Orientador: Arthur Bernardes Cecílio Filho / Coorientador: Priscila Lupino Gratão / Coorientador: Hilário Júnior de Almeida / Banca: André Rodrigues dos Reis / Banca: Rogério Falleiros Carvalho / Banca: Jairo Osvaldo Cazetta / Banca: Roberto Botelho Ferraz Branco / Resumo: O selênio (Se) é um elemento essencial para os seres humanos, no entanto, em plantas é considerado benéfico. O Se pode atuar como antioxidante de células vegetais, melhorando o crescimento das plantas. A aplicação de Se em culturas para fins de biofortificação tem sido uma estratégia eficaz para aumentar o fornecimento do elemento na dieta da população. Entretanto, para ter sucesso é fundamental estudar a aplicação de Se, conhecendo a fonte e concentração adequadas, de forma que não prejudique o crescimento, os aspectos nutricionais, bioquímicos e produtivos da planta. Assim, objetivou-se avaliar o efeito de fontes e concentrações de Se na biofortificação e aspectos nutricionais, fisiológicos e enzimáticos de plantas de couve-flor. O estudo foi realizado em ambiente protegido, com plantas de couve-flor cultivadas em sistema hidropônico, sob concentrações de Se (0, 5, 15, 30 e 60 μmol L-1) nas fontes selenato e selenito de sódio. Os tratamentos foram organizados em delineamento inteiramente casualizado, em esquema fatorial 5 x 2, com quatro repetições. Verificou-se que a aplicação de Se na concentração de 5 μmol L-1 na solução nutritiva promoveu crescimento das plantas e teor do elemento na inflorescência da couve-flor foi superior ao limite considerado adequado pelo Codex Alimentarius. O fornecimento de Se, na fonte selenato, pode ter contribuído para inibir a peroxidação lipídica e a produção de espécies reativas de oxigênio, uma vez que com esse elemento houve melhora na ta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Selenium (Se) is an essential element for humans, however, in plants is considered beneficial. The Se can act as an antioxidant of plant cells, improving plant growth. The application of Se in crops for biofortification has been an effective strategy to increase the supply of the element in the diet of the population. However, to be successful, it is essential to study the application of Se, knowing the proper source and concentration, so as not to harm the growth, nutritional, biochemical and productive aspects of the plant. Thus, objective was evaluate the effect of sources and concentrations of Se on biofortification and nutritional, physiological and enzymatic aspects of cauliflower plants. The study was carried out in a protected environment, with cauliflower plants cultivated in a hydroponic system, under concentrations of Se (0, 5, 15, 30 and 60 μmol L-1 ) in sources selenate and selenite sodium. The treatments were organized in a completely randomized design, in factorial scheme 5 x 2, with four replications. It was verified that the application of Se at the concentration of 5 μmol L-1 in the nutritive solution promoted plant growth and element content in the cauliflower inflorescence was higher than the limit considered adequate by the Codex Alimentarius. The supply of exits at the selenate source may have contributed to inhibit a lipid peroxidation and a production of reactive oxygen species, since this element is an improvement in the rate of photosynthesis and the regulation of the enzymatic activity of plants. At high concentrations, if supplied mainly in the selenite source, it adversely affects the nutritional balance of plants and, consequently, the growth, the physiological, enzymatic and productive aspects of the cauliflower, depreciating a quality of the product. / Doutor

El objetivo de esta tesis ha sido aportar información básica sobre el modo de acción de los enzimas carbohidrasas sobre los cereales viscosos o raciones convencionales no viscosas de maíz y soja. Partiendo de estos mecanismos, hemos valorado sus efectos sobre los parámetros productivos y digestivos en los pollos en crecimiento. En particular, en el caso de los cereales blancos (cebada y trigo) estudiamos la efectividad de b-glucanasas y arabinoxilanasas sobre los resultados de producción (consumo de alimento, ganancia media diaria) y digestibilidad de la ración en pollos en crecimiento; y los posibles mecanismos involucrados, como la cinética de tránsito y viscosidad de la digesta. Para alcanzar este objetivo se realizaron 2 ensayos experimentales. En el primero de ellos se utilizaron pollitos sexados machos que recibieron 4 raciones experimentales basadas en un 44% de cebada + trigo (2 x 2; con o sin enzimas, con o sin 2% de sepiolita) durante un periodo experimental de 36 días. Se realizaron dos balances de digestibilidad de la MO, PB y MG establecidos entre los días 21-22 y 41-42 de edad. Los resultados relativos a este experimento muestran que la suplementación de enzimas mejoró la ganancia media diaria de los animales y la eficiencia de conversión del alimento, sobre todo durante la primera fase de crecimiento (14%, P<0.001 y 10%, P<0.001; respectivamente). Las mejoras de los resultados productivos fueron fundamentalmente explicadas por una mejora significativa de la digestión y la retención de los componentes de la materia orgánica (5.6 up y 1.7 up.,), y en especial de la fracción lipídica (13 up., y 3.5 up.) en el día 22º y 42º, respectivamente. Teniendo en cuenta los resultados productivos obtenidos durante este primer ensayo, se planteó un segundo experimento en un diseño similar con el objetivo de caracterizar los posibles mecanismos determinantes de los resultados productivos. Entre los aspectos contemplados, se estudió i/ el ritmo de tránsito de la digesta (estimados a partir de la excreción fecal de un marcador indigestible o a partir de su cuantificación en el tracto digestivo de los animales en condiciones de equilibrio dinámico) y ii/ la viscosidad de la digesta del contenido del yeyuno e íleon.Los resultados productivos fueron similares a los del primer ensayo. No se observaron diferencias significativas en el consumo de alimento, aunque con la suplementación enzimática se incrementaron los ritmos de crecimiento (+5%, P<0.05), y se mejoraron los índices de conversión de la dieta (7%, P<0.01). Se observo una mejora en la digestión durante el día 21º de todas las fracciones de la MO (PB, 6 u.p., P<0.001; MG, 6 u.p., P<0.01 y carbohidratos, 2.5 up., P<0.05) y en consecuencia de la concentración energética de la ración (EMAn: 5.4% de 3110 a 3280 Kcal/ kg MS alimento). La suplementación enzimática redujo significativamente la viscosidad relativa al agua determinada en el intestino (yeyuno e íleon) y los tiempos de retención (MRT) de la digesta en el tracto digestivo.La suplementación enzimática en la alimentación de los broilers en la actualidad consiste mayoritariamente en la inclusión de b-glucanasas y arabinoxilanasas en raciones con cereales «viscosos». Sin embargo, recientemente, numerosas expectativas se están incorporando como son la búsqueda de estrategias enzimáticas dirigidas a mejorar el valor nutritivo de las leguminosas en general y la torta de soja en particular. En esta línea, nos planteamos como objetivo caracterizar «in vitro» e «in vivo» la efectividad de diferentes enzimas carbohidrasas sobre la digestión de los polisacáridos solubles e insolubles de la torta de soja.Para atender estos objetivos, se planteó un primer ensayo de caracterización de los carbohidratos de la soja (cotiledones y cascarilla). El fraccionamiento (soluble e insoluble en agua) de la cascarilla de la soja mostró la existencia de una estructura insoluble mayoritariamente constituida por celulosa (b-1,4-D-glucosa), mientras que la fracción soluble contiene una mayor proporción de manosa y galactosa (posiblemente b-mananos del tipo galacto- y glucómananos). En cuanto a los cotiledones, su fraccionamiento mostró la existencia de estructuras complejas compuestas por un entramado de pectinas, hemicelulosas y celulosa. La incubación "in vitro" con enzimas de actividad pectinasa, xilanasa y celulasa mostró un bajo porcentaje de hidrólisis de los carbohidratos, aunque se incrementó (hasta un 20% de liberación de monosacáridos) tras la extracción de diferentes componentes del entramado de pared vegetal.Puesto que es conocido el efecto del tostado de la soja sobre la solubilidad y la digestibilidad de la proteína, se planteó un segundo ensayo con el objetivo de establecer la influencia de un tostado suave o intenso sobre la eficiencia enzimática "in vitro", especialmente de la proteasa y diferentes carbohidrasas. Efectivamente, el tostado provocó un descenso gradual en la solubilidad tanto de la fracción proteica como de los carbohidratos, lo que sugiere cambios estructurales del entramado de carbohidratos en su relación con la proteína. Ambas fracciones, las solubles e insolubles en agua fueron utilizadas como sustrato susceptible de hidrólisis por los enzimas carbohidrasas (pectinasa, xilanasa, mananasa y celúlasa) en combinación o no con proteasa. La incubación de los substratos solubles con enzimas carbohidrasas (en especial, celulasa y pectinasa) redujo la proporción de rafinosa y estaquiosa. Actividad que no fue evidenciada con la mananasa en su combinación con proteasa. La incubación con carbohidrasas de las fracciones no extractables con agua liberó una baja cantidad de monosacáridos, aunque se incrementó sinérgicamente con la inclusión de proteasa. Sin embargo, la posible efectividad de los enzimas "in vivo" esta condicionada por aspectos asociados al animal, que no pueden ser simulados "in vitro". Por ello, decidimos iniciar el desarrollo de pruebas de crecimiento en broilers en las que estudiar las posibles mejoras productivas y digestivas asociadas a la inclusión de enzimas de pared vegetal. En concreto, nos planteamos probar, en un primer ensayo, el efecto de incorporar a una ración base de maíz-soja una mananasa (a dos niveles de inclusión), asociado o no con proteasa. Para ello, se planteó un experimento similar al descrito para las b-glucanasas y arabinoxilanasas. La suplementación con enzimas no provocó modificaciones significativas de los resultados productivos de los animales o de la digestibilidad de la ración. Sin embargo, la incorporación de mananasa a la dieta redujo significativamente la proliferación microbiana en íleon (medida como concentración de bases puricas) durante la primera fase de crecimiento de los pollitos. Aunque la experiencia fue realizada en jaulas, y no se observaron diferencias en productividad, nos planteamos como interesante valorar en el futuro las repercusiones productivas y sanitarias que este tipo de modificaciones microbianas pueden tener, especialmente en condiciones prácticas de producción. / The aim of this thesis is to obtain basic information on the activity of carbohydrase enzymes over viscous cereals or conventional no viscous diets based on corn and soybean meal. The effects of enzymes supplementation in broiler chickens productive and digestive parameters will be also evaluated. In particular, in the case of the white cereals (barley and wheat), we have studied initially the effectiveness of b-glucanase and arabinoxylanase on the productive performances (Feed intake, bodyweight gain) and nutrient digestion in growing chicks; and the possible mechanisms involved, like the digesta kinetics and viscosity. Two experimental trials are presented. Male chickens received 4 experimental diets based on 44% of barley + wheat (2 x 2; with or without enzymes, with or without 2% of sepiolite) during an experimental period of 36 days. Two balances of organic matter (OM), crude protein (CP) and fat (EE) were carried out on days 21-22 and 41-42 of age. The enzymes supplementation improved the daily weight gain of the animals and the food: gain ratios, mainly during the first phase (14%, P<0.001 and 10%, P<0.001; respectively). Productive results were simultaneous to significant increases of the whole-tract digestibility and retention of organic components, (5.6 and 1.7 percent units), especially the fatty fraction (13 and 3.5 percent units) on day 22 and 42, respectively.The second experiment aimed to characterise the possible mechanisms involved. They were studied i/ the digesta mean retention time (MRT estimated from the faecal excretion of an indigestible marker or from its quantification in the animal digestive tract in steady state conditions) and ii / the jejunum and ileum digesta viscosity.Productive results were similar to those of the first trial. Although, not significant differences were observed in food consumption, enzymes supplementation increased growth rates (+5%, P<0.05), and improved the FCR (7%, P<0.01). It was also observed a significant increase of the OM digestibility (CP, 6 percent units, P<0.001; EE, 6 percent units, P<0.05 and carbohydrates, 2.5 percent units, P<0.05) and metabolizable energy (EMAn: 5.4% from 3110 to 3280 Kcal / kg DM food). The enzymatic suplementación significantly reduced intestinal digesta viscosity (jejunum and ileum) and digesta MRT in the digestive tract.Enzymatic supplementation in broilers feeding has expanded mainly as the b-glucanase and arabinoxylanase inclusion in «viscous» cereal diets. However, recently numerous expectations are being incorporated in poultry feeding, addressed by the search of enzymatic strategies to improve the nutritive value of leguminous in general, and soybean meal in particular. In this line, we planed to characterise «in vitro» and «in vivo» the effectiveness of different carbohydrase enzymes on the digestion of soluble and insoluble soybean meal polysaccharides.We designed a first trial to characterise soybean meal carbohydrates (cotyledons and hulls). Fractionation (Water soluble and Water insoluble) of soybean hulls showed the existence of an insoluble structure mostly constituted by cellulose (b-1,4-D-glucose), while the soluble fraction contains a higher proportion of mannose and galactose (possibly b-manans of galacto- and gluco-manans type). Fractionation of cotyledons showed the existence of complex structures composed by a matrix of pectins, hemicellulose and cellulose. The "in vitro" incubation with pectinase, xylanase and cellulase enzymes released a low percentage of monosaccharides, but increased (until 20%) after cell wall fractionation.Knowing the effect of toasting on SBM protein solubility and digestibility, the second experiment aimed to establish relationships between toasting (low and severe) and the "in vitro" enzymatic efficiency, especially of protease and different carbohydases. Effectively, toasting soybean caused gradual decreases in protein and carbohydrate solubility, indicating structural changes of the carbohydrate matrix in their relationship with proteins. Both, water-soluble and water-insoluble fractions were incubated with carbohydrases (pectinase, xylanase, mannanasa and cellulase) in combination or not with protease. Incubation of the soluble substrates with carbohydrases (especially cellulase and pectinase) reduced raffinose and stachyose. Similar results were not observed with mannanase. Incubations of the water-insoluble fractions released a low monosaccharide quantity, but sinergycally increased with the simultaneous incubation with protease. However other aspects that cannot be simulated "in vitro" will affect efficiency of enzymes "in vivo" associated to the animal gastro-intestinal conditions. Then, we decided to check in "in vivo" trials the possibility of improving productive and digestive performances in broilers. In a first trial, we proved the effects of incorporating to a corn-soybean meal diet a mannanase enzyme (at two inclusion levels) associated or not with protease. Experimental design was similar to that described above for the b-glucanase and arabinoxylanase trial. Enzymes supplementation didn't cause significant modifications on productive or digestive results. However, mannanase incorporation significantly reduced the ileum microbial content (measured as purine bases concentration, during the first phase of chicken's growth). Although the experience was carried out in cages, and the not differences in the productivity observed, we think interesting to evaluate the productive and health impact of this effect on the microbial colonisation of small intestine digesta, especially under practical conditions.

Se ha aislado una enzima similar a trombina del veneno de la serpiente peruana Bothrops barnetti mediante dos pasos cromatográficos sobre CM Sephadex C-50 y Sephadex G-100, en ambos casos utilizando Acetato de Amonio 0,05 M a pH 5,0. La enzima fue purificada 45 veces con un rendimiento de 14% y por PAGE-SDS se obtuvo una sola banda proteíca de 52 kDa en condiciones reductoras con 2β-Mercaptoetanol y de 48 kDa en condiciones no reductoras, determinándose que la enzima es de una sola cadena polipeptidica con al menos un enlace disulfuro. El tratamiento con N- glicosidasa (PNGasa F) determinó que es una glicoproteína con un 45% de contenido total de carbohidratos. La enzima mostró tener actividad coagulante sobre plasma citratado y fibrinógeno y actividad amidásica sobre BApNA y Chromozym TH, La potencia coagulante sobre fibrinógeno bovino fue equivalente a 131 unidades NIH de trombina/mg. La enzima es inhibida por PMSF y por el inhibidor de tripsina de soya calificándola como una serinoproteasa; el pH óptimo para la actividad amidolítica fue de 8,0 y es estable hasta los 40 ºC. Se demostró mediante inmunoelectroforesis e inmunodifusión la antigenicidad de la enzima frente al suero antibotrópico polivalente sobre plasma citratado (DE: 250 µl de antiveneno/ mg de enzima). Palabras clave: Veneno, serpiente, enzima similar a trombina, Bothrops barnetti, coagulante. / A thrombin- like enzyme was purified from Bothrops barnetti peruvian snake venom using CM Sephadex C-50 followed by Sephadex G-100, in both two cases with 0,05 M Amonium Acetate buffer pH 5,0. The enzyme was purified 45 fold with 14% of yield and the PAGE-SDS showed only protein band of 52 kDa under reducing condition with 2β-Mercaptoetanol and 48 kDa under non reducing condition indicating that the enzyme has a single polypeptide chain with disulfide bond. The PNGase treatment showed that it is a basic glycoprotein containing 45% total carbohydrates. The enzyme has coagulant activity on fibrinogen and citrated plasma and amidolytic activity on BApNA and Chromozym TH. The coagulant potency was equivalent to 131 NIH thrombin Units/ mg. In addition the enzyme is inhibited by PMSF and soybean trypsin inhibitor suggesting that is a serine proteinase. The enzyme had optimal amidolytic activity pH was 8,0 and is stable until 40 ºC. The antigenicity and neutralization of enzyme was demonstrated by inmunodiffusion and inmunoelectrophoresis with the polyvalent antibothropic serum on citrated plasma (ED: 250 µl of antivenom/ mg of enzyme). Key words: Venom, snake, enzyme, thrombin like, Bothrops barnetti, coagulant.

Se han determinado las principales propiedades bioquímicas e inmunológicas de la enzima similar a trombina (EST) de la serpiente peruana Bothrops atrox (“jergón”). Para este fin, la enzima fue purificada hasta la homogeneidad utilizando tres pasos cromatrográficos en Sephadex G-75, CM-Sephadex C-50 y Agarosa-PAB. Asimismo, se determinó el peso molecular por PAGE-SDS y el porcentaje de carbohidratos asociados mediante hidrólisis y análisis de hexosas, hexosaminas y ácido siálico. Luego se ensayaron las actividades fibrinocoagulante, amidolítica y esterásica, sobre fibrinógeno bovino, BApNA y BAEE, respectivamente, así como la hidrólisis sobre los sustratos cromogénicos S-2238, S-2251 y S-2266 y finalmente se realizó la identificación de la enzima aislada mediante la técnica de peptide mass fingerprinting. Para el análisis inmunoquímico, se inmunizaron conejos albinos con 150 µg de la enzima purificada a fin de obtener un suero hiperinmune anti-EST de B. atrox. Posteriormente se analizaron los patrones de reactividad inmunológica del suero contra la enzima purificada y los venenos de Bothrops atrox, Bothrops brazili, Lachesis muta y Crotalus durissus mediante la técnica de ELISA. Como resultado del análisis bioquímico se determinó que la enzima constituye el 1.7% del veneno completo, siendo purificada 25.5 veces y con un rendimiento del 43.3% utilizando BApNA como sustrato. La enzima presenta un peso molecular de 29.6 kDa, del cual el 14.2% lo constituyen los carbohidratos asociados, asimismo la EST de B. atrox produjo coagulación del fibrinógeno bovino y presentó actividad sobre BAEE, BApNA, S-2238 y S-2266, siendo incapaz de hidrolizar el sustrato S-2251. El análisis mediante espectrometría de masas de los péptidos obtenidos por la hidrólisis de la EST de B. atrox permitieron relacionarla con la proteína venombina A, presentando una homología en secuencia del 75% con esta proteína. Al finalizar el protocolo de inmunización se obtuvo un suero hiperinmune anti-EST de B. atrox con un título de 64000, siendo evidencia del potencial inmunogénico de esta proteína. Por otro lado, los anticuerpos producidos reaccionaron de forma cruzada con los venenos completos de B. atrox (9.9%) y B. brazili (9.6%) y en menor intensidad con los de L. muta y C. durissus, con valores de 5.1% y 4.8%, respectivamente. / We have determinated the main biochemical and immunological properties of a thrombin-like enzyme (TLE) isolated from Bothrops atrox Peruvian snake venom (“jergón”). In this concern, TLE was purified until homogeneity using three chromatographical steps on Sephadex G-75, CM-Sephadex C-50 and Agarose-PAB. Furthermore, molecular weight was determinate by PAGE-SDS and associated carbohydrates by hydrolysis and analysis of hexoses, hexosamines and sialic acid. Then, fibrinocoagulant, amidolytic and sterasic activities were measured on bovine fibrinogen, BApNA and BAEE, respectively, hydrolysis on S-2238, S-2251 and S-2266 chromogenic substrates, and finally molecular identity of this enzyme was determinated by peptide mass fingerprinting technique. For the immunochemical analyses, white rabbits were immunized with 150 µg of EST and a hyperimmune serum anti-TLE was obtained. Patterns of immunological reactivity were determined between this serum against TLE and venoms of Bothrops atrox, Bothrops brazili, Lachesis muta y Crotalus durissus using ELISA technique. As a result of biochemical analysis, we determined that this enzyme represents 1.7% of total venom and was 25.5-fold purified with a 43.3% yield, using BApNA as substrate. This enzyme had 29.6 kDa, where 14.2% was associated carbohydrates. The TLE of B. atrox produced coagulation of bovine fibrinogen and had enzymatic activity on BAEE, BApNA, S-2238 and S-2266, being unable to act on S-2251. Mass spectrometry analysis of hydrolyzed EST of B. atrox results on a 75% sequence homology with venombin A protein. At the final of immunization protocol, we obtained an anti-TLE hyperimmune serum with a title of 64000, which showed the potential immunogenicity of this protein. On the other hand, raised antibodies cross-reacted with total venoms of B. atrox (9.9%) y B. brazili (9.6%) and with less intensity with those from L. muta and C. durissus (5.1% and 4.8%, respectively).

Para evaluar su uso como predictor de daño miocárdico en insuficiencia cardíaca se determinaron los niveles séricos de las enzimas Asparto aminotransferasa (AST), Creatina fosfokinasa (CK), isoenzima Creatina fosfokinasa – MB (CK-MB) y Lactato Deshidrogenasa (LDH) en perros adultos con enfermedad cardiovascular (ECV) y aparentemente sanos. Fueron estudiados 25 animales sin distinción de sexo y raza, Los cuales se dividieron en 2 grupos: 10 animales aparentemente sanos (G1) y 15 animales con enfermedad Cardiovascular (G2). Se muestrearon sueros de ambos grupos y se analizaron por espectrofotometría. Los valores obtenidos fueron procesados mediante la prueba estadística “el Test de la t de student para comparar dos muestras independientes”. Los resultados indican que los niveles séricos de la Isoenzima CK-MB tuvieron diferencia estadística significativa (p menor a 0.05) a favor de los animales con ECV; y el resto de enzimas (AST, CK Y LDH) no obtuvieron diferencia estadística significativa. Se concluye que la CK-MB puede servir como predictor de daño miocárdico progresivo en la insuficiencia cardíaca canina. Palabras Clave: Asparto aminotransferasa (AST), Creatina fosfokinasa (CK), isoenzima Creatina fosfokinasa – MB (CK-MB), Lactato Deshidrogenasa (LDH), Enfermedad cardiovascular, Perros adultos. / Serum levels of the enzymes Asparte amino transferase ( AST ), Creatine kinase ( CK ), the MB fraction of Creatine kinase ( CK - MB ) and Lactate Dehydrogenase ( LDH ) were measured in adult dogs with cardiovascular illness and apparently healthy; they could evaluate their use as predictor of myocardial damage in heart failure. 25 animals irrespective of sex and race were studied, them as they split in 2 groups: 10 apparently healthy animals (G1) and 15 animals with illness Cardiovascular (G2). We sampled serums in the 2 groups and they were examined for spectrophotometry. The obtained values were analyzed by means of the statistical test the Test of student's t to compare two independent samples. The results indicate than levels of CK-MB had statistical significant difference (p menor a 0.05) in favor to dogs with cardiovascular illness. The rest of enzymes (AST, CK and LDH) did not obtain statistical significant difference. We Conclude than CK- MB can serve as predictor of progressive myocardial damage in the heart failure. Key Words: Asparte amino transferase (AST), Creatine kinase (CK), the MB fraction of Creatine kinase (CK - MB) and Lactate Dehydrogenase (LDH), cardiovascular illness, Adult dog.

Se determinó los niveles de las enzimas antioxidantes: Glutation Peroxidasa (GPx), Superóxido Dismutasa (SOD) y Catalasa (CAT); y la concentración de Malondialdehído (MDA) como un indicador de la peroxidación lipídica, en eritrocitos de 60 personas aparentemente sanas: 30 residentes de la altura (Cerro de Pasco, 4340 m.s.n.m.) y 30 residentes de nivel del mar (Lima, 150 m.s.n.m.). Se encontró diferencia estadísticamente significativa entre los niveles eritrocitarios de Glutation peroxidasa de los nativos de altura (66,86 ± 8,97 U GPx / g Hb) y los de nivel del mar (37,78 ± 8,87 U GPx / g Hb), (p < 0,001). No se encontró diferencia estadísticamente significativa entre los valores obtenidos para la Superóxido dismutasa de la altura y de nivel del mar: 4140,56 ± 1147,69 U SOD / g Hb vs. 4104,35 ± 1917,9 U SOD / g Hb, respectivamente. La actividad de la enzima Catalasa fue menor en los sujetos de altura (275,27 ± 44,21 k CAT / g Hb) en relación con los de nivel del mar (414,29 ± 81,07 k CAT / g Hb), siendo esta diferencia estadísticamente significativa (p < 0,001). Los niveles de Malondialdehído en los nativos de altura también fueron menores (8,60 ± 1,91 nMol MDA / g Hb) que los obtenidos a nivel del mar (11,16 ± 1,55 nMol MDA / g Hb), (p < 0,001). / It was determined the levels of the antioxidants enzymes: Glutathione Peroxidase (GPx), Superoxide Dismutase (SOD) and Catalase (CAT); and the concentration of Malondialdehyde (MDA) like an indicator of the lipid peroxidation in erythrocytes of 60 seemingly healthy people: 30 residents at the high altitude (Cerro de Pasco, 4340 m above sea level) and 30 residents at sea level (Lima, 150 m above sea level). It was found significant difference stadistic between erythrocyte levels of Glutathione peroxidase higher in the native of high altitude (66,86 ± 8,97 U GPx / g Hb) and people living at sea level (37,78 ± 8,87 U GPx / g Hb), (p < 0,001). It was not found significant difference stadistic between the values obtained of Superoxide dismutase at high altitude and sea level: 4140,56 ± 1147,69 U SOD / g Hb vs. 4104,35 ± 1917,9 U SOD / g Hb, respectively. The Catalase enzyme activity was smaller in subjects of high altitude (275,27 ± 44,21 k CAT / g Hb) that in subjects at sea level (414,29 ± 81,07 k CAT / g Hb), this difference was significant (p < 0,001). The levels of Malondialdehyde in the native of high altitude were also smaller (8,60 ± 1,91 nMol MDA / g Hb) that in those obtained at sea level (11,16 ± 1,55 nMol MDA / g Hb), (p < 0,001).

Los objetivos del presente trabajo fueron: • Producir Ramnosidasa alcalina de Verticillum tenerum en medios con harina de soja y triptona como fuente de carbono y energía y fuente de nitrógeno respectivamente. • Buscar condiciones de cultivo que maximicen la producción de enzima. • Purificar parcialmente la enzima a partir del sobrenadante de los cultivos. • Determinar parámetros de la enzima tales como pH óptimo, estabilidad térmica, etc.

Objetivos: - Estudiar el efecto de la concentración de inoculo sobre la producción de PPasa-SE en cultivos líquidos de G. klebahnii. - Estudiar el efecto de la agitación del cultivo sobre la producción de la PPasa-SE. - Estudiar la maceración de cáscara de pomelo con PPasa-SE bajo diferentes condiciones de agitación, pH, concentración de buffer citrato y fuerza iónica. - Estudiar y cuantificar la liberación de azúcares, flavonoides y pectina provocada a partir de la maceración de albedo de pomelo.

Made available in DSpace on 2014-06-11T19:23:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-10-19Bitstream added on 2014-06-13T19:09:15Z : No. of bitstreams: 1 gargel_ca_me_sjrp.pdf: 289963 bytes, checksum: bef29e3ebeeee94612ad1bdd03849b34 (MD5) / Enzimas são de grande importância para a indústria de alimentos, a fim de facilitar e acelerar os processamentos. Pectinases são enzimas amplamente utilizadas no processamento de vinhos, facilitando o processo de maceração, clarificação e a filtração do mosto. Outra enzima de grande importância na fabricação de vinhos, é a tanase. A coloração do vinho se deve à presença de taninos; a oxidação destes compostos em contato com o ar pode causar uma turbidez indesejável e consequentemente perda da qualidade do produto final. Essa turbidez pode ser evitada com o emprego de tanases, que impedem a reação de oxidação. Como a maioria dos formulados comerciais destas enzimas são provenientes de fungos filamentosos nao pertencentes ao ambiente vinicola, estes formulados contêm também outras enzimas não adequadas para aplicação no vinho, produzindo alguns efeitos indesejáveis. Assim, a busca por leveduras autóctonas, isto é, do próprio ecossistema vínico, produtoras de tais enzimas faz-se necessária. Outra enzima de grande importância no processamento de vinhos é a invertase, que hidrolisa a sacarose liberando frutose e glicose. Ao lado da vinificação, a frutose é considerada 40% mais doce que a sacarose, sendo assim, de grande importância ao processamento de vinho. No Brasil, a região de Jales, no Noroeste Paulista, vem despontando como um importante centro de produção de uvas e recentemente alguns produtores começaram a processar vinho de maneira artesanal. Assim, o presente trabalho teve como objetivo a triagem de leveduras autóctonas isoladas de uma vinícola da região de Jales para a produção de poligalaturonases, tanases e invertases visando a aplicação no processamento e no melhoramento de vinhos. Foram rastreadas diferentes linhagens... / Enzymes are important for the food industry in order to facilitate and accelerate the processing of food. Pectinases are enzymes widely used in wine processing, facilitating the process of maceration, clarification and filtration of the must. Another enzyme of great importance in wine production is the Tanase. The color of the wine is due to the presence of tannins, the oxidation of these compounds in contact with air can cause an undesirable turbidity and consequently loss of product quality. This turbidity can be avoided by employing Tanase, which prevent the oxidation reaction. As most of these commercial enzymes are made from filamentous fungi, which do not belong to the winery environment, this formula also contains other enzymes that are not suitable for application in the wine, producing some side effects. Thus, the quest for autochthonous yeasts, ie, from the own wine ecosystem, producing such enzymes is necessary. Another enzyme of great importance in the processing of wine is invertase, which hydrolyzes sucrose releasing fructose and glucose. Besides the importance for winemaking, fructose is considered 40% sweeter than sucrose, therefore, of great importance to the food industry. In Brazil, the region of Jales, Sao Paulo in the Northwest, has emerged as an important center of production of grapes, and recently some farmer began to produce artisanal wine. Thus, this study aimed at screening of autochthonous yeasts isolated from a winery from Jales region, which are able to produce poligalaturonases, Tanase and invertase in order to apply in the processing of wines. From, thirteen different strains belonging to different species of yeast, there were no significant activities to Tanase and pectinases. However, one strain of Candida stellata produced invertase activity, reaching... (Complete abstract click electronic access below)

Made available in DSpace on 2013-08-07T19:03:39Z (GMT). No. of bitstreams: 1 000446130-Texto+Completo-0.pdf: 1910036 bytes, checksum: 59d877dc63ef59118f780b6e08cf8f9f (MD5) Previous issue date: 2012 / Tuberculosis remains the most common cause of death due to an infectious agent. Among targets identified in Mycobaterium tuberculosis genome, enzymes of the shikimate pathway deserve special attention. Shikimate kinase is the fifth enzyme of the shikimate pathway, which has been identified in fungi, apicomplexans, plants and prokaryotes. This metabolic route is composed of seven steps, which converts erythrose-4-phosphate and phosphoenol pyruvate to chorismic acid and is responsible for the biosynthesis of aromatic amino acids. Shikimate kinase has been shown to be essential to the survival of Mycobacterium tuberculosis, and since it is absent in human, this enzyme is considered to be a target for chemotherapeutic for development of antitubercular drugs. The aim here is to identify possible inhibitors, focusing on simulations of molecular docking in the ATP-binding site of the enzyme. The program used in the simulations was the Molegro Virtual Docker and protein-ligand interactions were tested in 12 crystallographic structures and then, it was choosen a protocol which generated docking RMSD values below 2 Å. Application of this docking protocol to a decoy dataset generated a enrichment factor of 24. 57, which is considered adequate for molecular docking simulations focused on kinases. The present docking protocol was then applied to a small-molecule database with over 80,000 entries. Analysis of the results identified 5 potencial shikimate kinase inhibitors. Examination of the intermolecular interaction between enzyme and the ligands identified the main structural features responsible for ligand-binding affinity. This is the first molecular docking study focused on the ATP-binding pocket of shikimate kinase. / A Tuberculose continua sendo a causa mais comum de morte em decorrência de um agente infeccioso. Entre os alvos identificados no genoma do Mycobaterium tuberculosis, enzimas da via do chiquimato merecem atenção especial. A chiquimato quinase é a quinta enzima da via do chiquimato, e tem sido identificada em fungos, organismos do filo apicomplexa, plantas e procariontes. Esta via metabólica é composta por sete passos, que catalisam sequencialmente a conversão de eritrose-4-fosfato e fosfoenolpiruvato em corismato; e é responsável pela biossíntese de aminoácidos aromáticos. Chiquimato quinase parece ser essencial para a sobrevivência do Mycobacterium tuberculosis, uma vez que é ausente no homem, esta enzima é considerada como um alvo para o desenvolvimento de quimioterápicos e medicamentos contra a tuberculose. O objetivo é identificar possíveis inibidores, focando as simulações de docking molecular no sítio de ligação do ATP da enzima. O programa usado nas simulações foi o Molegro Virtual Docker e a interação proteína-ligante foi testada em 12 estruturas cristalográficas e logo após, escolhido um protocolo de docking a partir de valores de RMSD abaixo de 2Å. O método foi validado usando o melhor protocolo de re-docking no Virtual Screening através do Fator de Enriquecimento que obteve resultado de 24,57%, que é considerado adequado para as simulações de docking molecular focados em quinases. O presente protocolo de docking foi aplicado em um banco de dados com mais de 80. 000 moléculas. A análise dos resultados identificaram 5 potenciais inibidores da chiquimato quinase. Na análise das interações intermoleculares entre a enzima e os ligantes foram identificadas características estruturais responsáveis pela afinidade da ligação pelo ligante. Este é o primeiro estudo de docking molecular focado no bolsão de ligação do ATP da chiquimato quinase.