Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-26Bitstream added on 2014-06-13T20:50:00Z : No. of bitstreams: 1 vieira_gal_me_araiq.pdf: 2780789 bytes, checksum: 822f36edcc6ae68080d4ff7d0cca2cdf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O protozoário hemoflagelado, Trypanosoma cruzi, transmitido principalmente pelo inseto triatomíneo, por infecção congênita ou por transfusão sanguínea, causa a tripanossomíase americana ou doença de Chagas, como é mais conhecida. Essa é uma séria doença parasitária que ocorre na América Latina, com considerável impacto social e econômico. Dois fármacos, nifurtimox e benzonidazol, são indicados no tratamento de pessoas infectadas, mas são pouco eficientes na fase aguda e praticamente ineficientes na fase crônica da doença. Devido a esses fatores, é de extrema importância que se encontre agentes quimioterápicos e/ou quimiopreventivos mais eficientes e eficazes. O presente trabalhou objetivou avaliar a influência no proteoma de T. cruzi de três substâncias extraídas de plantas da família Piperaceae, peperobtusina A e B de Peperomia obtusifolia e piplartina de Piper tuberculatum, através da eletroforese bidimensional (2D) – DIGE (Difference Gel Electrophoresis - GE Healthcare) associada à espectrometria de massas. O tratamento realizado com peperobtusina A de Peperomia obtusifolia em cepa Y de T. cruzi identificou proteínas de interesse, como proteína do bastão paraflagelar e triparedoxina peroxidase, importantes na composição flagelar e proteção natural do parasito ao estresse oxidativo, respectivamente. Os resultados obtidos com peperobtusina B de Peperomia obtusifolia na mesma cepa de T. cruzi mostraram proteínas que podem servir como interessantes alvos potenciais para futuro desenvolvimento de fármacos. São elas: calmodulina, tirosina aminotransferase e arginina quinase. Os resultados obtidos com o tratamento das cepas Y e Bol de T. cruzi pela piplartina de Piper tuberculatum demonstraram que apesar das diferenças de suscetibilidade dessas cepas em relação ao benzonidazol... / The protozoa hemoflagelate, Trypanosoma cruzi, transmitted mainly for the triatomíneo insect, congenital infection or sanguineous transfusion, cause American tripanosomiasis or Chagas disease, as more it is known. This is a serious parasitic illness that occurs in Latin America, with considerable social and economic impact. Two drugs, nifurtimox and benznidazol, are indicated in the treatment of infected people, but they are little efficient in the acute phase and practically inefficient in the chronic phase of the illness. Because of these factors, it is of extreme importance find efficient chemopreventive and/or chemotherapic agents. This work aims to evaluate the T. cruzi proteome influence of three substances extracted of plants belonging to Piperaceae family, being peperobtusine A and B of Peperomia obtusifolia and piplartine of Piper tuberculatum, through two dimensional electrophoresis (2D) - DIGE (Difference Gel Electrophoresis - GE Healthcare) and mass spectrometry. The treatment carried with peperobtusine A of P. obtusifolia in Y strain of T. cruzi identified interest proteins, as paraflagelar rod protein and triparedoxin peroxidase, important in the flagella composition and natural protection of oxidative stress, respectively. The results using peperobtusine B of the P. obtusifolia in Y strain of T. cruzi showed proteins that can be interesting potential targets for future development of drug. They are: calmodulin, tyrosine aminotransferase and arginine kinase. The results with the treatment of Y and Bol strains of T. cruzi with piplartine of P. tuberculatum demonstrated that although to the differences of susceptibility of these strains related to benznidazol, the piplartine was active for both strains as a similar way and modified the expression of enzymes involved in the protection of the parasitic to the... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-20Bitstream added on 2014-06-13T19:29:01Z : No. of bitstreams: 1 riul_aj_me_araiq.pdf: 887543 bytes, checksum: cec5191b12ce838bc6e308cecf5c08c1 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A tanase (EC 3.1.1.20) é uma enzima capaz de hidrolisar ligações éster e depsídicas de taninos hidrolisáveis obtendo-se como produtos a glicose e o ácido elágico ou ácido gálico, sendo este último, um importante substrato para as indústrias farmacêutica e química. Entre os diferentes organismos capazes de produzir tanases, os microrganismos, de modo especial os fungos filamentosos, vêm se destacando uma vez que são mais versáteis na degradação de diferentes tipos de taninos. Neste contexto, o objetivo geral deste trabalho foi estudar a produção de tanases intra e extracelulares por Aspergillus phoenicis em Fermentação Submersa (FSbm) e em Fermentação em Substrato Sólido (FSS), melhorando as condições de cultivo para obtenção destas enzimas em altos níveis, purificando e caracterizando-as bioquimicamente. Os maiores níveis enzimáticos em FSS foram obtidos utilizando-se folhas de caju e chá verde como fonte de carbono umedecidas com solução de sais Khanna (1:1, m/v), a 30ºC por 6 dias e em FSbm tendo 2% de ácido tânico como fonte de carbono, por 3 dias a 30ºC para ambas as formas enzimáticas. Fontes de nitrogênio e fosfato aumentaram a produção de tanases quando adicionadas em baixas concentrações. As tanases extra e intracelular produzidas em FSbm foram purificadas 22 e 17 vezes com recuperação de 9% e 16%, respectivamente, após dois passos cromatográficos, DEAE-Celulose e Sepharose CL6B. A forma extracelular possui massa molar nativa de aproximadamente 218,8 kDa com 65,1% de carboidratos. Já a enzima intracelular apresentou massa molar nativa de 154,9 kDa e 43,2% de carboidratos. A temperatura ótima de atividade para ambas as enzimas purificadas foi de 60ºC e o pH ótimo de atividade ficou estabelecido na faixa de 5,0 a 6,0 para ambas as formas tanásicas. As tanases se mostraram... / (EC 3.1.1.20) is an enzyme able to hydrolize ester and depside bonds of hydrolysable tannins obtaining as products glucose and ellagic acid or gallic acid. This last one is an important substrate for the pharmaceutical and chemical industries. Among different organisms able to produce tannases, the microorganisms, especially filamentous fungi, have been highlighted since they are more versatile in the degradation of different types of tannins. In this context, the aim of this work was to study the production of intracellular and extracellular tannase from Aspergillus phoenicis under Submerged Fermentation (SbmF) and Solid Substrate Fermentation (SSF), improving culture conditions to obtain these enzymes at high levels, purifying and characterizing them biochemically. The highest enzyme levels were obtained in SSF using cashew and green tea leaves wetted with Khanna salt solution (1:1, w/v) as carbon source at 30°C for 6 days and SbmF with 2% tannic acid as carbon source, for 3 days at 30°C, for both enzymatic forms. Nitrogen and phosphate sources increased the production of tannase when added at low concentrations. The extra and intracellular tannase produced in SbmF were purified 22 and 17 times with recovery of 9% and 16%, respectively, after two chromatographic steps, DEAE-Cellulose and Sepharose CL6B. The extracellular form has native molecular mass of 218.8 kDa with 65.1% of carbohydrate content. The intracellular enzyme showed native molecular mass of 154.9 kDa and 43.2% of carbohydrate content. The optimum temperature for both purified enzymes was 60°C, and the optimum pH activity was established at a range from 5.0 to 6.0 for both enzymatic forms. Tannase proved to be quite stable at both temperature and pH and the activities remained constant for all ions tested, except for... (Complete abstract click electronic access below)

Orientador: Luis Henrique Souza Guimarães / Banca: Hamilton Cabral / Banca: Adalberto Pessoa Junior / Resumo: A tanase (EC 3.1.1.20) é uma enzima capaz de hidrolisar ligações éster e depsídicas de taninos hidrolisáveis obtendo-se como produtos a glicose e o ácido elágico ou ácido gálico, sendo este último, um importante substrato para as indústrias farmacêutica e química. Entre os diferentes organismos capazes de produzir tanases, os microrganismos, de modo especial os fungos filamentosos, vêm se destacando uma vez que são mais versáteis na degradação de diferentes tipos de taninos. Neste contexto, o objetivo geral deste trabalho foi estudar a produção de tanases intra e extracelulares por Aspergillus phoenicis em Fermentação Submersa (FSbm) e em Fermentação em Substrato Sólido (FSS), melhorando as condições de cultivo para obtenção destas enzimas em altos níveis, purificando e caracterizando-as bioquimicamente. Os maiores níveis enzimáticos em FSS foram obtidos utilizando-se folhas de caju e chá verde como fonte de carbono umedecidas com solução de sais Khanna (1:1, m/v), a 30ºC por 6 dias e em FSbm tendo 2% de ácido tânico como fonte de carbono, por 3 dias a 30ºC para ambas as formas enzimáticas. Fontes de nitrogênio e fosfato aumentaram a produção de tanases quando adicionadas em baixas concentrações. As tanases extra e intracelular produzidas em FSbm foram purificadas 22 e 17 vezes com recuperação de 9% e 16%, respectivamente, após dois passos cromatográficos, DEAE-Celulose e Sepharose CL6B. A forma extracelular possui massa molar nativa de aproximadamente 218,8 kDa com 65,1% de carboidratos. Já a enzima intracelular apresentou massa molar nativa de 154,9 kDa e 43,2% de carboidratos. A temperatura ótima de atividade para ambas as enzimas purificadas foi de 60ºC e o pH ótimo de atividade ficou estabelecido na faixa de 5,0 a 6,0 para ambas as formas tanásicas. As tanases se mostraram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: (EC 3.1.1.20) is an enzyme able to hydrolize ester and depside bonds of hydrolysable tannins obtaining as products glucose and ellagic acid or gallic acid. This last one is an important substrate for the pharmaceutical and chemical industries. Among different organisms able to produce tannases, the microorganisms, especially filamentous fungi, have been highlighted since they are more versatile in the degradation of different types of tannins. In this context, the aim of this work was to study the production of intracellular and extracellular tannase from Aspergillus phoenicis under Submerged Fermentation (SbmF) and Solid Substrate Fermentation (SSF), improving culture conditions to obtain these enzymes at high levels, purifying and characterizing them biochemically. The highest enzyme levels were obtained in SSF using cashew and green tea leaves wetted with Khanna salt solution (1:1, w/v) as carbon source at 30°C for 6 days and SbmF with 2% tannic acid as carbon source, for 3 days at 30°C, for both enzymatic forms. Nitrogen and phosphate sources increased the production of tannase when added at low concentrations. The extra and intracellular tannase produced in SbmF were purified 22 and 17 times with recovery of 9% and 16%, respectively, after two chromatographic steps, DEAE-Cellulose and Sepharose CL6B. The extracellular form has native molecular mass of 218.8 kDa with 65.1% of carbohydrate content. The intracellular enzyme showed native molecular mass of 154.9 kDa and 43.2% of carbohydrate content. The optimum temperature for both purified enzymes was 60°C, and the optimum pH activity was established at a range from 5.0 to 6.0 for both enzymatic forms. Tannase proved to be quite stable at both temperature and pH and the activities remained constant for all ions tested, except for... (Complete abstract click electronic access below) / Mestre

Orientador: Pedro de Oliva Neto / Banca: Maria das Graças de Almeida Felipe / Banca: Eutímio Gustavo Fernández Núñez / Resumo: As amilases são enzimas pertencentes à classe das hidrolases, atuando na estrutura do amido com a hidrólise das ligações glicosídicas dos tipos α-1,4 e α-1,6, do interior das cadeias de amilose e amilopectina, respectivamente. Atualmente são responsáveis por cerca de 30% do mercado mundial de enzimas e apresentam uma vasta gama de aplicações industriais. Os fungos capazes de produzir enzimas amilolíticas podem ser cultivados mediante insumos de baixo custo empregados na fermentação em estado sólido (FES), o que possibilita a valorização dos subprodutos da agroindústria, bem como fornece suporte semelhante ao encontrado pelo micro-organismo no ambiente natural. O presente estudo visa produzir amilases por Rhizopus microsporus var. oligosporus em FES utilizando o farelo de trigo como substrato, purificar parcialmente o extrato bruto enzimático obtido, caracterizar bioquimicamente o extrato enzimático parcialmente purificado e posteriormente realizar a sacarificação pela hidrólise do amido presente na quirera de arroz e fermentação alcoólica por Saccharomyces cerevisiae para produção de etanol. A atividade enzimática do extrato bruto foi 39,8 U/mL o que equivale a 358 U/g de substrato. O índice de purificação, após as etapas de cromatografia foi parcial, mas suficiente para que a caracterização bioquímica do extrato produzido fosse realizada. Estes testes demonstraram faixa ótima no pH 4,0 e pH 5,5, indicando as condições ácidas como as melhores para este estudo. A estabilidade do pH foi ampla variando do pH 3,5 ao pH 8,5 com cerca de 40 a 60% de atividade relativa. A temperatura ótima de atividade enzimática determinada como ideal foi de 60 a 65 °C, porém a enzima mostrou-se termoestável até 60 °C. Os testes do efeito de íons na reação enzimática amilolítica demonstraram que os íons Cu+2, Zn+2, Al+2 e Na+2 comportaram-se como inibidores da atividade. O íon Mn+2 destacou-se por potencializar em... / Abstract: Amylases are enzymes belonging to the class of hydrolases acting on starch structure with the hydrolysis of glycosidic bonds of α-1,4 and α-1,6 types, in the interior of the chains of amylose and amylopectin, respectively. Currently they are responsible for about 30% of the world market enzymes and show a wide range of industrial applications. Fungi capable of producing amylases can grow by low cost inputs in solid-state fermentation (SSF), which enables the recovery of the agricultural industry by-products and provides support similar to that found by the microrganism in the natural environment. This study aims to produce amylase by Rhizopus microsporus var. oligosporus in solid-state fermentation using wheat bran as substrate partially to purify the crude enzyme extract obtained biochemically characterize the partially purified enzyme extract and subsequently to carry out the hydrolysis saccharification of starch present in broken rice and fermentation of it by Saccharomyces cerevisiae for ethanol production. Enzymatic activity of the raw extract was 39.8 U/mL equivalent to 358 U/g substrate. The purification ratio after the chromatography step was partial, but sufficient for the biochemical characterization of the produced extract was taken. These tests showed optimal range of pH 4.0 to pH 5.5 indicating the acidic condition as the best for this study. The pH stability was wide range of pH 3.5 to pH 8.5 with 40 to 60% relative activity. The optimum temperature for enzyme activity determined as optimum was 60 to 65 °C, but the enzyme was thermostable up to 60 °C. Ion effect of the amylolytic enzyme tests showed that the reaction Cu+2, Zn+2, Al+2 and Na+2 ions behaved like activity inhibitors. The Mn+2 ions distinguished for enhancing at about 60% relative enzymatic activity to hydrolysis without addition of ions. The enzymatic hydrolysis of broken rice using as substrate allowed the complete conversion of starch to reducing sugars ... / Mestre

digitalisering@umu.se

Genetic polymorphism is by definition the occurrence in the same population of two or more alleles at one locus,each with a frequency high enough not to be maintained by recurrent mutation only (1). Among the human plasmaproteins two major categories of polymorphism have been described, allotypic and electrophoretic heterogeneity. Allotypy is defined by Oudin as individual antigenic differences among proteins within a species (2). The first discovered polymorphism of this category was the Gm system of immunoglobulin G by Grubb (3). The first described electrophoretic heterogeneity in plasma proteins was theHp (haptoglobin) system discovered by Smithies (4). Sincethen genetic variants of several other human plasma proteins have been found. This dissertation is concerned with thegenetic and immunological aspects of the polymorphism of the third component of human complement, C3. / digitalisering@umu.se

No description available.

Magister Scientiae - MSc / The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common αββα structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the αββα-αββα fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm. The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 Å. Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost – either by specific proteolysis or autolysis - an active helix is formed. / South Africa

South Africa has failed to develop a viable Biotechnology industry despite strong life sciences facilities and academic training. Government has pursued various strategies for developing this industry since 1982 however none have succeeded. The most recent strategy entails the establishment of four Biotechnology Regional Innovation Centres (BRICs).Competitiveness at a nation level is arguably best described using Porter’s Diamond of National Competitiveness model which provides a framework for analysing an industry cluster. The South African National Biotechnology Strategy has been designed to stimulate cluster formation with the BRIC at the core. In order to assess the success of the BRIC strategy it is necessary to establish a baseline analysis of the biotechnology sector. To do this an analysis was performed by means of a quantitative email survey which assessed the South African biotechnology sector in terms of the four attributes of the Diamond model.The analysis presented here found that the South African biotechnology industry is deficient in all four attributes of the Diamond model. Positives do exist and can be leveraged to attempt to address the deficiencies. The most notable deficiencies identified were a skills shortage, poor access to funding and a poor understanding of the fundamentals of biotechnology. The establishment of a dedicated Biotechnology Park was found to be of interest to stakeholders. / Dissertation (MBA)--University of Pretoria, 2010. / Gordon Institute of Business Science (GIBS) / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc (Botany))--University of Pretoria, 2006. / Plant Science / unrestricted