A pele humana é o maior e um dos mais importantes órgãos, esta é a principal barreira entre o corpo e o meio externo, sempre que há um desequilíbrio no mecanismo de defesa inata, o risco de uma infecção é proeminente. A queimadura é um quadro clínico em que a barreira epitelial é danificada, resultado em uma defesa imunológica deficitária e uma grande abundância de nutrientes, o que favorece a proliferação de micro-organismos. No presente estudo foi desenvolvido e caracterizado uma suspensão inovadora de nanocápsulas poliméricas de poli(ε-caprolactona) com triclosan e α-bisabol, revestida com quitosana (NCCQ) para aplicação tópica em feridas de queimadura, com intuito de reduzir a contaminação microbiana. Foram realizados ensaios de caracterização físico-química, microbiológica e cutânea. Os resultados obtidos para os ensaios de caracterização mostraram que a suspensão de nanocápsulas apresentou diâmetro de partícula adequado, ausência de cristais ou contaminação micrométrica, potencial zeta catiônico, pH levemente ácido, com uma alta eficiência de encapsulação e estável no período de trinta dias. Os ensaios microbiológicos foram realizados frente Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans, através de microdiluição seriada e teste do desafio. O primeiro ensaio mostrou uma redução no valor da concentração inibitória mínima e melhores resultados para quase todas as cepas analisadas frente aos controles e dados da literatura, inclusive P. aeruginosa, a qual possui uma alta resistência ao triclosan. O teste do desafio serviu para comprovar que o efeito antimicrobiano da suspensão, se manteve ao longo de 28 dias, mesmo quando incorporada ao curativo de hemi-celulose. Os ensaios cutâneos de permeação/penetração e lavabilidade mostraram que a suspensão de nanocápsulas NCCQ teve uma retenção no estrato córneo, o que é desejável para uma formulação tópica. / The human skin is the largest and one of the most important organs, this is the main barrier between the body and the external environment, whenever there is an imbalance in innate defense mechanism, the risk of infection is prominent. Burn is a clinical condition in which the epithelial barrier is damaged, resulting in a deficient immune defense, also the abundance of nutrients favors to proliferation of microorganisms. In the present study it was developed and characterized a novel suspension of polymeric nanocapsules of poly (ε-caprolactone) with triclosan and α-bisabol coated with chitosan (NCCQ) for topical application in burn wounds, in order to reduce microbial contamination. Physical-chemical, microbiological and skin tests were performed in order to prove its applicability. The results obtained for the characterization tests showed that the suspension of nanocapsules had suitable particle diameter, no crystals or micrometric contamination cationic zeta potential, slightly acid pH with a high encapsulation efficiency and stable within thirty days. Microbiological tests were carried forward Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, via serial microdilution and challenge test. The first experiment showed a reduction in the value of the minimum inhibitory concentration and best results for nearly all strains analyzed when compared with controls and data from the literature, including P. aeruginosa, which has a high resistance to triclosan. The challenge test was used to demonstrate that the antimicrobial effect of the suspension was maintained over 28 days even when incorporated into the dressing hemi-cellulose. The skin permeation/penetration and washability tests of NCCQ nanocapsule suspension showed that it had retention in the stratum corneum, which is desirable for a topical formulation.

Dissertação (mestrado)—Universidade de Brasília, Faculdade em Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2017. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2017-03-29T19:02:21Z No. of bitstreams: 1 2017_LorenaFerreiraGomes_Parcial.pdf: 226967 bytes, checksum: d00af9fafbe2e2bd391079ffd56e7e47 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2017-04-18T18:33:11Z (GMT) No. of bitstreams: 1 2017_LorenaFerreiraGomes_Parcial.pdf: 226967 bytes, checksum: d00af9fafbe2e2bd391079ffd56e7e47 (MD5) / Made available in DSpace on 2017-04-18T18:33:11Z (GMT). No. of bitstreams: 1 2017_LorenaFerreiraGomes_Parcial.pdf: 226967 bytes, checksum: d00af9fafbe2e2bd391079ffd56e7e47 (MD5) / O Brasil possui a maior biodiversidade vegetal do mundo. Porém, apesar de tamanha riqueza, muitas espécies vegetais ainda não foram estudas química e biologicamente. Miconia chamissois (Melastomataceae) é uma espécie frequentemente encontrada no bioma Cerrado e é descrita basicamente pelos benefícios ecológicos. Seu potencial químico e biológico é pouco explorado. Assim, o objetivo desde trabalho foi estudar, preliminarmente, os compostos presentes no extrato aquoso das folhas de M. chamissois, sua ação citotóxica e atividades antioxidante, antimicrobiana e de inibição enzimática. O estudo fitoquímico foi realizado pelas técnicas de Cromatografia em camada delgada (CCD) e Cromatografia Líquida de Alta Eficiência (CLAE). Os teores de polifenóis e flavonoides foram determinados com reagente de Folin e cloreto de alumínio, respectivamente. A atividade antioxidante foi avaliada pelos métodos DPPH e fosfomolibdênio. Os métodos de microdiluição em caldo e difusão em disco foram utilizados para determinação da atividade antimicrobiana frente à Staphylococcus aureus, Escherichia coli e Candida albicans. A atividade de inibição da tirosinase e α-amilase foram determinadas. A citotoxicidade do extrato foi avaliada frente às linhagens celulares de queratinócito humano (HaCat), fibroblasto de camundongo (L929) e macrófago de camundongo (Raw 264.7) pelo método do MTT. É sugerido a presença de compostos devidos da vitexina. O extrato aquoso de M. chamissois apresentou atividade antioxidante, equivalente a 18,35 μg/mL de ácido ascórbico pelo método fosfomolibdênio e IC50 de 2,91 μg/mL pelo método DPPH. Inibição do crescimento de S. aureus foi observado em ambos os métodos e a inibição do crescimento de C. albicans foi observada apenas frente ao método de microdiluição. Os valores de IC50 encontrados para a inibição da tirosinase e α-amilase foram de 183,2 e 15,06 μg/mL, respectivamente. O extrato não apresentou citotoxicidade frente às linhagens avaliadas. Os resultados encontrados sugerem potencial biológico do extrato aquoso de M. chamissois, sendo necessários mais estudos para a elucidação dos compostos presentes na espécie. / Brazil has the largest plant biodiversity in the world. However, despite such wealth, many plant species have not yet been studied chemically and biologically. Miconia chamissois (Melastomataceae) is a species frequently found in the Cerrado biome and is basically described by its ecological benefits. Its chemical and biological potential is little explored. The aim of this work was to study, preliminarily, the compounds present in the aqueous extract of the leaves of M. chamissois, its cytotoxic action and antioxidant, antimicrobial and inhibitory enzymatic activities. The phytochemical study was performed by the techniques of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The total polyphenols and flavonoids contents were determined with Folin reagent and aluminum chloride, respectively. The antioxidant activity was evaluated by DPPH and phosphomolybdenum methods. Broth microdilution and disk diffusion methods were used to determine the antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans. The extract cytotoxicity was evaluated against human keratinocyte (HaCat), mouse fibroblast (L929) and mouse macrophage (Raw 264.7) cell lines by the MTT method. The presence of due vitexin compounds is suggested. The aqueous extract of M. chamissois presented antioxidant activity, equivalent to 18.35 μg/mL of ascorbic acid by the phosphomolybdenum method and presented IC50 of 2.91 μg/mL by DPPH method. Inhibition of S. aureus growth was observed in both methods and inhibition of C. albicans was observed only in the microdilution method. The IC50 values found for tyrosinase and α-amylase were 183.2 and 15.06 μg/mL, respectively. The extract did not present cytotoxicity against cell lines evaluated. The results suggest a biological potential of M. chamissois aqueous extract, and further studies are needed to elucidate the compounds present in the species.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T18:50:23Z : No. of bitstreams: 1 donato_pm_me_botfm.pdf: 1472808 bytes, checksum: dd94c6d1ccf0834bf27a62051a637836 (MD5) / Ministério da Saúde / As plaquetas são fragmentos do citoplasma do megacariócito e possuem papel fundamental na manutenção da hemostasia. Ao aderirem umas às outras, ativam-se, mudam de forma e liberam grânulos que contêm fatores de crescimento, proteínas que atuam na divisão celular. Os grânulos α- plaquetários secretam fatores de crescimento que regulam eventos, tais como, síntese de DNA, quimiotaxia e citodiferenciação. A estocagem prolongada das plaquetas a 22ºC é prejudicial à viabilidade e à função, mesmo quando estocadas em condições adequadas nos bancos de sangue. Observa-se diminuição da concentração do glutation intraplaquetário, representando diminuição da proteção contra o stress oxidativo, do qual resulta a perda do grupo sulfidril. As plaquetas desempenham funções com consumo de energia, como exemplo a diapedese, que pode ser recuperada após cinco dias de armazenamento pelo azul de metileno. Durante a divisão celular há necessidade de bloqueio das funções citoplasmáticas com aumento da produção de proteínas de adesão para que ocorra a fixação da célula que vai se dividir. Os resultados mostraram que quando adicionamos azul de metileno, a quantidade de células viáveis diminui com aumento de apoptose e necrose, muito provavelmente devido à liberação de energia / Platelets are megakaryocyte cytoplasmic fragments that play a fundamental role in maintaining hemostasis. When they adhere to each other, they become activated, change shape and release granules containing growth factors, proteins involved with cell division. Platelet α-granules secrete growth factors that regulate events such as DNA synthesis, chemotaxis and cytodifferentiation. Long platelet storage at 22ºC is harmful to their viability and function, even when they are stored under proper conditions at blood banks. A reduced concentration of intraplatelet glutathione is observed, representing decreased protection against oxidative stress, from which the loss of the sulfhydryl group results. Platelets perform actions with energy consumption, such as diapedesis, which can be recovered after five storage days by methylene blue. During cell division, cytoplasmic functions must be blocked by increased adhesion protein so that the fixation of the dividing cell can occur. Results showed that when methylene blue is added, the number of viable cells decreases with apoptosis and necrosis increase, which is very likely due to energy release

A pele humana é o maior e um dos mais importantes órgãos, esta é a principal barreira entre o corpo e o meio externo, sempre que há um desequilíbrio no mecanismo de defesa inata, o risco de uma infecção é proeminente. A queimadura é um quadro clínico em que a barreira epitelial é danificada, resultado em uma defesa imunológica deficitária e uma grande abundância de nutrientes, o que favorece a proliferação de micro-organismos. No presente estudo foi desenvolvido e caracterizado uma suspensão inovadora de nanocápsulas poliméricas de poli(ε-caprolactona) com triclosan e α-bisabol, revestida com quitosana (NCCQ) para aplicação tópica em feridas de queimadura, com intuito de reduzir a contaminação microbiana. Foram realizados ensaios de caracterização físico-química, microbiológica e cutânea. Os resultados obtidos para os ensaios de caracterização mostraram que a suspensão de nanocápsulas apresentou diâmetro de partícula adequado, ausência de cristais ou contaminação micrométrica, potencial zeta catiônico, pH levemente ácido, com uma alta eficiência de encapsulação e estável no período de trinta dias. Os ensaios microbiológicos foram realizados frente Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans, através de microdiluição seriada e teste do desafio. O primeiro ensaio mostrou uma redução no valor da concentração inibitória mínima e melhores resultados para quase todas as cepas analisadas frente aos controles e dados da literatura, inclusive P. aeruginosa, a qual possui uma alta resistência ao triclosan. O teste do desafio serviu para comprovar que o efeito antimicrobiano da suspensão, se manteve ao longo de 28 dias, mesmo quando incorporada ao curativo de hemi-celulose. Os ensaios cutâneos de permeação/penetração e lavabilidade mostraram que a suspensão de nanocápsulas NCCQ teve uma retenção no estrato córneo, o que é desejável para uma formulação tópica. / The human skin is the largest and one of the most important organs, this is the main barrier between the body and the external environment, whenever there is an imbalance in innate defense mechanism, the risk of infection is prominent. Burn is a clinical condition in which the epithelial barrier is damaged, resulting in a deficient immune defense, also the abundance of nutrients favors to proliferation of microorganisms. In the present study it was developed and characterized a novel suspension of polymeric nanocapsules of poly (ε-caprolactone) with triclosan and α-bisabol coated with chitosan (NCCQ) for topical application in burn wounds, in order to reduce microbial contamination. Physical-chemical, microbiological and skin tests were performed in order to prove its applicability. The results obtained for the characterization tests showed that the suspension of nanocapsules had suitable particle diameter, no crystals or micrometric contamination cationic zeta potential, slightly acid pH with a high encapsulation efficiency and stable within thirty days. Microbiological tests were carried forward Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, via serial microdilution and challenge test. The first experiment showed a reduction in the value of the minimum inhibitory concentration and best results for nearly all strains analyzed when compared with controls and data from the literature, including P. aeruginosa, which has a high resistance to triclosan. The challenge test was used to demonstrate that the antimicrobial effect of the suspension was maintained over 28 days even when incorporated into the dressing hemi-cellulose. The skin permeation/penetration and washability tests of NCCQ nanocapsule suspension showed that it had retention in the stratum corneum, which is desirable for a topical formulation.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-12T11:55:14Z No. of bitstreams: 2 DISSERTAÇÃO Amália Cristine Medeiros Ferreira.pdf: 1292292 bytes, checksum: cf06788df2c672bca8f740e4286d5815 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T11:55:14Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Amália Cristine Medeiros Ferreira.pdf: 1292292 bytes, checksum: cf06788df2c672bca8f740e4286d5815 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-02-28 / CNPq / A α-amilase é uma endocarboidrase que atua na hidrólise de ligações glicosídicas α(1→4) presentes em polissacarídeos como o amido e glicogênio, gerando como produtos oligossacarídeos, dextrinas e maltose. Esta enzima corresponde à segunda classe de enzimas mais utilizadas na indústria, apresentando diversas aplicações biotecnológicas, principalmente nas indústrias alimentícia e de detergentes. A α-amilase é encontrada em diversos organismos, e pode ser obtida através de bactérias, fungos e tecidos animais e vegetais. Com o intuito de reduzir o impacto causado pelo avanço da indústria pesqueira, essas e outras classes de enzimas vêm sendo extraídas através do reaproveitamento dos resíduos pesqueiros. Neste trabalho, α-amilases foram obtidas através dos cecos pilóricos e intestinos do tambaqui, Colossoma macropomum, caracterizadas e avaliadas quanto ao seu uso como aditivo de limpeza em detergentes comerciais. Para isso, o extrato bruto obtido foi submetido a fracionamento salino utilizando sulfato de amônio, a fração 30-60% apresentou maior atividade, foi em seguida dialisada e aplicada a uma coluna de cromatografia p-aminobenzamidine-agarose (Sigma®). Após esse processo de purificação, foram realizados zimogramas e eletroforese SDS-PAGE, os resultados obtidos sugerem uma purificação parcial, e presença de isoformas. As α-amilases apresentaram atividade ótima no pH 7.0 e 8.5, respectivamente para AMY-1 (intestinos) e AMY-2 (cecos pilóricos). Foram termoestáveis durante 30 minutos a 40°C, e apresentando temperatura ótima de 40 e 45°C, respectivamente. As α-amilases foram fortemente inibidas pelos íons Hg+3 e Cu+2. Quanto ao seu uso como aditivo de detergentes, as α-amilases extraídas do tambaqui, foram significativamente estáveis durante incubação de 60 minutos com agente oxidante (H2O2 15%), surfactantes coleato de sódio, tween 20, tween 80 e SDS, sendo também compatíveis e estáveis com todas as marcas de sabão utilizadas neste trabalho. Estes resultados sugerem que estas enzimas possuem potencial aplicação na indústria de detergentes, podendo ser utilizadas como aditivo de limpeza. Adicionalmente, pode-se concluir que o reaproveitamento dos resíduos do tambaqui corresponde a uma fonte inovadora de α-amilase com alta atividade e aplicabilidade industrial.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2016-09-26T14:16:09Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Simone_Pacheco_Frutos da família Myrtaceae_Caracterização físico-química e potencial .pdf: 1364587 bytes, checksum: 6df19769efc9216d28d9614070c8e6af (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-26T18:34:47Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Simone_Pacheco_Frutos da família Myrtaceae_Caracterização físico-química e potencial .pdf: 1364587 bytes, checksum: 6df19769efc9216d28d9614070c8e6af (MD5) / Made available in DSpace on 2016-09-26T18:34:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Simone_Pacheco_Frutos da família Myrtaceae_Caracterização físico-química e potencial .pdf: 1364587 bytes, checksum: 6df19769efc9216d28d9614070c8e6af (MD5) Previous issue date: 2015-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / As espécies vegetais Campomanesia xanthocarpa (guabiroba), Eugenia uniflora (pitanga), Eugenia pyriformis (uvaia), Psidium cattleianum (araçá) e Syzygium cumini (jambolão) estão presentes na Floresta Atlântica e são utilizadas pela população, para tratar diversas patologias, especialmente o diabetes melito tipo 2. Entretanto, a eficácia destes tratamentos e o mecanismo envolvido ainda não foram totalmente elucidados. Neste contexto, o presente estudo teve como objetivo principal avaliar o potencial dos compostos naturais destes frutos em inibir as enzimas α-amilase e α- glicosidase que estão envolvidas no metabolismo de carboidratos. Estes frutos também foram avaliados físico-quimicamente, realizando-se dentre outras análises a quantificação dos compostos fenólicos totais e a determinação da atividade antioxidante (métodos ABTS e DPPH). Os extratos metanólicos de P. cattleianum (acesso 44), S. cumini e E. pyriformis (acessos 11 e 15) inibiram de forma significativa a atividade da α-amilase. Os extratos metanólicos de P. cattleianum (acessos 44 e 87) também inibiram a atividade da α-glicosidase, utilizando-se os substratos maltose e sacarose. Em virtude da atividade antioxidante, da elevada quantidade de compostos fenólicos e da capacidade de inibição das enzimas digestivas do metabolismo de carboidratos, os frutos de P. cattleianum (acessos 44 e 87), S. cumini e E. pyriformis (acessos 11 e 15) podem apresentar potencial uso no manejo da hiperglicemia pós-prandial. / Campomanesia xanthocarpa (guabiroba), Eugenia uniflora (pitanga), Eugenia pyriformis (uvaia), Psidium cattleianum (araçá) and Syzygium cumini (jambolão) grow in the Brazilian Atlantic Forest and their fruits are commonly used as medicine to treat diseases related to carbohydrate metabolism, such as diabetes. The effectiveness of these treatments has not been demonstrated neither the biochemical mechanism involved. Therefore this study was devised to evaluate the potential of natural compounds of these fruits to inhibit key enzymes α-amylase and α- glucosidase involved in the carbohydrate metabolism. The fruits were also subjected to physicochemical characterization, quantification of phenolics compounds and antioxidant activity (ABTS and DPPH methods). The methanolic extracts of P. cattleianum (access 44), S. cumini, E. pyriformis (accesses 11 and 15) distinctively inhibited α-amylase activity. The methanolic extracts of P. cattleianum. (accesses 44 and 87) also inhibited α-glycosidase activity, with either maltose or sucrose as substrate. By having antioxidant activities, a fairly content of phenolic compounds, and capacity to inhibit carbohydrate digestive enzymes, P. cattleianum (access 44 and 87), S. cumini and E. pyriformis (accesses 11 and 15) could be good candidates to be used in the management of postprandial hyperglycemia.

Martensitic transformation is very important in austenitic stainless steels where the transformation induced plasticity phenomenon provides a combination of good mechanical properties, such as formability and strength. However, the difficulty of predicting the material behaviour is one of the major drawbacks of these steels. In order to model this behaviour it is of great importance to be able to characterize the morphology, crystallography and the amount of different types of martensite. The morphology and crystallography of thermal and deformation induced lath martensite in stainless steels were re-examined by means of optical microscopy and electron backscatter diffraction (EBSD) technique. The experiments were performed on AISI301, 304 and 204Cu austenitic stainless steels. Plastic deformation was carried out by means of uniaxial tensile tests at the strain rate of  to produce strain induced α’-martensite at a temperature ranging from 0 to 60ºC. An in-situ measurement of the martensite content was performed during the tensile testing using a Ferritescope to provide the necessary experimental values for modelling. Optical microscopy revealed the morphology of the strain induced α’-martensite as sets of thin parallel needles that go through the parent austenite grain and stop at the grain or annealing twin boundaries. Large amount of α’-martensite could be seen at the intersection of shear bands. However, considerable amount of α’-martensite was also observed when only one set of bands is activated. EBSD was successfully used to analyze the morphology and crystallography of martensite. The α’-martensite maintained the Kurdjumov-Sachs (K-S) orientation relationship with the austenite phase. Although all six possible variants did not appear within a single packet, one or two variants were often favoured out of six related to the specific {111} plane. The misorientations between the neighbouring variants were mainly <111> 60º or <110> 49.5º.

α-MSH immunoreactive peptides were fractionated and characterized in rat and human brain and rat pituitary by reversed phase high pressure liquid chromatographic techniques. α-MSH and deacetylated α-MSH were two major naturally existing peptides in both brain and pituitary gland. Subsequent experiments examined the roles of these two peptides in neuronal function. The α-MSH was clearly more effective than deacetylated α-MSH in improving performance on a visual discrimination task after intraperitoneal administration and in inducing excessive grooming after intraventricular administration. The difference in behavioral potency may be explained by the fact that α-MSH was much more resistant to peptidase degradation than was deacetylated α-MSH. N-acetylation of α-MSH may be an effective regulatory process for modulating the behavioral potency of the secretory product of α-MSH-containing pituitary cells and neurons.

De nombreuses méthodes in vitro d’évolution d'enzymes existent mais elles sont souvent coûteuses en temps et en main d'œuvre. Une approche alternative, l’évolution dirigée in vivo, permet la sélection de variantes d'enzymes améliorées ou modifiées à moindre coût et peut être appliquée à des enzymes de structure inconnue. Des dispositifs automatisés de culture continue (GM3), ont été développés et construits au Genoscope. Le GM3 a été conçu pour maintenir indéfiniment des cultures bactériennes en suspension tout en les soumettant à des régimes de culture sélectifs de type turbidostat et chémostat. Le principal objectif du présent travail était l’optimisation par évolution dirigée in vivo d’enzymes de la famille des dioxygénases à α-cétoglutarate et fer-dépendantes (α-KAO). Les α-KAOs catalysent l'hydroxylation d’un atome de carbone non substitué et sont donc d’intéressants outils pour la fonctionnalisation de molécules en synthèse organique. La dynamique de fixation d’une mutation bénéfique dans une population en croissance en fonction du régime de culture et du format d’expression a été étudiée. Des souches d’Escherichia coli ont été génétiquement modifiées pour que leur croissance dépende strictement d’une activité α-KAO. Des populations de ces bactéries exprimant une α-KAO ont été soumises à des protocoles d’évolution dirigée in vivo pour sélectionner: dans un premier temps, des variants d’expression optimisée sur le substrat naturel de l’enzyme, dans un second temps, des variants utilisant des analogues de substrat non naturels. Deux variantes de la L-isoleucine dioxygénase (E65K, A62P) ont été obtenues. L’étude des paramètres cinétiques déterminera l’effet de ces mutations sur l’activité de l’enzyme. En collaboration avec l’équipe du Dr. Tolonen, la culture continue a été appliquée à l’évolution de la bactérie cellulolytique Clostridium phytofermentans. Cette bactérie anaérobie stricte qui dégrade la lignocellulose en éthanol et H2 présente un intérêt applicatif pour la valorisation de la biomasse. La dégradation de la lignocellulose libère des composés phénoliques, en particulier l’acide férulique. C. phytofermentans a été adapté à résister à des concentrations croissantes de ce composé inhibiteur. Des isolats résistant au ferulate ont été analysés et leur génomes séquencés (Cerisy et al., 2017). / Numerous methods of directed evolution in vitro have been developed. These methods are often expensive and time-consuming. An alternative approach, directed evolution in vivo, allows the selection of protein variants at lower cost and does not require prior knowledge of the protein’s structure and mechanism. An automated continuous culture device, the GM3, has been developed and built at the Genoscope. The GM3 technology enables the maintenance of steady-state microbial growth over an extended period of time.The main objective of the present study was the optimization by directed evolution in vivo of enzymes of the family of the Iron(II)/α-ketoacid-dependent oxygenases (α-KAOs). The α-KAOs catalyze the hydroxylation of non-activated C-H bonds and thus are interesting tools for the functionalization of organic molecules in synthetic chemistry.The dynamics of fixation of beneficial mutations in a growing cell population depending on the expression format and the culture regime has been explored.Strains of Escherichia coli have been genetically modified such that their growth strictly depended on a α-KAO activity. Populations of these bacteria expressing a α-KAO were subjected to protocols of directed evolution in continuous culture in order to select, in a first round, variants showing optimized expression on natural substrates of the enzyme, and in a second round, variants with activity towards non-natural analogs of the substrate. Two variants of L-isoleucine dioxygenase (E65K, A62P) were obtained. The determination of the kinetic parameters will reveal the influence of these mutations on the activity of the enzyme variants.In collaboration with the group of Dr. Tolonen, directed evolution in continuous culture was applied to the cellulolytic bacteria Clostridium phytofermentans. This anaerobic bacterium degrades lignocellulose into ethanol and H2 and can thus be applied for biomass valorization. The degradation of lignocellulose liberates phenolic compounds like ferulic acid known to be toxic to the bacteria. C. phytofermentans was adapted in the GM3 to resist to growing concentrations of this inhibitor. Resistant bacteria were analyzed and their genomes sequenced (Cerisy et al., 2017).

Fabry disease (FD) is an X-linked recessive lysosomal storage disorder (LSD) known to affect approximately 1 in every 40,000 males, and a smaller number of females. FD results from a deficiency of functional α-galactosidase (α-GAL), which leads to the accumulation of terminally α-galactosylated substrates in the lysosome. The predominant treatment is Enzyme Replacement Therapy (ERT), requiring the regular infusion of recombinant human α-GAL. More than half of individuals receiving ERT experience a range of adverse infusion reactions, and it has been reported that as many as 88% of patients receiving ERT develop neutralizing IgG antibodies against the drug. In aim of designing a non-immunogenic treatment candidate for Fabry disease ERT, we have engineered the active sites of α-GAL and another homologous family 27 exoglycosylase named α-N-acetylgalactosaminidase (α-NAGAL) to have interconverted substrate specificities. 11 of 13 active site residues are conserved between these two enzymes, and we have shown that their substrate specificities can be interconverted by mutating the two non-conserved active-site residues. We report the kinetic properties of these two mutants along with wild type controls, and use western blotting to show that both mutant enzymes retain their respective wild type enzyme antigenicity. Structural data obtained by X-ray crystallography on the α-GAL mutant (called α-GALSA ) reveals the mechanism by which substrate specificity is dictated between these two proteins, and provides explanations for the mutant’s reduced catalytic efficiency.