Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-22T19:13:45Z No. of bitstreams: 1 SandraGasparini.pdf: 349340 bytes, checksum: 0464691b140faa8019f9a16a1192d667 (MD5) / Made available in DSpace on 2017-06-22T19:13:45Z (GMT). No. of bitstreams: 1 SandraGasparini.pdf: 349340 bytes, checksum: 0464691b140faa8019f9a16a1192d667 (MD5) Previous issue date: 2014-03-27 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Two experiments to determine the metabolizable energy in different ages and true digestibility of amino acids of rice bran (RB), broiler slow growth were performed. In the first experiment, 352 birds were distributed in a completely randomized design with three treatments and six replicates of eight or six birds per experimental unit as experimental age. The RB replaced 40% references diets formulated for three age groups (1-30, 31-60 and 61-90 days). The method used was the total collection and 1.0% iron oxide was used as a fecal marker. In the second experiment, 168 birds at 41 days of age fed a diet free of protein (DFP) and formulated with 55% of RB as the sole source of dietary crude protein and amino acids were used. All birds were killed by cervical dislocation and open the abdominal cavity to collect the contents at the terminal ileum portion. The samples were sent for amino acid analysis using HPLC. Was used 0.5% of chromic oxide as a marker for determining the digestibility of amino acids of the RB and the endogenous excretion of amino acids. The mineral content of the RB, with a value of 3.94%, below those content presented in the literature. The apparent metabolizable energy, true and true corrected for nitrogen balance, found for the RB in birds 70-79 days of age (2,801, 3,002 and 2,394 kcal / kg, respectively), statistically different (P <0.05) from values obtained with birds 40-49 days (2,415, 2,566 and 2,427 kcal / kg, respectively) and birds 10-19 days of age (2,131, 2,266 and 2,214 kcal / kg, respectively). The coefficients of apparent metabolism of dry matter (CMADM), with values of 65.80% (70 to 79 days), 59.20% (40 to 49 days) and 53.10% (10 to 19 days of age), statistically different (P <0.05). The coefficient of real gross energy metabolization corrected for nitrogen balance (CMVGEn), found in birds 70-79 days (56.40%), statistically different (P <0.05) of the value found in poultry 10 to 19 days of age (49.10%). Thus, in the formulation of rations, should be considered the differences in energy values of RB for young and adult birds, because the digestibility of dry matter and gross energy, increases with age. The average true digestibility of amino acids of the RB was 79.5%, as fed, where the methionine had the lowest coefficient of true digestibility, with a value of 78.12% and the highest arginine, with a value of 82.10 %. The true digestible values were 0.51% for lysine, 0.18% for methionine, 0.41% for methionine + cystine, 0.39% for threonine and 0.52% for valine. / Foram realizados dois experimentos com o objetivo de determinar a energia metabolizável em diferentes idades e a digestibilidade verdadeira dos aminoácidos do farelo de arroz integral (FAI), para frangos de corte de crescimento lento. No primeiro experimento, 352 aves foram distribuídas em delineamento inteiramente casualizado com três tratamentos e seis repetições de oito ou seis aves por unidade experimental, conforme idade experimental. O FAI substituiu em 40% as dietas referências, formuladas para três idades (1 a 30, 31 a 60 e 61 a 90 dias). O método utilizado foi o de coleta total de excretas e 1,0% de óxido de ferro foi utilizado como marcador fecal. No segundo experimento, foram utilizadas 168 aves com 41 dias de idade, alimentadas com uma dieta isenta de proteína (DIP) e uma dieta formulada com 55% de FAI como única fonte de proteína bruta e aminoácidos. Todas as aves foram abatidas por deslocamento cervical e abertas na cavidade abdominal para coleta do conteúdo na porção íleo terminal. As amostras foram enviadas para análise de aminoácidos utilizando o HPLC. Foi utilizado 0,5% de óxido de cromo como indicador para determinação dos coeficientes de digestibilidade dos aminoácidos do FAI e da excreção endógena de aminoácidos. O conteúdo mineral do FAI, com valor de 3,94%, apresentou teor abaixo daqueles descritos na literatura. Os valores de energia metabolizável aparente, verdadeira e verdadeira corrigida para o balanço de nitrogênio, encontrados para o FAI em aves de 70 a 79 dias de idade (2.801, 3.002 e 2,394 kcal/kg, respectivamente), diferiram estatisticamente (P<0,05) dos valores obtidos com aves de 40 a 49 dias (2.415, 2.566 e 2.427 kcal/kg, respectivamente) e aves de 10 a 19 dias de idade (2.131, 2.266 e 2.214 kcal/kg, respectivamente). Os coeficientes de metabolização aparente da matéria seca (CMAMS), com valores de 65,80% (70 a 79 dias), 59,20% (40 a 49 dias) e 53,10% (10 a 19 dias de idade), diferiram estatisticamente (P<0,05). O valor do coeficiente de metabolização verdadeiro da energia bruta corrigido para o balanço de nitrogênio (CMVEBn), encontrado em aves de 70 a 79 dias (56,40%), diferiu estatisticamente (P<0,05) do valor encontrado em aves de 10 a 19 dias de idade (49,10%). Assim, nas formulações de rações, devem ser consideradas as diferenças nos valores energéticos do FAI para aves jovens e adultas, pois a digestibilidade da matéria seca e da energia bruta, aumenta com a idade. O valor médio de digestibilidade verdadeira dos aminoácidos do FAI foi de 79,5%, na matéria natural, onde a metionina apresentou o menor coeficiente de digestibilidade verdadeira, com valor de 78,12% e a arginina o maior, com valor de 82,10%. Os valores digestíveis verdadeiros foram de 0,51% para lisina, 0,18% para metionina, 0,41% para metionina+cistina, 0,39% para treonina e 0,52% para valina.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-05-19T11:27:05Z No. of bitstreams: 1 2015 - Jos? Claudio Bezerra M. Junior.pdf: 1045163 bytes, checksum: d759d4bffb13bbf9696edda30c7326f2 (MD5) / Made available in DSpace on 2017-05-19T11:27:05Z (GMT). No. of bitstreams: 1 2015 - Jos? Claudio Bezerra M. Junior.pdf: 1045163 bytes, checksum: d759d4bffb13bbf9696edda30c7326f2 (MD5) Previous issue date: 2015-12-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The determination of digestible lysine requirements for Nile tilapia is essential to adjust the correct balance of amino acids in it?s food. Lysine is the amino acid reference for studies in ideal protein, which is currently an important concept in animal nutrition once it allows a reduction in the protein amount of the diet without compromising performance, and furthermore reducing the nitrogen excretion and eutrophication risks of the water. This study aimed to determine the digestible lysine requirement for Nile tilapia in the final phase of growth (500 to 600 g of body weight). Isocaloric and isoproteic experimental diets were utilized with different levels of digestible lysine in each formulation. The amounts of L-lysine HCl 78% used were 0.000; 0.388; 0.776; 1.164, and 1.552% replacing the ingredient glutamic acid 99%; which adjust the treatments to 0.932; 1.23; 1.53; 1.83, and 2.14% of digestible lysine. Nile tilapia weighing 519 g making a sum of 300 animals were utilized in this experiment. They were distributed in 25 water tanks each one measuring a 1000 liters using a renewable system of water. The pH, dissolved oxygen, temperature, salinity and conductivity were monitored daily and ammonia was measured weekly. Slaughters happened when fishes reached 28 and 50 days counting from the beginning of the experiment. The following animal science aspects were evaluated at the end: weight gain (WG), daily weight gain ratio (DWGR), specific growth rate (SGR), food intake (FI), consumption of digestible lysine (CDL), food conversion (FC) protein efficiency gain (PEG), lysine efficiency gain (LEG), nitrogen retention efficiency (NRE), protein deposition rate (PDR), Daily fat deposition rate (DFDR), protein retention efficiency (PRE), and survival rate (SR). The data was interpreted by analysis of variance with 5% probability. There was quadratic effect for GP, CA, TCE, ELG, EPG. The estimated value of lysine for both the largest GP and the best CA is 1.31% digestible lysine. There was no significant differency regarding the food intake, carcass or filet characteristics. This result is in part due to the fact of the gain being proportional throughout the body of the fish. In the second slaughter were obtained quadratic effect in the ether extract and carcass moisture. The Nile tilapia in the weight range of 500 to 600 grams of body weight presents the requirement for 1.31% of digestible lysine, corresponding to 5.31% of digestible dietary protein and 0.431% per Mcal of digestible energy for greater weight gain and better food conversion in it?s experiment conditions / A determina??o da exig?ncia de lisina para a til?pia-do-Nilo ? importante para a elabora??o de ra??es com adequado balanceamento de amino?cidos. A lisina ? o amino?cido refer?ncia nos estudos de prote?na ideal, um conceito relevante atualmente uma vez que permite a redu??o na quantidade de prote?na da ra??o sem comprometimento do desempenho, diminuindo ainda a excre??o de nitrog?nio para a ?gua e o risco de eutrofiza??o. O presente trabalho objetivou determinar a exig?ncia em lisina digest?vel para a til?pia-do-Nilo na fase de 500 a 600 g de peso vivo. Foram utilizadas dietas experimentais que consistiram em ra??es isocal?ricas, isoproteicas e com valores crescentes de lisina digest?vel. A L-lisina HCl 78% foi utilizada com valores crescentes (0,000; 0,388; 0,776; 1,164 e 1,552%), em substitui??o ao ingrediente ?cido glut?mico (99%), perfazendo os tratamentos com 0,932; 1,23; 1,53; 1,83 e 2,13% de lisina digest?vel. Foram utilizadas 300 til?pias-do-Nilo com peso m?dio de 519 g e distribu?dos em 25 caixas d??gua de 1000 L em sistema aberto de circula??o de ?gua. O pH, oxig?nio dissolvido, temperatura, salinidade e condutividade da ?gua foram monitorados diariamente e a am?nia foi medida semanalmente. Foram realizados 2 abates, aos 28 dias e aos 50 dias de experimento. Foram avaliados os seguintes ?ndices zoot?cnicos: ganho de peso (GP), ganho de peso di?rio (GPD), taxa de crescimento espec?fico (TCE), ingest?o alimentar (IA), consumo de lisina digest?vel (CLD), convers?o alimentar (CA), efici?ncia proteica para ganho (EPG), efici?ncia de lisina para ganho (ELG), efici?ncia de reten??o de nitrog?nio (ERN), taxa de deposi??o de prote?na (TDP), taxa de deposi??o di?ria de gordura (TDG), efici?ncia de reten??o de prote?na (ERP) e taxa de sobreviv?ncia (TS). Os dados foram interpretados por meio de an?lise de vari?ncia a 5% de probabilidade. Houve efeito quadr?tico para GP, CA, TCE, ELG, EPG, sendo o valor estimado de lisina tanto para o maior GP quanto para a melhor CA de 1,31% de lisina digest?vel. N?o houve diferen?a significativa para o consumo de ra??o, rendimento de carca?a, rendimento de fil?, caracter?sticas da carca?a e do fil?. Esses resultados se devem em parte ao fato do ganho ter sido proporcional em todo o corpo do peixe. No segundo abate houve efeito quadr?tico no extrato et?reo e na umidade da carca?a. A Til?pia-do-Nilo na faixa de peso de 500 a 600 gramas de peso vivo apresenta a exig?ncia de 1,31% de lisina digest?vel, que corresponde a 5,31% da prote?na digest?vel da dieta e a 0,431%/Mcal de energia digest?vel para o maior ganho de peso e melhor convers?o alimentar nas condi??es deste experimento.

Wong, Kin Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 86-98). / Abstract also in Chinese. / THESIS COMMITTEE --- p.i / ABSTRACT (ENGLISH) --- p.ii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGEMENTS --- p.v / DECLARATION --- p.vi / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.x / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Antibiotics market and β-lactam antibiotics --- p.1 / Chapter 1.2. --- Semi-synthetic cephems --- p.1 / Chapter 1.3. --- Conversion of CPC to 7-ACA --- p.3 / Chapter 1.4. --- Chemical production versus enzymatic bioconversion --- p.5 / Chapter 1.5. --- Industrial two-step bioconversion of CPC --- p.11 / Chapter 1.6. --- Phylogenetics and physiological roles of DAAO --- p.15 / Chapter 1.7. --- Yeast DAAOs are suitable candidates for enzymatic bioconversion --- p.17 / Chapter 1.8. --- Structural and mechanistic studies of DAAOs --- p.18 / Chapter 1.9. --- "Modifications of pkDAAO, RgDAAO and TvDAAO" --- p.25 / Chapter 1.10. --- Objectives of the study --- p.26 / Chapter CHAPTER 2 --- HOMOLOGY MODELLING / Chapter 2.1. --- Introduction --- p.27 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Sequence alignment and selection of homologs --- p.28 / Chapter 2.2.2. --- Generation of three-dimensional TvDAAO model --- p.28 / Chapter 2.3. --- Results --- p.29 / Chapter 2.4. --- Discussion --- p.33 / Chapter CHAPTER 3 --- "MUTAGENESIS, EXPRESSION, PURIFICATION AND SCREENING OF MUTANTS" / Chapter 3.1. --- Introduction --- p.38 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Cloning of TvDAAO mutants / Chapter 3.2.1.1. --- Preparation of competent E. coli --- p.39 / Chapter 3.2.1.2. --- Transformation of E. coli --- p.40 / Chapter 3.2.1.3. --- Agarose gel electrophoresis and gel-purification --- p.41 / Chapter 3.2.1.4. --- Plasmid extraction --- p.42 / Chapter 3.2.1.5. --- Site-directed mutagenesis of TvDAAO --- p.42 / Chapter 3.2.2. --- Heterologous expression and purification of mutants / Chapter 3.2.2.1 --- Shake flask fermentation --- p.45 / Chapter 3.2.2.2. --- Cell harvest and disruption --- p.45 / Chapter 3.2.2.3. --- Purification of WT and mutants --- p.47 / Chapter 3.2.2.4. --- Determination of protein concentration --- p.47 / Chapter 3.2.2.5. --- SDS-PAGE --- p.48 / Chapter 3.2.3. --- Screening of mutants --- p.48 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Preparation of purified TvDAAO mutants --- p.50 / Chapter 3.3.2. --- Evaluation of activity and thermostability --- p.50 / Chapter 3.4. --- Discussion --- p.53 / Chapter CHAPTER 4 --- ENZYME KINETICS / Chapter 4.1. --- Introduction --- p.57 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Standard assay --- p.58 / Chapter 4.2.2. --- Determination of kinetic parameters --- p.59 / Chapter 4.2.3. --- Inhibitory studies --- p.59 / Chapter 4.2.4. --- Effects of pH --- p.60 / Chapter 4.2.5. --- Heat treatments --- p.60 / Chapter 4.2.6. --- CD measurements --- p.61 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Time progress curve analysis --- p.61 / Chapter 4.3.2. --- Kinetics of WT and mutants --- p.62 / Chapter 4.3.3. --- Temperature-dependent and time-dependent thermostability --- p.67 / Chapter 4.3.4. --- Secondary structure measurements --- p.71 / Chapter 4.4. --- Discussion --- p.71 / Chapter CHAPTER 5 --- CONCLUSIONS AND PERSEPECTIVES --- p.83 / BIBLIOGRAPHY --- p.86

O presente estudo teve como objetivo avaliar níveis ótimos de valina digestível. Foram conduzidos três ensaios com frangos de corte da linhagem Cobb nas fases inicial (1-14 dias), crescimento (14-28 dias) e terminação (28-42 dias), com ensaios realizados separadamente para cada fase, tendo como base o método dose-resposta. Para os ensaios de 1 a 14dias e de 14 a 28 dias, 672 amimais foram distribuídos em delineamento inteiramente ao acaso, em oito tratamentos (7 níveis de valina e 1 controle), com 7 repetições, sendo cada unidade experimental composta por 12 aves , porém no ensaio de 28 a 42 dias foram utilizados 560 frangos, igualmente distribuídos como nos ensaios anteriores em 8 tratamentos com 7 repetições com 10 aves cada. Foram formuladas dietas basais pela técnica da diluição, deficientes em valina, contendo níveis de energia, minerais e vitaminas conforme recomendações de Rostagno et al. (2005), para cada fase. As recomendações dos níveis de valina digestível para cada fase, obtidas no presente estudo, foram realizadas com base nas respostas de CA. As recomendações obtidas pelo método dose resposta são de 0,917; 0,905 e 0,783% de valina digestível para frangos de 1 a 14 dias, 14 a 28 e 28 a 42 dias de idade, respectivamente. / This study was to evaluate optimal levels of digestible valine. Three trials were conducted with broiler chickens of Cobb in the initial phase (1-14 days), growth (14-28 days) and termination (28-42 days), with tests performed separately for each phase, based on the method dose-response. For tests 1 to 14 days and 14 to 28 days, 672 animals it were distributed in a completely randomized in eight treatments (seven levels of valine and 1 control), with 7 replicates, each replicate consisting of 12 birds but the test of 28 to 42 days 560 chickens were used, equally distributed as in previous trials in 8 treatments with 7 replicates of 10 birds each. Basal diets were formulated by the dilution technique, deficient in valine, containing levels of energy, minerals and vitamins as recommended by Rostagno et al. (2005), for each phase. The recommended levels of digestible valine for each phase, obtained in this study were based on responses from CA. The recommendations obtained by the dose response are 0.917, 0.905 and 0.783% of digestible valine for broilers from 1-14 days, 14-28 and 28-42 days of age, respectively.

Os argilominerais são empregados na área farmacêutica e cosmética tanto como excipientes quanto ingredientes ativos. Esses compostos inorgânicos são inertes quimicamente, apresentam estruturas definidas e alta estabilidade térmica, o que contribui para o uso nessas áreas. Atualmente a indústria farmacêutica busca modificações no sistema de entrega de drogas (melhorias no tempo, local e taxa de liberação), objetivando um aumento na estabilidade das drogas e a prevenção e diminuição de efeitos colaterais. Nesse sentido, surge a necessidade de desenvolver novas formulações farmacêuticas, novos métodos de preparação e novos materiais. Considerando o fato dos argilominerais incorporarem espécies diversas entre suas lamelas, é interessante explorar a possibilidade de uso dessas matrizes inorgânicas como carregadores de espécies bioativas. O principal objetivo do presente trabalho foi preparar e caracterizar argilas de uso farmacêutico e/ou cosmético intercaladas com espécies que apresentam potencial terapêutico. Para tanto, usou-se duas argilas esmectitas naturais do tipo montmorilonita (Cloisita Sódica e Veegum HS) e uma esmectita sintética do tipo hectorita (Laponita RD). Os aminoácidos L-lisina, L-arginina e L-ornitina, e o dipeptídeo L-carnosina foram imobilizados em argilas catiônicas, por meio de reação de troca iônica. Na preparação dos materiais híbridos, alguns parâmetros experimentais foram avaliados: concentração hidrogeniônica (pH) da suspensão de reação, proporção argila/aminoácido e tempo de reação. As argilas precursoras e os materiais híbridos obtidos foram caracterizados por difratometria de raios X, espectroscopia vibracional na região do infravermelho e Raman, análise termogravimétrica acoplada à espectrometria de massas e análise química de carbono. Os valores de distância interlamelar (d(001)) dos materiais sugerem que a cadeia carbônica das espécies orgânicas se orienta paralelamente em relação às lamelas de baixa densidade de carga dos argilominerais. Nos espectros vibracionais na região do infravermelho há predominância das bandas características da estrutura inorgânica, mas as bandas entre 1800 e 1400 cm-1 relativas aos grupos funcionais do aminoácido permitem inferir sobre o seu grau de protonação no material híbrido. A acidez de Brönsted gerada pela polarização das moléculas de água associadas à argila foi observada para as montmorilonitas empregadas neste estudo. Amostras preparadas em suspensões nas quais o valor do pH era maior que o valor da primeira constante ácida (pKa1) dos aminoácidos apresentam bandas atribuídas ao estiramento C=O de grupo carboxilato protonado. Os espectros Raman foram obtidos apenas para a argila sintética, uma vez que as naturais apresentam luminescência. O espectro Raman da L-carnosina imobilizada em Laponita indica a presença preponderante da espécie zwitteriônica; o deslocamento das bandas atribuídas aos grupos amida e carboxílico do dipeptídeo para região de menor energia sugere a formação de ligações de hidrogênio com os grupos silanol da Laponita. Os resultados de análise termogravimétrica acoplada à espectrometria de massas dos materiais híbridos são distintos daqueles observados para os aminoácidos livres. A temperatura de início de decomposição das espécies orgânicas não é praticamente modificada após imobilização nas argilas, mas os processos térmicos se estendem até regiões de maior temperatura, evidenciando a influência da estrutura inorgânica sobre a decomposição térmica dos aminoácidos. Através dos dados de quantidade de carbono e de água nas amostras, calculou-se a concentração de aminoácidos nos materiais híbridos (massa de aminoácido / 100 gramas de material). As maiores concentrações de aminoácido (entre seis e oito por cento) foram observadas para as amostras de Cloisita e Veegum HS, isoladas em condições nas quais predomina a interação eletrostática entre as lamelas e os aminoácidos com carga positiva. Nas condições experimentais empregadas neste trabalho não foi observada a saturação das argilas com os aminoácidos, ou seja, as cargas das lamelas não foram totalmente neutralizadas pelos íons orgânicos. / Clay minerals are used as excipients or active ingredients in the pharmaceutical and cosmetic fields. These inorganic compounds are chemically inert, have defined structures and high thermal stability, which make them useful for these areas. Currently the pharmaceutical industry seeks modifications in the drug delivery systems (improvements in the time, place and rate of release), aiming an increase in the stability of the drugs and also the prevention and reduction of side effects. In this way, it is a need to develop new pharmaceutical formulations, new preparation methods and new materials. Considering the fact that clay minerals incorporate various species between their layers, it is interesting to explore the possibility of using these inorganic matrices as carriers of bioactive species. The main aim of this work was to prepare and characterize clays of pharmaceutical and/or cosmetic usage intercalated with species of therapeutic potential. Two natural smectite clays of montmorillonite type (Sodium Cloisite and Veegum HS) and one synthetic smectite of hectorite type (Laponita RD) were employed. The amino acids L-lysine, L-arginine and L-ornithine, and the L-carnosine dipeptide were immobilized on cationic clays by ion exchange reaction. Some experimental parameters were evaluated in the preparation of hybrid materials: hydrogen ion concentration (pH) of reaction suspension, clay/amino acid proportion and reaction time. Pristine clays and hybrid materials were characterized by X-ray diffraction, infrared and Raman vibrational spectroscopies, thermogravimetric analyses coupled to mass spectrometry and chemical analysis of carbon. The materials values of interlayer distance (d(001)) suggest that the carbon chain of the organic species is oriented parallel to the layers of clay minerals. The infrared vibrational spectra are dominated by the inorganic structure bands; however the bands between 1800 and 1400 cm-1 related to the functional groups of the amino acid allow to infer about the protonation degree in the hybrid material. The Brönsted acidity generated by the polarization of water molecules associated with the clay was observed for montmorillonite samples used in this study. Materials prepared in suspensions in which the pH value was greater than the value of the first acid constant (pKa1) show bands assigned to the C=O stretching of protonated carboxylate group. Raman spectra were obtained only for the synthetic clay, since the natural ones luminesce. Raman spectrum of L-carnosine immobilized on Laponita indicates the presence of mostly zwitterionic species; the displacement of bands assigned to amide and carboxylic groups of the dipeptide to the lower energy region suggests the formation of hydrogen bonds with the Laponita silanol groups. The results of thermogravimetric analyses coupled to mass spectrometry of hybrid materials are different from those observed for the free amino acids. The onset temperature of the organic species decomposition is practically unmodified after the immobilization on clays, but thermal processes are postponed up to higher temperature, revealing the inorganic structure influence on the amino acids thermal decomposition. Data on the carbon and water amounts in the samples were used to calculate the concentration of amino acids in the hybrid materials (mass of amino acid / 100 grams of material). The highest concentrations of amino acid (between six and eight percent) was observed for Cloisite and Veegum HS samples, isolated under conditions in which the electrostatic interaction between the layers and the positively charged amino acids are predominant. Under the experimental conditions employed in this study no saturation of clay with amino acids was observed, i.e. the layer charges were not completely neutralized by the organic ions.

Neste trabalho foi realizada a síntese de amidas, bem como de ácidos e ésteres borônicos, derivados dos aminoácidos L-cistina e L-cisteína, através de rota sintética simples, curta e de baixo custo, com o intuito de busca e a identificação de novo(s) inibidor(es) do proteassomo 20S. Esta classe de compostos possui estrutura que permite a inserção de diversos grupos funcionais, o que confere versatilidade e a construção de biblioteca de compostos que contém partes hidrofílicas e hidrofóbicas importantes para posterior avaliação inibitória. Para tanto, empregou-se rota sintética química convencional e rota biocatalisada para a formação da ligação amida. Os compostos derivados de L-cisteína foram obtidos via síntese clássica de peptídeos a qual forneceu os compostos desejados em rendimentos de até 85%. Por outro lado, tentativas de obtenção das amidas via biocatálise não se mostraram efetivas. Já amidas derivadas de L-cistina foram obtidas em rendimentos de até 79%, via síntese tradicional e até 100% de conversão através de rota biocatalítica. A inserção do átomo de boro nas estruturas se deu utilizando-se metodologias sintéticas já bem estabelecidas na literatura. Os ésteres borônicos derivados de L-cisteína foram obtidos em bons rendimentos (até 78%), enquanto que não foi possível obter-se compostos de boro derivados de L-cistina. Por sua vez, os compostos contendo ácido borônico na estrutura foram sintetizados via reação de hidrólise dos respectivos ésteres borônicos, em rendimentos moderados (até 34%). Após a obtenção dos compostos contendo grupamentos organoboro realizou-se avaliação inibitória dos mesmos frente ao proteassomo 20S. Valores de IC50 iguais a 52 &#181;M foram obtidos para composto derivado de L-cisteína contendo grupamento éster borônico, que se mostraram inibidores moderados e reversíveis. Ácidos borônicos se mostraram sem capacidade de inibir o proteassomo 20S. Adicionalmente, realizaram-se estudos de modelagem molecular com a finalidade de elucidar os resultados obtidos experimentalmente. Inicialmente realizaram-se cálculos de modelagem molecular através da realização de docking de alguns compostos e após geração de modelo farmacofórico. De maneira geral observou-se que os inibidores derivados da L-cisteína não ocupam a mesma cavidade que o fármaco bortezomibe, o que pode explicar a diferença na atividade dos compostos frente à inibição do proteassomo 20S. Também se observou que, tendo-se a interação dos inibidores com a enzima, a vizinhança do átomo de boro tem grande influência na capacidade inibitória, uma vez que estes grupamentos determinam qual a região da cavidade do proteassomo 20S será ocupada pelo inibidor. / In our study, amides, boronic acids and esters derivatives from L-cysteine and L-cysteine were synthesized by simple, short and inexpensive synthetic route, in order to search for new inhibitor(s) of the 20S proteasome. This class of compounds has a structure that allows inclusion of various functional groups, giving it versatility and allowing the construction of library compounds containing hydrophilic and hydrophobic moieties, important for further evaluation. To this end, we used conventional chemical synthetic route and biocatalysis for peptide bond formation. The compounds derived from L-cysteine were obtained by classical synthesis of peptides which provided the desired compounds up to 85% yields. On the other hand, attempts to obtain the amides via biocatalysis were not effective. However, amides derived from L-cystine were obtained with up to 79% yields via chemical synthesis and conversion up to 100% using biocatalytic route. The insertion of the boron atom in the structures was possible using synthetic methodologies well established in literature. Boronic esters derived from L-cysteine were obtained in good yields (up to 78%), whereas it was not possible to obtain boron compounds derived from L-cystine. In turn, compounds containing boronic acids in the structure were synthesized by hydrolysis reaction of the respective boronic esters in moderate yields (up to 34%). With organoboron compounds in hand, we turned our attention to inhibitory assessment against the 20S proteasome. IC50 up to 52 &#181;M were obtained when L-cysteine boronic ester derivatives were evaluated. These compounds are moderate and reversible inhibitors. L-cysteine boronic acids derivatives have shown not ability to inhibit the 20S proteasome. Additionally, molecular modeling studies were carried out in order to elucidate the results obtained experimentally. Initially molecular modeling calculations were carried out by performing docking experiments of some compounds. Generation of pharmacophoric model calculations was also executed. In general, it was observed that inhibitors derived from L-cysteine do not occupy the same cavity that drug bortezomib, which may explain the difference in the activity of compounds against the inhibition of 20S proteasome. We also observed that, with the interaction of inhibitors and enzyme, the side chains around boron atom has a great influence on inhibitory capacity, since these groups determine which region of the 20S proteasome cavity is occupied by the inhibitor.

Protozoários do gênero Leishmania são parasitas digenéticos, com uma fase no tubo digestório de um hospedeiro invertebrado (promastigota), e uma fase parasita intracelular de macrófagos (amastigotas). Estudar a demanda de L-arginina no parasita é interessante, uma vez que o aminoácido é indispensável para a sobrevida do parasita e, ao mesmo tempo, serve de substrato para a produção de óxido nítrico, principal composto microbicida dos macrófagos. O objetivo deste trabalho foi caracterizar o transporte de arginina em Leishmania (Leishmania) amazonensis do ponto de vista fisiológico e caracterizar o gene que codifica o transportador do aminoácido, bem como a regulação da sua expressão em resposta a diferentes condições biológicas. Para medir o influxo de L-arginina em fagolisossomos, utilizamos macrófagos J774 infectados com L. (L.) amazonensis e desenvolvemos uma metodologia com citometria de fluxo com sorting para a purificação da organela. Validamos microscopicamente a presença do parasita na organela por sua fluorescência, e avaliamos a integridade da membrana externa dessa com marcador de pH ácido. Paralelamente, o gene que codifica o transportador de arginina do parasita foi caracterizado. Foram encontradas duas cópias em tandem que produzem dois transcritos (5.1AAP3 e 4.7AAP3), cujas regiões 5\'UTR e 3\'UTR são diferentes. Por meio de PCR quantitativo em tempo real, avaliamos a expressão desses transcritos e verificamos que 5.1AAP3 é mais expressa ao longo do desenvolvimento do parasita, com um máximo em fase estacionária. A determinação da meia-vida dos mRNA das duas cópias indicou uma duração de 32,6±5,0min para o mRNA de 4.7AAP3, enquanto que o de 5.1AAP3 não apresentou decaimento até 180min do estudo, evidenciando que a estabilidade maior pode ser a razão de sua maior abundância. A submissão de parasitas à privação de arginina levou a aumento na tomada do aminoácido concomitante ao aumento do transcrito 5.1AAP3. Mutantes nulos de arginase submetidos à privação de arginina respondem com uma taxa de incorporação mais baixa em relação aos parasitas selvagens, e mantém a resposta à privação mesmo com os parasitas em fase estacionária, diferente do observado nos parasitas selvagens. Esse conjunto de resultados nos levou a sugerir que a expressão do transportador pode ser regulada pela estabilidade do mRNA, e que o pool de arginina interno ao parasita pode controlar, num mecanismo de retroalimentação negativo, a expressão de seu transportador. / Protozoan of genus Leishmania are digenetic parasites that present a stage in the life in insect gut (promastigotes) and an intracellular phase (amastigotes) inside vertebrate host macrophages. The study of L-arginine influx consists in an interesting matter, since the amino acid is used on NO production pathway (the main macrophage microbial pathway) but are also important for parasites survival. The aim of this work was to perform a genetic and physiological characterization of the arginine transport in Leishmania (Leishmania) amazonensis. To verify how does the arginine uptake occurs in the phagolisosomes, we used J774 macrophages infected with L. (L.) amazonensis to establish a flow cytometry sorting protocol to purify the organelle. Microscopic validation of organelle integrity was achieved by acidic pH marker treatment and detection of fluorescent parasites. The arginine transporter coding gene was characterized. We found two copies in tandem that produces two transcripts, named 5.1AAP3 and 4.7AAP3, with distinct 5\'UTR and 3\'UTR. By quantitative real time PCR we found that 5.1AAP3 mRNA expression varies along parasite development. This copy was, also, more abundant than 4.7AAP3 mRNA. This last mRNA showed a half-life of 32.6±5.0 min, while the 5.1AAP3 mRNA did not decay until 180min. As response to arginine starvation, wild type parasites increase the uptake of arginine, as well as the abundance of 5.1AAP3 mRNA. Arginase null parasites starvation responses showed lower arginine uptake rates compared to wild type responses. Unlike wild type, the null mutants also respond to starvation in stationary phase. This data set allow us to propose that arginine internal pool can downregulate its transporter expression in a feed-back mechanism.

No description available.

There has been limited investigation as to whether a predominantly forage-based diet can provide adequate amounts of limiting amino acids (AA) to horses. The first objective was to determine if AA supplementation of AA believed to be limiting to protein synthesis in forage-based diets would affect measures of whole-body protein metabolism in sedentary mature horses. The effect of forage type (timothy or alfalfa) and AA supplementation (lysine, threonine or histidine) on plasma urea nitrogen (PUN) and AA concentrations and measures of whole-body phenylalanine kinetics were evaluated. There was no effect of either forage type or AA supplement on rates of whole-body protein synthesis (P > 0.05). The second objective was to determine the effects of either timothy or alfalfa hay supplemented with either a high or low protein ration balancer on measures of whole-body protein metabolism in yearling horses. The effect of forage type and the ration balancer protein level on concentrations of PUN, plasma AA and measures of wholebody phenylalanine kinetics were evaluated. There was no effect of treatment on average daily gain (P = 0.18). When horses consumed the alfalfa-based diets, rates of phenylalanine flux, oxidation and use for protein synthesis were greater than when they consumed timothy-based diets (P < 0.05). Phenylalanine use for protein synthesis was not affected by the protein level of the ration balancer (P = 0.3). Yearling horses achieve greater rates of protein synthesis when fed alfalfa-based diets, compared to timothy-based diets, supplemented with a low protein ration balancer.

Molecular dynamics simulations are an increasingly valuable tool to biochemical researchers: advances in computational power have expanded the range of biomolecules that can be simulated, and parameters describing these interactions are increasingly accurate. Despite substantial progress in force field parameterization, recent simulations of protein molecules using state-of-the-art, fixed-charge force fields revealed that the interactions among and within protein molecules can be too favorable, resulting in unrealistic aggregation or structural collapse of the proteins being simulated. To understand why these protein-protein interactions are so over-stabilized, I first assessed the ability of simulation force fields to represent accurately the interactions of individual amino acids, employing an osmotic pressure simulation apparatus that enabled direct comparison with experiment. Surprisingly, simulations of most of the amino acids resulted in behavior that was in strong agreement with experiment. A number of amino acids, however—notably those that contain hydroxyl groups and those that carry a formal charge—interacted in ways that were clearly inaccurate. Additionally, some commonly-used force fields failed to accurately represent the interactions of amino acids in a consistent manner. By further investigating the interactions of the functional groups of these amino acids, I was able not only to determine some of the root causes of individual amino acid inaccuracies, but also to implement simple modifications that brought the interactions of these small molecules and amino acids in stronger accord with experiment. These studies have highlighted some of the shortcomings in popular simulation force fields, and have proposed useful modifications to address them. Still, there is additional work that must be—and is being—conducted in order to correctly model the interaction behavior of proteins in simulation.