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Sistemas micelares de duas fases aquosas aplicados à purificação de enzimas; Aqueous two-phase micellar system for enzyme purification

Rangel-Yagui, Carlota de Oliveira
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 30/07/2003 PT
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A partição das enzimas glicose-6-fosfato desidrogenase (G6PD) e uroquinase (UK) em sistemas micelares de duas fases aquosas foi investigada, teórica e experimentalmente. Inicialmente, a partição de G6PD em sistema micelar de duas fases aquosas composto pelo tensoativo não-iônico C10E4 (óxido de n-deciltetraetileno) foi estudada. Observou-se uma partição governada primariamente por interações repulsivas estéricas, do tipo "volume de exclusão", sendo a G6PD recuperada preferencialmente na fase inferior do sistema, pobre em micelas, resultando em valores de KG6PD inferiores a 1,0. Os tensoativos catiônicos CnTAB (brometos de alquiltrimetilamônio, n = 8, 10 e 12) foram adicionados ao tensoativo C10E4, de modo a formar sistemas micelares mistos (não-iônico/catiônico) de duas fases aquosas e atrair a G6PD de carga efetiva negativa para a fase superior, rica em micelas positivamente carregadas. Os coeficientes de partição obtidos nos sistemas C10E4/CnTAB/tampão foram no mínimo 2,5 vezes maiores que os correspondentes no sistema C10E4/tampão mantendo-se o volume de exclusão constante. De uma forma geral, o sistema micelar misto C10E4/C10TAB/tampão forneceu o melhor KG6PD = 7,7, com um rendimento de G6PD na fase superior de 71%. A adição de ligantes de afinidade (inibidores enzimáticos) ao sistema de duas fases aquosas C10E4/tampão para purificação de UK e G6PD também foi estudada. Não foram observadas diferenças significativas no perfil de partição da UK na presença ou não do ligante p-aminobenzamidina...

Desenvolvimento de processo cromatográfico para purificação de fator VIII humano. Emprego de anticorpos contra fragmentos específicos da proteína na avaliação da pureza e estabilidade durante as etapas de purificação.; Process development for human factor VIII purification by chromatography, the use of specific antibodies against fragments of the protein for evaluation of purity and stability during purification processes.

Jinzenji, Daniela
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 31/10/2008 PT
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O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de "western blot" para detectar as cadeias de FVIII ou degradação.; Coagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography...

Desenvolvimento de métodos de purificação do Gálio-67 e Gálio-68 para a marcação de biomolécula; Development of methods for the purification of 67Ga and 68Ga for biomolecules labeling

Costa, Renata Ferreira
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 29/03/2012 PT
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Há mais de 50 anos os geradores de 68Ge/68Ga vêm sendo desenvolvidos, obtendo o 68Ga sem a necessidade da instalação de um cíclotron próximo à radiofarmácia ou ao centro hospitalar que tenha um PET/CT. O 68Ga é um emissor de pósitron com baixa emissão de fóton (β+, 89%, 1077 keV) e meia vida de 67,7 minutos, compatível com a farmacocinética de moléculas de baixo peso molecular, como peptídeos e fragmentos de anticorpos. Além disso, a química do Ga permite a ligação estável com agentes quelantes acoplados com peptídeos, como o DOTA. Todas estas características do 68Ga aliado a tecnologia PET/CT permitiram avanços em imagem molecular, como no diagnóstico de doenças de origem neuroendócrina. Entretanto, o eluato de 68Ga proveniente dos geradores de 68Ge/68Ga comerciais, ainda contém altos níveis de contaminantes, como o 68Ge e outros metais que competem quimicamente com o 68Ga, como o Fe3+ e Zn2+ e, como consequência, há redução do rendimento de marcação com biomoléculas. Quanto menor a quantidade de impurezas no eluato, a competição entre o peptídeo radiomarcado e peptídeo não marcado será menor e a qualidade de imagem será melhor, por isso existe a necessidade de diminuir a quantidade destes metais. Portanto...

Estratégia de purificação por IMAC de fragmento Fab de IgG humana adicionado a extrato proteico de soja; IMAC purification strategy of human IgG Fab fragments added to soy protein extract

Marcel Mafei Serracchiani
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 25/10/2013 PT
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A produção de proteínas recombinantes em plantas transgênicas tem se mostrado uma forma segura, eficiente e barata de obtenção em grande escala de várias proteínas de interesse, tais como vacinas, enzimas industriais, bioativos, biofarmacos e anticorpos e seus fragmentos. Contudo, para que a produção de proteínas recombinantes em plantas se torne viável é necessário o desenvolvimento de um processo de recuperação e separação da molécula alvo que seja eficiente e reprodutivo, pois a produção de biomoléculas em plantas transgênicas, assim como os métodos tradicionais de produção, apresentam componentes indesejáveis que devem ser removidos. Neste trabalho, avaliou-se a purificação de fragmentos Fab de IgG humana adicionados artificialmente (spiking) a extrato protéico de soja não transgênica utilizando-se a técnica de cromatografia de afinidade por íons metálicos imobilizados (IMAC). Estudou-se o efeito dos quelatos IDA-Ni(II) e TREN-Ni(II), dos sistemas tamponantes Tris-HCl, fosfato de sódio e Mes, na presença e na ausência de sal (NaCl) na purificação de fragmentos Fab. Primeiramente foram realizados experimentos cromatográficos com as proteínas nativas do extrato proteico do grão de soja. Dos resultados obtidos...

Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Marcos, João Carlos; Fonseca, Luís Pina; Ramalho, Maria Teresa; Cabral, J. M. S.
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2002 ENG
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Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350 – sodium citrate aqueous two phase systems was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, Sodium Citrate and NaCl concentrations of 15.1%, 11.0% and 8.52% respectively. However under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.

Low-cost purification of nisin from milk whey to a highly active product

Jozala, A. F.; Novaes, Letícia Celia de Lencastre; Mazzola, Priscila Gava; Nascimento, Laura Oliveira; Penna, T. C. V.; Teixeira, J. A.; Passarinha, L. A.; Queiroz, J. A.; Pessoa Júnior, Adalberto
Fonte: Institution of Chemical Engineers Publicador: Institution of Chemical Engineers
Tipo: Artigo de Revista Científica
Publicado em //2015 ENG
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Abstract Nisin is a natural peptide used as a preservative in a variety of food products, in which it inhibits mainly Gram-positive bacterial growth, including multidrug-resistant pathogens. However, its application range depends on the cost-effective production and purification of this molecule. Our group has previously produced nisin by Lactococcus lactis cultivation in milk whey, which is an industrial residue from dairy production. To our knowledge, no report used milk whey as a culture medium, although several investigators have purified nisin using different techniques. We thus aimed to establish a low-cost purification of nisin obtained by this process. Samples were diluted in ammonium sulphate, applied onto HIC columns (butyl sepharose CL 4B matrix), and eluted with Milli-Q water or PBS. Elution fractions were monitored for protein content and nisin antibacterial activity. Water elution resulted in purification factor values (270, commercial nisin; 775, nisin produced in-house) higher than those obtained with PBS elution. We concluded that purification of nisin does not require precipitation with ammonium sulphate, therefore allowing step/cost reduction. Moreover, purification from milk whey using HIC provides nisin with high activity and low salt content...

Smart macroporous structures for the purification of viral particles

Sousa, Margarida Bucho Nunes de
Fonte: Faculdade de Ciências e Tecnologia Publicador: Faculdade de Ciências e Tecnologia
Tipo: Dissertação de Mestrado
Publicado em //2014 ENG
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Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica; The increasing application of viral particles in vaccination and gene-based therapies, has led to the development of alternative and improved purification processes. Traditional purification methods include chromatographic techniques, however the chromatographic matrices used present limitations specially when aimed at the purification of large molecules. This work presents the preparation of chitosan-based monoliths using clean processes and easy functionalization techniques intending to improve Adenovirus serotype 5 (Ad5) purification. Monoliths were prepared by blending chitosan (CHT) with glycidylmethacrylate (GMA) or poly(vinyl alcohol) (PVA), using two preparation techniques, freeze-drying and a scCO2 – assisted drying process, and were subsequently functionalized with Q ligands by three different methods. In addition, monoliths blended with magnetic nanoparticles were also prepared using the same strategies to confer them a controlled magnetic response. The monoliths produced were characterized in terms of ligand immobilization yield, and evaluated for Ad5 purification. Two types of monoliths showed potential: the CHT/PVA(50:50) prepared by freeze drying and functionalized by the alternative plasma technique (M2) and the CHT/PVA(50:50) 7% monolith prepared by scCO2 – assisted drying process and functionalized by the epoxyactivation technique (M1). The amount of ligand Q immobilized on the supports was monitored by titration assays...

Purification of alpha-galactosidase from seeds of Sesbania marginata

Falco,A.L.P.; Durrant,L.R.; Franco,T.T.
Fonte: Brazilian Society of Chemical Engineering Publicador: Brazilian Society of Chemical Engineering
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2000 EN
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Alpha-galactosidase taken from a raw extract of Sesbania marginata legume seeds was purified by partitioning in aqueous two-phase systems (ATPS). Initially, galactomannan/dextran 2,000,000 systems were used for the purification, and the partition coefficients of alpha -galactosidase varied from 1.5 to 4.0. However, mass transport in these systems was poor due to the high viscosity of the employed polymers. Therefore, partitioning in polyethyleneglycol (PEG)/ sodium phosphate systems and the effect of sodium chloride upon the enzyme purification and the yield of alpha -galactosidase were also investigated. The purification achieved in a single-step was 5.7 with a recovery of 144% of alpha -galactosidase, possibly due to the removal of materials which inhibited alpha -galactosidase activity before the purification. The removal of the main protein contaminants and the highest yields were achieved in PEG 4,000/ sodium phosphate + 6% NaCl system at pH 5.0. Further purification by preparative on-exchange chromatography was also developed.

Therapeutic effects of blood purification in treatment of fulminant hepatic failure

Pu,Yunchuan; Yang,Daokun; Mao,Yanqun; Zhang,Ying; Chen,Kaihong
Fonte: Brazilian Society of Infectious Diseases Publicador: Brazilian Society of Infectious Diseases
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/08/2013 EN
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OBJECTIVES: To evaluate the clinical effects of blood purification for treating fulminant hepatic failure (FHF). METHODS: Thirty-three severe FHF patients with hepatic encephalopathy (HE) above grade III were subjected to a combined blood purification treatment in addition to the comprehensive liver protection therapy. Patients underwent continuous hemofiltration on a daily basis during the daytime followed by sequential treatment with plasma exchange or hemodialysis every 2-3 days. The therapeutic effects of this treatment were evaluated. RESULTS: After treatment with blood purification, restoration of consciousness (those who abandoned the treatment without restoration of consciousness were excluded) was achieved in 6 of 8 cases (75%) in acute liver failure (ALF) group, 3 of 3 cases (100%) in subacute liver failure (SALF) group, and 9 of 14 cases (64.29%) in acute/subacute on chronic liver failure (A/SCLF) group. Of all cases, 11 patients restored consciousness after 7 days in a coma. The rate of long-term survival (those who abandoned the treatment were excluded) was 3/7 (42.86%) for ALF group, 2/2 (100%) for SALF group, and 1/11 (9.09%) for A/SCLF group. The levels of hemoglobin and platelet in peripheral blood were significantly reduced after blood purification. CONCLUSIONS: Treatment of FHF patients with daily continuous hemofiltration during the daytime is effective in treating HE and in improving health status in the early stages of the disease. Long-term prognosis also benefits from this treatment. The rate of consciousness recovery and long-term survival is highest in SALF group followed by ALF group. This treatment is less effective in A/SCLF patients. It should be noted that blood purification procedure may cause damage to blood cells.

Purification of C-phycocyanin from Spirulina platensis in aqueous two-phase systems using an experimental design

Antelo,Francine Silva; Costa,Jorge Alberto Vieira; Kalil,Susana Juliano
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/2015 EN
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C-phycocyanin from Spirulina platensis was purified in aqueous two-phase systems (ATPS) of polyethylene glycol (PEG)/potassium phosphate, varying the molar mass of the PEG. Results using a full factorial design showed that an increase in the concentration of salt and decrease in the concentration of PEG caused an increment in the purification factor for all the ATPS studied. Optimization of the conditions of the purification was studied using a central composite rotatable design for each molar mass of PEG. The ATPS composed of 7% (w/w) PEG 1500 or 4% (w/w) PEG 8000 (g/gmol) and 23 or 22.5% (w/w) of phosphate resulted a purification factor of 1.6-fold for C-phycocyanin, with total and 57% recovery, respectively. Process conditions were optimized for the purification factor for the system with PEG 1500. The ATPS with 4% (w/w) PEG 4000 or 4% (w/w) PEG 6000 and 21% (w/w) phosphate resulted purification factors of 2.1 and 2.2-fold, recovering 100% and 73.5%, respectively of C-phycocyanin in the top phase.

Design Strategies for Integrated b-Galactosidase Purification Processes

Lemes, Ailton Cesar; Machado, Juliana Ribeiro; Brites, Marcela Lazzare; Di Luccio, Marco; Kalil, Susana Juliano
Fonte: Universidade Federal do Rio Grande Publicador: Universidade Federal do Rio Grande
Tipo: Artigo de Revista Científica
ENG
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The operational conditions for an aqueous two-phase system (ATPS) for b-galactosidase purification were optimized and applied to the design of a purification strategy as an alternative to the primary purification steps. The ATPS proved to be suitable for the recovery and primary enzyme purification. The purification process design developed by ATPS, diafiltration, ion exchange, and diafiltration/ultrafiltration was successful, yielding a more than tenfold purification. The purification strategy design resulted in a powerful integrated purification and recovery process, an evidence of the potential for a scale-up of the b-galactosidase purification process.

Extraction, purification et caractérisation d’isoformes d’hexokinase du tubercule de pomme de terre (Solanum tuberosum)

Moisan, Marie-Claude
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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L’hexokinase (HK) est la première enzyme du métabolisme des hexoses et catalyse la réaction qui permet aux hexoses d’entrer dans le pool des hexoses phosphates et donc par le fait même la glycolyse. Bien que le glucose soit son principal substrat, cette enzyme peut aussi phosphoryler le mannose et le fructose. Malgré son importance dans le métabolisme primaire, l’HK n’a jamais été purifiée à homogénéité sous forme native. Le but de ce travail était donc de purifier une isoforme d’HK à partir de tubercule de Solanum tuberosum et par la suite de caractériser ses propriétés cinétiques. Bien avant que je commence mon travail, un groupe de recherche avait déjà séparé et partiellement purifié trois isoformes d’HK de S. tuberosum. Un protocole d’extraction était donc disponible, mais l’HK ainsi extraite était peu stable d’où le besoin d’y apporter certaines modifications. En y ajoutant certains inhibiteurs de protéases ainsi qu’en modifiant les concentrations de certains éléments, le tampon d’extraction ainsi modifié a permis d’obtenir un extrait dont l’activité HK était stable pendant au moins 72h après l’extraction, en empêchant la dégradation. À l’aide du tampon d’extraction optimisé et d’une chromatographie sur colonne de butyl sépharose...

Purification par affinité et marquage isotopique spécifique pour études d’ARN fonctionnels

Dagenais, Pierre
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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Il existe un lien étroit entre la structure tridimensionnelle et la fonction cellulaire de l’ARN. Il est donc essentiel d’effectuer des études structurales de molécules d’ARN telles que les riborégulateurs afin de mieux caractériser leurs mécanismes d’action. Une technique de choix, permettant d’obtenir de l’information structurale sur les molécules d’ARN est la spectroscopie RMN. Cette technique est toutefois limitée par deux difficultés majeures. Premièrement, la préparation d’une quantité d’ARN nécessaire à ce type d’étude est un processus long et ardu. Afin de résoudre ce problème, notre laboratoire a développé une technique rapide de purification des ARN par affinité, utilisant une étiquette ARiBo. La deuxième difficulté provient du grand recouvrement des signaux présents sur les spectres RMN de molécules d’ARN. Ce recouvrement est proportionnel à la taille de la molécule étudiée, rendant la détermination de structures d’ARN de plus de 15 kDa extrêmement complexe. La solution émergeante à ce problème est le marquage isotopique spécifique des ARN. Cependant, les protocoles élaborées jusqu’à maintenant sont très coûteux, requièrent plusieurs semaines de manipulation en laboratoire et procurent de faibles rendements. Ce mémoire présente une nouvelle stratégie de marquage isotopique spécifique d’ARN fonctionnels basée sur la purification par affinité ARiBo. Cette approche comprend la séparation et la purification de nucléotides marqués...

Application of response surface methodology to the modeling of α-amylase purification by aqueous two-phase systems; Application of response surface methodology to the modeling of alpha-amylase purification by aqueous two-phase systems

Zhi, W.; Song, J.; Ouyang, F.; Bi, J.
Fonte: Elsevier Science BV Publicador: Elsevier Science BV
Tipo: Artigo de Revista Científica
Publicado em //2005 EN
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Mathematical models concerning the purification of greek small letter alpha-amylase from the cultivation supernatant of Bacillus subtilis in a polyethylene glycol–citrate aqueous two-phase system (ATP) are established with response surface methodology. The PEG3350, citrate and sodium chloride concentrations were selected as variables to evaluate the purification impact factors in aqueous two-phase system, including partition coefficients of greek small letter alpha-amylase, total protein, purification factor and greek small letter alpha-amylase yield. An experimental space with two-fold purification and over 90% yield of greek small letter alpha-amylase is achieved through the optimized condition basing on the model. Two systems with low viscosity within said space were further selected to perform greek small letter alpha-amylase purification and the experimental results coincide well with the calculation of the models, which indicates that the model provides a promising tool for experimental design of protein purification by aqueous two-phase system.; http://www.elsevier.com/wps/find/journaldescription.cws_home/505515/description#description

Análisis de ciclo de vida de los procesos de extracción y purificación de la salmuera del proceso cloro-álcali; Life cycle assessment of salt mining and brine purification for the chlor-alkali process

Herrero González, Marta
Fonte: Universidade de Cantabria Publicador: Universidade de Cantabria
Tipo: Dissertação de Mestrado
SPA
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RESUMEN: La industria cloro-álcali hace referencia a aquella que produce cloro, hidróxido sódico, e hidrógeno como subproducto. Esta industria tiene una gran importancia a nivel productivo y económico, alcanzando en el año 2014 en Europa las 9.6 Mt/año de Cl2 en las 73 plantas de cloro-álcali, localizadas en 21 países diferentes. España también cuenta con una relativa importancia en este sector, con 9 plantas situadas mayoritariamente en el norte de la península (Euro Chlor, 2015). El proceso cloro-álcali comienza con la obtención de la sal (NaCl), la cual es transportada a la planta y utilizada en la preparación de la salmuera, que debe ser purificada antes de introducirse en electrólisis. La obtención la sal puede realizarse por diversas vías: minería de sal de roca, extracción por disolución, evaporación solar de sal, purificación de residuos de KCl, y producción de sal a vacío (Euro Chlor, 2013). Respecto a la electrolisis, se pueden emplear tres tecnologías diferentes: mercurio, membrana y diafragma. En dichas celdas, mediante la aplicación de una corriente eléctrica, se consigue la ruptura de la molécula de NaCl. Existen diversos estudios que realizan un análisis de ciclo de vida al proceso cloro-álcali (Euro Chlor...

Le «travail du négatif» comme purification dans les Leçons sur la philosophie de la religion de Hegel

Genest, Benoit
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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La purification est une métaphore désignant le moteur de la philosophie de la religion de Hegel. Elle est d’abord à l’œuvre dans la création de la Nature qui se consume pour produire la conscience de soi divine à travers l'esprit humain. En second lieu, elle s’opère dans l’objectivation des productions spirituelles de l’homme qui sont purifiées jusqu'à ce que l’Esprit soit auprès de soi dans le christianisme. La troisième purification est morale et trouve son fondement dans la Genèse, le judaïsme étant le premier à avoir identifié l'unité des natures humaine et divine. Le mythe témoignera également de la culpabilité en tant que l'homme n'exprime pas immédiatement sa divinité, mais sa finitude. La réalisation du divin impliquera donc la purification de la naturalité au profit de la substantialité. Le christianisme explicitera cette tâche par l’héroïsme de Jésus et cet héroïsme se perpétuera jusqu’à ce qu’émergent un individualisme moderne et une religion assurant la cohésion sociale : le protestantisme luthérien. Cet individualisme sera toutefois défectueux puisqu’il produira éventuellement davantage d’égoïsme que de réconciliation, ce qui donnera lieu à certaines critiques de l’analyse hégélienne du christianisme. En effet...

Adaptations de la méthode de purification d’ARN par affinité avec l’étiquette ARiBo

Salvail-Lacoste, Alix
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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Dans les dernières années, une explosion de la recherche sur les ARN a eu lieue à cause de nombreuses découvertes démontrant l’importance de l’ARN dans plusieurs processus biologiques. Ainsi, de grandes quantités d’ARN sont devenues indispensables au bon déroulement de plusieurs études, notamment pour la biologie structurale et la caractérisation fonctionnelle. Cependant, il existe encore peu de méthodes de purification simples, efficaces, fiables et produisant un ARN sous forme native. Dans les dernières années, le laboratoire Legault a mis au point une méthode de purification par affinité utilisant une étiquette ARiBo pour la purification d’ARN transcrits in vitro par la polymérase à ARN du phage T7. Cette méthode de purification d’ARN a été spécifiquement développée pour maximiser la pureté et le rendement. De plus, elle est très rapide et fonctionne avec plusieurs types d’ARN. Cependant, comme plusieurs autres méthodes de purification, cette méthode produit des ARN avec des extrémités 5′ hétérogènes. Dans ce mémoire, des solutions sont proposées pour remédier au problème d’hétérogénéité en 5ʹ′ des ARN transcrits avec la polymérase à ARN du phage T7 et purifiés par la méthode ARiBo. La première solution consiste à choisir la séquence en 5′ parmi celles des 32 séquences testées qui ne présentent pas d’hétérogénéité en 5ʹ′. La seconde solution est d’utiliser une étiquette clivable en 5ʹ′ de l’ARN d’intérêt...

Análise da viabilidade econômica da purificação da bromelina das folhas de curauá em sistema bifásico aquoso PEG/Fosfato; Economic viability of bromelain purification from curaua using an aqueous two phase system PEG/phosphate

Dalva Sbruzzi
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 07/06/2010 PT
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O curauá (Ananas Erectifolius L.B. Smith), espécie vegetal de porte herbáceo, é uma monocotiledônea pertencente à família Bromeliaceae, nativa da região norte do Brasil, especialmente da Amazônia, de onde pode ser extraída a bromelina. Essa faz parte de um grupo de enzimas proteolíticas, usadas na indústria alimentícia e como medicamento, pois oferece amplo espectro de eficácias terapêuticas: antiedemas, anti-inflamatórias, antitrombóticas e atividades fibrinolíticas. O desenvolvimento de novos processos de extração e purificação de proteínas é muito importante, uma vez que essa é uma etapa limitante na produção de bioprodutos. Sistemas de duas fases aquosas são amplamente utilizados para a separação e purificação de biomoléculas. As vantagens da extração em duas fases aquosas, em comparação com outros métodos de purificação, são o elevado rendimento, a faixa de trabalho próxima do equilíbrio, a fácil ampliação de escala e o uso em processos contínuos. Esta pesquisa propõe a caracterização da bromelina do curauá e a sua purificação por extração líquido-líquido em duas fases aquosas, formadas por um polímero (polietilenoglicol) e um sal (fosfato de potássio). Apresenta também um estudo sobre os custos do processo e o preço de venda estimado...

The Purification Offering of Leviticus and the Sacrificial Offering of Jesus

Vis, Joshua Marlin
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2012
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The life, death, resurrection, and ascension of Jesus are not often read against the backdrop of the sacrificial system of Leviticus, despite the fact that the Letter to the Hebrews and other New Testament texts do exactly this. Until recently, Hebrew Bible scholars had little insight into the function of many of the sacrifices of Leviticus. However, over the last thirty years, Jacob Milgrom has articulated the purgative function of the purification offering of Leviticus, the principal sacrifice offered for wrongdoing. The blood of the purification offering, which contains the animal's ,nefesh, best understood as the animating force of the animal, acts as a ritual cleanser. Milgrom has insisted that the purification offering only cleanses the sanctuary, never the offerer. This conclusion likely has kept many New Testament scholars from seeing the impact this sacrifice had on various New Testament authors. Thus although Milgrom's work has had a profound impact on Hebrew Bible scholarship, it has had little effect on New Testament scholarship on the sacrifice of Jesus.

Using source criticism and a close reading of the relevant Hebrew Bible texts and New Testament texts, this study argues that the purification offering of Leviticus can purge the offerer...

Preliminary purification of anthocyanins from blue corn by adsorption and electrophoresis

Cerón-Montes,G.I.; San Martin-Martinéz,E.; Yañez-Fernández,J.; Quezada-Cruz,M.; Castro-Muñoz,R.
Fonte: UAM, Unidad Iztapalapa, División de Ciencias Básicas e Ingeniería Publicador: UAM, Unidad Iztapalapa, División de Ciencias Básicas e Ingeniería
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2015 EN
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Anthocyanins pigments are natural antioxidants of interest in food, pharmaceutical and cosmetic industries. In this study a process for preliminary purification of anthocyanins from blue corn (Zea mays) was implemented. The anthocyanin purification advancement was analysed in terms of Total Organic Carbon (TOC), Total Anthocyanin Content (TAC), and reverse phase High Performance Liquid Chromatography (HPLC). Microfiltration and ultrafiltration steps were used as pre-treatment of the extracts. Purification of anthocyanins was performed by adsorption and electrophoresis. The adsorption was achieved using a (SP-207) styrene-divinylbenzene resin, the results were correlated using Freundlich and Langmuir models. The best condition for purification of anthocyanins in electrophoresis was at pH 3 and 220 V. The purification process was improved using; electrophoresis resulting in a decreasing on TOC from 59.52±0.53 mg to 3.966±0.257 mg per milligram of anthocyanins. The process implemented was successfully used in red cabbage (Brassica oleracea var. Capitata) and blueberries (Vaccinium corymbosum). In conclusion, proposed process can be useful as a preliminary purification process of anthocyanins from differeni sources.