This thesis deals with optimization of capillary electrophoresis conditions for the separation of unreacted substrate and products (cellobiose, glucose) generated by the enzymatic cleavage of cellotriose with enzyme cellobiase. The optimal separation electrolyte was shown to be a solution containing 30 mmol·dm-3 sodium hydroxide with addition of 1.0 mmol·dm-3 sodium phosphate and 1.0 mmol·dm-3 sodium phosphate dibasic (pH = 12.38). With this separation electrolyte calibration curves and repeatability were measured and the limits of detection and quantification were determined. The relative standard deviations of peak areas ranged up to 6.1 % and they did not exceed 0.2 % for the migration time. Limits of detection were determined in the range from 0.020 to 0.026 mmol·dm-3 and limits of quantification from 0.066 mmol·dm-3 to 0.085 mmol·dm-3 . Subsequently the enzyme reaction was monitored in offline setup. Furthermore, the offline procedure was automated and the relative standard deviation of cellodextrines after 30 minutes of hydrolysis was ranging up to 13.0 % of glucose and cellotriose and 3.0 % cellobiose using this automated method, which makes it suitable for semiquantitative and comparative measurements. The last part of the experiment was to carry out enzymatic reaction in online setup....

L'activité cellulolytique est recherchée chez des champignons filamenteux microscopiques isolés de sols environnant les sources thermales des régions Guelma (Hammam Debagh) et de Mila (Hammam Grouz-Atmania et Hammam Safsaf-Teleghma). 88 souches fongiques sont isolées, appartenant à six genres différents : Aspergillus, Alternaria, Emericella, Fusarium, Penicillium et Trichoderma. Leur sélection (test au papier filtre et le test des plaques à trous), montre que seule la souche J2 possède une activité cellulolytique importante, comparable à celle de la souche T. reesei Rut C-30. Cet isolat appartient à l'espèce Trichoderma longibrachiatum Rifai. A 35°C, notre isolat ne montre pas de différences significatives des activités papier filtre et endoglucanase par rapport à T. reesei Rut C-30, mais l'activité β-glucosidase produite par notre isolat est deux fois plus que celle produite par cette dernière. Avec un taux d'ensemencement de 106spores/ml, la souche est en activité maximale. Un bon rendement en enzyme est obtenu avec des spores âgées de 6 jours. La souche D choisie du sous-clonage, est cultivée en fermenteur de 4 litres sur le milieu minéral Mandel Avicel 1%, et produit un maximum d'activité papier filtre 1.88UI/ml, d'activité endoglucanase 11.22UI/ml et d'activité β-glucosidase 0.64UI/ml après 128 heures, 144 heures et 120 heures d'incubation, respectivement. Les enzymes papier filtre et endoglucanase sont optimalement active à 60°C et 55°C, respectivement. Un pH optimum de 4.0 et 5.0 pour l'activité papier filtre, alors que l'activité endoglucanase à un pH optimum de 4.0. L'activité endoglucanase est thermostable, elle résiste à un traitement thermique pendant 5 heures à 70°C, 80% de son activité originale est maintenue. La demi-vie de l'activité papier filtre est de 3 heures à 60°C. Le substrat CMC améliore la stabilité thermique de ces enzymes. Ces enzymes sont stables à 50°C pendant 5 heures dans une gamme de pH de 3.0 à 6.0 et 4.0 à 6.0, respectivement. L'EDTA (5 mM), provoque une forte diminution des enzymes, alors que, le β-mercaptoethanol (5 mM) conduit à leur activation. Les cations divalents calcium (Ca2+) et zinc (Zn2+), provoquent une augmentation et une amélioration des activités enzymatiques en présence de l'EDTA. L'extrait enzymatique brut est capable d'hydrolyser les substrats cellulosiques insolubles, cette enzyme peut ainsi être classée comme un type endo et exo de la cellulase. Par ces caractéristiques, production de l'enzyme, thermostabilité et pH acide, notre souche sauvage Trichoderma longibrachiatum peut être attractive en industrie pour la production de la cellulase.Mots clés : Moisissures, isolement, écosystèmes extrêmes, cellulase, thermostabilité. / Filamentous fungi found in soils surrounding the hydrothermal stations of regions in the east of Algeria: Guelma (Hammam Debagh) and Mila (Hammam Grouz-Atmania, Hammam Safsaf-Teleghma), are screened for the presence of cellulase activity. 88 fungal strains were isolated and identified from six kinds: Aspergillus, Alternaria, Emericella, Fusarium, Penicillium and Trichoderma. Their selection (filter paper test and test of perforated plates) shows that only the strain J2 has a significant cellulolytic activity. This isolate belongs to the species of Trichoderma longibrachiatum Rifai. At 35°C, our isolate shows equivalent activities filter paper and endoglucanase than T. reesei. On the other hand the β-glucosidase activity of our isolate was until twice more important than T. reseei one. With an inoculum size of 106 spores/ml the strain produces the maximum enzyme activities. A good yield of enzyme is obtained with spores aged of six days. The strain D allowed from subcloning cultivated in 4 liter fermenter on Mandels medium with cellulose Avicel (1%) produces maximum activities of filter paper (1.88UI/ml), endoglucanase (11.22UI/ml) and β-glucosidase (0.66UI/ml) after 128 hours, 144 hours and 120 hours, respectively. The optimum temperatures were 55°C and 60°C for endoglucanase and FPA, respectively. The endoglucanase was optimally active at pH 4.0, and the FPA was optimal at pH 4.0 and 5.0. The endoglucanase was thermostable at 70°C after 5 hours incubation, preserved 80% of the original activity. The half-life of the filter paper activity appeared to be 3 hours at 60°C. These activities were stable at 50°C after 5 hours incubation in a pH range of 3.0 to 6.0 and 4.0 to 6.0, respectively. The EDTA (5mM) causes a significant diminution of the enzymes, while the β-mercaptoethanol (5mM) leading to their activation. Divalent cations calcium (Ca2+) and zinc (Zn2+) cause an increase and improvement of enzymes activities in the presence of EDTA (5mM). The crude enzyme extract is able to hydrolyse insoluble cellulosic substrates. This enzyme can be classified as an endo and exo type of the cellulase. These results suggested that the no-mutated strain Trichoderma longibrachiatum should be an attractive producer for cellulases production.Keywords: Fungi; isolation; extreme ecosystems; cellulase; thermostability.

>Magister Scientiae - MSc / Drought is a major abiotic stress which causes not only differences between the mean yield and the potential yield but also yield variation from year to year. Although selection for genotypes with improved productivity under drought environments has been a central goal of numerous plant breeding programs, the molecular basis for plant tolerance towards drought stress is still poorly understood. Exposure of plants to this abiotic stress is known to trigger excessive formation of reactive oxygen species (ROS), which induce cell death and reduce growth. Part of the mechanism of plant responses to drought involves alterations in the expression of antioxidant enzymes and biosynthesis of different compatible solutes such as proline. Sorghum is regarded as generally more drought tolerant than maize, and it is a potential key model system for investigating the physiological and molecular mechanisms conferring drought tolerance. Comparative studies in crop plants to decipher differences in drought tolerance are essential for crop improvement to sustain a higher level of production, which in turn will improve food security, under severe drought conditions resulting from climate change. On this basis, the aim of this study is to determine molecular differences between Zea mays and Sorghum bicolor in response to drought stress in an attempt to identify novel biomarkers for drought tolerance. The physiological and molecular responses of maize and sorghum were studied for changes in growth, chlorophyll content, relative water content, ROS content, lipid peroxidation level, proline content, and antioxidant enzyme activity. Spectral Count Label-free Quantitation analysis was conducted to reveal the changes in protein profiles under drought in attempt to identify drought-responsive molecular mechanisms in the leaves of the two plant species. In this study, water deficit triggered mechanisms that resulted in overproduction of ROS in both Zea mays and Sorghum bicolor. However, Sorghum bicolor showed less oxidative damage under water stress compared to Zea mays. Drought-induced proline accumulation in the roots of Sorghum bicolor was associated with enhanced water retention. Significant changes were identified in the antioxidant enzyme activity between the two plant species in response to drought conditions. Proteomics results showed differing patterns for drought-responsive proteins in the two species. Together with the physiological, biochemical and proteomic profiling results between Zea mays and Sorghum bicolor, potential proteins and/or metabolic pathways underlying drought tolerance were identified. The findings obtained through this study provide insight towards understanding the molecular basis of crop drought tolerance.

Com a grande demanda por derivados da cana-de-açúcar no Brasil a expansão de área para produção, assim como o aumento da produtividade tem sido foco para áreas agrícolas na região centro-sul. Portanto áreas que anteriormente eram dedicadas a pastagem estão tornando-se cultivo de cana-de-açúcar. Essa mudança está ocorrendo com uma grande rapidez e por esse motivo preocupações com a sustentabilidade do solo estão sendo levantadas. O objetivo deste trabalho foi verificar como a atual mudança de uso da terra de pastagem para cultivo de canade- açúcar impacta atributos microbiológicos quantitativos e funcionais do solo em um transecto edafoclimático na região centro-sul do Brasil tendo a vegetação nativa como referência. Para isso foram feitas análises do carbono e nitrogênio da biomassa microbiana do solo segundo Vance, Brookes e Jenkinson (1987) e análises da atividade enzimática da β-glicosidase, fosfatase ácida e alcalina segundo Tabatabai (1994). Verifica-se a tendência de reduzir a fertilidade na pastagem em relação a mata nativa e posteriormente aumentar com o uso de fertilizantes na cana-de-açúcar. O estoque de C tem um menor valor na pastagem quando não manejada em Lat S 17º, diminui na sequencia de mudança dos sistemas de uso da terra em Lat S 21º, porém uma recuperação na mudança para cultivo de cana-de-açúcar e é maior em Lat S 23º quando a textura do solo é mais argilosa. Existe uma diminuição do estoque de CBM das matas nativas para as pastagens e um aumento nos cultivos de cana-de-açúcar em Lat S 17º e Lat S 21º. Em Lat S 23º a pastagem apresenta maior CBM do que os outros sistemas de uso da terra. Nos locais de estudo a menor relação CBM/C Total foi de 2,54% na mata nativa em Lat S 17º e a maior de 5,74% na pastagem de Lat S 23º. O N é um elemento muito sensível, em todos os atributos analisados a mudança de uso da terra causou uma grande diminuição, e a degradação do solo é o fator que causa o grande impacto nesses atributos. A textura argilosa do solo garante maior atividade enzimática, porém em cultivos de cana-de-açúcar a atividade diminui independente da classe textural. Os mesmos usos da terra em diferentes locais não podem ser analisados como repetições. Sendo assim pode-se concluir que nos casos analisados o impacto da mudança de uso da terra de pastagem para cultivo de cana-de-açúcar se manifestou de forma diferente em cada local, não permitindo uma generalização. / The great demand for derivatives of sugarcane in Brazil is the motive for the area expansion for production as well as the increase of productivity have been the focus of agricultural areas in the center-south region. So areas that were previously pastures have been changing for sugarcane cultivation. This change has occured with great speed and therefore concerns about the sustainability of soil have been raised. The aim of this study was to determine how the current change from pasture to sugarcane cultivation impacts quantitative and functional microbiological attributes of soil in a soil climatic zone in the central-south region of Brazil considering the native vegetation as reference. For it were made analysis of soil microbial biomass carbon and nitrogen according to Vance, Brookes and Jenkinson (1987) and analysis of enzymatic activity of β-glucosidase, acid and alkaline phosphatase according to Tabatabai (1994). There is a tendency to reduce fertility in pasture compared to native vegetation and then increase with the use of fertilizers in sugarcane cultivation. The stock of C has a lower value in the pasture when there is no fertilizers in Lat S 17º, decreases in the land use change in Lat S 21º, but there is a recovery in the switch over to sugarcane cultivation and it is the biggest in Lat S 23º when the soil texture is clayey. There is a decrease in the stock of CBM from native forests to pastures and an increase in sugarcane cultivation in Lat S 17º and Lat S 21º. In Lat S 23º the pasture has higher CBM stock than other land use systems. In the study sites the reason between CBM and C Total was 2.54% in the native vegetation in Lat S 17º and the highest 5.74% in the pasture of Lat S 23º. The N is a very sensitive element, in all attributes analyzed, the land use change caused a large decrease, and soil degradation is the factor that causes serious impact on those attributes. The clayey soil ensures greater enzymatic activity, but in sugarcane cultivation decreases the activity independent of the texture. The same land uses in different locations can\'t be analyzed as repetitions. Therefore in the cases analyzed the impact of the change from pasture to sugarcane cultivation was differently in each place, not allowing a generalization.

O fósforo (P) é limitante tanto na produtividade da cana-de-açúcar (Saccharum officinarum L.) quanto na atividade da microbiota do solo. Em solos de regiões tropicais, devido à elevada saturação por óxidos de ferro e alumínio, o P se encontra normalmente indisponível às plantas. Portanto, o manejo da adubação de P, baseado no uso de microrganismos capazes de promover uma melhor ciclagem do P, se faz necessário. Assim, o objetivo do estudo foi avaliar as mudanças na microbiota do solo e na produtividade da cana-de-açúcar, em condições de campo, em resposta ao manejo orgânico associado à inoculação de bactérias solubilizadoras de fosfato (BSF). Para tanto, foi utilizado um composto, obtido previamente por meio da compostagem de subprodutos da indústria sucroenergética (torta de filtro e cinzas), enriquecido com fosfatos de rocha (fosfato de Araxá-FA ou fosfato de Bayóvar-FB). No campo foram estabelecidos sete tratamentos, sendo um destes o tratamento controle, adubado com superfosfato triplo. Seis tratamentos compreendiam áreas adubadas com composto, sendo duas com composto que continha FA, duas com FB e duas somente com composto sem enriquecimento com P (C). A metade dessas áreas recebeu também inoculação com BSF, contendo os tratamentos FA+I, FB+I e C+I. A inoculação foi feita com Bacillus simplex BACBR04, Bacillus sp. BACBR06 e Rhizobium sp. RIZBR0. As avaliações foram realizadas durante o primeiro ano de cultivo (cana planta) em dois períodos (aos seis e 12 meses). As mudanças na microbiota foram acessadas por meio da atividade das enzimas fosfatases (ácida e alcalina), fitases e β-glucosidase. Mudanças na estrutura da comunidade bacteriana e fúngica foram acessadas por meio do T-RFLP. A abundância dos genes 16S RNAr, phoD (relacionado à solubilização de P) e ITS foram avaliadas por meio de PCR quantitativo. A atividade das fosfatases e β-glucosidase aumentaram com a inoculação na cana adubada com composto enriquecido com fosfato de Araxá (FA+I) e também no solo adubado com composto sem enriquecimento (C+I). Nos tratamentos FA+I e C+I houve incremento do fósforo disponível e aumento de 10% na produtividade, em relação ao tratamento controle (M). As comunidades bacteriana e fúngica do tratamento C+I se estruturaram de forma distinta em relação ao tratamento controle (M). Nos solos inoculados houve menor abundância do gene ITS aos seis meses, enquanto que, para o gene 16S RNAr, os solos inoculados apresentaram menor abundância no período de 12 meses. Verificou-se que o consórcio bacteriano inoculado, associado com a aplicação de composto, superou, no primeiro ano de cultivo, o manejo rotineiramente utilizado em cana-de-açúcar (adubação com superfosfato triplo). É possível que haja efeito residual no decorrer dos ciclos da cana-de-açúcar, o que ainda reforçaria a importância do manejo orgânico associado à inoculação com BSF. / Phosphorus (P) is limiting both the yield of sugarcane (Saccharum officinarum L.) and the activity of the soil microbiota. In tropical soils, due to the high saturation of iron and aluminum oxides, P is normally unavailable to plants. Therefore, the management of P fertilization, based on the use of microorganisms that promote better P cycling is necessary. Thus, the objective of this study was to evaluate changes in the soil microbiota and in sugarcane yield under field conditions, in response to the organic management associated with the inoculation of phosphate solubilizing bacteria (PSB). In our field experiment, our fertilizer was an organic compost, previously obtained by the composting process of by-products of the sugarcane industry (filter cake and ashes), enriched with rock phosphates (Araxá phosphate-AP or Bayóvar phosphate-BP). Seven treatments were established in the field, one of which was the control treatment, fertilized with triple superphosphate. Six treatments comprised compost fertilized areas, two with compost containing AP, two with BP, and two only with compost, and without any enrichment with P (C). Half of these areas were also inoculated with PSB, containing the treatments AP+I, BP+I and C+I. Field inoculation was done with Bacillus simplex BACBR04, Bacillus sp. BACBR06 and Rhizobium sp. RIZBR0. We performed evaluations during the first year of cultivation at two periods (at six and twelve months after planting). We accessed the changes in the microbiota through the activity of acid and alkaline phosphatases, phytases and β-glucosidase. Changes in bacterial and fungal community structure were accessed through T-RFLP. We evaluated the abundance of the 16S RNAr, phoD (related to P solubilization) and ITS genes by quantitative PCR. The phosphatases and β-glucosidase activity increased with the inoculation in sugarcane fertilized with compost and enriched with Araxá phosphate (AP+I) and in the soil fertilized with compost, but without any enrichment (C+I). In these treatments (AP+I and C+I), we found an increase in available phosphorus and a 10% increase in productivity, in relation to the control treatment (M). The bacterial and fungal communities of the treatments C+I presented a different structure in relation to the control treatment (M). In the soils that received bacterial inoculations, there was a lower abundance of the ITS gene at six months, whereas for the 16S RNAr gene the inoculated soils presented lower abundance at 12 months. We verified that the inoculated bacterial consortium, associated with the application of compost, overcame the conventional management in sugarcane (triple superphosphate fertilizer) in the first year of cultivation. In addition, it is possible residual effect during the sugarcane cycles, which would further reinforce the importance of organic management associated with BSF inoculation.

Duas enzimas do clone PL14.H10 de biblioteca metagenômica de consórcio microbiano degradador de óleo diesel, EST3, da família IV, e EST5, da família V das enzimas lipolíticas, foram caracterizadas. Os modelos estruturais mostraram cap-domínio e grande cavidade interna. A EST3 hidrolisou pNP-ésteres de até 12 carbonos, com Kcat/Km para pNP-acetato foi 1533,27 s-1.mM-1. Já a EST5 hidrolisou pNP-ésteres de 2 a 14 átomos de carbono, com maior atividade em pNP-valerato, e Kcat/Km de 1732,3 s-1.mM-1. Ambas apresentaram o fenômeno da ativação térmica. A atividade máxima da EST5 se deu a 45°C e em pH 7,5. A EST3 exibiu atividade máxima em pH 6,0, a 41°C. A atividade das enzimas aumentou na presença de quase todos os íons testados, e a EST5 quase não foi influenciada por detergentes. Foram relativamente estáveis em solventes orgânicos. Um dos cristais da proteína EST5 difratou a 2,311 Å e espera-se resolver a estrutura desta proteína. Os resultados deste trabalho revelaram interessantes características da EST3 e EST5 para possíveis aplicações biotecnológicas. / Two enzymes of PL14.H10 clone from a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation, EST3, from family IV, and EST5, from the V of lipolytic enzymes were characterized. Structural models showed cap-domain and large internal cavity. EST3 hydrolyzed pNP-esters of up to 12 carbons, with Kcat/Km in pNP-acetate of 1533.27 s-1.mM-1. EST5 hydrolyzed pNP-esters from 2 to 14 carbon atoms, with greater activity on pNP-valerate, and Kcat/Km of 1732.3 s-1.mM-1. Both showed the thermal activation phenomenon. EST5 activity was optimal at 45°C and pH 7.5. EST3 exhibited maximum activity at pH 6.0, 41°C. The esterases activity increased in the presence of almost all ions tested, and EST5 was not influenced by detergents. They were relatively stable in organic solvents. One of the crystals of EST5 protein diffracted at 2.311 Å and is expected to solve the structure of this protein. The results of this study revealed interesting features of the EST3 and EST5 proteins for possible biotechnological applications.

O objetivo do estudo foi avaliar a inclusão de enzimas exógenas nas rações sobre o desempenho, rendimento de carcaça e cortes, digestibilidade de nutrientes, peso relativo do pâncreas, atividade endógena de enzimas e expressão de RNA mensageiro de enzimas pancreáticas de frangos de corte alimentados com dietas contendo enzimas exógenas, no período de 1 a 42 dias de idade. Para tanto, foram conduzidos dois experimentos, o primeiro com a utilização da protease e o segundo, com o uso da α-amilase exógenas. No primeiro experimento, foram utilizados quatro tratamentos com níveis crescentes de inclusão da protease monocomponente à dieta: 0,50%, 100%, 150% da inclusão recomendada pelo fornecedor. As formulações foram a base de milho e farelo de soja e formuladas de acordo com as recomendações nutricionais sugeridas por Rostagno et al. (2011). Não foram observados efeitos significativos da adição da enzima sobre os parâmetros de desempenho, rendimento de carcaça e de cortes, digestibilidade de nutrientes e sobre o peso relativo do pâncreas de frangos de corte. O uso da protease exógenas não alterou (P>0,05) a atividade da tripsina pancreática nas idades avaliadas (21 e 42 dias), porém observou-se o aumento (P<0,05) da expressão de RNA mensageiro da tripsina pancreática em aves aos 42 dias de idade, em função do aumento da inclusão da enzima na dieta. A produção da tripsina pancreática foi maior (P<0,05) nas aves aos 42 dias do que nas aves jovens (21 dias de idade). No segundo experimento, foram utilizados quatro tratamentos com o uso da -amilase exógena, sendo o controle sem o uso de enzimas, 2 tratamentos com a inclusão de α-amilase (50% e 100% da dosagem recomendada pelo fornecedor) e um tratamento com a inclusão da amilase exógena (100% da dosagem recomendada) combinada com o uso de 500 FTU de fitase por quilograma de ração. As formulações foram a base de milho e farelo de soja e formuladas de acordo com as recomendações nutricionais sugeridas por Rostagno et al. (2011). Não foram observados efeitos significativos da adição da α-amilase sobre os parâmetros de desempenho, rendimento de carcaça e de cortes, digestibilidade de nutrientes e sobre o peso relativo do pâncreas de frangos de corte. A suplementação de -amilase nas rações aumentou (P<0,05) a atividade da amilase pancreática e reduziu (P<0,05) a atividade da tripsina endógena em frangos na fase inicial (1 a 21 dias de idade). A expressão de RNA mensageiro da amilase e tripsina pancreática não foram alteradas com o uso das enzimas nas rações. Com o avançar da idade, observou-se o aumento significativo da atividade e da expressão do RNA mensageiro da amilase pancreática, sendo maior aos 42 dias do que aos 21 dias de idade da ave. / The objective of the study was evaluate the inclusion of exogenous enzymes on performance, carcass yield and commercial cuts, nutrient digestibility, relative weight of pancreas, endogenous activity of enzymes and mRNA expression of pancreatic enzymes of broilers fed with diets containing exogenous enzymes, from 1 to 42 days of age. To this end, two experiments were conducted, the first with the use of protease and the second, with the use of exogenous α-amylase. In the first experiment, four treatments with increasing levels of inclusion of single-component protease were used in the diet: 0,50%, 100%, 150% of the inclusion recommended by the supplier. The formulations were based on corn and soybean meal and formulated according to the nutritional recommendations suggested by Rostagno et al. (2011). Significant effects were not observed on the addition of enzyme on performance, carcass yield and commercial cuts, digestibility of nutrients and on the relative weight of the pancreas of broilers. The use of exogenous protease did not change (P>0.05) the activity of pancreatic trypsin in evaluated ages (21 and 42 days), however the increase was observed (P<0.05) in mRNA expression of pancreatic trypsin in 42-days-old birds, due to the increased inclusion of the enzyme in the diet. Pancreatic trypsin production was higher (P<0.05) in 42-days birds than the young birds (21 days of age). In the second experiment, four treatments with the use of exogenous α-amylase were used, being the control without the use of enzymes, 2 treatments with the inclusion of α-amylase (50% and 100% of the dosage recommended by the supplier) and a treatment with the inclusion of exogenous amylase (100% of recommended dosage) combined with the use of 500 FTU of phytase per kilogram of feed. The formulations were based on corn and soybean meal and formulated according to the nutritional recommendations suggested by Rostagno et al. (2011). Significant effects were not observed on the addition of -amylase on performance, carcass yield and commercial cuts, digestibility of nutrients and on the relative weight of the pancreas of broilers. The -amylase supplementation in feed increased (P<0.05) the activity of amylase and reduced (P<0.05) the activity of endogenous trypsin in initial phase chickens (1 to 21dias). mRNA expression of amylase and pancreatic trypsin have not changed with the use of enzymes in feed. As the age increased, there was a significant increase in activity and expression of mRNA of pancreatic amylase, being higher at 42 days than at 21 days of age of the bird.

Com a grande demanda por derivados da cana-de-açúcar no Brasil a expansão de área para produção, assim como o aumento da produtividade tem sido foco para áreas agrícolas na região centro-sul. Portanto áreas que anteriormente eram dedicadas a pastagem estão tornando-se cultivo de cana-de-açúcar. Essa mudança está ocorrendo com uma grande rapidez e por esse motivo preocupações com a sustentabilidade do solo estão sendo levantadas. O objetivo deste trabalho foi verificar como a atual mudança de uso da terra de pastagem para cultivo de canade- açúcar impacta atributos microbiológicos quantitativos e funcionais do solo em um transecto edafoclimático na região centro-sul do Brasil tendo a vegetação nativa como referência. Para isso foram feitas análises do carbono e nitrogênio da biomassa microbiana do solo segundo Vance, Brookes e Jenkinson (1987) e análises da atividade enzimática da β-glicosidase, fosfatase ácida e alcalina segundo Tabatabai (1994). Verifica-se a tendência de reduzir a fertilidade na pastagem em relação a mata nativa e posteriormente aumentar com o uso de fertilizantes na cana-de-açúcar. O estoque de C tem um menor valor na pastagem quando não manejada em Lat S 17º, diminui na sequencia de mudança dos sistemas de uso da terra em Lat S 21º, porém uma recuperação na mudança para cultivo de cana-de-açúcar e é maior em Lat S 23º quando a textura do solo é mais argilosa. Existe uma diminuição do estoque de CBM das matas nativas para as pastagens e um aumento nos cultivos de cana-de-açúcar em Lat S 17º e Lat S 21º. Em Lat S 23º a pastagem apresenta maior CBM do que os outros sistemas de uso da terra. Nos locais de estudo a menor relação CBM/C Total foi de 2,54% na mata nativa em Lat S 17º e a maior de 5,74% na pastagem de Lat S 23º. O N é um elemento muito sensível, em todos os atributos analisados a mudança de uso da terra causou uma grande diminuição, e a degradação do solo é o fator que causa o grande impacto nesses atributos. A textura argilosa do solo garante maior atividade enzimática, porém em cultivos de cana-de-açúcar a atividade diminui independente da classe textural. Os mesmos usos da terra em diferentes locais não podem ser analisados como repetições. Sendo assim pode-se concluir que nos casos analisados o impacto da mudança de uso da terra de pastagem para cultivo de cana-de-açúcar se manifestou de forma diferente em cada local, não permitindo uma generalização. / The great demand for derivatives of sugarcane in Brazil is the motive for the area expansion for production as well as the increase of productivity have been the focus of agricultural areas in the center-south region. So areas that were previously pastures have been changing for sugarcane cultivation. This change has occured with great speed and therefore concerns about the sustainability of soil have been raised. The aim of this study was to determine how the current change from pasture to sugarcane cultivation impacts quantitative and functional microbiological attributes of soil in a soil climatic zone in the central-south region of Brazil considering the native vegetation as reference. For it were made analysis of soil microbial biomass carbon and nitrogen according to Vance, Brookes and Jenkinson (1987) and analysis of enzymatic activity of β-glucosidase, acid and alkaline phosphatase according to Tabatabai (1994). There is a tendency to reduce fertility in pasture compared to native vegetation and then increase with the use of fertilizers in sugarcane cultivation. The stock of C has a lower value in the pasture when there is no fertilizers in Lat S 17º, decreases in the land use change in Lat S 21º, but there is a recovery in the switch over to sugarcane cultivation and it is the biggest in Lat S 23º when the soil texture is clayey. There is a decrease in the stock of CBM from native forests to pastures and an increase in sugarcane cultivation in Lat S 17º and Lat S 21º. In Lat S 23º the pasture has higher CBM stock than other land use systems. In the study sites the reason between CBM and C Total was 2.54% in the native vegetation in Lat S 17º and the highest 5.74% in the pasture of Lat S 23º. The N is a very sensitive element, in all attributes analyzed, the land use change caused a large decrease, and soil degradation is the factor that causes serious impact on those attributes. The clayey soil ensures greater enzymatic activity, but in sugarcane cultivation decreases the activity independent of the texture. The same land uses in different locations can\'t be analyzed as repetitions. Therefore in the cases analyzed the impact of the change from pasture to sugarcane cultivation was differently in each place, not allowing a generalization.

A total maximum daily load (TMDL), which is the calculated total amount of pollutant that a waterbody can receive from point and non-point sources, is established for streams that do not meet their designated use criteria. Physical, chemical, and biological water quality parameters are used to attempt to identify pollution sources. Microbial enzyme activity (mg/mL) is used to monitor the changes in the microbial community by identifying changes in their metabolism. The health of a stream can be monitored by the presence and absence of microorganisms due to the response of the microbial community to prolonged pollution exposure. To fully understand the metabolic activity, MEA data are compared to other factors including biochemical oxygen demand (BOD), nitrogen, phosphate, total coliform and Escherichia coli concentrations. Dissolved oxygen refers to the amount of oxygen used by microorganisms to degrade organic carbon, which reduces the survivability of aerobic organisms. Nitrogen and phosphate compounds from anthropogenic sources are readily dissolved into water and are limiting nutrients for microorganisms. Total coliforms and E. coli determine the fecal contamination in the waterbody. All of these factors are used to determine point and nonpoint pollution sources. From February 2014 to January 2016, water and sediment samples were collected monthly from 16 sites along Sinking Creek in Northeast Tennessee. During the two-year study, physical and field parameters were measured. Water samples were analyzed for chemical parameters including alkalinity, hardness, and BOD. Using an ion chromatograph, the concentration of phosphates and nitrates in the samples was measured. For biological parameters, the water sample was used to obtain total coliform and E. coli data using the Colilert enzyme substrate test. MEA data were collected from triplicate sediment samples collected at each site. These sediment samples were tested for acid and alkaline phosphatase, glucosidase, galactosidase, and dehydrogenase enzymes. The substrate for these enzymes was added to the respective sample, incubated for approximately 24 hours, and analyzed by colorimetric spectrophotometry to obtain absorbance. The enzyme concentration was calculated by comparing the absorbance to a generated standard curve. The results for the measured parameters were compared to identify correlation between the MEA concentrations and other biological and chemical parameters. The microbial activity should show a holistic view of changes in the waterbody, which should correlate with the other factors. A direct relationship between E. coli, total coliforms, nitrate, phosphate concentrations, and MEAs was expected. A correlation should be seen between MEAs and BOD data, due to an increase in oxygen demand during microbial metabolism. By examining multiple parameters together, the results would provide the necessary information to determine remediation efforts for the waterbody.

Phosphoinositide-dependent protein kinase-1 (PDK1) plays an integral role in signaling cellular growth and proliferation, one that's dependent on its ability to autophosphorylate Ser-241 in its T-loop. This process appears to have a strict requirement for its C-terminal pleckstrin homology (PH) domain. Thus, the overall objective of this work was to determine the mechanism by which the PH domain induces an active kinase conformation in unphosphorylated PDK1, capable of Ser-241 autophosphorylation. First, computational modeling and protein cross linking studies were combined with site-directed mutagenesis and kinetic assays in order to provide initial assessment of how the PH domain scaffolds Ser-241 autophosphorylation. A significant number of contacts were identified between the enigmatic "N-bud" region of the PH domain and the kinase domain. Specifically, these studies implicated Glu-432 and Glu-453 of the N-bud region of the PH domain that bind and serve as mimics of the phosphorylated Ser-241 in the T-loop and the phosphorylated C-terminal tail of PDK1 substrates, respectively. Next, a novel method for protein trans-splicing of the regulatory and catalytic kinase domains of PDK1 was developed. The method utilizes the N- and C-terminal split inteins of the gene dnaE from Nostoc punctiforme [(N)NpuDnaE] and Synechocystis sp. strain PCC6803 [(C)SspDnaE], respectively. The cross-reacting KINASE(AEY)-(N)NpuDnaE-His6 and GST-His6-(C)SspDnaE-(CMN)PH fusion constructs generated full length spliced-PDK1 with kobs = (2.8 +- 0.3) x 10-5 s-1. Finally, NMR was used to further characterize the structural and dynamical properties of the PH domain in both its isolated form and in full length PDK1. Whereas, it was not possible to obtain chemical shift assignments of any backbone or side chain nuclear resonances, methods were optimized for 2H,13C,15N-isotopic labeling of the recombinant PH domain. Furthermore, the protein trans-splicing method was significantly improved and utilized for segmental isotopic labeling of the PH domain in full length PDK1. These new findings and developments may provide specific insight and technological improvements towards future studies aimed to better understand and target autoinhibited conformations of PDK1 for translational purposes.