La síndrome de Sjögren és una malaltia autoimmune amb una prevalença força elevada a la població general (entre el 0,5% i el 3%). Tal com succeeix en d'altres malalties d'aquesta naturalesa, la presència d'anticossos antinuclears (ANA) està àmpliament acceptada com a prova important en el seu diagnòstic. En el caso de la síndrome de Sjögren, el principal marcador serològic és la presència d'anticossos dirigits contra l'antigen SS-B (La), tot i que en alguns casos també s'ha descrit la presència única d'anticossos dirigits contra l'antigen SS-A (Ro), format per dues proteïnes (Ro60 i Ro52) associades a diferents RNA citoplasmàtics.En el treball que es presenta, es descriu el procediment d'obtenció de les proteïnes antigèniques SS-B (La) i SS-A (Ro), per tal de poder ser utilitzades posteriorment en el desenvolupament de dues enzimoimmunoanàlisis (ELISA anti-SS-B (La) i ELISA anti-SS-A (Ro)) útils per a la detecció d'anticossos dirigits contra elles. L'estrategia que es va seguir fou la següent: es van preparar tres vectors d'expressió en Escherichia coli de les tres proteïnes (SS-B, Ro60 i Ro52) mitjançant la tecnologia del DNA recombinant, es van expressar les tres proteïnes recombinants en els microorganismes procariotes en forma de proteïnes de fusió, es van purificar les proteïnes per cromatografia d'afinitat, es van immobilitzar per adsorció passiva en microplaques de poliestirè, i es van optimitzar les diferents variables dels assajos de tipus ELISA. Els dos assajos desenvolupats es van avaluar mitjançant l'estudi de les constants diagnòstiques i analítiques i l'estudi de les característiques analítiques. Per a l'ELISA anti-SS-B (La), es van obtenir els següents resultats: sensibilitat diagnòstica del 96%, especificitat diagnòstica del 97%, coeficient de repetibilitat del 2,1%, coeficient de reproductibilitat del 10,0%, i absència de reaccions creuades i interferències. Per a l'ELISA anti-SS-A (Ro) els resultats foren els següents: sensibilitat diagnòstica del 88%, especificitat diagnòstica del 91%, coeficient de repetibilitat de l'1,9%, coeficient de reproductibilitat del 10,6%, i absència de reaccions creuades i interferències. / Sjögren's syndrome is an autoimmune disease with a high prevalence (between 0.5% and 3% of population). As other autoimmune diseases, the presence of antinuclear antibodies (ANA) in sera of Sjögren's syndrome patients is widely accepted as an important evidence for its diagnostic. The main serological marker of Sjörgren's syndrome is the presence of anti-SS-B (La) antibodies, although sometimes the presence of antibodies against the antigen SS-A (Ro) has also been described. The latter antigen is composed by two proteins (Ro60 and Ro52) binded to different cytoplasmatic RNA, so that antibodies against both proteins have been described in patient's sera.In that thesis, we describe the procedure for obtaining SS-B (La) and SS-A (Ro) proteins in order to be used as antigens in two enzymoimmunoassays (ELISA anti-SS-B (La) and ELISA anti-SS-A (Ro)) useful for the detection of antibodies against these proteins. We used the following strategy: first of all, three Escherichia coli expression vectors for the proteins (SS-B, Ro60 and Ro52) were constructed by means of recombinant DNA technology. The proteins were expressed in these prokaryote cells as fusion proteins, were purified by affinity cromatography and the proteins were immobilized by passive adsortion in polystyrene microplates. Finally, we optimized different variables of both ELISA and we evaluated the assays by calculating the diagnostic and analytical constants. The results obtained with the anti-SS-B (La) ELISA were the following: diagnostic sensibility 96%, diagnostic specificity 97%, intraserial CV 2.1%, interserial CV 10.0% and absence of cross-reactions and interferences. With the anti-SS-A (Ro) ELISA, the results obtained were: diagnostic sensibility 88%, diagnostic specificity 88%, intraday CV 1.9%, interday CV 10.6% and absence of cross-reactions and interferences.

Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (In Czech)

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.