Made available in DSpace on 2014-08-20T14:38:49Z (GMT). No. of bitstreams: 1 Dissertacao_ Juliana_ Klug_ Nunes.pdf: 4118597 bytes, checksum: 3c27dbdcf97e2cfc50cbf822af01cb11 (MD5) Previous issue date: 200-11-20 / 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.

Les bactéries lactiques sont largement utilisées en tant que ferments dans l'industrie laitière. Leur production s’effectue généralement dans des milieux semi-définis ou complexes dans lesquels certains nutriments peuvent être apportés par des extraits de levure (EXLs). Ce projet de thèse, qui associe deux partenaires industriels, les groupes Lesaffre et Sacco, ainsi que l’INRA, s’est focalisé sur l’effet des peptides de deux EXLs (EXL1 et EXL2) sur une souche industrielle de Streptococcus thermophilus, un levain lactique d’intérêt économique majeur. L’hypothèse sous-jacente était que ces peptides pourraient avoir un double rôle de nutrition et de régulation de fonctions cellulaires pouvant présenter un intérêt technologique. Afin d’explorer cette question, une stratégie expérimentale à deux niveaux a été élaborée : i) caractérisation et suivi cinétique de la fraction peptidique des deux EXLs par spectrométrie de masse (peptidomique) durant la fermentation de S. thermophilus en bioréacteurs, et ii) suivi cinétique parallèle du transcriptome et du protéome de la bactérie. L’objectif final était de croiser ces deux niveaux d’information afin de corréler des différences de contenu peptidique avec des différences d’activation de systèmes participant aux performances globales du levain.La caractérisation et le suivi du peptidome des EXLs en cours de fermentation a nécessité un important travail de développement méthodologique ayant abouti in fine à l’élaboration d’un outil analytique complet, combinant analyse peptidomique à haut-débit des échantillons et traitement bioinformatique et statistique des données. Cet outil a permis d’identifier environ 4000 peptides différents composant les deux EXLs. Le suivi cinétique a notamment permis de préciser la spécificité du transporteur d’oligopeptides de la bactérie (Ami). En particulier, il s’est avéré qu’une charge nette positive était le facteur prévalent pour le transport des peptides chez S. thermophilus. En complément de cette approche semi-quantitative, des analyses quantitatives ont été réalisées sur des fractions peptidiques des EXLs (dosages différentiels par HPLC des acides aminés avant et après hydrolyse). Elles ont notamment permis de révéler d’importantes différences de teneurs en oligopeptides entre les deux EXLs.En parallèle, le suivi transcriptomique et protéomique réalisé durant la croissance de la bactérie a révélé deux faits marquants. Le premier fait a trait à la surexpression dans l’EXL1 d’un locus génétique régulé par un mécanisme de quorum sensing utilisant un peptide phéromone comme signal moléculaire. Le deuxième fait marquant concerne diverses voies de biosynthèse (acides aminés et purines) différentiellement affectées par les deux EXLs. L’origine de ces dynamiques pourrait être au moins pour partie le fait de différences de contenu peptidique entre les deux substrats. Notamment, certaines voies de biosynthèse pourraient avoir été modulées différentiellement sous l’action de régulateurs centraux tels que CodY, dont l’activité est corrélée au contenu peptidique du milieu, ou encore YebC, un régulateur CodY-like dont le lien fonctionnel avec CodY reste encore inconnu chez S. thermophilus. Tous ces résultats ouvrent d’intéressantes perspectives pour mieux explorer le lien entre peptides et métabolisme bactérien. A terme, cette démarche pourrait se traduire par l’identification de biomarqueurs de performances dans les EXLs, et l’élaboration à façon de produits permettant de maximiser le potentiel technologique des ferments lactiques. / Lactic acid bacteria are widely used as starters in dairy industry. They are generally produced in complex fermentation media containing a wide array of nutrients that can be provided by yeast extracts (YEs). The main goal of this thesis project, involving two industrial partners, Lesaffre and Sacco, as well as INRA, was to investigate the effect of the peptide fraction of two YEs (YE1 and YE2) on an industrial Streptococcus thermophilus strain, a major lactic acid starter. The underlying hypothesis of this whole project was that YE peptides could have a role in nutrition but also regulate cellular functions of technological relevance. In order to explore this question, a two-step strategy was elaborated: i) mass spectrometry characterization (peptidomics) of both YE peptide fractions and time course analysis of their relative abundance during the growth in bioreactors of S. thermophilus, and ii) parallel time course analysis of the strain transcriptome and proteome. The final objective was to cross these two levels of information in order to correlate differences of peptide content with differentially activated systems related to technological performances.YE peptidome characterization and kinetic analysis first required an important methodological development. It eventually resulted in a complete analytical tool that combines high throughput peptidomic analysis as well as bioinformatic and statistical data processing. This powerful tool was able to identify around 4,000 different peptides in both YEs. Then, the time course analysis also clarified the in vivo substrate specificities of the oligopeptide transport system of the bacterium (Ami). A peptide positive net charge notably turned out to be the leading factor governing peptide transport. In addition to this semi-quantitative approach, quantitative analyses were carried out on YE peptide fractions (differential HPLC analyses of amino acids before and after sample hydrolysis). They notably revealed significant differences in oligopeptides content between both YEs.Meanwhile, genome scale transcriptomic and proteomic analyses performed during the strain growth highlighted two significant events. The first one concerns the overexpression in YE1 of a quorum sensing-based genetic locus that uses a pheromone peptide as molecular signal. The second event relates to several biosynthesis pathways (amino acids and purines) that were differentially affected by both YEs. These dynamics could result from differences in peptide content between both substrates. In particular, some pathways could have been differentially modulated by central regulators such as CodY, whose activity is correlated to the medium peptide richness, or YebC, a CodY-like regulator whose functional link with CodY is still unknown in S. thermophilus. All these results open avenues for a better understanding of the interplay between peptides and bacterial metabolism. In the future, this whole approach could lead to the identification of performance biomarkers in YEs, which in turns may eventually translate into the conception of new customized products granting high technological performances to dairy starters.

<p>The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.</p><p>Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.</p><p>Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.</p><p>In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.</p><p>Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.</p>

Existe um número muito limitado de estudos referentes à produção de componentes antimicrobianos ou bacteriocinas produzidas por espécies de bifidobactérias. Nesse âmbito, o objetivo deste trabalho foi avaliar a produção de bifidobacteriocina em leite desnatado (LD). Para tanto, o estudo foi dividido em três etapas. A primeira etapa constituiu na preparação dos meios de cultura Man, Rogosa e Sharpe (MRS), Bifidus Selective Medium (BSM) e LD suplementado com 1% (p/v) de Tween 80 (T80), Inulina (I) ou Extrato de levedura (YE). Nesta etapa, os processos fermentativos foram conduzidos em shaker, nas condições: 50 rpm/37ºC/48h. Foram realizadas análises de pH, concentração de açúcares e ácidos, crescimento celular e determinação da atividade da bifidobacteriocina pelo método de difusão em ágar contra L. monocytogenes. Na segunda etapa, e baseado nos resultados obtidos, foi desenhado um delineamento composto central (CCD) construído a partir dos seguintes parâmetros: temperatura (34, 37, 40 °C) e concentração de YE (0,5; 1,0; 1,5 g/L). Na terceira etapa do trabalho, foram realizados os cultivos em biorreator de 2 L, contendo 10% de leite desnatado, nas seguintes condições: 200 rpm, 36°C, 2,0 g/L de YE, 48h de incubação em anaerobiose. Obteve-se em LD suplementado com YE (1%), combinado ao método de difusão em placa modificado (prévia refrigeração das placas por 12h), contra L. monocytogenes (2130 AU/mL), com uma fase exponencial de 24h, &#181;m de 0,604/h. A otimização feita através do CCD permitiu atingir níveis de atividade de 3.000 AU/mL a 3.100 AU/mL (ensaios 7, 11 e 14, blocos 3 e 1) contra L. monocytogenes, em condições ótimas de crescimento de YE: 2,0 g/L1 e T°C: 36°C. A análise de regressão mostrou ser estatisticamente significativa a relação entre as variáveis: \"concentração de \"YE e temperatura\". Os resultados indicaram que o leite desnatado é um meio adequado para produção de bifidobacteriocina. / There are few publications that have been reported about bacteriocin production by Bifidobacterium species. Therefore, the aim of this work was measure bacteriocin production in skim milk by B. lactis. Consequently, this work was divided in three stages. First, MRS, BSM and LD medium were tested with additives (Tween 80 (T80), Inuline (I) or Yeast extract (YE)) for bacteriocin production and cellular growth. Fermentation processes were conducted in shaker under specific conditions: 50 rpm/37ºC/48h. pH; sugars; acids; biomass, and bacteriocin activity against L. monocytogenes, L. plantarum, E. coli, L. sakei e S. aureus strains were analyzed . In the second stage, based on the obtained results, a central composite design (CCD) was created using the parameters: temperature (34, 37, 40 ºC), and concentration of YE (0.5, 1.0, 1.5 g/L). After, the activity was measured by two methods of plates pre-diffusion (cooling and addition of Tween 20). Third step consisted of 2 L bioreactor cultivations containing 10% skim milk diluted in 1.5 L of water (6.5 pH), under 200 rpm, 36 ºC, 2.0 g/L of YE, 48h, under anaerobic condition. Finally, the cultures supplemented with LD and YE (1%) with a modified plate diffusion method (cooling plates for 12 h) showed bacteriocin activity against L. monocytogenes (2130 AU/mL) with an exponential phase of 24 h, &#181;m of 0.604/h. The optimization performed using CCD resulted in a higher level of activity 3000 AU/mL to 3100 AU/mL mL (Run 7, 11 and 14, blocks 3 and 1) against L. monocytogenes, also with ideal growth conditions of YE: 2,0 g/L1 and T °C: 36 °C. The pH value varied between 6.4 and 4.0. Concentration of produced acid lactic varied from 3.03 to 4.72 g/L and biomass concentration from 3.4 to 11.1 Lg UFC/mL. Regression analysis was significant to the variables: YE concentration and temperature. Results indicated that skim milk is a proper medium for \"Bifidobacteriocin\" production.

Existe um número muito limitado de estudos referentes à produção de componentes antimicrobianos ou bacteriocinas produzidas por espécies de bifidobactérias. Nesse âmbito, o objetivo deste trabalho foi avaliar a produção de bifidobacteriocina em leite desnatado (LD). Para tanto, o estudo foi dividido em três etapas. A primeira etapa constituiu na preparação dos meios de cultura Man, Rogosa e Sharpe (MRS), Bifidus Selective Medium (BSM) e LD suplementado com 1% (p/v) de Tween 80 (T80), Inulina (I) ou Extrato de levedura (YE). Nesta etapa, os processos fermentativos foram conduzidos em shaker, nas condições: 50 rpm/37ºC/48h. Foram realizadas análises de pH, concentração de açúcares e ácidos, crescimento celular e determinação da atividade da bifidobacteriocina pelo método de difusão em ágar contra L. monocytogenes. Na segunda etapa, e baseado nos resultados obtidos, foi desenhado um delineamento composto central (CCD) construído a partir dos seguintes parâmetros: temperatura (34, 37, 40 °C) e concentração de YE (0,5; 1,0; 1,5 g/L). Na terceira etapa do trabalho, foram realizados os cultivos em biorreator de 2 L, contendo 10% de leite desnatado, nas seguintes condições: 200 rpm, 36°C, 2,0 g/L de YE, 48h de incubação em anaerobiose. Obteve-se em LD suplementado com YE (1%), combinado ao método de difusão em placa modificado (prévia refrigeração das placas por 12h), contra L. monocytogenes (2130 AU/mL), com uma fase exponencial de 24h, &#181;m de 0,604/h. A otimização feita através do CCD permitiu atingir níveis de atividade de 3.000 AU/mL a 3.100 AU/mL (ensaios 7, 11 e 14, blocos 3 e 1) contra L. monocytogenes, em condições ótimas de crescimento de YE: 2,0 g/L1 e T°C: 36°C. A análise de regressão mostrou ser estatisticamente significativa a relação entre as variáveis: \"concentração de \"YE e temperatura\". Os resultados indicaram que o leite desnatado é um meio adequado para produção de bifidobacteriocina. / There are few publications that have been reported about bacteriocin production by Bifidobacterium species. Therefore, the aim of this work was measure bacteriocin production in skim milk by B. lactis. Consequently, this work was divided in three stages. First, MRS, BSM and LD medium were tested with additives (Tween 80 (T80), Inuline (I) or Yeast extract (YE)) for bacteriocin production and cellular growth. Fermentation processes were conducted in shaker under specific conditions: 50 rpm/37ºC/48h. pH; sugars; acids; biomass, and bacteriocin activity against L. monocytogenes, L. plantarum, E. coli, L. sakei e S. aureus strains were analyzed . In the second stage, based on the obtained results, a central composite design (CCD) was created using the parameters: temperature (34, 37, 40 ºC), and concentration of YE (0.5, 1.0, 1.5 g/L). After, the activity was measured by two methods of plates pre-diffusion (cooling and addition of Tween 20). Third step consisted of 2 L bioreactor cultivations containing 10% skim milk diluted in 1.5 L of water (6.5 pH), under 200 rpm, 36 ºC, 2.0 g/L of YE, 48h, under anaerobic condition. Finally, the cultures supplemented with LD and YE (1%) with a modified plate diffusion method (cooling plates for 12 h) showed bacteriocin activity against L. monocytogenes (2130 AU/mL) with an exponential phase of 24 h, &#181;m of 0.604/h. The optimization performed using CCD resulted in a higher level of activity 3000 AU/mL to 3100 AU/mL mL (Run 7, 11 and 14, blocks 3 and 1) against L. monocytogenes, also with ideal growth conditions of YE: 2,0 g/L1 and T °C: 36 °C. The pH value varied between 6.4 and 4.0. Concentration of produced acid lactic varied from 3.03 to 4.72 g/L and biomass concentration from 3.4 to 11.1 Lg UFC/mL. Regression analysis was significant to the variables: YE concentration and temperature. Results indicated that skim milk is a proper medium for \"Bifidobacteriocin\" production.

Na indução de resistência a planta possui mecanismos de defesa físicos e químicos para impedir a entrada e o desenvolvimento de patógenos e parasitas, incluindo fungos, bactérias, vírus, nematóides e até insetos. Estes mecanismos são ativados por infecções prévias ou pelo tratamento com agentes indutores (eliciadores) bióticos ou abióticos. Entre os agentes indutores bióticos, destaca-se a S. cerevisiae, que além da importância biotecnológica, tem demonstrado em estudos prévios potencial para o controle de doenças em várias plantas de importância econômica. Produtos à base de S. cerevisiae, como por exemplo o Agro-Mos® (carboidratos da parede celular da levedura) estão disponíveis no mercado, mas não como indutores de resistência. Já o indutor químico registrado como Bion® vem sendo comercializado e utilizado na indução de resistência em diversas espécies de plantas contra vários patógenos. Os objetivos deste trabalho foram purificar parcialmente frações de S. cerevisiae indutoras de resistência em pepineiro contra antracnose, causada por Colletotrichum lagenarium, e avaliar o efeito do extrato bruto autoclavado de S. cerevisiae, Agro-Mos® e Bion® na indução de resistência contra o inseto T. absoluta em tomateiro, o nematóide M. incognita em pepineiro, como também, verificar o efeito destes agentes na interação simbiótica entre soja e B. elkanii. Os resultados mostraram que o extrato bruto aquoso de S. cerevisiae autoclavado por 4 h foi o mais efetivo na redução da antracnose. Dessa maneira, o mesmo foi submetido à precipitação etanólica e o sobrenadante da precipitação foi fracionado utilizando-se Cromatografia de Troca Aniônica - CTA. Obtiveram-se quatro picos, sendo que os picos I (frações não ligada à resina DEAE-celulose) e II (frações ligadas à resina DEAE-celulose) foram os mais efetivos na proteção de plântulas de pepineiro reduzindo a severidade de antracnose em 80% e 72%, respectivamente. A aplicação foliar do extrato bruto aquoso de S. cerevisiae, Agro-Mos® e Bion® não afetou o desenvolvimento do inseto em tomateiro, como também, não interferiu significativamente na multiplicação do nematóide em raízes de pepineiro. Na interação simbiótica da soja com B. elkanii, os agentes testados não afetaram a nodulação por B. elkanii em raízes e o desenvolvimento vegetativo das plantas. Porém, a aplicação foliar do extrato bruto autoclavado de S. cerevisiae aumentou a quantidade de nitrogênio total da parte aérea das plantas. Finalmente, conclui-se que frações de S. cerevisiae induziram resistência em pepineiro contra C. lagenarium. Por sua vez, os agentes testados são foram eficientes no controle do inseto herbívoro e do nematóide e não demonstraram efeito negativo na interação soja - rizóbio. / In the resistance induction, the plant has physical and chemical defense mechanisms to avoid the entrance and the development of pathogens and parasites, including fungi, bacteria, virus, nematodes and even insects. These mechanisms are activated by previous infections or by the treatment with biotic and abiotic inducer agents. Among the biotic agents there is S. cerevisiae, that besides the biotechnological importance, was shown in previous studies to control diseases in several plants of economical importance. Products made of S. cerevisiae, as for exemple, the Agro-Mos® (formulated with carbohydrates from the cellular wall of the yeast) are available in the market, but not resistance inducers. The chemical inducer known as Bion® is already marketed and used to induced resistance in several plant species against several pathogens. The objectives of this work were to partially purify fractions of S. cerevisiae able to induce resistance in cucumber against anthracnose, caused by Colletotrichum lagenarium, and also evaluate the effect of the autoclaved crude aqueous extract from S. cerevisiae, Agro-Mos® and Bion® in the resistance induction against the insect T. absoluta in tomato plants, the nematode M. incognita in cucumber plants, as well as to verify the effect of the agents in the symbiotic interaction envolving soybean and B. elkanii. The results showed that the crude aqueous extract of S. cerevisiae autoclaved for 4 h was the most effective out in the reduction of cucumber anthracnose. Thus, the same extract was submitted to ethanolic precipitation and the obtained supernatant was fractioned by using Anion Exchange Chromatography - AEC. For peaks were obtained and peak I (non-adsorbed fraction to DEAE-Cellulose) and II (fraction adsorbed to DEAE-Cellulose) were the most effective out in the protection of the cucumber seedling by reducing anthracnose severity in 81% and 72% ,respectively. The application of the autoclaved extract of S. cerevisiae, Agro-Mos® and Bion® did not affect the development of the insect in tomato plants as well as did not interfere significantly in the multiplication of the nematode in cucumber roots. In the symbiotic interaction of soybean and B. elkanii, the tested agents did not affect the formation of nodules in soybean roots and the vegetative development of the plants. However, the foliar application of the autoclaved crude extract of S. cerevisiae significantly increased the amount of total nitrogen in the aerial part of the plants. Finally, it is concluded that the fractions (peaks I and II) of S. cerevisiae induced resistance of the cucumber plants. However the tested agents were not efficient in the control of the herbivore insect and the nematode and did not exhibit negative effects in the symbiotic interaction soybean and rhizobium.

Na indução de resistência a planta possui mecanismos de defesa físicos e químicos para impedir a entrada e o desenvolvimento de patógenos e parasitas, incluindo fungos, bactérias, vírus, nematóides e até insetos. Estes mecanismos são ativados por infecções prévias ou pelo tratamento com agentes indutores (eliciadores) bióticos ou abióticos. Entre os agentes indutores bióticos, destaca-se a S. cerevisiae, que além da importância biotecnológica, tem demonstrado em estudos prévios potencial para o controle de doenças em várias plantas de importância econômica. Produtos à base de S. cerevisiae, como por exemplo o Agro-Mos® (carboidratos da parede celular da levedura) estão disponíveis no mercado, mas não como indutores de resistência. Já o indutor químico registrado como Bion® vem sendo comercializado e utilizado na indução de resistência em diversas espécies de plantas contra vários patógenos. Os objetivos deste trabalho foram purificar parcialmente frações de S. cerevisiae indutoras de resistência em pepineiro contra antracnose, causada por Colletotrichum lagenarium, e avaliar o efeito do extrato bruto autoclavado de S. cerevisiae, Agro-Mos® e Bion® na indução de resistência contra o inseto T. absoluta em tomateiro, o nematóide M. incognita em pepineiro, como também, verificar o efeito destes agentes na interação simbiótica entre soja e B. elkanii. Os resultados mostraram que o extrato bruto aquoso de S. cerevisiae autoclavado por 4 h foi o mais efetivo na redução da antracnose. Dessa maneira, o mesmo foi submetido à precipitação etanólica e o sobrenadante da precipitação foi fracionado utilizando-se Cromatografia de Troca Aniônica - CTA. Obtiveram-se quatro picos, sendo que os picos I (frações não ligada à resina DEAE-celulose) e II (frações ligadas à resina DEAE-celulose) foram os mais efetivos na proteção de plântulas de pepineiro reduzindo a severidade de antracnose em 80% e 72%, respectivamente. A aplicação foliar do extrato bruto aquoso de S. cerevisiae, Agro-Mos® e Bion® não afetou o desenvolvimento do inseto em tomateiro, como também, não interferiu significativamente na multiplicação do nematóide em raízes de pepineiro. Na interação simbiótica da soja com B. elkanii, os agentes testados não afetaram a nodulação por B. elkanii em raízes e o desenvolvimento vegetativo das plantas. Porém, a aplicação foliar do extrato bruto autoclavado de S. cerevisiae aumentou a quantidade de nitrogênio total da parte aérea das plantas. Finalmente, conclui-se que frações de S. cerevisiae induziram resistência em pepineiro contra C. lagenarium. Por sua vez, os agentes testados são foram eficientes no controle do inseto herbívoro e do nematóide e não demonstraram efeito negativo na interação soja - rizóbio. / In the resistance induction, the plant has physical and chemical defense mechanisms to avoid the entrance and the development of pathogens and parasites, including fungi, bacteria, virus, nematodes and even insects. These mechanisms are activated by previous infections or by the treatment with biotic and abiotic inducer agents. Among the biotic agents there is S. cerevisiae, that besides the biotechnological importance, was shown in previous studies to control diseases in several plants of economical importance. Products made of S. cerevisiae, as for exemple, the Agro-Mos® (formulated with carbohydrates from the cellular wall of the yeast) are available in the market, but not resistance inducers. The chemical inducer known as Bion® is already marketed and used to induced resistance in several plant species against several pathogens. The objectives of this work were to partially purify fractions of S. cerevisiae able to induce resistance in cucumber against anthracnose, caused by Colletotrichum lagenarium, and also evaluate the effect of the autoclaved crude aqueous extract from S. cerevisiae, Agro-Mos® and Bion® in the resistance induction against the insect T. absoluta in tomato plants, the nematode M. incognita in cucumber plants, as well as to verify the effect of the agents in the symbiotic interaction envolving soybean and B. elkanii. The results showed that the crude aqueous extract of S. cerevisiae autoclaved for 4 h was the most effective out in the reduction of cucumber anthracnose. Thus, the same extract was submitted to ethanolic precipitation and the obtained supernatant was fractioned by using Anion Exchange Chromatography - AEC. For peaks were obtained and peak I (non-adsorbed fraction to DEAE-Cellulose) and II (fraction adsorbed to DEAE-Cellulose) were the most effective out in the protection of the cucumber seedling by reducing anthracnose severity in 81% and 72% ,respectively. The application of the autoclaved extract of S. cerevisiae, Agro-Mos® and Bion® did not affect the development of the insect in tomato plants as well as did not interfere significantly in the multiplication of the nematode in cucumber roots. In the symbiotic interaction of soybean and B. elkanii, the tested agents did not affect the formation of nodules in soybean roots and the vegetative development of the plants. However, the foliar application of the autoclaved crude extract of S. cerevisiae significantly increased the amount of total nitrogen in the aerial part of the plants. Finally, it is concluded that the fractions (peaks I and II) of S. cerevisiae induced resistance of the cucumber plants. However the tested agents were not efficient in the control of the herbivore insect and the nematode and did not exhibit negative effects in the symbiotic interaction soybean and rhizobium.

The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells. Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation. Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics. In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity. Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well. / <p>QC 20101129</p>