Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. How Haa1 is activated by weak acids is not clear. This study proposes that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. YRO2, one of the targets of Haa1, shows increase in expression during stationary phase. This increase is due to basal activity of Haa1 and another, unknown, transcription factor. This study proposes that Gsm1 is another transcription factor that regulates YRO2 expression in the stationary phase. Finally, the mechanism of regulation of YRO2 by Haa1 is largely unknown. This study identifies two possible Haa1-medated cis-acting elements in the YRO2 promoter.

Doctor of Philosophy / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Previous data suggest that greater amounts of postruminal protein increase small intestinal starch digestion in cattle. Duodenally and ileally cannulated steers were used in 5 studies to measure responses in small intestinal starch digestion to amino acids (AA) or casein. Flows of starch to the ileum from the diet were small. Small intestinal starch digestibility was 34.0% when raw cornstarch was continuously infused into the duodenum. Infusion of casein linearly increased (P ≤ 0.05) small intestinal starch digestibility, and small intestinal starch digestion adapted to infusion of casein in 6 d. Ethanol-soluble starch and unpolymerized glucose flowing to the ileum increased linearly (P ≤ 0.05) with increasing infusion of casein. Plasma cholecystokinin was not affected by casein infusion, but circulating levels of glucose increased linearly (P ≤ 0.05). In another study, 5 steers were fed a low-starch diet and provided continuous duodenal infusion of raw cornstarch in combination with AA or casein in order to measure response of small intestinal starch digestion. Duodenal infusion of casein increased (P ≤ 0.05) small intestinal starch digestion. When a mixture of AA with a profile similar to casein (CASAA) was infused, small intestinal starch digestion was similar (P = 0.30) to casein infusion. Infusion of only non-essential AA tended to increase (P = 0.14) small intestinal starch digestion relative to control; however, infusion of essential AA alone did not affect (P = 0.84) small intestinal starch digestion. Additionally, infusion of casein or essential AA increased ileal flows of ethanol-soluble starch, but non-essential AA alone were not different than the negative control. Duodenal infusion of Glu increased (P ≤ 0.05) small intestinal starch digestion, whereas a mixture of Phe, Trp, and Met (PTM) did not. Neither Glu nor PTM increased ileal flow of ethanol-soluble starch, but Glu and PTM provided together tended (P = 0.07) to increase ileal flows of ethanol-soluble starch. Our data suggest that Glu alone can increase small intestinal starch digestion in cattle similar to casein, but increases in small intestinal starch digestion in response to Glu are not associated with an increase in ileal flows of ethanol-soluble starch.

Master of Science / Department of Food Science / Karen A. Schmidt / People who suffer from swallowing disorders are diagnosed with dyphasgia. The beverage for the dyphagia patients should have the apparent viscosity in the range of nectar-like (51 to 350 mPa•s) or honey-like (351 to 1750 mPa•s). Due to the swallowing problems, dysphagia patients usually consume beverages slowly. Thus, the apparent viscosity of beverage for such patients should be high enough to be in the suitable range during the entire time of consumption. Three ratios of α-lactalbumin (α-la)/β-lactoglobulin (β-lg) (3:8, 1:1 and 8:3) were used to prepare the milk systems. These ratio adjusted milk systems were either processed at 70, 80, and 90ºC for 30 min or at 25ºC, and cooled to 25 ± 1ºC. After the process was completed, the milk systems were set quiescently 120 min at 25 ±1ºC. Physical and chemical properties were assessed at various time. For the milk systems at 0 min, the apparent viscosity increased in all 90°C processed-samples, and the increase was in the order of 8:3 (15.96%), 1:1 (6.38%) and 3:8 (2.11%) compared with the 25ºC samples at each ratio. When the milk systems set for 120 min, apparent viscosity increased slightly by 3.7%. The maximum apparent viscosity was 2.18 mPa•s, which was less than nectar-like. Therefore, xanthan gum was added at 0.15 w/w % to enhance rheological properties of the milk systems. α-La/β-lg ratio adjusted milk systems either with or without xanthan gum were prepared, and processed at 90ºC or 25ºC, and cooled to 25 ± 1ºC. Apparent viscosity increased by 48.61 and 89.61% in 3:8 and 8:3 milk systems, respectively for those at 0.15% xanthan gum concentration and processed at 90ºC compared with at 25ºC. Apparent viscosity of 8:3 milk systems at xanthan gum concentration of 0.15% processed at 90°C was 58.7 ± 2.12 mPa•s which was within the nectar-like range. When the samples were set for 120 min, no changes were found in the apparent viscosity of the milk systems. If the rheological properties of the milk systems can be controlled by ingredients interactions, this can be used to develop nutritious products with different forms for dysphagia patients.

A doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva, caracterizada pela perda de neurônios corticais e subcorticais. Padrões patológicos da doença de Alzheimer incluem a presença de lesões neurofibrilares que consistem no acúmulo da proteína ?- amilóide e dos emaranhados neurofibrilares, decorrentes da hiperfosforilação da proteína Tau. Apenas dois grupos principais de fármacos são utilizados para o tratamento da DA, os inibidores colinesterásicos e antagonistas do receptor N-metil-D-aspartato. Entretanto, a fosforilação de proteínas pelas proteínas cinases constituem um dos principais mecanismos pelos quais as células se utilizam para regular seu metabolismo e demais funções e desequilíbrios nestas atividades estão relacionados a uma infinidade de doenças. O número elevado de isoformas de proteínas cinases 1 (CK1) encontradas na DA e sua associação em marcadores de lesões neurodegenerativas indicam sua participação nas etapas finais da degeneração, comum tanto à DA quanto a outras desordens neurodegenerativas. A abordagem da proteína CK1 como alvo terapêutico para a DA é promissora uma vez que os compostos usuais utilizados para diminuir a produção de ?-amilóide também bloqueiam a quebra de outras proteínas, causando graves efeitos colaterais. Cada vez mais as ferramentas de bioinformática vêm sendo utilizadas como auxílio na redução de custos e tempo para as pesquisas. Dentre estas técnicas destaca-se a triagem virtual, que reúne um conjunto de técnicas utilizadas de forma sequencial com o objetivo de selecionar compostos protótipos para os alvos desejados. Neste trabalho, foram utilizadas técnicas de triagem virtual baseada em ligantes e estrutura, a partir de uma base de 500 mil compostos, selecionando 35 compostos com perfil de atividade inibitória para a enzima CK1. Destes, os compostos 24, 25, 36, 39 41 e 42 apresentaram resultados significativos com relação ao potencial de inibição da enzima CK1?. Entre aqueles que já foram submetidos a ensaios de inibição enzimática, 25 apresentou seletividade para CK1?, com 40% de inibição. Para aqueles compostos com melhor potencial de inibição à concentração de 10 ?M será determinado o IC50 e uma extensa análise dos resultados será realizada futuramente. / Alzheimer\'s disease (AD) is a progressive neurodegenerative disorder characterized by loss of cortical and subcortical neurons. Pathological patterns of Alzheimer\'s disease include the presence of neurofibrillary lesions consisting of the accumulation of amyloid-? protein and the neurofibrillary tangles, resulting of hyperphosphorylated Tau protein. Only two major groups of drugs are used for treatment of AD, cholinesterase inhibitors and antagonists of Nmethyl- D-aspartate receptor. The phosphorylation of proteins by protein kinases constitute one of the major mechanisms which cells use to regulate their metabolism and imbalances in these activities are related to a series of diseases. The high number of isoforms of protein kinase 1 (CK1) found in AD and its association with neurodegenerative markers of indicate their participation in the final stages of degeneration, common to both AD and other neurodegenerative disorders. The approach of CK1 protein as a therapeutic target for AD is promising as the usual compounds used to reduce the production of amyloid-? also block the breakdown of other proteins, causing severe side effects. Increasingly, bioinformatics tools have been used as an aid in reducing costs and time to research. Among these techniques highlights the virtual screening, which includes a set of techniques used sequentially with the aim of select compounds prototypes for the desired target. Ligand and structure-based virtual screening techniches from a 500 thousand database resulted in 35 selected with inhibitory profile for CK1 enzyme were used in this study. The compounds 24, 25, 36, 39 41 and 42 had significan potential for CK1? enzyme inhibition. Among those submitted to enzyme inhibition assays, compound 25 showed selectivity for CK1? , with 40 % inhibition. For those compounds with the best inhibitory concentration at 10 mM and the IC50 will be given an extensive analysis of the results will be held in the future .

Os objetivos gerais do presente estudo foram avaliar o efeito do polimorfismo genético da kappa-caseína, da raça e da estação do ano sobre as características físico-químicas (acidez, pH e crioscopia), composição (gordura, lactose, sólidos totais, contagem de células somáticas, uréia, proteína bruta, proteína verdadeira, nitrogênio não protéico e nitrogênio não caseinoso) e estabilidade do leite. Foram selecionados 11 rebanhos leiteiros comerciais, sendo cinco deles da raça Holandesa e seis da raça Girolanda, dos quais foram amostradas em média 122 vacas em lactação por rebanho, totalizando 1350 vacas amostradas em três períodos: 2 no período seco e 1 no período chuvoso. As vacas selecionadas foram analisadas quanto a composição e propriedades físico-químicas do leite, assim como para a determinação do polimorfismo de kappa-caseína. O presente trabalho foi dividido em dois estudos, no primeiro avaliou-se os efeitos da raça e estação do ano sobre características físico-químicas, composição e estabilidade do leite. Foi observado efeito de raça sobre acidez titulável, pH, lactose, uréia e estabilidade do leite. O efeito de sazonalidade mostrou-se significativo sobre pH, crioscopia, teores de lactose, uréia, proteína bruta, sólidos totais, contagem de células somáticas, proteína verdadeira, caseína, nitrogênio não protéico e estabilidade térmica do leite. Em animais da raça Girolanda, foram observados no período seco maiores teores de sólido totais. O ponto crioscópico do leite sofreu efeito do período de coleta somente em animais da raça Holandesa, onde valores mais altos foram observados durante o período seco. Os teores de lactose apresentaram maiores médias no período seco em animais da raça Holandesa. Os teores de uréia sofreram efeito de raça e período de coleta estudado. A proteína bruta do leite sofreu efeito dos fatores raça e período de coleta, sendo que vacas da raça Holandesa e Girolanda apresentam maiores teores no período seco. A concentração de caseína do leite sofreu efeito significativo do período de coleta. A estabilidade térmica do leite sofreu influência da raça, pois o leite de vacas da raça Holandesa mostraram-se mais estáveis do que os da raça Girolanda. A sazonalidade é um fator determinante para a estabilidade do leite, pois o leite mostrou-se mais estável no período chuvoso que em período seco. No segundo estudo foram avaliados o polimorfismo genético da kappa-caseína em vacas Holandesas e Girolandas e o efeito deste sobre características físico-químicas, de composição e estabilidade térmica do leite. A freqüência do alelo A foi maior do que a do alelo B no que diz respeito ao gene da kappa-caseína. Com relação às características físico-químicas do leite, não houve efeito do polimorfismo do gene da kappa-caseína sobre teores de gordura, sólidos totais, lactose, contagem de células somáticas e uréia do leite. Não foram observados efeitos do polimorfismo genético do gene para kappa-caseína sobre a composição protéica do leite, sendo que teores de proteína bruta, nitrogênio não protéico, nitrogênio não caseinoso e proteína verdadeira não sofrem influência. A estabilidade do leite frente à prova do álcool não sofreu influencia do polimorfismo genético de kappa-caseína / The objective of this study were to determine the effects of kappa-casein gene polymorphisms, breed and season on physical-chemical properties (acidity, pH and cryoscopy), composition (fat, lactose, total solids, somatic cells count, urea, crude protein, true protein, non protein nitrogen and non casein nitrogen) and stability of milk. For this aim 11 dairy herds were selected, six of them composed of Girolando cows and five from Holstein cows, in average milk samples were taken of 112 cows from each herd, collected three times: twice in dry season and once on rainy season. Each cow were analyzed for milk composition, physico-chemical properties, and to determine its kappa-casein polymorphism. This study was divided in two, the first one analyzed possible breed and season effects over milk physico-chemical characteristics, composition and stability. Breed effect was observed over acidity, pH, lactose, urea and milk stability. Season effects was significant for pH, cryoscopy, lactose, urea, CP, total solids, SCC, TP, casein, EqNPN and heat milk stability. In cows Girolando it was observed highest total solids in dry season. Milk cryoscopy had season effects only on Holstein cows, with highest results in dry season. Lactose concentration was greatest in Holstein cow in dry season. Urea concentration showed breed and season effect on this study. Crude protein was affected by breed and season, in dry season the concentration were highest for Holstein and Girolanda. Casein milk concentration demonstrated season effect. Heat milk stability showed breed stability, milk from Holstein cow were more stable that milk from Girolanda cows. Season is a determinant factor for milk stability, milk showed more stable in rainy season than during dry period. The second study analyzed: kappa-casein gene polymorphisms in Holstein and Girolando cows and its effects over milk physico-chemical characteristics, milk composition and milk heat stability. Kappa-casein allele A had a higher frequency, than allele B, in Girolando and Holstein cows in comparison with other polymorphisms. No effect of kappa-casein polymorphism was observed on milk phisico-chemical characteristics and on milk fat, total solids, lactose, SCC, and milk urea. There were no difference for milk protein composition (EqNPN, NCNC, TP, casein and EqNCN) for kappa-casein polymorphism. Milk alcohol stability did not showed effect of gene polymorphism

A doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva, caracterizada pela perda de neurônios corticais e subcorticais. Padrões patológicos da doença de Alzheimer incluem a presença de lesões neurofibrilares que consistem no acúmulo da proteína ?- amilóide e dos emaranhados neurofibrilares, decorrentes da hiperfosforilação da proteína Tau. Apenas dois grupos principais de fármacos são utilizados para o tratamento da DA, os inibidores colinesterásicos e antagonistas do receptor N-metil-D-aspartato. Entretanto, a fosforilação de proteínas pelas proteínas cinases constituem um dos principais mecanismos pelos quais as células se utilizam para regular seu metabolismo e demais funções e desequilíbrios nestas atividades estão relacionados a uma infinidade de doenças. O número elevado de isoformas de proteínas cinases 1 (CK1) encontradas na DA e sua associação em marcadores de lesões neurodegenerativas indicam sua participação nas etapas finais da degeneração, comum tanto à DA quanto a outras desordens neurodegenerativas. A abordagem da proteína CK1 como alvo terapêutico para a DA é promissora uma vez que os compostos usuais utilizados para diminuir a produção de ?-amilóide também bloqueiam a quebra de outras proteínas, causando graves efeitos colaterais. Cada vez mais as ferramentas de bioinformática vêm sendo utilizadas como auxílio na redução de custos e tempo para as pesquisas. Dentre estas técnicas destaca-se a triagem virtual, que reúne um conjunto de técnicas utilizadas de forma sequencial com o objetivo de selecionar compostos protótipos para os alvos desejados. Neste trabalho, foram utilizadas técnicas de triagem virtual baseada em ligantes e estrutura, a partir de uma base de 500 mil compostos, selecionando 35 compostos com perfil de atividade inibitória para a enzima CK1. Destes, os compostos 24, 25, 36, 39 41 e 42 apresentaram resultados significativos com relação ao potencial de inibição da enzima CK1?. Entre aqueles que já foram submetidos a ensaios de inibição enzimática, 25 apresentou seletividade para CK1?, com 40% de inibição. Para aqueles compostos com melhor potencial de inibição à concentração de 10 ?M será determinado o IC50 e uma extensa análise dos resultados será realizada futuramente. / Alzheimer\'s disease (AD) is a progressive neurodegenerative disorder characterized by loss of cortical and subcortical neurons. Pathological patterns of Alzheimer\'s disease include the presence of neurofibrillary lesions consisting of the accumulation of amyloid-? protein and the neurofibrillary tangles, resulting of hyperphosphorylated Tau protein. Only two major groups of drugs are used for treatment of AD, cholinesterase inhibitors and antagonists of Nmethyl- D-aspartate receptor. The phosphorylation of proteins by protein kinases constitute one of the major mechanisms which cells use to regulate their metabolism and imbalances in these activities are related to a series of diseases. The high number of isoforms of protein kinase 1 (CK1) found in AD and its association with neurodegenerative markers of indicate their participation in the final stages of degeneration, common to both AD and other neurodegenerative disorders. The approach of CK1 protein as a therapeutic target for AD is promising as the usual compounds used to reduce the production of amyloid-? also block the breakdown of other proteins, causing severe side effects. Increasingly, bioinformatics tools have been used as an aid in reducing costs and time to research. Among these techniques highlights the virtual screening, which includes a set of techniques used sequentially with the aim of select compounds prototypes for the desired target. Ligand and structure-based virtual screening techniches from a 500 thousand database resulted in 35 selected with inhibitory profile for CK1 enzyme were used in this study. The compounds 24, 25, 36, 39 41 and 42 had significan potential for CK1? enzyme inhibition. Among those submitted to enzyme inhibition assays, compound 25 showed selectivity for CK1? , with 40 % inhibition. For those compounds with the best inhibitory concentration at 10 mM and the IC50 will be given an extensive analysis of the results will be held in the future .

Process cheese models were prepared by blending acid or rennet casein, milk fat, sodium chloride, 2.5% emulsifying salt and water and heating to 80 C. Acid casein cheese models were subjected to sodium hydroxide conditioning at 65 C in the cooker. Model process cheeses were acidified with lactic acid and treated by addition of undenatured and heat-denatured whey protein, four different emulsifying salts and sodium oxalate. Meltability and toughness of the model cheese increased to a maximum with increased sodium hydroxide conditioning of acid casein to pH 7.20. These same properties decreased with addition of undenatured and heat-denatured whey protein to both casein cheese models. Loss of emulsion occurred during the meltability test of rennet casein cheese models with 3.0 and 4.5% added whey protein. Emulsifying salts affected the models differently. Disodium phosphate and tetrasodium pyrophosphate in rennet casein models eliminated the melting property. These same salts in acid casein models produced excellent meltability. Trisodium citrate produced cheeses with good meltability in both acid and rennet casein cheese models. Acid casein cheese models prepared with sodium aluminum phosphate had fair meltability and were very tender (no rupture upon compression). Chelation of calcium by sodium oxalate in rennet casein cheese emulsified with disodium phosphate or tetrasodium pyrophosphate improved meltability with a corresponding increase in toughness. Scanning electron micrographs of model process cheeses indicated a direct relationship between extent of emulsification and poor meltability of rennet and pH conditioned acid casein model cheeses. Acid casein model cheeses prepared with different emulsifying salts did not exhibit this same relationship. Addition of whey protein concentrate to rennet case~n model cheese produced fibrous structures around the fat globules. No structural abnormalities were noted in the acid casein cheeses prepared with whey protein concentrate.

The problem of age gelation in ultra-high temperature (U1IT) sterilized milk retentate (ultrafiltered 3x concentrated) is investigated in this work. Transmission electron microscopy (1EM), utilizing the microcube encapsulation technique and protocols for immunolocalization of milk proteins, provides insight into the phenomenon of age gelation ofUHT-sterilized, ultrafiltered (UF) milk retentate. Primary antibodies (specific for the native as well as the complexed forms of milk proteins) and secondary antibodies (conjugated to gold probes) are used to elucidate the positions of the milk proteins in various samples of milk from the stage of milking through UHT sterilization and storage for 12 months, by which time gelation had occurred. The movement of the milk proteins is charted and these data are used to determine the role of the proteins in age gelation of UHT-sterilized UF milk retentate. Heat-denatured β-lactoglobulin and α-lactalbumin form complexes within the serum as well as with the casein components of the micelles. UHT sterilization not only denatures β-lactoglobulin and α-lactalbumin, but catalyzes the reaction of these whey proteins and K-casein, leading to the successful formation of the complex. Complexing of β-lactoglobulin and K-casein competitively weakens the complex of K-casein to other casein fractions of the micelle. This leads to migration of K-casein from the micelle to the serum, compromising the role of K-casein in stabilizing the casein proteins within the micellar moiety. The time-dependent loss of K-casein from the micelle would expose the calcium-insoluble micellar αs1-casein and β-casein to the serum calcium. Subsequent to this, some αs1-casein and β-casein are also released from the micelles, and gelation of the milk occurs. No information was obtained on location of αs2-casein. The release of K-casein from the micelles thus apparently represents the critical factor in the phenomenon of age gelation in UHT-sterilized milk concentrates.

Resumo: Objetivo: O objetivo do estudo foi comparar os efeitos de uma dose-única de caseína micelar (MCa) com a ingestão de caseína micelar associada à proteína do soro do leite (whey protein) (1:1) sobre a resposta aminoacidêmica e a via de sinalização do alvo da rapamicina (mTOR) em músculos esqueléticos de ratos durante a fase de inatividade (período de luz ambiente). Métodos: Após 10h de jejum durante a fase ativa, os ratos foram alimentados com MCa ou PB (5,6g proteína por kg de massa corporal) por gavagem e a água foi usada como veículo (grupo controle, PLA). Em 30 e 450 min após a suplementação das proteínas, os animais foram sacrificados e as amostras de sangue e do músculo gastrocnêmio foram coletadas para análises bioquímicas. Resultados: Os níveis plasmáticos dos aminoácidos de cadeia ramificada (BCAA) aumentaram após as suplementações de MCa (3 vezes) e PB (3,2 vezes). Ainda mais relevante, os níveis estimulatórios da fosforilação da mTOR e do seu alvo downstream p70S6K foram maiores 30 min após MCa (2,6 e 2,9 vezes, respectivamente) e PB (2,8 e 3,8 vezes, respectivamente) quando comparado com PLA. As concentrações plasmáticas de leucina forma correlacionadas com a ativação da mTOR (r = 0,60; p < 0,05) e p70S6K (r = 0,77; p < 0,05) em 30 min. Não existiu diferença para as concentrações plasmáticas de BCAA e a via de sinalização da mTOR em 450 min. Conclusão: Nós concluímos que a suplementação de MCa e PB resultaram em um efeito anabólico semelhante no músculo esquelético ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objective: The aim of the study was to compare the effects of single-dose supplementation of a protein blend (PB) composed of micellar casein and whey protein (1:1) with isolated micellar casein (MCa) on aminoacidemic response and the mammalian target of the rapamycin (mTOR) signaling pathway 30 and 450 min after the beginning of the inactive phase in Wistar rats. Methods: After 10h of fasting during the active phase, rats were fed with MCa or PB (5.6g protein per kg of body mass) by gavage and water was used as the vehicle (PLA, placebo group). At 30 and 450 min after protein supplementation, the animals were euthanized and blood and gastrocnemius muscle samples were collected for biochemical and immunoblot analysis. Results: Plasma BCAA levels increased after MCa (3-fold) and PB (3.2-fold) supplementations. More importantly, the stimulatory phosphorylation levels of mTOR and its downstream target ribosomal protein S6 kinase (p70S6K) were higher 30 min after MCa (2.6 and 2.9-fold, respectively) and PB (2.8 and 3.8-fold, respectively) when compared with PLA. Plasma leucine levels were correlated with activation of mTOR (r = 0.60, p < 0.05) and p70S6K (r = 0.77; p < 0.05) at 30 min. There were no differences for plasma amino acids levels and the mTOR signaling pathway at 450 min. Conclusions: MCa and PB supplementations resulted in a similar anabolic milieu in rat skeletal muscle by inducing a transient increase in BCAA plasma levels and activation of the mTOR/p70S6K axis. / Mestre

Ces dernières années, les méthodes hybrides QM/MM combinant la mécanique quantique (QM) et la mécanique moléculaire (MM) se sont revélées des méthodes de choix pour l'étude de systèmes chimiques et biochimiques contenant plus d'une centaine d'atomes. Nous avons mis en évidence les apports et difficultés liés à leur utilisation à travers des systèmes variés: modélisation de ligands phosphines, réactivité d'une protéine de type acide phosphatase pourpre, modélisation de l'interaction protéine-ligand.