Dissertationen zum Thema „Extrakce EKG“

In the past years, utilization of ECG for verification and identification in biometry is investigated. The topic is investigated in this thesis. Recordings from ECG ID database from PhysioNet and our own ECG recordings recorded using Apple Watch 4 are used for training and testing this method. Many of the existing methods have proven the possibility of using ECG for biometry, however they were using clinical ECG devices. This thesis investigates using recordings from wearable devices, specifically smart watch. 16 features are extracted from ECG recordings and a random forest classifier is used for verification and identification. The features include time intervals between fiducial points, voltage difference between fiducial points and PR intervals variability in a recording. The average performance of verification model of 14 people is TRR 96,19 %, TAR 84,25 %.

The aim of this master thesis is to create a 3D electroanatomical model of a heart atria, which would be able to perform atrial fibrillation. To control the model, the differential equations of the FitzHugh-Nagumo model were chosen. These equations describe the change of voltage on the cell membrane. The equations have established parameters. The modification of them leads to changes in the behavior of the model. The simulations were performed in the COMSOL Multiphysics environment. In the first step, the simulations were performed on 2D models. Simulations of healthy heart, atrial flutter and atrial fibrillation were created. The acquired knowledge served as a basis for the creation of a 3D model on which atrial fibrillation was simulated on the basis of ectopic activity and reentry mechanism. Convincing results were obtained in accordance with the used literature. The advantages of computational modeling are its availability, zero ethical burden and the ability to simulate even rarer arrhythmias. The disadvantage of the procedure is the need to compromise between accuracy and computational complexity of simulations.

This thesis uses one of Brain Computer Interface (BCI) products, NeuroSky headset, to design a prototype model to control window blind by using headset’s single channel electrode. Seven volunteers performed eight different exercises while the signal from the headset was recorded. The dataset was analyzed, and exercises with strongest power spectral density (PSD) were chosen to continue to work with. Matlabs spectrogram function was used to divide the signal in time segments, which were 0.25 seconds. One segment from each of these eight exercises was taken to form different combinations which were later classified.The classification result, while using two of proposed exercises (tasks) was successful with 97.0% accuracy computed by Nearest Neighbor classifier. Still, we continued to investigate if we could use three or four thoughts to create three or four commands. The result presented lower classification accuracy when using either 3 or 4 command thoughts with performance accuracy of 92% and 76% respectively.Thus, two or three exercises can be used for constructing two or three different commands.

Orientador: Márcia Justino Rossini Mutton
Banca: Flávia Cecílio Ribeiro Bregagnoli
Banca: Francisco Vicente Gaiotto Cleto
Resumo: As indústrias sucroenergéticas têm como preocupação o controle de contaminantes da fermentação, responsáveis por afetar a viabilidade da levedura, provocando diversos transtornos no processo, comprometendo a eficiência fermentativa e o rendimento industrial. Dentre as alternativas para o controle das contaminações, destacam-se o uso de antimicrobianos sintéticos. Sua utilização continua pode favorecer o desenvolvimento de cepas resistentes, contribuindo para o incremento do custo de produção, além da possibilidade de incorporação de resíduos no produto final. Objetivou-se avaliar o efeito do biocida convencional (monensina sódica) e biocidas naturais preparados à base de própolis (Extrato Hidroalcoólico de Própolis - EHP e Extrato Oleoso de Própolis- EOP) sobre a fisiologia das leveduras, o controle dos contaminantes do processo fermentativo e composição do destilado. O delineamento experimental utilizado foi o Inteiramente Casualizado com parcelas subdivididas, com 4 repetições. Os Tratamentos Principais foram: Testemunha, EOP, EHP e monensina sódica (Kamoran WP). Os Tratamentos Secundários constituíram-se nos 10 ciclos fermentativos. Avaliaram-se as características químico-tecnológicas do caldo, mosto e vinho, parâmetros microbiológicos das leveduras e composição do destilado obtido. Os resultados obtidos demonstraram que os biocidas avaliados apresentaram efeito similar, sendo efetivos no controle dos contaminantes da fermentação, não afetando negativamente suas características fisiológicas. Não afetaram a composição a composição do destilado final obtido
Abstract: The control of fermentation contaminants is one of the sugar mills concerns. The fermentation contaminants are responsible to affect the yeast viability, generating several overturns to the process, compromising the fermentative efficiency as well the industrial yield. Among the alternatives to control contamination, the use of synthetic antimicrobials can be highlighted. Its progressed use may favor the development of resistant strains, contributing in production cost improving, besides the possibility of residues incorporation into the final product. This work aimed evaluate the effect of conventional biocides (sodic monensin) and natural ones based on propolis (Propolis Hydroalcoholic Extract - EHP and Propolis Oily Extract - EOP) under the yeasts physiology, the fermentative process contaminants control, and the distilled composition. The experimental design used was the split-plot with four replications. The main treatments were: Control, EOH, EHP, and sodic monensin (Kamoran WP). The secondary treatments were the 10 fermentative cycles. The evaluated characteristics were: juice, must, and wine chemical-technical characteristics, yeasts microbiologic parameters, and the distillated obtained composition. The results obtained showed that the evaluated biocides presented similar effect, being effectives to control the fermentation contaminants, not affecting negatively its physiologic characteristics. They did not affect the composition of the distilled obtained
Mestre

Orientador: Maria Regina Barbieri de Carvalho
Banca: José Fernando Durigan
Banca: José Paschoal Batistuti
Resumo: Visando ampliar o aproveitamento nutricional e tecnológico do amendoim realizou-se este trabalho, com o objetivo de elaborar extrato aquoso, com diferentes processamentos, e verificar a aceitação do extrato fermentado com Streptococcus thermophilus e Lactobacillus delbrueckii ssp bulgaricus e com adição de leite em pó desnatado. O procedimento para obtenção do extrato consistiu no aquecimento dos grãos em solução de bicarbonato de sódio a 0,5% (1:4, p/v) até ebulição, com posterior drenagem, lavagem, desintegração e filtração. Foram avaliadas duas temperaturas (75 ºC e 97 ºC) e duas proporções de grão: água (1:5 e 1:8, p/v) para a desintegração dos grãos. Os produtos fermentados, com 0%, 2% e 4% de leite em pó, foram avaliados sensorialmente. Água a 75 ºC produziu extrato com o menor conteúdo de lipídeos (5,87%) e maior de carboidratos (2,31%). Os componentes dos extratos foram significativamente diluídos com a maior proporção de água (1:8 p/v), que permitiu o maior rendimento (1:6,92 kg), a maior extração de sólidos totais e proteína e a menor perda de sólidos no resíduo, sendo este procedimento selecionado para elaboração do extrato fermentado. O aquecimento dos grãos a 97 ºC propiciou proteína com maior digestibilidade (80,6%). Os resultados mostraram a possibilidade de se elaborar um produto adequado, fermentando-se o extrato de amendoim adicionado de leite em pó. Apresentou pH 4,5, 0,5% de ácido lático, 4,86% proteína e 2,36% de lipídeos e características sensoriais aceitáveis. A adição de leite em pó desnatado favoreceu a fermentação (4 horas e meia), melhorou a consistência e a aceitação geral do produto fermentado. O extrato aquoso fermentado de grãos de amendoim é uma alternativa tecnológica viável à elaboração de alimentos para a população, pela qualidade nutricional de seus componentes e a sua fácil disponibilidade.
Abstract: Aiming to increase nutritional and technological utilization of the peanut, this research was realized to produce an aqueous extract, with variations in processing, and to check the acceptance of the fermented extract with Streptococcus thermophilus and Lactobacillus delbrueckii ssp bulgaricus and with the addition of powder milk. The procedure to obtain the extract consisted of heating the beans until boiling (1:4 w/v) 0.5% of sodium bicarbonate, draining, washing, disintegration and filtration. It was evaluated two temperatures (75 ºC and 97 ºC) and two amounts of grain: water (1:5 and 1:8, w/v) in disintegration of the grains. Fermented products, with 0%, 2% and 4% of powder milk, were evaluated. Water at 75 ºC produced extract with lower content of lipids (5.87%), and higher of carbohydrates (2.31%). The extracts components of the were significantly diluted with the proportion 1:8 w/v, that permited the highest yield of process (1:6.92 kg) and better total solids and protein extraction and lower loss of solids in the waste. It was therefore selected for preparation of the fermented extract. The heating of the grains at 97 ºC, due to better digestibility of protein (80.6%). The results showed that is possible to obtain a fermented product with peanut extract added at milk powder. It presented pH 4.5, 0.5% lactic acid, 4.86% protein and 2.36% lipids and sensorial quality. The addition of powder milk favored fermentation (4 hours) and improved the consistency and general acceptance of the fermented product. The aqueous extract fermented of peanut grains represents a viable technological alternative in the preparation of food for population, due to nutritional quality of its components and its easy availability.
Mestre

Orientador: Cezinande de Meira
Banca: Sony Dimas Bicudo
Banca: Cássia Maria Barroso Orlandi
Resumo: O presente estudo visou avaliar a resposta ovariana e a taxa de recuperação embrionária em éguas tratadas com EPE nas doses de 6, 8 e 12,5 mg sendo o tratamento iniciado após a aplicação de prostaglandina F2 no oitavo ou sexto dia após a ovulação com a finalidade de reduzir o tempo e custo do tratamento. Foram realizados dois experimentos, para o experimento 1, 40 ciclos estrais de éguas Mangalarga Marchador, entre cinco e 24 anos, e para o segundo experimento 30 ciclos estrais de éguas mestiças entre quatro e 12 anos de idade, foram estudados. Foi aplicada 7,5 mg i.m.de prostaglandina F2 (Dinoprost Trometamina) no oitavo (experimento 1) ou sexto (experimento 2) dia após a ovulação. Nesse momento as éguas foram distribuídas aleatoriamente em cinco grupos para o experimento 1: (n=8 ciclos estrais/grupo): Grupo F20-23mm 6mg e F20-23mm 8mg receberam 6 e 8mg, respectivamente, de EPE por via i.m. a cada 12 horas, a partir da detecção de folículo(s) entre 20-23mm de diâmetro. Nos Grupos D8 - 6mg e D8 - 8mg foi aplicado 6 ou 8 mg de EPE, respectivamente, a cada 12 horas por via i.m. a partir do D8 (concomitante a PGF2 ). No Grupo F20-23mm Salina (Controle): as éguas receberam solução salina respeitando os mesmos intervalos que os grupos tratados com EPE a partir da detecção de folículo(s) entre 20-23mm de diâmetro. No experimento 2, a metodologia foi similiar ao experimento 1, contudo, as éguas receberam 12,5 mg de EPE, e o tratamento foi iniciado no sexto dia após a ovulação para o Grupo D6-12,5 mg (n=9), ou quando detectou-se folículo(s) entre 20-23mm, Grupo F20-23mm 12,5 mg (n=10) e F20-23mm Salina (Controle, n=10). Em todos os grupos (Exp. 1 e 2) o tratamento (EPE ou salina) foi mantido até 12 horas anterior a detecção de folículo(s) com diâmetro 35 mm, nesse momento a ovulação foi induzida com única dose de 2500 U.I. de hCG, i.v. (Vetecor®)... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present study aimed to evaluate the ovarian response and embrionary recovery in mares treated with doses of 6, 8 and 12.5 mg of EPE. The treatment was started after application of prostaglandin F2 for reduce the period and cost of treatment. Two experiments were conducted, for the experiment one, 40 estrous cycles of Mangalarga Marchador mares and ranged in age from 5 to 24 yrs, and for the second experiment 30 estrous cycles of crossbreed mares ranged in age from four to 12 yrs was used. In the eighth (experiment 1) or sixth (experiment 2) after ovulation was applied 7.5 mg i.m. of prostaglandin F2 (Dinoprost Trometamina). In this moment, all mares in experiment 1 was randomly assigned to treatment and control groups as follows: Groups 1 and 2 (n=8 cycles/group) received 6 mg of EPE, i.m. twice daily, beginning when largest follicle (s) was (were) 20-23 mm (Group F20-23mm 6mg) or regardless of follicle size on Day 8 (Group D8 - 6 mg); Groups 3 and 4 (n=8 cycles/group), received 8 mg of EPE, i.m. twice daily, beginning when largest follicle was 20-23 mm (Group F20-23 mm 8 mg) or regardless of follicle size on Day 8 (Group D8 - 8mg); Group F20-23mm Saline (Control, n=8 cycles) received saline, i.m. beginning when largest follicle (s) was (were) 20-23 mm in the same range of groups treated with EPE. In the second experiment was used the same methodology of the experiment 1, however, the mares received 12.5mg of EPE, and the treatment began in the sixth day after ovulation for the Group D6- 12.5mg (n=9), or when the largest follicle (s) was (were) 20-23 mm, Group F20-23mm 12,5 mg (n=10) e F20-23mm Saline (Control, n=10). Treatments with EPE or saline continued until 12 hours before detection of follicle (s) reached 35 mm, at which time a single dose of hCG (2500 U.I., i.v., Vetecor®) was given. Groups were compared using ANOVA and mean differences among groups were... (Complete abstract click electronic access below)
Mestre

One of the major limitations on research into the control of eukaryotic DNA replication has been the lack of any cell-free system that initiates DNA replication in vitro. The first part of the disseration describes the establishment of a eukaryotic system, derived from the activated eggs of the South African clawed toad, Xenopus laevis, that efficiently initiates and completes DNA replication in vitro. Using a variety of biochemical techniques I show that DNA added to the extract in the form of sperm nuclei is efficiently replicated over a period of 4 - 6 hours. Replication of nuclear DNA represents a single round of semiconservative, semidiscon-tinuous replication. The extract will also replicate naked DNA incubated in it, regardless of sequence, though less efficiently than nuclear templates. This is probably related to the unusual ability of the egg extract to assemble apparently normal interphase nuclei from any DNA molecule incubated in it Evidence is presented that initiation, rather than chain elongation, is the rate-limiting step for replication in vitro. In this and in other ways the cell-free system behaves as though it were an early embryo blocked in a single cell cycle. The second part of the dissertation describes experiments that examine the control of DNA replication in the extract The first set of experiments suggest that on replication, DNA is marked in some way so that it can no longer act as a substrate for further initiation. This provides a mechanism by which the template DNA is replicated precisely once per incubation in vitro (or per cell cycle in vivo). The second set of experiments investigate the relationship between nuclear assembly and the initiation of DNA replication in vitro. A novel method for quantifying DNA replication in intact nuclei using the nucleotide analogue biotin-11-dUTP is described. This technique reveals that although they are in the common cytoplasm of the egg extract, different nuclei start to replicate at different times. Entry into S-phase is characterised by a burst of many synchronous or near-synchronous initiations within individual nuclei. This means that nuclei act as independent and integrated units of replication in the cell-free system, and suggests a fundamental role for nuclear assembly in controlling DNA replication in vitro.

Resumo: O objetivo deste trabalho foi avaliar as características químicas das amostras recolhidas nas etapas do processamento do "leite" de soja e utilizar o Controle Estatístico de Processo - CEP (gráficos de média e desvio-padrão) para determinar os limites de variabilidade para controle de processo da produção do mesmo. Os materiais utilizados foram: soja em grão descascado cozido, água de cozimento do grão da soja descascada, calda para preparo do "leite" de soja, resíduo da soja (okara) e "leite" de soja pasteurizada processado pela Unidade de Produção e Desenvolvimento de Derivados da Soja - UNISOJA. Foram analisadas 329 amostras, coletadas em 15 dias, em pontos da linha de processamento de "leite" de soja. Destas 329 amostras, 75 amostras foram retiradas do grão de soja cozido descascado para análise de proteína e umidade; 75 da água de cozimento do grão descascado para análise de proteína; 29 de calda para análise de sólidos solúveis; 75 de resíduo (okara) para análise de proteína, lipídeos e cinzas e, finalmente, 75 amostras de "leite" de soja pasteurizado para análise de cinza, cor, lipídeos, proteína e sólidos solúveis. Essas análises foram feitas em duplicatas. Esse estudo mostrou que os gráficos da ferramenta da qualidade "Controle Estatístico de Processo" são eficientes para detectar possíveis falhas durante as diferentes etapas do processamento do "leite" de soja. Entretanto, ainda há necessidade de treinamento dos funcionários e padronização no processo de fabricação do "leite", em todas as etapas. Além disso, foi possível construir, para as etapas críticas do processamento, os limites de processo em gráficos de desvio padrão e média para a produção de "leite" de soja.
Abstract: The purpose of this study was to evaluate the chemical characteristics of the samples collected from soybeans "milk" processing stages as well as using statistical process control (mean and standard deviation graphs) to determine the variability limits to control its production process. The used materials were: decorticated cooked soybeans water in grain, cooking the grain of peeled soybean, syrup preparation for soybeans "milk", the soybean residue (okara) and pasteurized soybean "milk" processed by the Unidade de Produção e Desenvolvimento de Derivados da Soja - UNISOJA. Three hundred and twenty nine (329) samples were analyzed, collected in 15 days from several points of the soybeans milk processing line. Among these 329 samples, 75 samples were taken from cooked grain strip for protein analysis and moisture; other 75 samples were taken from the cooking water of grain strip for protein analysis; 29 samples were taken from syrup for analysis of soluble solids; 75 samples were taken from residue (okara) for protein, lipids and ash analysis; and, finally, 75 samples were taken from soybean pasteurized "milk" for gray, color, lipids, protein and soluble solids analysis. All these analyses were performed in duplicate. The study showed that the graphics of the Statistical Process Control are efficient to detect possible failures during different stages of soy "milk" processing but there is still a need for training of officials and for standardization in the "milk" production process at the different stages. Moreover, it was possible to build the limits of the process in graphics of standard deviation and means for the critical processing stages in the soybeans "milk" production.
Orientador: José Paschoal Batistuti
Coorientador: Elizeu Antonio Rossi
Banca: José Paschoal Batistuti
Banca: Elizeu Antonio Rossi
Banca: João Bosco Faria
Banca: Alice Yoshiko Tanaka
Mestre

Orientador: Roberto Lyra Villas Bôas
Banca: Antonio de Pádua Sousa
Banca: Ana Cláudia Pacheco Santos
Resumo: O presente trabalho teve por objetivo avaliar os efeitos de diferentes doses de extrato de Ascophyllum nodosum na produção de mudas de Eucalytus urograndis e Eucalyptus urophilla durante a fase de enraizamento. Os experimentos foram conduzidos em casa de vegetação, pertencentes às empresas VEC Florestal e Monte Flora, produtoras de microestacas de eucalipto e localizadas na cidade de Bofete, estado de São Paulo. O experimento foi conduzido em quatro fases no período de abril de 2009 a março de 2010, utilizando-se dois clones de E. urograndis (Euca 103 e Euca 105) e um clone de E. urophilla (I144), dois substratos (Brasil Minérios e Carolina Soil) e aplicação de diferentes doses de extrato de A. nodosum (EAN) 0, 0,5, 1, 1,5, 2, 3, 4, 8 e 16 mL L-1. O delineamento experimental adotado foi em blocos ao acaso com 4 repetições e 86 plantas por parcela. As aplicações do extrato de A. nodosum ocorreram aos 0, 7, 14, 21 e 28 dias após o estaqueamento variando conforme a fase. Foram avaliados a massa fresca de raiz (MFR), massa seca de raiz (MSR), comprimento de raiz (CR), massa fresca de parte aérea (MFA), massa seca de parte aérea (MAS), diâmetro do caule (D) e quantidade de raiz (Q) aos 30 e 45 2 dias após dias após o estaqueamento e análise química da planta. As avaliações permitiram observar que houve resposta diferenciada do EAN em relação aos substratos e ao material genético. O tratamento na dose de 3 mL de EAN para o clone I144 proporcionou melhor enraizamento das microestacas. Observou-se também que a forma de aplicação do produto interfere nos resultados
Abstract: The objective of this work was to evaluate the effects of different rates of extract of Ascophyllum nodosum in the production of microshoots Eucalyptus urograndis and Eucalyptus urophilla during the rooting. The experiments were conducted in a greenhouse belonging to companies VEC Florestal and Monte Flora, two producers of microcuttings eucalyptus and located in the city of Bofete, State of São Paulo. The experiment was conducted in four phases between April 2009 and March 2010, using two clones of E. urograndis (Euca 103 e Euca 105) and one clone of E. urophilla (I144), two substrates (Brasil Minérios and Carolina Soil) and application of different rates of extract A. nodosum (EAN) 0, 0,5, 1, 1,5, 2, 3, 4, 8, 16 mL L-1. The experimental design used was randomized blocks with four replications and 86 plants per plot. The applications of the extract A. nodosum occur at 0, 7, 14, 21 and 28 days after the cutting. It were evaluated root fresh weight (MFR), root dry mass (MSR), root length (CR), fresh weight of shoot (MFA), shoot dry mass (MAS), stem diameter (D) and quality of root (Q) at 30 and 45 days after the cutting. The evaluations allowed to note that there was differential response of EAN in relation to substrates and genetic material, The dose of 3 mL of ean for clone I144 provided better microcuttings rooting. It was also observed that the way of the product application affect the results
Mestre

Orientador: Marco Antonio Alvarenga
Resumo: Vários estudos têm relatado que a égua é refratária a todas as drogas rotineiramente utilizadas, visando superovulação em outras espécies, como o FSH e o eCG (Mc Cue, 1996). O extrato de pituitária eqüina (EPE) é um preparado parcial de gonadotrofina eqüina, e o único composto que consistentemente induz ovulações múltiplas em éguas (Squires et al, 1999); contudo, a resposta superovulatória tem sido baixa (1-3 ovulações/égua) (Mc Cue, 1996). Estudos mais recentes demonstram uma melhora no percentual de ovulações múltiplas em éguas superovuladas com a administração do EPE duas vezes ao dia (4-7ovulações/égua), entretanto com baixa taxa de recuperação embrionária (Scoggin et al., 2002; Alvarenga et al., 2001). Este fato pode estar relacionado a produção de oócitos de baixa qualidade (anormalidade na maturação folicular e oocitária) (Dippert et al., 1994; Palmer et al., 1993), a falha do folículo em liberar o oócito e a dificuldade na captação do mesmo para o interior do oviduto, assim como o trânsito deste ou do embrião pelo oviduto (Dippert et al., 1994). O extrato de pituitária eqüina (EPE) foi preparado no laboratório do Departamento de Reprodução Animal e Radiologia Veterinária da Universidade Estadual Paulista (UNESP), campus de Botucatu - SP, através do método proposto por Guillon & Combarnous, (1983). Seis éguas em bom estado nutricional, com idade variando entre quatro e 15 anos e massa corpórea de 400 a 500 Kg, além de um bom histórico reprodutivo, foram monitoradas diariamente durante os períodos compreendidos entre os meses de novembro (2001) a março (2002). Foram utilizados dois ciclos estrais de cada égua subdivididos em dois grupos: doses constantes e doses decrescentes; porém entre os tratamentos esperou-se um intervalo de dois ciclos estrais. O experimento foi conduzido no posto de monta... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Several studies have reported the is unresponsive to all the routinely used drugs aiming the superovulation in other species, such as the FSH and the eCG. The equine pituitary extract (EPE) is a partial equine gonadotropin preparation, and the only compound which consintenty induces multiple ovulation in mares; however, the superovulatory response has been low (1-3 ovulations/mare). More recent studies demonstrate some improvement in the percentage of multiple ovulation in superovulated mares with the EPE administration twice a day (4-7 ovulations/mare), nevertheless with a low rate of embryo recovery. This fact can be related to the low quality oocyte production (abnormality in the follicular and oocytary maturation), the follicular failure in releasing the oocyte and its collection towards the inner part of the oviduct, as its or the embryo traffic through the oviduct. The improvement on embryo recovery rate has been shownin cows with the utilization of decreasing doses of FSH. The present experiment aimmed to compare the ovulatory answer and embryo production of mares treated twice daily with constant and decreasing doses of EPE. Six mares in good nutrition status, with ages ranging from four and 15 years old and weighing from 400 to 500 Kg, together with a good reproductive history, were monitored daily through the preiods between the months of november/2001 to march/2002. Two estrous cytrous cycles were used from each mare and subdivided into two groups: constant doses and decresing doses;however between the treatments na interval of two estrous cycles was given. The experiment was carried out in the riding station (UNESP), placed in Lageado, a farm in the city of Botucatu - SP. For both groups, the treatment was started in the seventh post-ovuatory day, presenting a very evident corpora lutea and follicles equal or under 25 mm of diameter in the... (Complete abstract, click electronic address below)
Mestre

Orientador: Vera Maria Barbosa de Moraes
Banca: Maria Cristina Thomaz
Banca: Alexandre Oba
Resumo: O objetivo do presente trabalho foi o de determinar a digestibilidade do extrato de leveduras, avaliação do desempenho, rendimento de carcaça, morfometria e ultra-estrutura da mucosa intestinal e resposta imune humoral de frangos de corte criados em diferentes temperaturas e que receberam, na fase pré-inicial, ração contendo ou não extrato de leveduras e/ou prebiótico. Foram utilizados 1440 pintainhos Cobb-500® machos de um dia de idade, criados sob diferentes temperaturas a partir do primeiro dia de vida. As rações acrescidas ou não com extrato de leveduras e/ou prebiótico foram oferecidas somente na fase pré- inicial (1-7 dias) e a partir do 8º dia todas as aves receberam a mesma ração. Conclui-se que o extrato de leveduras apresenta 92,49% de matéria seca, 48,07% de proteína bruta, 4.883 Kcal/kg de energia bruta, e apresenta, em média, um coeficiente de digestibilidade verdadeira dos aminoácidos de 99,42%, sendo rico em ácido glutâmico, leucina, ácido aspártico, serina, glicina. A alta temperatura ambiente prejudicou o desempenho, rendimento de carcaça, morfometria intestinal e resposta imune humoral. A inclusão de prebiótico na ração pré-inicial resultou em maior ganho de peso e melhor conversão alimentar nas aves criadas sob alta temperatura, ao final de 42 dias de idade, além de aumentar a viabilidade criatória até os 21 dias de idade. Observou-se também que a inclusão dos produtos nas temperaturas quente e fria produziu efeito benéfico sobre as vilosidades frente ao estresse ambiental e que os aditivos estudados não produziram efeito sobre a resposta imune humoral para VDN e VDG ao longo do ciclo produtivo do frango de corte.
Abstract: The aim of this experiment was to determine the digestibility of the yeast extract, estimate the performance, carcass yield, intestinal mucosa structure and ultra structure and humoral immune response of broiler reared in different temperatures and that received in started diet containing or not extract of yeasts or prebiotic. 1440 one-day old male Cobb-500® were reared in different temperatures from first day of life. The diets with or without yeast extract and/or prebiotic were provided only in starter diet (1 to 7 days), forward of 8 days all broiler were fed with same commercial diet. The yeast extract contain 92.49% of dry matter, 48.07% of crude protein, 4,883 kcal/kg of crude energy /kg, and, on average a coefficient of digestibility true of amino acids of 99.42%. The amino acids digestibilities are glutamic acid, leucine, aspartic acid, serine, glycine. The high temperature damages the performance, carcass yield, intestinal mucosa structure, villus densities and antibody titters. The prebiotic inclusion in started diet resulted in increase body weight and improved feed conversion in the birds reared in high temperature to the 42 days of age and besides increase the viability until the 21 days of age. It was also observed that the inclusion of the two products in the hot and cold temperatures produced beneficial effect on the villus front in environmental stress and that the yeast extract and prebiotic did not produce effect on immune humoral response along the productive cycle of broiler.
Mestre

Le fractionnement du jaune d’œuf est une façon judicieuse d’étendre les domaines d’application de cet ingrédient dans les industries alimentaire et nutraceutique. Le but de ce projet a été de mettre au point un fractionnement non-toxique du jaune d’œuf, par centrifugation, dans l’objectif d’obtenir un produit enrichi en extrait naturel de folate. De par sa teneur élevée en cholestérol, le jaune d’œuf a été considéré comme un sous-produit du procédé de séparation des œufs. Cependant, le jaune d'œuf contient aussi une forme précieuse et biodisponible d'acide folique. La dilution du jaune d’œuf et son fractionnement en granules et plasma par une technique de centrifugation (centrifugeuse de laboratoire et à échelle pilote) a permis l’obtention de granules d’une richesse en folate trois fois plus importante que celle du jaune d’œuf natif. Aussi, les granules obtenus ont présenté une concentration protéique deux fois plus élevée que celle du jaune d'œuf natif, et une concentration de lipides et cholestérol trois fois inférieure à celle du jaune d'œuf natif. Les granules sont apparus non solubles et de structure très compacte. Afin d’augmenter encore la concentration en folate, nous avons tenté de remettre les granules en suspension et de les séparer par centrifugation en utilisant des prétraitements tels qu'augmentation de la force ionique et traitements mécaniques (ultrasons puissants et pression hydrostatique élevée). L’application d’ultrasons puissants et l’augmentation de la force ionique n’ont que peu amélioré la concentration en folate. Les granules soumis à une force ionique de 0.15 M de NaCl et un traitement par ultrasons puissants de 10 minutes ont présenté des concentrations de 21 μg de folate / g de granulés. Une augmentation de la force ionique au delà de 0.15 M de NaCl a conduit à une concentration plus faible en folate, du fait de la déformation de la structure des granules et de la séparation de leur fraction soluble. Les observations ont indiqué qu'il pourrait y avoir une association entre la structure protéique des granules et leur contenu en folate. Les variations de solubilité et la modification du réseau structurel des granules par augmentation de la force ionique ont affecté la teneur en folate des structures granulaires. Les granules ont cependant présenté une structure difficilement modifiable sous ultrasons puissants et après augmentation de la force ionique. Le traitement à pression hydrostatique élevée (HHP), technique puissante, a été utilisé pour étudier l'effet des hautes pressions sur la concentration en folate des granules. Après 5 minutes de traitement à 600 MPa, la concentration en folate a été mesurée dans les granules et le plasma, séparé des granules par centrifugation. Le plasma issu des granules contenait des concentrations en folate plus importantes que celles des granules précipités. Une analyse SDS-PAGE a permis de vérifier le profil protéique des granules sous l'effet du traitement HHP. Il est intéressant de noter que pour le plasma séparé des granules après traitement à haute pression, les migrations sur gel SDS-PAGE présentaient une bande protéique principale correspondant à la phosvitine. Les résultats de notre étude nous ont encouragés à proposer un modèle schématique de la structure des granules. Ces granules contiennent des HDL, LDL et phosvitine, et conservent le contenu total en folate du jaune d’œuf. Les protéines des granules sont principalement phosphorylées et il existe une forte liaison entre les apoprotéines de HDL et la phosvitine du fait de ponts de phosphate de calcium. Des traitements mécaniques ont libérés des particules de LDL qui pourraient être piégées dans le réseau structurel des granules. Les interconnexions entre les apoprotéines de HDL, la phosvitine et le folate pourraient se faire par des ions calcium. Nos résultats mettent en évidence le potentiel du procédé afin de produire un concentré riche en folate à partir des jaune d’œuf. Cependant, d’autres travaux seront nécessaires afin de trouver des utilisations aux co-produits (plasma) et rendre le procédé viable à l’échelle commerciale.
Fractionation of egg yolk is a smart way to expand the application of egg yolk ingredient in food and nutraceutical industry. The goal of this project was to develop non-toxic fractionation process of egg yolk by using centrifugation in order to prepare a natural folate-enriched extract. The egg yolk has been considered as a by-product of egg separation process due to its high cholesterol content. However, egg yolk contains valuable and bioavailable form of folate. Dilution of egg yolk and its fractionation into granule and plasma by centrifugation technique (lab- and pilot-scale centrifuge) resulted in separation of a granule fraction being rich in folate which was 3 fold higher than native egg yolk. This granule fraction was also characterized by high protein concentration (2 fold higher than protein content of yolk) and lower lipid and cholesterol (3 fold) content compared to non-treated egg yolk. The granule fraction appeared to be non-soluble with a very compact structure. By using the pre-treatments techniques such as increasing ionic strength and mechanical treatments (ultrasound and high hydrostatic processes), we attempted to re-suspend granules and separate them by centrifugation in order to further increase folate concentration. Results demonstrated that ultrasound and increased ionic strength did not largely change folate concentration. At ionic strength 0.15 M NaCl and after 10 min of ultrasound treatment granule contained 21 μg folate/g granules. By increasing ionic strength higher than 0.15 M NaCl the folate concentration was lower in granule due to the disruption of granule structure and separation of soluble fraction of granule. The observations denoted that there might be association between granular protein structure and folate content. Changes in solubility and disruption of granule network structure by increasing ionic strength affected folate content of granule structure. However, granules appeared to have very stable structure under the ultrasound and after increasing ionic strength, and their modifications were not easily possible. The high hydrostatic pressure processing (HHP) was used as an innovative and powerful technique in order to study the effect of high and drastic pressure on the concentration of folate in granules. After 5 min of 600 MPa HHP treatments, the folate concentration was measured in granule and separated plasma from granule after centrifugation. Plasma from granule contained higher concentration of folate compared to the precipitated granule. SDS-PAGE analysis was used in order to verify the granule protein profiles as a function of HHP treatment. Interestingly, the plasma separated from granule after HHP treatment contained phosvitin as a leading protein band separate in SDS-PAGE gel. The results of our study allowed proposing a schematic model for the granule structure which contains HDLs, phosvitin and LDLs. Beside; granule contains large amount of folate. Proteins of granule are mostly phosphorylated and strong connection between apoproteins of HDLs and phosvitin exists through calcium phosphate bridges. LDL particles were liberated through mechanical treatments and might be entrapped in the granular network. The interconnection between the apoproteins of HDLs, phosvitin and folate could be through calcium ions. Our results provided highly promising evidences concerning the recovery of high-concentration folate extract from hen egg yolk. Our fractionation technique is also clean but it generates plasma as co-product that is still usable in food formulation. Such applications still need to be developed before the technology can be viable at commercial scale.

Orientador: Pedro de Oliva Neto
Banca: Iolanda Cristina Silveira Duarte
Banca: Maria das Graças de Almeida Felipe
Banca: Eleonora Cano Carmona
Banca: Crispin Humberto Garcia Cruz
Resumo: O presente trabalho teve por objetivo otimizar a autólise de levedura fresca de cervejaria (Saccharomyces cerevisiae), visando a extração máxima de ácido ribonucléico da biomassa na produção do extrato de levedura. As variáveis estudadas foram pH, temperatura, % de NaCl, % de NH3, tempo de processo e, métodos de recuperação de RNA do autolisado. Os experimentos foram realizados por meio de quatro ensaios delineados segundo Box & Benken (1989) e avaliado pela Metodologia da Superfície de Resposta, utilizando-se o Software Statística 5.1 e a análise estatística ANOVA. A otimização foi concluída por meio do quinto ensaio com a produção do extrato nas condições otimizadas (55,2ºC, 9,8% de NaCl em pH=5,1 por 24 horas e, 12,2% de NH3 a 60ºC sob agitação a 200 rpm/15minutos. Três métodos foram avaliados para recuperação do RNA e das frações de extrato e parede celular: 1) autólise/plasmólise; 2) choque térmico por 1 minuto a 68ºC seguido da autólise/plasmólise 3) hidrólise química alcalina. Pelo processo de autólise em combinação com 9,8% de NaCl, a taxa de extração de RNA em 24 horas foi de 89,7%, com um rendimento de 51,3% em massa de extrato com 57,9% de proteína e, 48,7% de parede celular desidratada com 21,7 % de proteína. A utilização de 12,2% de NH3 em base seca de levedura permitiu o aumento na taxa de extração de RNA de 89,7 para 93,6%, mas um forte escurecimento foi verificado no extrato obtido. Na recuperação do RNA após precipitação protéica em pH 4,3 com posterior uso de 2 volumes de etanol em pH=2, recuperou-se 15,47%, 13,80% e 7,42% de RNA respectivamente com purezas de 49,85%, 51,70% e 38,70%. As taxas de extração de RNA da biomassa foram de 87,45% para o método 1; 91,40% para o método 2 e 78,80% para o terceiro método, indicando uma boa alternativa para redução do teor de RNA da biomassa e produção do extrato rico... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present work had for objective to optimize the autolysis of fresh brewery's yeast (Saccharomyces cerevisiae), aiming the maximum extraction of ribonucleic acid of biomass in the yeast extract production. The studied variables were pH, temperature, % of NaCl, % NH3, processing time and RNA recovering methods from autolysed. The experiments were accomplished by mean of four delineated assays according to Box & Benken (1989) and evaluated by Surface Methodology of Answer, utilizing the software Statistica 5.1. and the analysis statistics "ANOVA". The optimization was concluded by mean of the fifth assay with an extract production in the optimized conditions (55.2ºC, 9.8% of NaCl in pH 5.1 for 24 hours and, 12.2% of NH3 at 60ºC under agitation at 200 rpm/15 minutes. Tree methods were evaluated for RNA recovering and of the extract fractions and cell wall: 1) autolysis/plasmolysis; 2) thermic shock during 1 minute at 68ºC followed of autolysis/plasmolysis; 3) alkaline chemical hydrolysis. The process of autolysis in combination with 9.8% of NaCl, the RNA extraction yield in 24 hours was of 89.7%, with a yield of 51.3% in extract mass with 57.9% of protein and, 48.7% of dehydrated cell wall with 21.7% of protein. The utilization of 12.2% of NH3 in dried base of yeast allowed the increase in the RNA yield extraction from 89.7 to 93.6%, but a strong darkness was observed in the obtained extract. The RNA recovering after 4.3 pH proteic precipitation with posterior use of 2 ethanol volumes in pH 2.0, it was recovered 15.47%, 13.8% and 7.42% of RNA respectively with purities of 49.85%, 51.70% and 38.70%. The RNA extraction yields of biomass were of 87.45% for the method 1; 91.40% for the method 2 and 78.80% for the third method, indicating a good alternative for RNA content reduction of biomass and rich extract production in nucleotides. The extract fractions were evaluated... (Complete abstract click electronic access below)
Doutor

Orientador: Marcia Carneiro Valera
Banca: Carlos Henrique Ribeiro Camargo
Banca: Brenda Paula Figueiredo de Almeida Gomes
Resumo: A proposta desta pesquisa foi avaliar se o preparo biomecânico (PBM) com extrato glicólico de gengibre 20% e hipoclorito de sódio 2,5% (NaOCl), seguido da medicação intracanal com clorexidina gel 2%, hidróxido de cálcio, hidróxido de cálcio associado à clorexidina gel 2% são efetivos sobre Candida albicans, Enterococcus faecalis, Escherichia coli e sua endotoxina em canais radiculares.Foram utilizados 72 dentes humanos unirradiculados, divididos em 6 grupos experimentais (n= 12) de acordo com a solução irrigadora (gengibre 20% ou NaOCl 2,5%) utilizada no preparo biomecânico e medicação intracanal (hidróxido de cálcio + soro fisiológico; hidróxido de cálcio + clorexidina gel 2%; clorexidina gel 2%).Foram realizadas coletas do conteúdo do canal radicular para confirmação de contaminação (coleta de confirmação), imediatamente após a instrumentação (1ª coleta), após 7 dias do preparo biomecânico (2ª coleta), imediatamente após 14 dias da ação da medicação intracanal (3ª coleta) e 7 dias após remoção da medicação (4ª coleta). Para todas as coletas foram realizados os seguintes testes: a) avaliação da atividade antimicrobiana pela semeadura e contagem UFC/mL de cada microrganismo; b) análise do conteúdo de endotoxina verificada pelo teste lisado de amebócitos de Limulus. Todos os resultados foram submetidos à análise de variância ANOVA, com nível de significância de 5%, e pelo teste de Dunn. Verifica-se que o NaOCl foi capaz de eliminar os microrganismos após PBM; O gengibre reduziu significantemente o número de bactérias e eliminou Candida albicans. As duas soluções irrigadoras (NaOCl e gengibre) reduziram significantemente endotoxinas mas não foram capazes de eliminá-las. As medicações intracanais foram eficazes na redução de microrganismos... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The purpuse of this research was to evaluate the biomechanical preparation (PBM) with glycolic extract of ginger and 20% sodium hypochlorite 2.5% (NaOCl), followed by intracanal medication with 2% chlorhexidine gel, calcium hydroxide, hydroxide calcium associated with chlorhexidine gel 2% is effective on Candida albicans, Enterococcus faecalis, Escherichia coli and endotoxin in root canals. Seventy two single-rooted human teeth was used and divided into 6 experimental groups (n = 12) according to the irrigating solution (ginger 20% or NaOCl 2.5%) used in the biomechanical preparation and intracanal medication (calcium hydroxide + saline, calcium hydroxide + 2% chlorhexidine gel, chlorhexidine gel 2%). Sampling was done of the contents of the root canal to confirm contamination (collect of confirmation), immediately after the instrumentation (1st collect), after 7 days of biomechanical preparation (2nd collect) and after 14 days of the action of intracanal medication (3rd collect) and 7 days after removal of the medication (4th collect ). For all collections were performed the following tests: a) evaluation of antimicrobial activity by sowing and counting CFU / mL of each microorganism b) analyzing the content of endotoxin checked the test of amebócitos of Limulus lysate. All results were submitted to analysis of variance ANOVA, with significance level of 5%, and the test of Dunn. It appears that the NaOCl was able to eliminate the microorganisms after PBM; The ginger significantly reduced the number of bacteria and Candida albicans eliminated. Both irrigating solutions (NaOCl and ginger) significantly reduced endotoxin but were unable to eliminate them. The intracanais medications were effective in the reduction of microorganisms, eliminating... (Complete abstract click electronic access below)
Mestre

Resumo: A fêmea da espécie eqüina é considerada monovulatória sazonal, o que é "ator limitante a produção de embriões ao longo do ano. Esta limitação poderia inimizada se houvesse uma resposta superovulatória eficiente em melhorar a ção de embriões. Protocolos mais recentes desenvolvidos em nosso atório utilizando-se o Extrato de Pituitária Eqüina (EPE) têm permitido uma esposta superovulatória. Contudo o número de embriões recuperados ainda sido inferior ao das ovulações, em conseqüência de fatores ainda não inados. O presente estudo teve por objetivo: verificar se o tratamento com o - administrado duas vezes ao dia, altera a maturação nuclear e citoplasmática I ito, avaliar o ambiente folicular mensurando os níveis de 17(3-estradiol, E5~)tlerona, progesterona, inibina e óxido nítrico, bem como o perfil eletroforético eínas no fluido folicular entre éguas superovuladas e não superovuladas. amos também o transporte do oócito para o oviduto. Este trabalho foi o em quatro experimentos: Experimento I "Estudo do transporte dos oócitos oviduto de éguas superovuladas com extrato de pituitária eqüina"; ento II "Efeito da superovulação na recuperação de oócitos quando da ~-es foliculares guiadas por ultra-sonografia"; Experimento 111 "Avaliação do e folicular de éguas superovuladas"; Experimento IV "Avaliação da '"'ação oocitária de éguas superovuladas com extrato de pituitária equina". O ento I" foi desenvolvido na Universidade de Rio Cuarto (Argentina), foram _za.:las 22 éguas de 3 - 12 anos, (09 éguas controles; 13 éguas tratadas _ . Estes animais foram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The equine female is considered a seasonal mono-ovulatory specie, which is a restrictive factor in respect to embryo production throughout the year. This limitation could be minimized if an efficient superovulatory response and consequent improvement of embryo production were possible. More recent protocols developed in our lab using EPE (equine pituitary extract) have allowed a good superovulatory response. However, the number of embryos recovered has been inferior to the number of ovulations detected due to unknown factors. The present study has the following objectives: Verify if the EPE treatment administered twice daily would alter the oocyte cytoplasmic and nuclear maturation; and to evaluate the follicular environment by measuring estradiol 17-, testosterone, progesterone, inhibin and nitric oxide. The electrophoresis pattern of proteins in follicular fluid from superovulated and non- superovulated mares was determined. In addition, the oocyte transport through the oviduct was investigated. The present study was divided into four experiments. Experiment I: Study of oocyte transport to the oviduct in superovulated mares using equine pituitary extract. Experiment II: Effect of superovulation on oocyte recovery using transvaginal ultrasound guided follicular aspiration. Experiment III: Evaluation of follicular environment in superovulated mares. Experiment IV: Oocyte maturation in superovulated in mares using equine pituitary extract. Experiment I was performed at Rio Cuarto University, Argentina. In the related study, 22 mares aging from 3 to 12 years were used (9 control mares, 13 EPE treated mares). These mares were monitored daily by ultrasound until the presence of a follicle ≥ 30mm in diameter, being then examined twice daily. The superovulation protocol used consisted in 25mg of EPE twice a day, intramuscularly, starting at day 7 post- ovulation... (Complete abstract click electronic access below)
Orientador: Marco Antonio Alvarenga
Coorientador: Fernanda da Cruz Landim e Alvarenga
Banca: Cezinande de Meira
Banca: Frederico Ozanam Papa
Banca: Rubens Pais de Arruda
Banca: Carlos Antonio de carvalho Fernandes
Doutor

Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.

Resumo: A exigência por produtos de melhor qualidade é cada vez maior por parte dos consumidores. Produzir um alimento sem qualquer resíduo de hormônios como os utilizados na reversão sexual de tilápias e, além disso, conseguir cultivar um peixe capaz de utilizar adequadamente os carboidratos de uma dieta sem deficiências nem excessos, pode significar um maior retorno econômico para o produtor e maior aceitação no mercado, garantindo a compra de um produto mais saudável e seguro ao meio ambiente. O objetivo deste trabalho foi avaliar os aspectos produtivos, econômicos e de composição corporal de juvenis de tilápia-do-nilo, revertidos ou não sexualmente, originados de recria utilizando-se dois tipos de alimentos (alimento natural, através de fertilização orgânica com esterco suíno ou dieta extrusada comercial) e alimentados com dietas extrusadas contendo três teores de extrativo não nitrogenado. Utilizaram-se 432 juvenis, estocados em 36 tanques e que receberam dietas contendo três teores de extrativo não nitrogenado (34, 38 ou 42 %), durante 123 dias de experimento. Foram calculados os principais parâmetros de desempenho, eficiência de utilização de nutrientes, variáveis morfo-somáticas e realizada uma avaliação econômica, utilizando-se a metodologia dos orçamentos parciais, estimando-se os efeitos das alterações nas despesas e receitas entre os tratamentos. O consumo diário de dieta foi significativamente maior para os juvenis que receberam alimento natural. No entanto, os juvenis alimentados com dieta comercial durante a fase de recria apresentaram maior eficiência de retenção de proteína. A inclusão de 42 % de ENN na dieta resultou em valores médios estatisticamente maiores para o ganho em peso e consumo diário de dieta em relação ao nível de 38 %. O teor de 42 % de ENN melhorou o crescimento dos peixes, mas levou a um aumento no índice... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The demand for better quality products is increasing among consumers. Produce a product without any residue of hormones such as those used in the sex reversal of tilapia and, besides, to grow a fish able to properly utilize the carbohydrates from a diet without deficiencies or excesses, may mean a higher economic return for the producer and largest market acceptance, assuring the purchase of a product healthier and safer for the environment. The aim of this study was to evaluate the productive and economic aspects and body composition of juvenile nile tilapia, sex reversed or mixed sex, which was previously produced with two types of food (natural food through organic fertilization with swine manure or extruded commercial diet) and fed with extruded diets containing three levels of non-nitrogenous extract (NNE). It was used 432 juveniles stocked in 36 tanks and fed with diets containing three levels of non-nitrogenous extract (34, 38 or 42 %) during 123 days of experiment. It was calculated the main parameters of performance, efficiency of nutrient use, morphological and somatic variables and performed an economic evaluation, using the methodology of the partial budgets, estimating the effects of changes in costs and revenues among the treatments. The food intake was significantly greater for the juveniles who received natural food. However, fishes fed with commercial diet during the growing phase showed higher efficiency of protein retention. The inclusion of 42% of NNE in the diet resulted in statistically higher mean values for weight gain and food intake in relation to the level of 38%. The content of 42% NNE improved fish growth, but led to an increase in the hepatosomatic index, which may mean fat accumulation in the liver. The diet with 34% of NNE has the lowest production cost, however, any change in the level of NNE in the diet from 42 to 34 or 38 % worse economic... (Complete abstract click electronic access below)
Orientador: Dalton José Carneiro
Coorientador: Maria Célia Portella
Banca: Maria Inêz Espagnolli Geraldo Martins
Banca: Eduardo Gianini Abimorad
Mestre

Orientador: Waldemar Gastoni Venturini Filho
Banca: Regina Marta Evangelista
Banca: Marta Helena Fillet Spoto
Resumo: O objetivo deste trabalho foi desenvolver bebida mista de extrato hidrossolúvel de soja e suco de amora, para atender aos interesses dos associados do Assentamento Rural Dandara, localizado no município de Promissão-SP. As matérias-primas foram soja (variedade BRS 213), amora (gênero Morus), pectina cítrica e açúcar. A bebida mista foi fabricada em diferentes proporções de extrato hidrossolúvel de soja e suco de amora (1:1; 1:1,5 e 1:2; respectivamente; m/m) e diferentes concentrações de sólidos solúveis (10, 12 e 14 °Brix). Os extratos hidrossolúveis de soja, os sucos de amora e as bebidas mistas foram analisadas quimicamente, além da quantificação dos valores energéticos. As bebidas mistas foram analisadas sensorialmente pelo teste de escala hedônica e seus resultados foram submetidos à análise de variância e posterior análise de regressão. As bebidas produzidas foram comparadas com bebidas similares comerciais. Todas as bebidas mistas produzidas apresentaram pH inferior a 4,5, sem adição de acidulantes. O tratamento térmico realizado foi suficiente para hidrolisar toda a sacarose, utilizada na correção do açúcar dessas bebidas. Essa correção do açúcar resultou na diminuição do teor de umidade, aumento no teor de carboidratos e valor energético das bebidas. Com relação às bebidas comerciais com soja e frutas, as bebidas mistas deste estudo, em média, foram mais ricas em todos os componentes da composição centesimal, particularmente em proteínas. Com relação às análises sensoriais, as diferentes proporções de extrato hidrossolúvel de soja e suco de amora interferiram no aroma e na aparência das bebidas produzidas, porém, não existiu uma relação direta entre essas proporções e a aceitabilidade desses atributos sensoriais. Já, a concentração de açúcar influenciou no atributo sabor e na preferência global... (Resumo completo clicar acesso eletrônico abaixo)
Abstract: The objective of this work was to develop a mixed drink of soymilk and mulberry juice, to serve the interests of the associated members of the Rural Settlement Dandara, located in Promissão-SP. The raw materials was soybeans (variety BRS 213), mulberry (genre Morus), citrus pectin and sugar. The mixed drink was made with different proportions of soymilk and mulberry juice (1:1, 1:1,5 and 1:2, respectively, m/m) and different concentrations of soluble solids (10, 12 and 14 °Brix). The soymilk, the mulberry juices and the mixed drinks were chemically analyzed, and the energy values was quantified too. The mixed drinks were analyzed by sensory evaluation, the test used was the hedonic scale and results were submitted to variance analysis and subsequent regression analysis. The beverages produced were compared with similar commercial beverages. All mixed drinks produced had pH below 4.5, without addition of acidifier. The thermic treatment performed was sufficient to hydrolyze all the sucrose of the sugar used in the correction of these drinks. This sugar correction resulted in reduction of moisture content, increase in content of carbohydrates and energy value. With regard of the commercial soy and fruit drinks, the mixed drink of this study, on average, were richer in all components of proximate composition, particularly in proteins. With regard to sensory evaluation, the different proportions of soymilk and mulberry juice interfered with the aroma attribute and the appearance attribute of beverages produced, however, there wasn't a direct relationship between these proportions and acceptability of sensory attributes. Nevertheless, the sugar concentration affected the taste attribute and the global preference. The preference of consumers, were both in taste and global preference, in the overall, were to the sweetest drink, with a direct relationship between sugar content and the... (Complete abstract click electronic access below)
Mestre

Orientador: Vera Lucia Borges Isaac
Banca: Pedro Alves da Rocha Filho
Banca: Roni Antonio Mendes
Banca: Adélia Emília de Almeida
Banca: André Gonzaga dos Santos
Resumo: Os frutos de Spondias dulcis, popularmente conhecidos no Brasil como cajá-manga ou cajarana, são de formato ovóide, de polpa amarela e sementecaracterística. Essa espécie tem despertado grande interesse devido ao seu potencial funcional, principalmente por apresentar compostos fenólicos de capacidade antioxidante, podendo ser úteis na prevenção do envelhecimento precoce da pele, provocado pelo estresse oxidativo, podendo assim ser utilizada em formulação de uso tópico com potenciais efeitos benéficos para a pele. Foram produzidos 14 extratos diferentes a partir da polpa do fruto, estabilizada e convenientemente seca. Os extratos foram avaliados quanto ao teor de fenólicos totais (Folin-Ciocalteau) e atividade antioxidante com os radicais DPPH • e ABTS • . O extrato obtido por maceração 2:10 m/v (M20) utilizando etanol 70° GL apresentou maior rendimento, teor de fenólicos totais e atividade antioxidante, além de não apresentar toxicidade nos ensaios de micronúcleo e citotoxicidade. A análise do extrato M20 etanólico 70° GL por cromatografia líquida de alta eficiência e por cromatografia em camada delgada permitiu a identificação, pela primeira vez na espécie, de rutina e de ácido clorogênico, substâncias com conhecida atividade antioxidante, confirmando os resultados preliminares obtidos nos ensaios de triagem fitoquímica (positiva para flavonoides). O extrato M20 foi incorporado em três concentrações distintas em formulação cosmética cuja estabilidade foi avaliada sob condições de estresse térmico e físico, analisando-se os seguintes parâmetrosdurante o período de 90 dias: pH, densidade e viscosidade. Os resultados apontam para o potencial uso de formulação fitocosmética estável, contendo extrato seco da polpa de S. dulcis capaz de minimizar os efeitos deletérios provocados... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The fruits of Spondias dulcis, popularly known in Brazil as cajá-manga or cajarana, are ovoid, with yellow pulp and characteristic seeds.This species contain phenolic compounds and may be useful in preventing premature skinaging caused by oxidative stress.Thus, it could be used in topical formulation with potential benefical effects on skin. Fourteen different extracts were produced from the stabilized and dried pulp. The phenolic content and antioxidant activity (DPPH • and ABTS) of extracts were evaluated. The extract 2:1 m/v (M20) obtained with ethanol 70 o GL presented higher yield, phenolic content and antioxidant activity. Additionaly, this extract have not presented toxicity in both micronucleus and citotoxicity assays. Rutin and chlorogenic acid (antioxidant compounds) were identified in M20 by high performance liquid chromatography and thin layer chromatography analysis. The extract M20 was incorporated (three different concentrations) in cosmetic formulations and the stability of formulations were evaluated (pH, viscosity and density) for a period of 90 days. The results of this work suggest the potential use of S. dulcis pulp fruit in a stable phytocosmetic formulation with antioxidant... (Complete abstract click electronic access below)
Doutor

Resumo: O experimento teve por objetivo avaliar a influência dos aditivos fitogênicos (AF) e ácidos orgânicos (AO), isolados ou associados, sobre a metabolizabilidade dos nutrientes da dieta bem como, avaliar o desempenho e características de carcaça de frangos de corte. Foram realizados dois ensaios em delineamento inteiramente casualizado em esquema fatorial 2 x 2 + 1 com cinco tratamentos sendo constituídos de dieta controle (DC); DC + AF; DC + AO; DC + AF + AO; DC + avilamicina + monensina sódica. No experimento I foram realizados dois ensaios de metabolismo para determinar os coeficientes de metabolizabilidade dos nutrientes da dieta nas fases inicial e crescimento, utilizando 125 frangos de corte machos. No experimento II foram utilizados 2520 pintos de um dia de idade alojados em 40 unidades experimentais e, avaliou-se o desempenho e características de carcaça. Os aditivos fitogênicos e ácidos orgânicos isolados ou associados melhoram a metabolizabilidade dos nutrientes da dieta e substituem os antibióticos melhoradores de desempenho. O uso de ácidos orgânicos isoladamente ou associados aos aditivos fitogênicos em dietas de frangos de corte melhoram o desempenho das aves em relação a dietas isentas de antibióticos melhoradores de crescimento aos 42 dias de idade. Aditivos fitogênicos e ácidos orgânicos isolados e associados proporcionam melhores características de carcaça
Abstract: The experiment evaluated the influence of isolated or associated phytogenics additive (FA) and organic acids (OA) on nutrient metabolization, performance and carcass characteristics of broiler chickens. Two experiments were conducted in a completely randomized design with 2 x 2 + 1 factorial arrangement of treatments. There were five treatments described as follows: control diet (CD), CD+FA, CD+OA, CD+FA+OA and CD + avilamicin + monesin sodium. In the first experiment a total of 125 male broilers were divided in two metabolism trials to determine the coefficients of metabolizability of the nutrients of starter and grower diets. In the second experiment, 2520 one-day-old chicks were housed in 40 experimental units to evaluate the performance and carcass characteristics. The phytogenics additive and organic acids, isolated or combined, improve the nutrient metabolization of the diet and replace the antibiotics for growth promotion. The use of isolated organic acids or combined with phytogenics additive in diets for broilers improve the chicken performance compared with free growth promoter diets at 42 days old. Isolated or associated phytogenics additive (FA) and organic acids (OA) provided better carcass characteristics
Orientador: José Roberto Sartori
Coorientador: Elisabeth Gonzales
Banca: Marcos Barcellos Café
Banca: Alfredo Sampaio Carrijo
Banca: Adriano Sakai Okamoto
Banca: Antonio Carlos Pezzato
Doutor

The E3 ubiquitin ligase \(CRL4^{Cdt2}\) targets proteins for destruction during DNA replication and following DNA damage (Havens and Walter, 2011). Its substrates contain "PIP degrons" that mediate substrate binding to the processivity factor PCNA at replication forks and damage sites. The resulting PCNA-PIP degron complex forms a docking site for \(CRL4^{Cdt2}\), which ubiquitylates the substrate on chromatin. Several \(CRL4^{Cdt2}\) substrates are known, including Cdt1, multiple CDK inhibitors, Drosophila E2f1, human Set8, S. pombe Spd1, and C. elegans \(Pol\eta\) (Havens and Walter, 2011). An emerging theme is that \(CRL4^{Cdt2}\) targets proteins whose presence in S phase is toxic. Here, I used Xenopus egg extract to characterize a new \(CRL4^{Cdt2}\) substrate, thymine DNA glycosylase (TDG). TDG is a base excision repair protein that targets G-U and G-T mispairs, which arise from cytosine and 5-methylcytosine deamination (Cortazar et al., 2007). Thus, TDG may function in epigenetic gene regulation via DNA demethylation, in addition to its canonical DNA repair function. A yet unknown E3 ubiquitin ligase triggers TDG destruction during S phase (Hardeland et al., 2007). Understanding TDG proteolysis in S phase is relevant to the regulation of DNA replication, DNA repair, and epigenetic control of gene expression. I discovered that TDG contains a variant of the "PIP degron" consensus and that TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and degron-specific manner during DNA repair and DNA replication in Xenopus egg extract. I further characterized what features of TDG contribute to its proteolysis. Interestingly, I could not identify any defects during DNA replication or during Xenopus embryonic development in response to a non-degradable form of TDG. Additionally, I examined how interactions between \(CRL4^{Cdt2}\) and multiple subunits of the PCNA homotrimer contribute to \(CRL4^{Cdt2}\) function. In a popular model, PCNA functions as a "tool belt" on DNA, binding three separate proteins through its individual subunits to facilitate rapid exchange of DNA replication and repair proteins as they are needed on DNA. To address this model, I generated a single chain polypeptide with three PCNA subunits connected through flexible linker sequences. I used this tool to determine how multiple PCNA subunits contribute to \(CRL4^{Cdt2}\) function. I found that a single wildtype subunit is sufficient for modest destruction of the \(CRL4^{Cdt2}\) substrate Cdt1, but complete Cdt1 destruction requires two separate wildtype subunits. Additionally, a single subunit was sufficient for leading strand elongation, challenging the "tool belt" model during DNA replication. I also discuss implications and future use of the single-chain PCNA.

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This study aimed to evaluate the effect of graded levels (0, 1, 2 and 3%) of dietary inclusion of an yeast-extract product2 on internal and external quality of eggs and on performance of layers. A total of 240-Hy Line W36 layers (47 to 75 weeks of age) were allocated in 60 cages (4 birds per cage), divided into 15 cages per treatment. Feed intake, body weight, egg production, egg weight, egg mass, feed conversion (per dozen), feed conversion (per mass), specific gravity, eggshell weight and thickness, albumen height, Haugh units and yolk and albumen weights were evaluated. Data were subjected to ANOVA and polynomial regression. A P<0.05 was required for statements of significance. Results indicated that addition of 1,5% NuPro® resulted in better eggshell quality. The remaining variables were not statistically influenced by dietary treatments.
Esta pesquisa teve por objetivo avaliar o efeito dos níveis crescentes (0, 1, 2 e 3%) de suplementação dietética do extrato de levedura1 sobre o desempenho produtivo e a qualidade externa e interna de ovos. Um total de 240 poedeiras Hy Line W36, no período de 47 a 75 semanas de idade, foram distribuídas em 60 gaiolas, sendo quatro aves por gaiola, divididas em 15 repetições por tratamento. As características avaliadas foram consumo de ração, peso corporal produção de ovos, peso do ovo, massa de ovo, conversões alimentares por dúzia e por massa de ovo, gravidade específica, peso e espessura da casca, altura do albúmen, unidade Haugh e pesos da gema e do albúmen. Os dados foram submetidos à análise de variação e regressão polinomial. O valor de P<0,05 foi estabelecido para a determinação de significância. Os resultados indicaram que a adição de 1,5% do extrato de levedura resultou melhor qualidade de casca dos ovos. As demais variáveis não foram influenciadas estatisticamente pelos tratamentos.

O ovo é consumido in natura ou processado, e usado como matéria-prima em diversos produtos. A pasteurização e a atomização são os principais processamentos aplicados ao ovo. Diferentes condições de processamento, embalagem e estocagem podem afetar a estabilidade oxidativa lipídica e reduzir a proteção antioxidante natural do ovo. O uso de embalagens onde não haja contato do ovo com a luz e oxigênio, estocagem em temperaturas baixas e uso de antioxidantes podem prevenir a oxidação lipídica. Atualmente, os antioxidantes sintéticos são cada vez mais substituídos por antioxidantes naturais ou feita uma associação entre eles. O alecrim (Rosmarinus officinalis L.) e o chá verde (Camelia sinensis L) constam entre os vegetais com grande potencial antioxidante. Com base no exposto, os objetivos do trabalho foram: a) Padronizar o método do fosfomolibdênio para avaliação da capacidade antioxidante total da fração lipídica (CATL) do ovo; b) Investigar o efeito da pasteurização e da atomização do ovo no que se refere à capacidade antioxidante e à estabilidade oxidativa da fração lipídica do ovo; c) Avaliar a estabilidade oxidativa de ácidos graxos e a capacidade antioxidante do ovo integral pasteurizado atomizado, estocado a 5ºC, por 90 dias; d) Verificar por meio de teste de concentração de antioxidantes, a estabilidade oxidativa lipídica do ovo integral pasteurizado atomizado adicionado da mistura de extrato de alecrim, extrato de chá verde e BHA (butilato de hidroxianisol); e) Otimizar a concentração da mistura de extrato de alecrim, extrato de chá verde e BHA a ser adicionada ao ovo líquido integral pasteurizado, posteriormente atomizado, pelo modelo matemático proposto pela metodologia de superfície de resposta; f) Investigar o efeito da mistura otimizada de extrato de alecrim, extrato de chá verde e BHA na estabilidade oxidativa lipídica do ovo integral pasteurizado atomizado, estocado sob as temperaturas de 5°C e 25ºC, por 90 dias. O método do fosfomolidênio para medir a CATL do ovo apresentou adequação analítica, indicada pela equação de regressão (y = 13,705x + 0,0808), coeficiente de determinação (R2 = 0,9931), limite de detecção (0,005mg α-tocoferol/ mL), limite de quantificação (0,017mg α-tocoferol/ mL), coeficiente de correlação (r = 0,9965), sendo que a precisão não indicou dispersão ao redor da média. A CATL diminuiu com o progresso do processamento e o inverso foi observado quanto aos lipídios, 7-CETO (7-cetocolesterol) e TBARS (substâncias reativas ao ácido tiobarbitúrico). O ovo integral pasteurizado atomizado (OIPA) mantido sob condições de estocagem consideradas ideais permaneceu estável em relação à hidratação, a CATL e as TBARS. Pelo teste de concentração de antioxidantes, foram ensaiadas dez misturas (alecrim, chá verde e BHA) no OIPA e refletiu na sua estabilidade oxidativa lipídica, verificada pelas TBARS, CATL, CT-F (capacidade redutora pelo reativo de Folin - Ciocalteau), AGL (ácidos graxos livres) e AS-233 (substâncias absorvidas a 233 nm). Os resultados mostraram que a metodologia de superfície de resposta (RSM) foi adequada para descrever a formação dos AGL e AS-233 no OIPA, podendo serem usadas para fins preditivos. A otimização da mistura de antioxidantes baseou-se no modelo matemático obtido com as AS-233, onde foi proposto como sistema antioxidante 150ppm de BHA, 600ppm de alecrim e 300ppm de chá verde. As condições de estocagem adotadas e a adição dos antioxidantes foram efetivas para manter estável o OIPA, sendo mais efetivo quando estocado a 5°C. Concluíu-se que o método do fosfomolibdênio apresentou adequação analítica. A pasteurização não afetou os parâmetros analisados (lipídios, TBARS, CATL e 7-CETO), mas a atomização provocou diminuição significativa da CATL, e elevação dos lipídios, TBARS e 7-CETO. Foi mantida a hidratação e a estabilidade oxidativa do OIPA estocado por 90 dias a 5°C, indicando que as condições de embalagem e estocagem foram efetivas. Pelo modelo matemático proposto pela RSM foi constatado que apenas os AGL e AS-233 podem ser usados para fins preditivos, optando-se pela otimização da concentração da mistura de antioxidantes, usando apenas o modelo matemático obtido nas AS-233. Com a presença ou não de antioxidantes, o OIPA estocado a 5°C, ao longo dos 90 dias, apresentou-se mais estável quanto à hidratação e à oxidação lipídica.
Egg is consumed in natura or processed and is used as raw material in various food products. The pasteurization and spray-drying processes are the main processing applied to the egg. Different processing conditions, packaging and storage can affect the lipid oxidative stability and may reduce the natural antioxidant protection of this product. The use of packages in which the egg has no contact with light and oxygen, storage at low temperatures, and the use of antioxidants can prevent lipid oxidation. Currently, synthetic antioxidants have been increasingly replaced by natural antioxidants or an association between synthetic and natural antioxidants is adopted by food companies. Rosemary (Rosmarinus officinalis L.) and green tea (Camellia sinensis L) are among the vegetables that present the highest antioxidant potential. Based on the above-mentioned considerations, the objectives of this research were: a) To standardize the method of phosphomolybdenum to evaluate the total antioxidant capacity of lipid fraction (CATL) egg; b)To investigate the effect of pasteurization and spray-drying on the antioxidant capacity and oxidative stability of the lipid fraction of egg; c) To assess the oxidative stability of fatty acids and the antioxidant capacity of whole egg subjected to pasteurization followed by spray-drying, stored at 5ºC for 90 days, d) To evaluate the by testing the concentration of antioxidants, oxidative stability of the lipid fraction of egg subjected to pasteurization and spray drying, added with a mixture of rosemary and green tea extracts and BHA (butylated hydroxyanisole); e) To optimize the concentration of the mixture composed of rosemary and green tea extracts and BHA to be added to pasteurized liquid egg (and further subjected to spray-drying) by using Response Surface Methodology; f) To investigate the effect of the addition of this optimized mixture of antioxidants on of the lipid fraction of spray-dried egg, stored at temperatures of 5 and 25°C for 90 days. The phosphomolybdenum method CATL of egg presented analytical suitability once it presented a high coefficient of determination (R2 = 0,9931), a regression equation expressed as y = 13.705 + 0.0808 x, a limit of detection of 0.005 mg α- tocopherol/mL, a limit of quantitation of 0.017 mg α-tocopherol/mL, a significant and high correlation coefficient (r = 0.9965), and the accuracy did not indicate dispersion around the mean. The CATL decreased with the progress of processing and the reverse was observed for the lipids, 7-CETO (7-ketocholesterol) and TBARS (thiobarbituric acid reactive substances). The pasteurized spray-dried egg (OIPA), which was kept under ideal storage conditions, remained stable in relation to moisture content, CATL and TBARS. In order to test the concentration of antioxidants to be added to OIPA, a total of 10 mixtures (rosemary, green tea and BHA) were assayed. The addition of antioxidants resulted in a higher oxidative stability of the lipid fraction as measured by TBARS, CATL, CT-F (reduction capacity by using the Folin-Ciocalteau reagent), AGL (free fat acids) and AS-233 (substances that absorb at 233 nm). The results showed that the response surface methodology (RSM) was adequate to describe the development of free fat acids and AS-233 in OIPA, and the RSM model proposed for AS-233 can be used for prediction purposes. The optimization of the antioxidant mixture based on the mathematical model proposed for AS-233 indicated that the mixture of 150ppm of BHA, 600ppm of rosemary extract and 300ppm of green tea extract was the best combination of antioxidants. The adopted storage conditions and the addition of antioxidants to OIPA were effective to maintain the response variables stable, and the stability was higher when OIPA was stored at 5°C. It was concluded that the method of phosphomolybdenum suitability presented analytically. The pasteurization did not affect the analyzed parameters (lipids, TBARS, CATL and 7-CETO), but the spray-drying caused a significant decrease CATL, and an increase in lipids, TBARS and 7-CETO. The hydration and the oxidative stability of OIPA remained stable for 90 days at 5°C, indicating that the packaging and storage conditions were effective. The RSM models proposed for free fat acids and AS-233 could be used for predictive purposes; however, the optimization procedure was performed taken into account only the AS-233 model. The OIPA stored at 5°C for 90 days, with or without the addition of the antioxidant mixture, was more stable in relation to hydration and lipid oxidation.

Aurora A (AurA) is an important mitotic kinase mainly studied for its involvement in cell cycle progression, centrosome maturation, mitotic spindle pole organization and bipolar spindle formation. It localizes to duplicated centrosomes and spindle microtubules (MTs) during mitosis where it regulates various factors participating in metaphase spindle formation. AurA is degraded late in mitosis suggesting that it might also have a function in anaphase. In this study we focused in understanding AurA function during anaphase in two different experimental systems. First, we kept AurA active in cycled Xenopus egg extracts and found that MTs maintained their mitotic organization longer throughout mitotic exit. We also observed chromosome segregation defects and problematic nuclear envelope formation. These observations indicate that AurA activity needs to be down-regulated for the transition from metaphase back to interphase. To get insights into the role of AurA during metaphase-anaphase transition we initially asked whether its kinase activity is still necessary for the maintenance of the metaphase spindle. We saw that the inhibition of AurA kinase activity in metaphase resulted to a collapse of the established metaphase spindle in HeLa cells. Indicating that AurA activity is necessary for the metaphase spindle maintenance. Then, we looked whether AurA kinase activity is still necessary during anaphase. We inhibited AurA at the onset of anaphase in Hela cells and found that anaphase spindles were smaller. We also observed that the MT structure responsible for anaphase spindle elongation, the central spindle, was defectively assembled and organized. Moreover, in cells where AurA was inhibited segregation of chromosomes was defective. These results indicate that AurA kinase activity is necessary for anaphase spindle elongation, central spindle assembly and organization and chromosome segregation. To understand further how AurA regulates anaphase spindle formation we looked known AurA substrates. We depleted TACC3, a known AurA substrate involved in MT formation earlier in mitosis and observed that TACC3 depletion phenocopied AurA inhibition. This indicates that TACC3 has a function in MT organization and chromosome segregation during anaphase and this function could possibly be regulated by AurA. In this study we have demonstrated that AurA activity is essential for metaphase spindle maintenance. We also found that during anaphase when AurA is either maintained active or inhibited MT organization is greatly affected and chromosome segregation is defective. Suggesting that AurA activity needs to be tightly controlled during anaphase for a correct completion of mitosis.
Aurora A (AurA) es una quinasa mitótica importante que se ha estudiado principalmente en su papel durante la progresión del ciclo celular, la maduración del centrosoma, la organización y la formación del polo y del huso mitótico. Durante la mitosis, AurA se localiza en los centrosomas duplicados y en los microtúbulos (MTs) del huso y se ha observado que regula varios factores que participan en la formación del huso mitótico. AurA se degrada al final de la mitosis indicando que pueda tener una función durante la anafase. En este estudio nos hemos centrado en la comprensión de la función de AurA durante la anafase en dos sistemas experimentales diferentes. En primer lugar, utilizando extractos de huevos de Xenopus hemos mantenido AurA activa durante la transición de metafase a anafase y hemos visto que los MTs del huso mitótico mantienen su organización durante más tiempo. También hemos observado que cuando AurA se mantiene activa existen defectos en la segregación cromosómica y la formación de la membrana nuclear. Esto indica que la actividad de AurA tiene un papel regulador sobre los MTs y la chromatina durante la transición de la metafase a la interfase. Para entender cual es la función de AurA durante la transición de metafase a anafase primero hemos estudiado si la actividad de la quinasa es necesaria para el mantenimiento del huso mitótico. Hemos visto que la inhibición de la actividad quinasa AurA resultó en el colapso del huso durante la metafase en células HeLa. Esto indica que la actividad de AurA es necesaria para el mantenimiento del huso mitótico de metafase. A continuación hemos analizamos si la actividad quinasa de AurA sigue siendo necesaria para la anafase. Para ello hemos inhibido AurA en células Hela al inicio de la anafase. En estas condiciones los husos de la anafase son más pequeños y la estructura de los MTs responsable del alargamiento del huso mitótico durante la anafase, el huso central, se organiza defectuosamente. Además, se encontraron errores durante la segregación de los cromosomas. Estos resultados indican que la actividad quinasa de AurA es necesaria para el alargamiento del huso durante la anafase y la organización y segregación cromosómica. Para entender el mecanismo de la función de AurA durante la anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 , un sustrato conocido de AurA que participa en la formación de MTs en las fase iniciales de la mitosis hemos encontrado que su eliminación de células HeLa produce el mismo fenotipo que la inhibición de AurA. Esto indica que TACC3 tiene una función en la organización de MT y la segregación de cromosomas durante la anafase y que esta función podría estar regulada por la quinasa AurA. En este estudio hemos demostrado que la actividad quinasa de AurA es esencial para el mantenimiento del huso mitótico. También hemos encontrado que durante la anafase cuando la quinasa AurA se mantiene activa o se inhibe la organización de los MTs del huso mitótico se ve muy afectada y los cromosomas se segregan defectuosamente. Por tanto los resultados de este estudio indican que la actividad quinasa de AurA está estrechamente controlada durante la anafase para el correcto cumplimiento de la mitosis.

碩士
國立中興大學
動物科學系所
101
Cell cycle of donor cells would affect the efficiency of somatic cell nuclear transfer (SCNT), as well as the nuclear reprogramming efficiency. Recent researches have demonstrated that the Xenopus laevis egg extracts (XEE) treated cells, called extract treated cells (ETCs), could undergo reprogramming and regain the expressions of pluripotent genes, such as oct4, sox2 and nanog. Therefore, the aims of this study were to investigate the effects of cell cycle on the efficiency of reprogramming into the pluripotent stage by treating mouse somatic cells with XEE and assess the possibility to define the embryonic grem like (EG-like) cells from the reprogrammed cells. Experiment 1, a mouse NIH/3T3 cell line and mouse embryonic fibroblasts (MEFs) were incubated with 0.2% or 0.5% bovine serum (BS) for 3 or 5 days would significantly stay at G0/G1 phase, while NIH/3T3s and MEFs treated with various concentrations of colchicine (0.5, 1.0 or 1.5 μg/ml) for 1 or 2 days, would significantly stay at G2/M phase. Experiment 2, NIH/3T3s were treated 0.5% BS for 3 days or 0.5 μg/ml colchicine for 1 day before treatment with XEE. On Day 7 and 8 after XEE treatment, all groups showed the expressions of tumor suppressors (p53, p21 and p16), pluripotent markers (oct4, sox2 and nanog) and primordial germ cells (PGCs) markers (blimp1 and stella), although the expressions were low and unstable. However, there showed more round morphology cells, and the expressions of tumor suppressors, pluripotenct and PGC markers were increased in colchicine-treated NIH/3T3s. The expressions of Oct4 and Nanog protein were also shown by analysis of immunocytochemical (ICC) staining. MEFs treated with 0.2% BS for 5 days or 0.5 μg/ml colchicine for 2 days before XEE treatment showed pluripotent markers on Day 7 to Day 9, but the expressions of tumor suppressors, pluripotent and PGC markers still remained at low level. Furthermore, the expressions of PGC markers were low. Experiment 3, cell cycle-regulated NIH/3T3s were treated with XEE for 7 or 8 days, then incubated in PGCM for 24 h. For groups changed to PGCM on Day 7, colchicine-treated NIH/3T3s had significantly higher expressions of Blimp1, Stella and Sox2, while for groups changed to PGCM on Day 8, serum-starved NIH/3T3s had significantly higher expression of Blimp1. After cultured in PGCM for 24 h, the cells were passaged and seeded on feeder layer and then cultured in 2i-LIF culture system. It was found that groups changed to PGCM on Day 7, the colchcine group formed more EG-like colonies, although the expressions of pluripotent and PGC markers in the EG-like colonies showed no differences with ES cells in the goups changed to PGCM on Day 7 and Day 8. By analysis of immunocytochemistry, all groups of EG-like colonies expressed pluripotent markers, including Oct4, Nanog, SSEA1 and a PGC-specific marker Stella. These studies showed that after serum starvation and colchicine treatment, NIH/3T3s and MEFs could significantly stay at G0/G1 and G2/M, respectively. Further, we treated cell cycle-regulated cells by XEE, we found colchicine had more round morphology cells and Oct4/Nanog expressions in NIH/3T3s; but there were less round morphology cells and Oct4/Nanog expressions in MEFs. Then we changed to 2i-LIF culture system, the d7 colchicine formed more EG-like colonies, otherwise, all groups of colonies expressed PGC marker Stella and pluripotent markers Oct4, Nanog and SSEA1 in NIH/3T3s.

碩士
國立臺灣大學
微生物與生化學研究所
93
In this study, mouse lymphoma cell L5178Y (tk+/-) assay was employed to detect the genotoxicity of the egg oil samples and the acetone extract of Monascus-fermented red rice. Three kinds of egg yolk oil samples and the acetone extract of Monascus-fermented rice and its silica gel fractions were dissolved in absolute ethanol and DMSO, respectively. In cytotoxicity tests, each sample with various concentrations was added into RPMI1640 medium with 5% horse serum containing 1 × 107 L5178Y tk+/- cells to incubate for three hours. Among the three kinds of egg oil samples, the PYO revealed the most cytotoxicity. When cells were incubated with 1 mg/mL of all three egg yolk oil samples, there were no cells survivals. When cells were incubated with 0.625 mg/mL of PYO, thirty percent of cells were dead. In acetone extract of Monascus-fermented rice, the ethyl acetate fraction of silica gel fractionation could cause 40% cell death at 0.055 mg/mL. In genotoxicity tests, a thymidine analogue TFT (5-trifluorothymidine) was applied to detect the mutation by 12-days incubation after the 2 days expression period followed the treatment. As the results, whether the three kinds of egg oil samples were treated with the S9 mix or not, no significant genotoxicity was shown. Similar non-genotoxic results were also observed in the acetone extract of Monascus-fermented red rice and its silica gel fractions.

To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.

The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.

Dissertation