Le Laboratoire de génie tissulaire est reconnu pour ses réalisations en ce domaine. Le principal défi soulevé par cette approche est le choix de la matrice des tissus reconstruits. Mes travaux ont consisté à établir une technologie de synthèse de collagène humain recombinant à des fins expérimentales et cliniques. Ce collagène sera utilisé éventuellement pour produire des substituts du ligament croisé antérieur (LCA) du genou, par génie tissulaire. Ces substituts ligamentaires pourraient être une alternative de remplacement des LCA rupturés. Le Dr. Nazrul Islam a conceptualisé une stratégie moléculaire pour construire un plasmide incluant les gènes codant pour les deux chaînes du collagène humain de type 1 et les deux sous-unités de l'enzyme prolyl-4-hydroxylase. Des cellules d'insecte ont été transfectées avec ce vecteur, en exploitant le système de bacul ovi rus, pour synthétiser le collagène recombinant. J'ai participé à chaque étape et à la mise au point des protocoles optimisé à grande échelle de cette nouvelle technologie, pour ensuite purifier le collagène et le caractériser biochimiquement. Mes superviseurs et moi-même considérons que ces travaux ont réussi et que bientôt, des substituts ligamentaires humains seront greffés pour évaluer leur intégration dans une articulation du genou in vivo.

L'utilisation thérapeutique de glycoprotéines recombinantes produites dans le système baculovirus-cellules d'insectes reste limitée par le potentiel de glycosylation de ces cellules, qui produisent essentiellement des structures O- et N-glycanniques courtes. Notre travail s'inscrit donc dans un effort global d'"humanisation" de la glycosylation des protéines produites dans les cellules de Lépidoptères.<br />Nous nous sommes d'abord attachés à comprendre la voie de maturation des N-glycannes dans des cellules Sf9. L'utilisation d'inhibiteurs de la maturation ou du trafic intracellulaire nous a permis d'identifier des intermédiaires clés et de confirmer l'hypothèse que la maturation des N-glycannes dans les cellules d'insectes et de mammifères suivent un chemin métabolique parallèle jusqu'à la formation de l'espèce GlcNAcMan3[Fuc]GlcNAc2. Chez les insectes, cette structure est ensuite substrat d'une β-N-acétylglucosaminidase qui produit l'espèce finale Man3[Fuc]GlcNAc2.<br />Cette voie de maturation peut néanmoins être déviée vers la synthèse de N-glycannes de type complexe par l'addition de glycosyltransférases absentes : ainsi, l'expression d'une β1,4-galactosyltransférase permet la synthèse de l'espèce GalGlcNAcMan3[Fuc]GlcNAc2.<br />Notre intérêt s'est ensuite porté sur l'ingénierie de la sialylation dans les cellules d'insectes, compliquée par l'absence du donneur d'acides sialiques, le CMP-Neu5Ac. Notre stratégie a été d'exprimer la trans-sialidase de Trypanosoma cruzi sur la membrane plasmique des cellules, afin qu'elle puisse sialyler les glycoprotéines sécrétées en utilisant des donneurs du milieu. La construction exprimée grâce à un vecteur baculovirus code une enzyme active, dont une partie est retrouvée sur la membrane plasmique et sur l'enveloppe des particules virales, tandis qu'une partie, croissante avec l'infection, est soluble. Néanmoins, le système permet une sialylation quantitative d'accepteurs exogènes.<br />Notre étude contribue à montrer que l'ingénierie de la glycosylation dans le système baculovirus-cellules d'insectes est envisageable. Pour résoudre le problème de la sialylation, la trans-sialidase est une alternative possible aux sialyltransférases.

Recentemente, vários arbovírus têm acometido a população de países emergentes ocasionando sérios problemas de saúde pública, como as doenças causadas pelos vírus da dengue, Chikungunya, Zika e febre amarela. Um vírus emergente e já circulante no Brasil, chamado Mayaro (MAYV), do mesmo gênero do Chikungunya (Alphavirus), possui potencial prejudicial semelhante a esses já estabelecidos. Seu vetor de transmissão é o mosquito do gênero Haemagogus, característico de regiões isoladas, principalmente florestas. Entretanto, estudos demonstraram que o Aedes aegypti é um competente vetor desse agente, o que possibilita sua disseminação em regiões urbanas. O presente trabalho avaliou a expressão das proteínas estruturais do vírus Mayaro (E1, E2, E3, C e 6K), utilizando dois sistemas de expressão distintos, um baseado na levedura Pichia pastoris, e outro derivado de Baculovírus (BEVS). Essa estratégia foi estabelecida para que a expressão dessas proteínas promova a formação de partículas semelhantes ao vírus (virus like particles), estruturas multiprotéicas que mimetizam a conformação de uma partícula viral podendo ser utilizada como um candidato vacinal. O trabalho evidenciou a correta obtenção de organismos recombinantes em ambos os sistemas, com a avaliação da expressão sendo feita com técnicas de dot blot, western blot e imunofluorescência indireta (IFI). Com o sistema baculovírus, foram avaliadas as linhagens Sf-9 e Hi-5, sendo evidenciada a expressão de proteínas do MAYV em ambas, utilizando MOI 10 e tempos pós-infecção de 96 e 72 h, respectivamente. A correta expressão das proteínas de MAYV também foi evidenciada com a levedura Pichia pastoris, com cultivo a 30 &deg;C e tempo de análise 48 h após indução. A geração de VLPs foi avaliada em amostras de sobrenadantes de ambos os sistemasapós a concentração por ultracentrifugação em gradiente de iodixanol, e análise por microscopia eletrônica de transmissão, sendo observadas nos dois sistemas com tamanhos variando entre 30-60 nm. Os resultados desse projeto podem gerar ferramentas importantes no desenvolvimento de kits diagnósticos e métodos vacinais contra o MAYV. / Recently, several arboviruses have affected emerging countries causing serious public health problems, such as diseases caused by dengue viruses, Chikungunya, Zika and yellow fever. An emerging and already circulating virus in Brazil, called Mayaro (MAYV), of the same genus of Chikungunya (Alphavirus), has harmful potential similar to those already established. Its transmission vector is the mosquito of the genus Haemagogus, characteristic of isolated regions, mainly forests. However, studies have demonstrated that Aedes aegypti is a competent vector of this agent, which would allow its dissemination in urban areas. The present work evaluated the expression of the structural proteins of the Mayaro virus (E1, E2, E3, C and 6K) using two distinct expression systems, one based on the yeast Pichia pastoris and another derived from Baculovirus (BEVS). The strategy of expressing structural proteins has been established so that the expression of these proteins promotes the formation of viruslike particles or VLPs, multiprotein structures that mimic the conformation of a viral particle and can be used as a vaccine candidate. The work evidenced the correct obtaining of recombinant organisms in both systems, with the evaluation of the expression being done with dot blot, western blot and indirect immunofluorescence (IFI) techniques. With the baculovirus system, the Sf-9 and Hi-5 strains were evaluated, and the expression of the MAYV proteins was evidenced in both, using MOI 10 and the time of post-infection analysis of 96 and 72 h, respectively. Correct expression of MAYV proteins was also evidenced with yeast Pichia pastoris, with culture at 30 &deg; C and analysis time 48 h after induction. The generation of VLPs was evaluated in both supernatants after concentration by iodixanol gradient ultracentrifugation and transmission electron microscopy analysis, being observed in both systems with sizes ranging from 30-60 nm. The results of this project can generate important tools in the development of diagnostic kits and vaccine methods against the MAYV.

Submitted by Verena Bastos (verena@uefs.br) on 2015-09-21T13:18:11Z No. of bitstreams: 1 DISSERTACAO FINAL ALINE THOMAZ.pdf: 1680438 bytes, checksum: 85bf2d7886a394df745618b6fd50d435 (MD5) / Made available in DSpace on 2015-09-21T13:18:11Z (GMT). No. of bitstreams: 1 DISSERTACAO FINAL ALINE THOMAZ.pdf: 1680438 bytes, checksum: 85bf2d7886a394df745618b6fd50d435 (MD5) Previous issue date: 2015-09-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The vitamin D receptor (VDR) is a cytoplasmic transcription factor and when activated by its ligand, translocates to the nucleus and interacts with DNA in the promoter regions with which has affinity, acting as a modulator of gene transcription and thereby producing multiple biological effects. Its main activities are the regulation and maintenance of plasma levels of calcium and phosphorus, as well as presenting immunomodulatory activities, such as the suppression of T cell activation, formation of secretion patterns of cytokines, modulation of proliferation and interference in the apoptosis. So this receptor is an important target for drugs that can help in the discovery of new immunomodulators. The present work had the objective to produce the recombinant vitamin D receptor in its soluble form for conducting future assays of drug screening of potential immunomodulators and drug-receptor interaction studies. For this, we used initially Escherichia coli expression system transformed with the plasmid HS_VDR_EC1-PQE T7, but it was only possible to obtain the protein in the insoluble fraction, even varying the temperature, time of induction and IPTG concentration. In an attempt to obtain soluble VDR, we used a eukaryotic expression system in insect cells using the baculovirus as a vector. It was built a vector, pFASTBacHT_VDR, which had the sequence of this protein cloned from pCMX.VDR. From there, it was possible to obtain recombinant bacmids used in transfection of insect cells, generating recombinant baculovirus, to then proceed with the expression of VDR. / O receptor de vitamina D (VDR) ? um fator de transcri??o g?nica citoplasm?tico e, quando ativado pelo seu ligante, transloca-se para o n?cleo e interage com regi?es promotoras no DNA com a qual apresenta afinidade, atuando como fator modulador da transcri??o g?nica e assim produzindo m?ltiplos efeitos biol?gicos. Suas principais atividades s?o a regula??o e a manuten??o dos n?veis plasm?ticos de c?lcio e f?sforo, e apresenta atividades imunomoduladoras, como a supress?o da ativa??o de c?lulas T, forma??o de padr?es de secre??o de citocinas, a modula??o da prolifera??o e interfer?ncia na apoptose. Sendo assim, esse receptor representa um importante alvo de drogas que pode contribuir na descoberta de novos f?rmacos com a??o imunomoduladora. O presente trabalho teve, como objetivo, produzir o VDR recombinante na forma sol?vel para a realiza??o de futuros ensaios de triagem de potenciais drogas com a??o imunomoduladora e estudos de intera??o droga-receptor. Para isso, foi utilizado, inicialmente, o sistema de express?o em Escherichia coli, utilizando o plasm?deo HS_VDR_EC1-PQE T7, por?m s? foi poss?vel obter a prote?na na fra??o insol?vel, mesmo variando a temperatura, o tempo de indu??o e a concentra??o de IPTG. Na tentativa de obter o VDR sol?vel, foi utilizado um sistema de express?o eucari?tico em c?lulas de inseto, utilizando como vetor o baculov?rus. Foi constru?do um vetor pFASTBacHT_VDR, o qual teve a sequ?ncia desta prote?na clonada a partir do pCMX.VDR. A partir da?, foi poss?vel obter bacm?deos recombinantes, utilizados na transfec??o de c?lulas de inseto, gerando baculov?rus recombinantes, para, ent?o, seguir com a express?o do VDR.

Le VHB est un problème majeur puisque les 350 millions de porteurs chroniques existant ont un risque accru de développer une cirrhose ou un carcinome hépatocellulaire. Compte tenu du manque de système d'étude du VHB in vitro qui soit facile d'accès et pleinement satisfaisant, l'objectif était d'améliorer l'un de ceux qui utilisent des baculovirus VHB recombinants pour délivrer le génome VHB dans des cellules hépatocytaires. La pertinence de ce système pour réaliser des tests phénotypiques et étudier le « fitness » des mutants résistants aux antiviraux a ensuite été démontrée. Enfin, l'utilisation de ces baculovirus VHB recombinants dans des cellules HepaRG a permis de mettre en évidence une réponse IFN efficace de l'hépatocyte suite à la synthèse des protéines du VHB. Ceci constitue une donnée nouvelle dans l'étude des interactions virus/cellules puisque le VHB était considéré jusqu'à présent comme un virus silencieux vis-à-vis de la réponse innée cellulaire. L'ensemble de ces résultats a des implications importantes dans la compréhension des mécanismes de persistance du VHB et le développement de nouveaux modèles cellulaires d'infection.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

No description available.

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by a defect in alpha-N-acetylglucosaminidase (NAGLU), a lysosomal enzyme involved in the degradation of heparan sulphate. Dysfunctional NAGLU gives rise to a clinical phenotype of severe and progressive mental retardation, often accompanied by hyperactivity and aggressive behaviour. At present, there is no effective treatment for MPS IIIB. However, cloning of the human NAGLU cDNA has made the potential production of human recombinant enzyme for use in enzyme replacement therapy (ERT) a viable option. The work outlined herein focuses on attempts to produce human recombinant NAGLU (rNAGLU) using both yeast and insect cell based expression systems; with the major focus on yeast based expression. Use of a humanized yeast strain, codon optimisation of a portion of the NAGLU gene, selection of Mut+, MutS and multiple integrant strains, and growth at decreased temperature were explored to optimise NAGLU expression in the methylotrophic yeast, Pichia pastoris. As none of these measures resulted in abundant NAGLU production, Sf9 and Tni insect cell lines were investigated as an alternate expression system. Additionally, a protein transduction domain (PTD) was fused to NAGLU (NTAT) to circumvent current problems faced in delivering therapeutic enzymes to the brain. NAGLU protein, with and without a fused PTD, were expressed using stable transfection and baculovirus infection techniques. Small scale experiments utilizing the baculovirus expression vector system (BEVS) have yielded promising results, generating functionally active NAGLU and NTAT protein of the expected approximately 80-85 kDa molecular mass. This preliminary success indicates the BEVS may be an attractive option for the large scale production of rNAGLU and rNTAT.

L'auteur n'a pas fourni de résumé en français / L'auteur n'a pas fourni de résumé en anglais

Made available in DSpace on 2015-03-26T13:40:00Z (GMT). No. of bitstreams: 1 texto completo.pdf: 984419 bytes, checksum: 33605a6daae90efa42a31afbfa576ba6 (MD5) Previous issue date: 2013-07-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The use of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) based biopesticide has the potential to control Spodoptera frugiperda (Lepidoptera: Noctuidae), but large scale production depends on maximizing virus production from infected larvae. The suitable temperature, time of exposure to the pathogen, the age at which the larvae are inoculated with the virus, the viral solution concentration and isolate used are important for viral replication. The experiments were performed in the Biological Control Laboratory of Insects at Embrapa Maize and Sorghum Research Center (CNPMS), located in Sete Lagoas, Minas Gerais, Brazil. The first study aimed to verify the effect of inoculation and incubation (multiplication) temperatures in viral production and the inoculation period. Cannibalism among the larvae of S. frugiperda was similar (around 4%) in different periods and temperatures baculovirus inoculation. The inoculation period did influence mortality and viral production. The higher mortalities of larvae were achieved at 28 °C, however, the production of polyhedral inclusion bodies (PIB) per larvae were higher at 25 °C, reducing the number of larvae needed for the production of a dose of baculovirus expressed larval equivalent (LE/ha). The total production of polyhedral inoculation at the different temperatures was similar, but the incubation at 25 °C produced a larger amount of virus (24.56 x 109 PIB at 25 °C and 20.81 x 109 PIB at 28 °C). In the second study, the lethal concentrations were determined for larvae SfMNPV six, seven and eight days and the best age to perform the inoculation of SfMNPV order to maximize viral production. The lethal concentration (LC50) ranged from 1.89 x 105 to 5.01 x 106 PIB/mL. The LC50 I6 was higher than the I19 to six days old larvae of similar for seven days and for those less than eight days. Total polyhedra production was higher with eight days old larvae for the isolate I6. The highest yields were obtained in viral concentrations above 1.00 x 107 PIB/mL. The production of PIB/larvae increased when I6 was used in eight days old larvae, which reduces the number of larvae needed to achieve dose of baculovirus (LE/ha). The third study aimed to verify the production parameter which is more correlated with viral production, mass, or the number of dead larvae by SfMNPV and also determine the parameters weight equivalent per hectare (estimated larval weight necessary to achieve a dose of baculovirus, based on the application of 2.00 x 1011 PIB/ha) and larvae dead weight due to SfMNPV infection caused by I6. All correlations were positive and significant for the total weight (r= 0.958, n= 60, P< 0.0001) even when considered in each age or concentration separately. For the number of tracks the correlation was also significant when considering the amount of treatment (r= 0.723, n= 60, P< 0.0001) within each age group and in each concentration was significant only at concentrations of 1.00 x 106 PIB/mL (r= 0.643, n= 12, P< 0.0242) and 1.00 x 108 (r= 0.657, n= 12, P< 0.0202), but the correlations are less strong. Larvae of S. frugiperda, killed by SfMNPV inoculated at eight days old had the highest average mass. The equivalent weight per hectare did differ among different viral concentrations of solutions used. The maximization of viral production baculovirus for control of S. frugiperda should be made with the isolated I6 in larvae eight days old maintained at a temperature of 25 °C and the weight of dead larvae by baculoviruses can be considered a more reliable parameter than the number of larvae to produce a commercial product based SfMNPV in view that shows strong and significant correlation with the total polyhedra production. / A utilização de bioinseticida a base de Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) tem potencial para o controle de Spodoptera frugiperda (Lepidoptera: Noctuidae), porém seu uso em larga escala depende de se maximizar sua produção a partir de larvas infectadas. A temperatura adequada, o tempo de exposição ao patógeno, a idade na qual as lagartas são inoculadas com o vírus, a concentração da suspensão viral e o isolado são importantes para a replicação viral. Os experimentos foram realizados no Laboratório de Controle Biológico de Insetos do Centro Nacional de Pesquisas com Milho e Sorgo (CNPMS) da Embrapa em Sete Lagoas, Minas Gerais, Brasil. O primeiro estudo objetivou verificar o efeito das temperaturas de inoculação e incubação (multiplicação) na produção viral e do tempo de inoculação. O canibalismo entre as lagartas de S. frugiperda foi semelhante (em torno de 4%) nos diferentes tempos e temperaturas de inoculação do baculovírus. O tempo de inoculação não influenciou a mortalidade e a produção viral. A mortalidade de lagartas foi maior a 28 °C, porém, a produção de corpos poliédricos de inclusão (PIB) por lagarta foi maior a 25 °C, o que reduz o número de lagartas para a produção de uma dose de baculovírus formulado (dose para um hectare). A produção total de poliedros, nas diferentes temperaturas de inoculação, foi semelhante, mas a incubação a 25 °C produziu uma maior quantidade de vírus (24,56 x 109 PIB a 25 °C e 20,81 x 109 PIB a 28 °C). No segundo estudo as concentrações letais de SfMNPV foram determinadas para lagartas de seis, sete e oito dias e a melhor idade para se realizar a inoculação de SfMNPV visando a maximização de sua produção. As concentrações letais médias (CL50) variaram de 1,89 x 105 a 5,01 x 106 PIB/mL. A CL50 do isolado 6 (I6) foi maior que a do isolado 19 (I19) para lagartas de seis dias de idade, semelhante para lagartas de sete dias e menor para aquelas de oito dias. A produção total de poliedros foi maior com lagartas inoculadas aos oito dias de idade com o I6. As maiores produções virais foram obtidas nas concentrações acima de 1,00 x 107 PIB/mL. A produção de PIB/lagarta foi maior com o I6 em lagartas de oito dias, o que reduz o número de lagartas para a produção de uma dose de baculovírus formulado (dose para um hectare). O terceiro estudo objetivou verificar o parâmetro de produção mais correlacionado com a produção viral, a massa, ou o número de lagartas mortas por SfMNPV e determinar os parâmetros peso equivalente por hectare (massa estimada de lagartas necessárias para obtenção de uma dose de baculovírus formulado, tendo como base a aplicação de 2,00 x 1011 PIB/ha) e peso por lagarta morta devido à infecção pelo I6 de SfMNPV. Todas as correlações foram positivas e altamente significativas para o peso total (r= 0,958, n= 60 e P< 0,0001), mesmo quando consideradas dentro de cada idade ou concentração, separadamente. A correlação para o número de lagartas foi, também, significativa considerando-se o total de tratamentos (r= 0,723, n= 60 e P< 0,0001) e por idade. Por concentração, foi significativa apenas nas concentrações de 1,00 x 106 PIB/mL (r= 0,643, n= 12, P< 0,0242) e 1,00 x 108 PIB/mL (r= 0,657, n= 12, P< 0,0202), porém, as correlações foram menos fortes. Lagartas de S. frugiperda, mortas por SfMNPV, inoculadas aos oito dias de idade apresentaram a maior massa média. O peso equivalente de SfMNPV a ser utilizado por hectare não diferiu entre as concentrações de suspensões virais. A produção viral de baculovírus, para o controle de S. frugiperda, deve ser feita com o isolado I6 em lagartas de oito dias na temperatura de 25 oC. O peso de lagartas mortas por baculovírus pode ser considerado um parâmetro mais confiável que o número de lagartas para a produção de um produto comercial a base de SfMNPV, devido a correlação mais forte e significativa com a produção total de poliedros.