The objectives of this study) are: (1) partial purification and characterization of rice β-glucosidase, (2) determination of the physiological role of the enzyme during rice germination, and (3) histochemical localization of the enzyme. The method for partial purification of the enzyme was based on that of Schliemann (1984), which included differential solubility, cryoprecipitation, and cation exchange chromatography. The enzymes were characterized with respect to their molecular weights, pI value, pli and temperature profile of activity and stability, activity in the presence of selected denaturants and organic’ solvents, substrate specificity, and inhibition by several known β-glucosidase inhibitors. To examine the physiological role of rice β-glucosidase, histochemical localization of the enzyme in dry seeds and application of inhibitors of the enzyme to the germinating seeds were carried out. The seeds were soaked in the presence or absence of β-glucosidase inhibitors, and the number of germinating seeds, growth and development of coleoptile and roots, and enzymatic activity of β-glucosidase and α-amylase were studied. To study histochemical localization of rice β-glucosidase, the chromogenic substrates were used. The substrates were incubated with cross and longitudinal sections of whole seeds and seedlings, tissue sections, protoplast and plastid preparations from 5-6-day-old coleoptiles. The development of the colors were observed under the light microscopes. Among the cation exchange chromatography fractions, two distinct peaks of oNPGase and pNPgase activity were found: fraction-1 (Fr-1) and fraction-2 (Fr-2) forms. It was found that the two forms of rice β-glucosidase are different with respect to susceptibility to denaturation by SDS, substrate specificity and some physico-chemical properties. Fr-1 is susceptible to denaturation by SDS, and catalyzes specifically the hydrolysis of several β-galactosides (pNPGal, X-gal, and 6-BNGal) but not gentiobiose and cellobiose, and is stable over pH range (4 to 10). Fr-2, on the other hand, is more resistant to denaturing agents, catalyzes the hydrolysis of gentiobiose and cellobiose, but not any of the β-galactosides mentioned above; it is relatively stable at pH 9, and less stable at high temperatures than Fr-1. Both Fr-1 and Fr-2 are 120 kD native dimers, made up of 60 kD monomers. In rice dry seeds, β-glucosidases were distributed in the aleurone layers and embryo parts. β-glucosidase inhibitors suppressed germination at the activation stage. The inhibitors Suppressed the expression of α-amylase and β-glucosidase during germination detected at the activity level. It is proposed, therefore, that the pre-existing f-glucosidase is involved in the regulation of availability and activity of a hormone (gibberellin) at the early step of germination that controls expression of hydrolytic enzymes such as α-amylase. In mature seeds, the Fr-1 is found mainly in the scutellum region and aleurone layers, while the Fr-2 form is in the axis of the embryo. In the seedling, the Fr-1 form is found in the scutellum, shoot and coleoptile, while the Fr-2 form is in the root. In young tissue of shoot and coleoptile, the enzyme is localized in the epidermis and vascular bundles. At the subcellular level it is localized to the plastids. / Ph. D.

Lactococcus is an economically important group in lactic acid bacteria (LAB) that are often used in the dairy industry as starters for cheese production. Good starter strains should possess the ability to grow, ferment milk sugar, and produce desirable flavor compounds during cheese making. Therefore, it is essential to understand the physiology of these starters during cheese processing in order to obtain high-quality cheese products. Cheese manufacturing compromises several stress factors that affect the growth of starter lactococci. Among these stressed environmental parameters, sugar starvation is the most important one to overcome to obtain energy for cellular processes. It is known that degradation of arginine produces energy. In this study, we investigated arginine utilization by Lactococcus lactis ssp. lactis strain ML3 via the arginine deiminase (ADI) pathway to see its influence on cellular physiology after exhaustion of a primary energy source. During the cell growth in a carbohydrate-limited environment, we observed that metabolic pathways switched between lactose utilization arginine degradation. The statistical model described in this study suggested lactose and arginine were co-metabolized during cell growth. These results initially showed arginine was a good candidate of secondary energy source after exhaustion of primary energy source (lactose). To confirm these observations, cell counts, cellular ATP levels, ADI enzyme activities, and total protein expression were compared in arginine-positive L. lactis ssp. lactis ML3 and arginine-negative L. lactis ssp. cremoris Sl grown in medium containing 0.2% lactose and 2% arginine. Results showed ATP levels remained high in strain ML3, in which a transition stage of protein expression pattern was also observed. This physiological evidence highlights the important roles of arginine degradation in starved ML3, perhaps by producing extra ATP and modulating external pH. The genes involved in the ADI pathway of strain ML3 were cloned, sequenced, and characterized. Genes involved in this pathway formed a unique multi-operon cluster structure that we termed MOC. It was organized as arcA, arcBD1, arcC1C2, and arcTD2. The influence of different environmental parameters including pH, various amino acids, and phosphate (organic and inorganic) on the expression of the ADI MOC was tested. No single factor regulated the entire MOC simultaneously. It is concluded that the unique structure of the MOC appears to allow the ADI pathway to occur in discrete sections in response to fluctuated external conditions, such as sugar starvation and low environmental pH.

Made available in DSpace on 2016-06-02T20:21:28Z (GMT). No. of bitstreams: 1 4218.pdf: 11765337 bytes, checksum: cdae8703c8d7792c7037dde48c22c345 (MD5) Previous issue date: 2012-04-04 / Financiadora de Estudos e Projetos / Plant cell wall confers to the cell plant, structural support as well as protection against pathogens and phytophagous. Among the cell wall polysaccharides includes pectic substances, which are composed of partially methyl-esterified galacturonic acid residues linked by α-1,4 glycosidic bonds. These enzymes are often synthesized by phytophatogenic micro-organisms for invasion of host plant or by own plant for modeling plant cell wall. The pectic substances are the major component of middle lamella and are natural degraded by pectinases action. Pectin methylesterase (PME) catalysis removes methyl-ester groups, and the Endo-polygalacturonase (Endo-PG) promoves the randomly hydrolysis reaction of α-1,4 bonds. One of the most importante agricultural pest species of the family Curculionidae (Coleoptera: Curculionidae) is Sphenophorus levis, the sugarcane weevil. The larvae of this insect penetrate into the rhizome and build galleries in the stem, decreasing productivity and causing the death of the plant. Large damages to the crop like that are significant in the costs of products derived from sugarcane. Considering the impact of this pest in the sugarcane crop and the absence of efficient method for control, new strategies for controlling are still necessary. The analysis of the cDNA library of S. levis larvaes shows the presence of one PME and one Endo-PG genes that we called Sl-PME and Sl-EndoPG respectively. Considering the importance of studies of insect pests and the extensively use of theses pectinases in different industry fields, we performed the characterization of genomic sequences coding for S. levis pectinases (Sl-Pectinases). It was also carried out the production and characterization of a Sl-PME and a Sl-EndoPG recombinant, expressed in heterologous system. We also accomplished analysis of gene expression by qRTPCR in different stages of development as well as different tissues, and phylogenetic studies between Sl-Pectinases and other pectinases from different kingdoms. The Sl- Pectinases sequences identified, show more similar to homologous insect genes deposited in the GenBank, especially with Sitophilus oryzae. The phylogenetic analysis indicates that the insect group is more correlated with bacteria group and fungi group respectively to PMEs and EndoPGs sequences. Pectinases genomic sequences revealed two introns for Sl-EndoPG gene with 53 and 166 bp, but no one for Sl-PME gene. Both of Sl-Pectinases Recombinant showed catalytic activity. The recombinant Sl-EndoPG shows optimal activity at pH 5,06 ± 0,27 and 49,74 ± 2,49 oC, but extremely low thermostability. For the polygalacturonic acid no-methylated as substract, the enzyme revealed Km = 3,88 mg.mL-1, Vmax = 21.96 μM.s-1 e Kcat = 3.137 s-1; for the citrus pectin partially methylated as substract, the enzyme presented Km = 4,98 mg.mL-1, Vmax = 17,19 μM.s-1 e Kcat = 2.456 s-1. Results in expression analysis suggest that S. levis pectinases have a digestive enzymes role, actting on the midgut. The present work represents the first pectinases of insect produced in Pichia pastoris heterologous system and characterized as optimal conditions of activity, thermostability and kinetic parameters. / A parede celular vegetal confere à célula vegetal suporte estrutural, proteção contra patógenos e fitófagos. Dentre os polissacarídeos da parede celular vegetal são inclusas as substâncias pécticas, as quais são compostas por resíduos de ácido galacturônico, parcialmente esterificados, ligados em série via ligações glicosídicas α- 1,4. As substâncias pécticas são o maior componente da lamela média e são naturalmente degradadas pela ação enzimática das pectinases. A Pectina metilesterase (PME) catalisa a remoção dos grupos metil-ester, e a Endo- Poligalacturonase (Endo-PG) promove a reação de hidrólise aleatória das ligações α- 1,4. Essas enzimas são comumente sintetizadas por micro-organismos fitopatógenos para invasão ao hospedeiro ou pelas próprias plantas para modelamento da parede celular vegetal. Na agricultura, uma das mais importantes espécies pragas da família Curculionidae (Coleoptera: Curculionidae) é o Sphenophorus levis, o bicudo da canade- açúcar. As larvas deste inseto penetram no rizoma e constroem galerias ao longo do colmo, causando queda na produtividade ou até mesmo a morte da planta. Grandes danos na cultura como esses são significativos nos custos de produtos derivados da cana-de-açúcar. Considerando o impacto dessa praga na cultura e a ausência de eficientes métodos de controle, novas estratégias ainda são necessárias no combate à praga. A análise de uma biblioteca de cDNA de larvas do inseto S. levis mostrou a presença de genes codificantes para uma PME e uma Endo-PG, os quais nomeamos de Sl-PME e Sl-EndoPG respectivamente. Devido a importância nos estudos de insetos pragas e a extensa aplicação das pectinases em diversos campos industriais, foi promovida a caracterização das sequências genômicas codificantes para as pectinases de S. levis (Sl-Pectinases). Também foi realizada a produção e caracterização de uma Sl-PME e uma Sl-EndoPG recombinantes, expressas em sistema heterólogo. Além disso, foram conduzidas análises de expressão gênica por qRT-PCR em diferentes estágios de desenvolvimento e diferentes tecidos; e estudos filogenéticos entre as Sl-Pectinases e outras pectinases de diferentes reinos. As sequências das Sl-Pectinases identificadas apresentaram maior similaridade com genes homólogos de insetos depositados no GenBank, principalmente como o Sitophilus oryzae. A análise filogenética indicou que o grupo dos insetos é mais correlacionado com o grupo das bactérias e com o grupo de fungos, respectivamente para as sequências PMEs e Endo-PGs. As sequências genômicas das pectinases revelaram dois introns para Sl-EndoPG com 53 e 166 pb, mas nenhum para o gene Sl- PME. Ambas as Sl-Pectinases Recombinantes apresentaram atividade catalítica. A Sl- EndoPG recombinante mostrou maior atividade em pH 5,06 ± 0,27 e 49,74 ± 2,49 oC, mas baixa termoestabilidade. Para o substrato ácido poligalacturônico não metilado, a enzima revelou Km = 3,88 mg.mL-1, Vmax = 21.96 μM.s-1 e Kcat = 3.137 s-1, para o substrato pectina de citrus parcialmente metilada, a enzima apresentou Km = 4,98 mg.mL-1, Vmax = 17,19 μM.s-1 e Kcat = 2.456 s-1. Os resultados da análise de expressão sugerem que as pectinases de S. levis são enzimas digestivas atuantes no intestino médio. Este trabalho representa as primeiras pectinases de inseto produzidas em sistema heterólogo de Pichia pastoris e caracterizadas quanto a condições ótimas de atividade, termoestabilidade e parâmetros cinéticos.

The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...

The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...