plasma. The relation between these amino acids concentrations and the schizophrenia needs more clarification. Therefore, an analytical tool is necessary to provide which of these amino acids are related with this mental disease. The aim of this research was to develop and to validate an analytical methodology using High Performance Liquid Chromatography with fluorescence detection for simultaneous analysis of the main human amino acids, which are: glutamate, aspartate, serine, glycine, arginine and lysine in plasma of schizophrenic patients. Protein precipitation was the extraction technique chosen for the amino acids determination using acetonitrile as organic solvent. The derivatization was conducted by the reaction between the amino acids and ortho-phthalaldehyde in the presence of B-mercaptoethanol. The separation was achieved using the analytical column LiChroCART® RP-18 (Merck, 125mm, 4mm ID, 5mm) by gradient elution in 18 minutes. The mobile phase was composed by sodium acetate buffer 0.1 mol / L (pH 7.0; A) and acetonitrile: water (B) (70:30, v/v). The detection was performed by fluorescence (excitation 340 nm, emission 450 nm). The method was validated according to criteria established by the regulatory agency (ANVISA). The detector response was found linear in the range 9.6 to 192 pmol / mL for all amino acids. The detection limit was set at 7.68 pmol / mL and the limit of quantification was 9.6 pmol / mL. The recovery was above 70%. The precision and accuracy intra-assay ranged from 3.6% to 5.3% and 3.1% to 8.3%, respectively. The precision and accuracy inter-assay ranged from 3.6% to 7.1% and 3.1% to 11.4%, respectively. The specificity was determined for the following possible interferents: lorazepam, diazepam, clonazepam, alprazolam, haloperidol, levomepromazine, propranolol, ranitidine, fluoxetine, olanzapine, carbamazepine, diclofenac, amiodarone, trimethoprim. Plasma hemolyzed, lipemic and normal were evaluated. This method was adequate to simultaneous determination of amino acids, therefore it can be applied to samples of schizophrenic patients and consequently, providing information of the main amino acids involvement with this psychiatric disorder.

Calbindin-D28K is a cytosolic calcium binding protein found in a variety of cells that transport calcium. The chorioallantoic membrane and yolk sac of oviparous squamate reptiles (lizards and snakes) transport calcium from the eggshell and yolk to the developing embryo. I used immunohistochemistry to localize calbindin-D28K expression in the chorioallantoic membrane and yolk sac of two species of oviparous scincid lizards, Plestiodon fasciatus and Saproscincus mustelinus. Calbindin-D28K was detected in the chorioallantoic membrane and yolk sac of both lizard species by a polyclonal anti-snake calbindin antibody and a monoclonal anti-cow calbindin antibody. Calbindin-D28K was localized in the chorionic epithelium and allantoic epithelium of the chorioallantoic membrane and in endodermal cells scattered throughout the yolk mass of both species. This is the first demonstration of calbindin-D28K in allantoic epithelium and in endodermal cells of the yolk sac of lizards.

Angiogenesis, the formation of new vessels from a preexisting vasculature, is critical for supplying a healing myocardium with oxygen and nutrients to sustain metabolism post myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart post-MI, is considered essential for angiogenesis in tumor growth and metastasis, arthritis, endometriosis, and wound healing. Matrix metalloproteinases (MMPs) are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Vascular endothelial growth factors (VEGFs) play a vital role in angiogenesis because of their involvement in the recruitment and proliferation of endothelial cells. The current study explores IL-1β-stimulated regulation of angiogenic genes in cardiac microvascular endothelial cells (CMECs), the signaling mechanisms involved, and the implications in the processes of angiogenesis. DNA microarray analysis indicated IL-1β modulates the expression of numerous angiogenesis-related genes, notably upregulating MMP-2 and downregulating VEGF-D expression. RT-PCR and Western blot analyses confirmed the differential expression in response to IL-1β. In-gel zymographic analysis demonstrated IL-1β-stimulated increase in MMP-2 activity. IL-1β activated ERK1/2 and JNKs, not p38 kinase, and activated PKCα/β1 independent of MAPKs. IL-1β inactivated GSK3β via ERK1/2. Pharmacological inhibition of these signaling cascades indicated IL-1β-stimulated regulation of MMP-2 and VEGF-D occurs via ERK1/2, JNKs, and PKCα/β1-dependent mechanisms. In addition, inactivation of GSK3β inhibited basal VEGF-D expression. H2O2 significantly increased MMP-2 protein levels while IL-1β-induced VEGF-D downregulation was further potentiated by ROS scavenging compounds and inhibition of NF-κB. Phalloidin-FITC stain indicated a sharp reduction in fibrillar actin in the cytoskeleton of IL-1β-stimulated cells. Wounding assays revealed that IL-1β induced CMEC migration but prevented cell-to-cell contact and restoration of the monolayer. Flow cytometric analysis revealed a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells, indicative of decreased proliferation. IL-1β inhibited three-dimensional in vitro tube formation by CMECs. Lastly, IL-1β inhibited microvessel sprouting from aortic rings, an assay examining the collective response of multiple cell types. Collectively, the data presented in this study provide evidence that IL-1β differentially regulates important angiogenesis-related genes in CMECs. This differential regulation may lead to interruptions in the processes of angiogenesis, ultimately creating a dysfunctional phenotype for myocardial vessel formation.

Lactic acid bacteria are essential as flavor producers of cheese and fermented products. They are capable of catabolizing aromatic, branched chain, and sulfur amino acids to flavor compounds. During cheese ripening the numbers of lactococcal colonies decrease, but lactococci survive without replication in culture. This prompted an investigation into possible mechanisms of catabolism of branched chain amino acids into branched chain fatty acids and the physiological relevance of amino acid catabolism to the bacteria. We hypothesized that lactococci catabolize branched chain amino acids to branched chain fatty acids during nonculturability. Lactococci, lactobacilli, and brevibacteria catabolized both branched chain amino acids and keto acids into branched chain fatty acids. Lactococci survived carbohydrate-limited conditions for over 4 yrs. Their survival was represented by maintaining intracellular ATP, enzyme activity, membrane integrity, capability of ATP- and PMF-dependent substrate transport, transcription, and catabolism of amino acids to fatty acids. Assays conducted with NMR spectroscopy coupled with in silico analysis showed that branched chain substrates are catabolized via keto acids, HMG-CoA, and acetyl-CoA to branched chain fatty acids. A short list of candidate genes was identified for the pathway by gene expression analysis coupled to NMR analysis. The expression of these genes and the presence of the related catabolites were identified in long-term starved cultures of nonculturable lactococci. This verified that catabolism of branched chain amino acids to branched chain fatty acids occurred during the nonculturable state only and in conditions of carbohydrate deprivation. The pathway also facilitated fixation of carbon by lactococci, revealing the mechanism of survival of lactococci over 4 yrs in culture without the addition of external carbon sources. Between strains the availability of carbohydrate and acid stress played significant roles in modulating their ability to produce branched chain catabolites. The ability of lactococci to catabolize branched chain amino acids during sugar starvation represents a shift in carbon catabolic routes. The identified pathway also represented a balance between catabolism and anabolism, suggesting that the bacteria were in a homeostatic state during nonculturability. We accepted the hypothesis that nonculturable lactococci catabolized branched chain amino acids to branched chain fatty acids during starvation./p>

Abstract not available