O Experimento foi realizado com o objetivo de avaliar a substituição parcial e total de plasma bovino por levedura hidrolisada na dieta de leitões desmamados no período de 21 a 47 dias de idade. Foram utilizados 1600 leitões da linhagem PIC, distribuídos em blocos ao acaso, com quatro tratamentos. As dietas foram divididas em quatro fases (pré-inicial I 22 a 28 dias; pré-inicial II 29 a 35 dias; inicial I - 36 a 47 dias e inicial II 48 a 63 dias). A inclusão percentual, Plasma: Levedura, nas dietas foi: T1 (6:0; 4:0; 2:0 e 0:0); T2 (3: 4; 2: 3; 1: 2 e 0: 0); T3 (1,5: 6; 1: 4,5; 0,5: 3 e 0: 0) e T4 (0: 8; 0: 6; 0: 4 e 0: 0). Cada tratamento teve 10 repetições (cinco de machos e cinco de fêmeas) totalizando 40 unidades experimentais com 40 animais cada. As variáveis zootécnicas avaliadas durante o período experimental foram: consumo de ração, ganho de peso, e conversão alimentar. Foram tomadas amostras de sangue de 5 animais por tratamento no dia de início do experimento, e de 10 animais por tratamento 25 dias depois. Foram quantificadas a IgA e a IgG. Os dados obtidos foram submetidos a análise de variância ANOVA, utilizando-se o teste Tukey para comparação das médias ao nível de significância de 5% utilizando o pacote estatístico SAS. Não houve diferença estatística nas quantidades de Ig A e Ig G circulante, nem na variação destas imunoglobulinas no tempo, entre os tratamentos. Considerando a análise dos dados conclui-se que nas condições estudadas, a utilização da relação percentual de (1,5: 6; 1: 4,5; 0,5: 3 e 0: 0) de plasma: levedura hidrolisada resultou um maior consumo de ração e ganho de peso dos animais, em comparação às outras proporções, e consequentemente podendo trazer um maior lucro para o produtor / The experiment was performed with the objective of evaluating the partial and complete substitution of bovine plasma for hydrolyzed yeast in the diet of weaned piglets from 21 to 47 days old. 1600 piglets of PIC lineage were used and randomly distributed in blocks where they received four treatments. Their diets were divided into four phases (pre-initial I: 22 to 28 days; pre-initial II- 29 to 35 days; initial I- 36 to 47 days and initial II-48 to 63 days). The percentage inclusion Plasma:Yeast in the diets were: T1 (6:0; 4:0; 2:0 and 0:0); T2 (3: 4; 2: 3; 1: 2 and 0: 0); T3 (1.5: 6; 1: 4.5; 0.5: 3 and 0: 0) and T4 (0: 8; 0: 6; 0: 4 and 0: 0). Each treatment had 10 repetitions (five times with the males and five times with the females), totaling 40 experimental units with 40 animals each. Husbandry variables evaluated during the trial period were: feed intake, body weight gain and feed conversion. Blood samples of 5 animals per treatment were taken at the beginning of the experiment day, and 10 animals per treatment 25 days later. IgA and IgG were quantified. The data obtained were analyzed by ANOVA analysis of variance, using the Tukey test to compare means at the 5% significance level, using the statistical package SAS. There was no statistical difference in the quantities of Ig A and Ig G circulating, or the variation of these immunoglobulins in the time, between treatments. Considering the analysis of the data it was concluded that the conditions studied, the percentage utilization ratio of (1.5: 6; 1: 4.5: 0.5: 3 and 0: 0) plasma: hydrolyzed yeast resulted in a higher feed intake and weight gain of animals compared to other proportions, and consequently can bring higher profits to the producer

Existe atualmente uma restrição muito forte ao uso de antibióticos como promotores de crescimento na alimentação avícola, uma vez que tais produtos passaram a ser vistos como fatores de risco para a saúde humana. Estudos com produtos alternativos tornaram-se necessários e, neste sentido, as leveduras vem sendo exploradas, principalmente, seu conteúdo celular. Dessa forma, o presente estudo teve como objetivo avaliar o uso de levedura hidrolisada, fonte de nucleotídeos, sobre o desempenho e imunidade de frangos de corte, suplementados no período de 1 a 14 dias de idade. Foram utilizados 576 pintos de corte, machos, linhagem Ross 308, criados até 42 dias de idade sobre cama reutilizada. O delineamento foi inteiramente casualizado com 6 tratamentos: controle negativo (sem suplementação); controle positivo (com antibiótico) e diferentes níveis de adição de levedura hidrolisada (0,25%; 0,50%; 0,75% e 1,00%), sendo 8 repetições/tratamento. Considerando-se o período total de criação, foram observados efeitos significativos para os parâmetros de ganho de peso (GP) e conversão alimentar (CA). Por sua vez, o parâmetro de consumo de ração (CR) não apresentou efeitos significativos entre os tratamentos. Com relação à imunidade, a suplementação com levedura hidrolisada apresentou efeito sobre a resposta a vacinação contra a doença de Newcastle, produção de água oxigenada, óxido nítrico e influenciou também no peso relativo dos órgãos linfóides, com destaque para o nível 0,75% de suplementação. Diante dos resultados obtidos no presente estudo, pode-se concluir que a levedura hidrolisada, suplementada durante o período de 1 a 14 dias de idade, promove efeitos significativos no desempenho durante todo o período de criação e também influencia na imunidade das aves. / There is currently a strong restriction on the usage of antibiotics as growth promoters in broiler feed, since such products were seen as risk factors for human health. Research with alternative products has became necessary, and the yeast has been exploited, particularly its cellular content. Thus, this experiment was aimed at evaluating the use of hydrolyzed yeast, source of nucleotides on performance and immunity of broiler chickens supplemented during the period of 1 to 14 days old. A total of 576 male chicks, Ross 308 were raised up to 42 days old, on reused litter. The design was completely randomized with six treatments: negative control (no supplementation), positive control (with antibiotic) and different levels of hydrolyzed yeast (0.25%; 0.50%; 0.75% e 1.00%), with 8 replicates per treatment. Considering the whole breeding period, significant effects were observed for parameters of weight gain (WG) and feed conversion (FC). However, the parameter of feed consumption (FC) had no significant effect among the treatments. Regarding to immunity, the hydrolyzed yeast supplementation had an effect on the response to vaccination against Newcastle disease, production of hydrogen peroxide, nitric oxide and also influenced the relative weight of lymphoid organs, especially the 0.75% level of supplementation. With the results obtained in this experiment it is possible to conclude that the hydrolyzed yeast supplementation, during the period of 1 to 14 days old, promotes a significant effect on performance throughout the whole rearing period and also influences the immunity of the broiler chicks.

Orientador: Dirlei Antonio Berto / Coorientador: Fabiana Ribeiro Caldara / Banca: Marcos Lívio Panhoza Tse / Banca: Luciano Hauschild / Resumo: Os objetivos desta pesquisa foram avaliar os efeitos da utilização de nucleotídeos (NU) e ácido glutâmico (AG) na dieta de leitões sobre o desempenho e perfil de lipídeos plasmáticos (colesterol total - CT, triglicérides - TRI, HDL colesterol e LDL+VLDL colesterol) (experimento I) e turnover do 13C na mucosa do duodeno e jejuno (meia-vida do 13C) (experimento II). Em ambos os experimentos foi utilizado delineamento experimental de blocos ao acaso, com esquema fatorial 2x2 dos tratamentos, sendo dois níveis de NU (0 e 0,1%) e dois níveis de AG (0 e 1%), com sete repetições e três animais por parcela. O primeiro experimento teve duração de 35 dias e foram utilizados 84 leitões desmamados aos 21 dias de idade e com peso médio inicial de 6,54±0,70kg. No segundo experimento, nos dias 3, 6, 9, 14, 21, 34 e 49 após o desmame, foram abatidos três leitões por tratamento e no dia do desmame (dia zero) foram abatidos três leitões, totalizando 87 animais com peso inicial de 6,28±0,13kg. Não houve interação dos fatores nas variáveis analisadas e nem efeitos de NU e do AG no consumo diário de ração, ganho diário de peso, CT, HDL colesterol, TRI, LDL + VLDL colesterol e no turnover do 13C na mucosa intestinal. O uso de NU na dieta piorou (P≤0,05) a conversão alimentar no período de 0 a 27 dias, mas esta variável não foi influenciada no período total de experimento (0 a 35 dias). Ocorreu redução (P<0,0001) na concentração plasmática de todas as variáveis estudadas aos 56 dias em relação aos 21 dias de idade e na mucosa do jejuno a meia-vida do 13C foi maior nos leitões mais leves (P≤0,05). Conclui-se, portanto, que a adição de NU e AG nas dietas não interferem no desempenho, no perfil lipídico plasmático e no turnover do 13C na mucosa do duodeno e jejuno de leitões recém-desmamados no período total de creche / Abstract: The objectives of this research were to evaluate the effects of nucleotides (NU) and glutamic acid (AG) in the diet of pigs on performance and plasma lipid profile (total cholesterol - CT, triglycerides - TRI, HDL cholesterol and LDL + VLDL cholesterol ) (experiment I) and turnover of 13C in duodenal and jejunum mucosa (13C half-life) (experiment II). In both experiments we used experimental design of randomized blocks, with 2x2 factorial treatments, with two levels of NU (0 and 0.1%) and two levels of AG (0 and 1%), each treatament had seven replicated and three pigs per pen. The first experiment lasted 35 days and were used 84 piglets weaned at 21 days of age and average initial weight of 6.54 ± 0,70kg. In the second experiment, on days 3, 6, 9, 14, 21, 34 and 49 after weaning, were slaughtered three pigs per treatment and the day of weaning (day zero) were slaughtered three piglets, totaling 87 animals with initial weight 6.28 ± 0,13kg. There was no interaction of factors in the analyzed variables and not effects of NU and AG in daily feed intake, daily weight gain, CT, HDL cholesterol, TRI, LDL + VLDL cholesterol and 13C turnover in the intestinal mucosa. The use of NU in the diet has worsened (p≤0.05) feed conversion in the period 0-27 days, but this variable was not affected in the total period (0-35 days). We observed a decrease (P<0.0001) in plasma concentration of all variables after 56 days compared to 21 days of age and in the jejunal mucosa the 13C half-life was higher in lighter piglets (p≤0.05). It can be concluded therefore that the addition of AG and NU in the diets did not affect performance, the plasma lipid profile and 13C turnover in the duodenum and jejunum mucosa of weaned piglets in total nursery period / Mestre

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 121-137).

We present research on the design, development and application of algorithms for DNA sequence analysis, with a focus on environmental DNA (metagenomes). We present an overview and primer on algorithm development for bioinformatics of metagenomes; work on frameshift detection in DNA sequencing data; work on a computational pipeline for the assembly, feature prediction, annotation and analysis of bacterial genomes; work on unsupervised phylogenetic clustering of metagenomic fragments using Markov Chain Monte Carlo methods; and work on estimation of bacterial genome plasticity and diversity, potential improvements to the measures of core and pan-genomes.

G protein coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins and downregulation. The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension. How GRKs recognize and are activated by GPCRs are not known, in part because the critical N-terminus and the kinase C-terminal extension were not resolved in GRK2 and GRK6 structures. The long-term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1), which represents a model system for studying phosphorylation-dependent desensitization of activated GPCRs. Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide-ligand binding states. One of the (Mg²⁺)₂·ADP·GRK1 structures is the most high-resolution structure (1.85 Å) of a GRK to date. In one (Mg²⁺)₂·ATP·GRK1 structure, almost the entire N-terminal region (residues 5-30) is observed. In addition, different segments of the kinase C-terminal extension are ordered in the various nucleotide-bound structures. Together, these two elements form a putative receptor-docking site adjacent to the hinge of the kinase domain. Based on these structures, a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase. Two novel phosphorylation sites were also identified at the N-terminus. The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS) homology domain of GRK1 was assessed using site-directed mutagenesis. In addition to mediating interaction with activated GPCRs, the N-terminus of GRKs also forms a binding site for calcium sensing proteins. Although its physiological significance is debated, the structures of these complexes could lend further insights into the conformation of the N-terminus of GRKs. The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes. Finally, the RH domain of GRK2 binds G[alpha subscript q], G[alpha]₁₁, and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors. The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆, a member of G[alpha subscript q] family.

published_or_final_version / Paediatrics / Master / Master of Philosophy

The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

Nucleotides, such as ATP and its derivatives, are released at high concentrations at sites of inflammation and modulate the immune response. When cultured in the presence of ADP or stable analogue ADP?S, DC surface expression of MHC-II and co-stimulatory molecules, CD40 and CD86 was unchanged. When DCs were pre-treated with ADP or ADP?S, there was no change in their ability to activate naïve CD4+ T cells. However, when CD4+ T cells were activated in the presence of ADP or ADP?S, activation and proliferation were significantly decreased. This correlated with a significant reduction in IL-2 secretion and CD25 surface expression, which may be due to decreased ERK and Akt phosphorylation. CD8+ T cell proliferation was unaffected by the addition of ADP or ADP?S, but secretion of IFN-? was significantly reduced. By demonstrating that ADP inhibits CD4+ T cell responses, we have identified a potential target of immune modulation by clinical intervention.

During periods of intense exercise the adenine nucleotide pool in skeletal muscle becomes partially depleted. Ribose has been shown to increase rates of both purine salvage and adenine nucleotide de novo synthesis in rat skeletal muscle. However, to date no research has been conducted on the effects of ribose on adenine nucleotide levels in human skeletal muscle. Therefore, the purpose of this investigation was to determine the effects of ribose supplementation on adenine nucleotide levels in human skeletal muscle during high-intensity exercise. To do this, an 11-day supplementation of either ribose (20 g.d-1) or placebo (glucose 20 g•d-1) was given to 16 healthy male subjects. After 72 hours of supplementation, the subjects performed five-days of high-intensity exercise designed to elicit significant reductions in adenine nucleotides. A 65 hour recovery period was completed following the exercise protocol. Muscle biopsies were performed at four different time points during the supplementation/exercise period.The exercise protocol elicited significant decreases in skeletal muscle adenine nucleotide levels of both the ribose and placebo groups (p<0.05). However, ribose supplementation was shown to partially attenuated the adenine nucleotide decrease. The placebo group demonstrated a 39% decrease, while the ribose group dropped 23% in TAN levels (p<0.05). The largest decreases were observed in ATP for both groups; however, the decreases were significantly greater in the placebo group (p<0.05). Both groups displayed a similar amount of replenishment in adenine nucleotides 65 hours following the 5-day exercise period. No differences were demonstrated in ADP or AMP for either the ribose and placebo groups throughout the investigation. Both groups displayed an increase in E MP post-exercise, but the increase was only significant in the ribose group (p<0.05). No differences in mean power, peak power, and fatigue were observed between the ribose and placebo group. However, the ribose group consistently displayed a non-significantly greater percent change (3.1 %) in mean power. / School of Physical Education