Os primeiros vasos sanguíneos do embrião de vertebrados, formados de novo a partir de células originadas da mesoderme, originam os vasos linfáticos em um processo denominado vasculogênese. Já no adulto, novos vasos são formados principalmente através da angiogênese (ou linfoangiogênese) a partir da vasculatura pré-existente. Em indivíduos saudáveis, a arquitetura vascular é relativamente estática, sendo que o excesso ou a insuficiência de vasos são comumente relacionados à angiogênese patológica, vinculada a diversas doenças, como câncer, degeneração macular relacionada à idade, isquemia de membros e muitas outras. Dessa forma, o controle local da densidade de vasos sanguíneos torna-se interessante para o tratamento de condições patológicas visando melhoria de prognóstico e cura. Dentre os diversos fatores de crescimento conhecidos, o fator de crescimento endotelial vascular, VEGF, destaca-se como o principal regulador do processo de angiogênese através de isoformas pró-angiogênicas (VEGFxxx) e antiangiogênicas (VEGFxxxb) do gene VEGF-A. Consequentemente, as proteínas codificadas por esse gene constituem um alvo com alto potencial terapêutico. No presente estudo, propusemos a produção das isoformas proteicas recombinantes rhVEGF165, rhVEGF165b e rhVEGF121, oriundas do gene VEGF-A humano, visando à geração de biofármacos que podem ser utilizados para terapia molecular e engenharia tecidual. As sequências codificadoras das isoformas rhVEGF165 e rhVEGF121 foram amplificadas a partir do cDNA total sintetizado de amostras de RNA total de pulmão humano, enquanto que a da isoforma rhVEGF165b foi gerada através da mutação sítio-dirigida da sequência de rhVEGF165. As sequências foram clonadas no vetor de clonagem pGEM®-T Easy, sendo em seguida subclonadas no vetor pLV-eGFP, um vetor plasmidial de transferência lentiviral, que permite a expressão de transgenes em células de mamíferos e, também, da proteína repórter eGFP. Células humanas HEK293 em cultura aderente foram independentemente cotransfectadas com cada uma das construções geradas (pLV-rhVEGF165, pLV-rhVEGF165b e pLV-rhVEGFl21) juntamente com o vetor pTK-Hyg na proporção de 40:1 (m/m), viabilizando a seleção dos transfectantes com o antibiótico higromicina B, além da detecção de eGFP. Os clones celulares superprodutores das proteínas de interesse foram avaliados quanto à cinética de expressão em meio carente de soro e adaptados para a cultura em suspensão estática na presença de meio na ausência de componentes derivados de animais, demostrando a capacidade de expressão das isoformas rhVEGFs nestas condições de cultivo. Para o nosso conhecimento, este é o primeiro trabalho a descrever a expressão de isoformas de VEGF-A em células HEK293 mantidas em suspensão. As isoformas rhVEGF165 e rhVEGF165b foram purificadas por cromatografia de afinidade a heparina, a partir do meio condicionado pelos clones superprodutores gerados. Ensaios in vitro, utilizando o AngioPhaseTM Kit, e in vivo, através do ensaio da membrana corioalantoide em embriões de galinha (CAM Assay), ambos próprios para avaliação da atividade pró- e antiangiogênica de diferentes compostos, demonstraram que a isoforma rhVEGF165 possui atividade biológica, enquanto a isoforma rhVEGF165b não apresentou a atividade esperada (inibição da angiogênese). Estas isoformas foram testadas em modelo murino de engenharia tecidual do intestino curto, com indícios de que poderiam contribuir para o uso terapêutico neste contexto. A purificação da isoforma rhVEGF121, bem como as análises estruturais das proteínas produzidas, estão em processo de otimização. / The first blood vessels of the vertebrate embryo are formed de novo from mesoderm-derived cells and give rise to lymph vessels in a process termed vasculogenesis. In the adult, new blood vessels are formed mainly through angiogenesis (or lymphangiogenesis) from the pre-existing vasculature. In healthy individuals, the vascular architecture is fairly static, and both the excess and the insufficiency of vessels comprise a pathological angiogenic state, to which is credited the onset and/or progression of several diseases such as cancer, age-related macular degeneration, limb ischemia, and many others. Therefore, locally controlling the blood vessel density becomes interesting for the treatment of pathological conditions aiming at prognosis improvement and cure. Among the various known growth factors, the vascular endothelial growth factor, VEGF, stands out as the major regulator of the angiogenic process. This process is mediated through the action of pro- (VEGFxxx) and antiangiogenic (VEGFxxxb) isoforms, which are derived from the VEGF-A gene. Consequently, the proteins encoded by this gene are potential therapeutic targets. In this work, we set out to produce the recombinant protein isoforms rhVEGF165, rhVEGF165b, and rhVEGF121, which originate from the human VEGF-A gene, with the aim of generating biopharmaceuticals to be used for molecular therapy and tissue engineering. The rhVEGF165 and rhVEGF121 coding sequences were amplified from total cDNA sythesized from human lung total RNA. Conversely, the rhVEGF165b coding sequence was generated by site-directed mutagenesis of the rhVEGF165 sequence. The sequences were cloned into the pGEM®-T Easy cloning vector. These cDNAs were then subcloned into pLV-eGFP, a plasmid lentiviral transfer vector that allows for expression of transgenes and the eGFP reporter protein in mammalian cells. Human HEK293 cells cultivated under adherent conditions were independently co-transfected with each of the obtained constructs (pLV-rhVEGF165, pLV-rhVEGF165b, and pLV-rhVEGF121) and the pTK-Hyg vector at a proportion of 40:1 (m/m), enabling for the selection of transfectants with hygromycin B, apart from the detection of eGFP. The cell clones overexpressing the proteins of interest were evaluated for expression kinetics in serum-deprived conditioned media and adapted to static suspension culture in medium free of animal-derived components, demonstrating that expression of the protein isoforms was possible in these culture conditions. To the best of our knowledge, this is the first work that describes the expression of VEGF-A isoforms in HEK293 cells in suspension culture. The rhVEGF165 and rhVEGF165b isoforms were purified by affinity chromatography from media previously conditioned by the overexpressing cell clones. The biological activity of rhVEGF165 was demonstrated in vitro, by the AngioPhaseTM Kit assay, and in vivo with the CAM (chorioallantoic membrane) assay, both of which are suitable for evaluating the pro- and antiangiogenic activity of different compounds. The rhVEGF165b did not show the expected antiangiogenic activity. These isoforms were tested in a model of murine tissue-engineered small intestine, indicating a possible contribution to therapeutic use in this context. The purification of rhVEGF121, as well as the structural analysis of all three proteins, are in the process of being optimized.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2018-04-19T17:09:39Z No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-23T11:47:10Z (GMT) No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-23T11:47:10Z (GMT). No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-13 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Paracoccidioidomycosis (PCM) is an important mycosis of Latin America, caused by thermodymorphic fungi of the genus Paracoccidioides. Homeostasis of metals such as copper, zinc and iron is important for the survival of fungi in the host environment. In this context, copper is an important cofactor for several enzymes, such as as superoxide dismutases and cytochrome c oxidase. The excess free copper in the cell promotes accumulation of reactive oxygen species, causing damage to nucleic acids, lipids and proteins. Thus, organisms shall to maintain cytoplasmic levels of that micronutrient at non-toxic levels, just sufficient for cell growth and vital metabolic processes. The metabolism of that metal is strictly controlled by high and low affinity uptake systems. The objective of this work is to perform heterologous expression of the Atx1 protein, described in the literature as a copper chaperone, and to analyze its cellular location, as well as the protein behavior upon copper deprivation in Paracoccidioides spp. Heterologous expression was performed on electrocompetent cells of Escherichia coli strain BL21. Identity of the recombinant protein was confirmed by LC-MS / MS. BALB/c mice were immunized to obtain anti-Atx1 polyclonal antibodies. After performing immunoblotting the produced antibodies were used in immunofluorescence assays. Analysis by qRT-PCR allowed us to evaluate the levels of the transcript encoding the Atx1 protein during copper deprivation in Paracoccidioides spp. / A paracoccidioidomicose (PCM) é uma importante micose da América Latina, causada por fungos termodimórficos do gênero Paracoccidioides. A homeostase de metais como cobre, zinco e ferro é importante para a sobrevivência dos fungos no ambiente do hospedeiro. Nesse contexto, o cobre é um importante cofator para várias enzimas, como as superóxido dismutases e a citocromo c oxidase. O excesso de cobre livre na célula promove o acúmulo de espécies reativas de oxigênio, causando danos a ácidos nucléicos, lipídeos e proteínas. Assim, os organismos devem manter concentrações citoplasmáticas desses micronutrientes em níveis não tóxicos, apenas suficiente para o crescimento celular e processos metabólicos vitais. O metabolismo desse metal é rigorosamente controlado por sistemas de captação de alta e baixa afinidade. O objetivo do trabalho é realizar a expressão heteróloga da proteína Atx1, descrita na literatura como uma chaperona citosólica de cobre e analisar a sua localização celular, bem como o comportamento diante da privação de cobre em Paracoccidioides spp. A expressão heteróloga foi realizada em células eletrocompetentes de Escherichia coli, cepa BL21. A identidade da proteína recombinante foi confirmada por LC-MS/MS. Camundongos BALB/c foram imunizados para obtenção de anticorpos policlonais anti-Atx1. Após realização de imunoblotting, os anticorpos produzidos foram utilizados para ensaios de imunofluorescência em células leveduriformes. Análise por qRT-PCR nos permitiram avaliar os níveis do transcrito codificante da proteína Atx1 durante a privação de cobre em Paracoccidioides spp.

A biomassa vegetal pode ser uma importante fonte de obtenção de diversos produtos a partir da desestruturação de suas frações por um vasto grupo de enzimas. No entanto, a geração de compostos de alto valor agregado a partir da biomassa lignocelulósica requer o desenvolvimento de novos sistemas enzimáticos. Pensando nisso, a prospecção e a caracterização de novas enzimas que estão presentes no secretoma de fungos degradadores da biomassa lignocelulósica tem sido fonte de pesquisa por pesquisadores do mundo todo. O objetivo deste trabalho foi prospectar, clonar e expressar de maneira heteróloga o gene codificante de uma enzima putativa do fungo termofílico T. terrestris em cepas do fungo filamentoso A. nidulans A773 e promover sua caracterização bioquímica e biofísica. O gene da enzima foi amplificado, clonado e inserido no vetor pEXPYR antes de ser inserido no sistema de expressão do A. nidulans. Os transformantes obtidos foram induzidos em meio mínimo de cultivo contendo 3% (m/v) de maltose e 1% (m/v) de glicose em meio estacionário para a produção, seguido da purificação da enzima. Estudos bioquímicos foram realizados para determinar o pH e a temperatura ótima de reação, bem como, a especificidade aos substratos e a determinação dos parâmetros cinéticos. A termoestabilidade da enzima também foi avaliada por estudo de dicroísmo circular (DC). Além disso, foi avaliado o efeito colaborativo entre uma xilanase GH10 e a enzima em estudo na hidrólise do bagaço de cana-de-açúcar pré-tratado. A enzima obtida por expressão heteróloga foi caracterizada como uma xilo-oligossacarídeo oxidase (XylO). Por meio da análise filogenética das sequências de aminoácidos entre a enzima expressa e outras enzimas oxidativas, a XylO foi classificada como pertencente a família das flavoproteínas e subfamília das BBE. A enzima TtXylO demonstrou ter especificidade em oligossacarídeos de C5 apresentando boa atividade enzimática em substratos complexos de xilana. A enzima possui pH ótimo de 5,5 e temperatura ótima de 25 ºC. As análises de DC indicaram temperatura de desnaturação de 62,7 ºC, caracterizando esta enzima como termofílica. Contudo, novos estudos ainda são necessários para avaliar os produtos gerados a partir da oxidação dos diferentes xilo-oligossacarídeos pela XylO e seu potencial uso na indústria. / Plant biomass is an important source for generation of several products obtained from enzymatic cleavage of its fractions by a large group of enzymes. However, the generation of high value compounds from lignocellulosic biomass requires the development of new enzymatic systems. Considering that, prospection and characterization of enzymes present in the biomass-degrading fungi secretome has been a source of study by researchers around the world. The aim of this work was to prospect, clone and heterologously express a putative enzyme encoding gene from the thermophilic fungus T. terrestris in A. nidulans A773 strains and to promote its biochemical and biophysical characterization. The gene was amplified, cloned and inserted into the pEXPYR vector before being inserted into A. nidulans expression system. The transformants were induced by culture in minimal médium containing 3% (w/v) maltose and 1% (w/v) glucose by stationary culture for the production, followed by enzyme purification. Biochemical analyses were performed to determine optimum pH and temperature as well as the substrate specificities and kinetic parameters. The enzyme thermostability was also evaluated by circular dichroism (CD). In addition, the collaborative effect between the enzyme and a GH10 on hydrolys of pre-treated sugarcane bagasse was evaluated. The enzyme obtained by heterologous expression was characterized as a xylooligosaccharide oxidase (XylO). Phylogenetic analysis between amino acid sequences of expressed enzyme and other oxidative enzymes classified XylO as belonging to flavoproteins family and subfamily of BBE. TtXylO has been shown to have specificity on C5 oligosaccharides exhibiting good enzymatic activity on complex xylan substrates. The enzyme has an optimum pH of 5.5 and optimum temperature of 25 ºC. DC analyses showed melting temperature of 62.7 ºC, characterizing this enzyme as thermophilic. In general, further studies are still needed to evaluate the products generated from oxidation of xylooligosaccharides by XylO and their potential use in the industry.

La symbiose mycorhizienne à arbuscule (MA) est une association mutualiste s’établissant entre les racines des plantes et des champignons du sol appartenant à l’embranchement des Gloméromycètes. Dans cette association, le champignon agit comme un fertilisant naturel, fournissant à la plante divers minéraux (phosphore, mais aussi azote et soufre …) en échange de sources de carbone indispensables à son développement. Une caractéristique originale de ces champignons est leur très large spectre d’hôte : de l’ordre de 80% des espèces végétales ont l’aptitude à former cette symbiose, et certains espèces de champignons MA ne semblent pas avoir de limitation de spectre d’hôte. Les champignons MA possèderaient-ils des « clés universelles » de compatibilité cellulaire avec leur hôte, ou de contournement de l’immunité végétale ? Pour aborder cette problématique, nous avons entrepris l’étude du sécrétome du champignon MA Rhizophagus irregularis dont plusieurs données génomiques étaient disponibles. Les microorganismes eucaryotes sécrètent en effet dans leur environnement des protéines agissant sur leurs structures exogènes (paroi cellulaire), leur environnement, et pouvant agir sur l’immunité des cellules hôtes. Ces protéines sécrétées (SPs) sont dans ce dernier cas appelées « effecteurs ». Sur la base de deux assemblages différents, un catalogue de 872 SPs de R. irregularis (RiSPs) a été défini pour lesquelles les profils d’expression dans trois plantes hôtes ont été comparés. Nous avons également comparé ces SPs à celles que nous avons définies sur une autre espèce de champignon MA, Gigaspora rosea. Après enrichissement du catalogue de RiSPs avec des séquences de petite taille identifiées sur un assemblage transcriptomique propre, puis sélection des candidats dont les cadres de lecture sont robustes et présentant un niveau d’expression élevé (FC>10) dans les 3 hôtes testés, un jeu de 33 RiSPs d’intérêt a été défini, dont 18 ont été sélectionnées pour effectuer des analyses fonctionnelles. En absence de protocole de transformation de ces champignons, l’analyse fonctionnelle a porté sur la cytolocalisation de protéines de fusion RiSP:citrine dans les cellules végétales par surexpression dans des feuilles de tabac et des racines de luzerne tronquée. Différents compartiments cellulaires sont ciblés par ces RiSPs, très souvent le compartiment vacuolaire. Des approches par surexpression in planta de plusieurs candidats RiSP ont permis d’identifier une activité stimulatrice de 3 RiSPs sur l’établissement de la symbiose. Parallèlement, des essais de stimulation de la symbiose MA par apport exogène de différents SPs sur plantules en cours de mycorhization en chambre ont été initiés. Ils devront être poursuivis sur les 3 candidats issus du crible de surexpression. En perspective, l’évaluation de la spécificité d’action de ces SPs sur la symbiose MA comparativement à d’autres interactions plante-champignon ouvrira la voie à des essais d’application au champ. / Arbuscular mycorrhizal (AM) symbiosis is a mutualistic association established between plant roots and soil fungi belonging to the phylum Glomeromycota. In this association, the fungus acts as a natural fertilizer, supplying the plant with various minerals (phosphorus, but also nitrogen and sulfur) in exchange to carbon sources essential for its development. An original feature of these fungi is their very broad host spectrum: c.a. 80% of plant species have the ability to form this symbiosis, and some species of AM fungi do not seem to have a restrictive host spectrum. Would MA fungi possess "universal keys" for cell compatibility with their host, or to by-pass plant immunity? To address this problem, we studied the secretome of the AM fungus Rhizophagus irregularis from which several genomic data were available. Eukaryotic microorganisms indeed secrete in their environment proteins acting on their exogenous structures (cell wall), on their environment (nutrient recruitment), and even on host plant cell immunity. These last secreted proteins (SPs) are defined as effectors. Based on two different assemblies, a repertoire of 872 SPs of R. irregularis was defined for which transcriptional expression profiles obtained in three hosts were compared, as well with SPs from another species of AM fungus, Gigaspora rosea. After adding sequences of small size identified from an in-house transcriptomic assembly, screening unambiguous open reading frame, and selecting strongly expressed candidates (FC> 10) in the 3 plant hosts analyzed, a set of 33 RiSPs of interest was defined, of which 18 were selected for functional analysis. As genetic transformation protocol is unavailable for AM fungi, RiSP:citrine fusion proteins were overexpressed in tobacco leaves and barrel medic roots for plant cell localization. Different cell compartments were targeted by these RiSPs, and often localised in the vacuolar compartment. In planta overexpression of several candidates allowed identifying 3 RiSPs that stimulate the establishment of the symbiosis. In the same time, attempts to enhance MA symbiosis by addition of exogenous RiSPs on seedlings during mycorrhizal establishment were initiated. Such assays should be pursued on the 3 active candidates revealed by overexpression assays. Evaluating the specificity of action of these RiSPs on AM symbiosis compared to other plant-fungus interactions will open the way to field trials.

<p>A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB).</p><p>The C-terminus of the Alpha class enzymes is an α-helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the β-carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of k_cat and k_cat/K_m with glutathione and CDNB as the varied substrates. </p><p>The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 ˚C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1.</p>

A number of active site mutants of human Alpha class glutathione transferase A1-1 (hGST A1-1) were made and characterized to determine the structural determinants for alkenal activity. The choice of mutations was based on primary structure alignments of hGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, from three different species and crystal structure comparisons between the human enzymes. The result was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB). The C-terminus of the Alpha class enzymes is an α-helix that folds over the active site upon substrate binding. The rate-determining step is product release, which is influenced by the movements of the C-terminus, thereby opening the active site. Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine 9 and phenylalanine 10, positioning the β-carbon of the cysteinyl moiety of glutathione. The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studied by the viscosity dependence of kcat and kcat/Km with glutathione and CDNB as the varied substrates. The compatibility of slightly different subunit interfaces within the Alpha class has been studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4. The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 ˚C. The heterodimers did not show strictly additive activities with alkenals and CDNB. This result combined with further studies indicates that there are factors at the subunit interface influencing the catalytic properties of hGST A1-1.

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.

No description available.

La production biologique d'hydrogène (H2) représente une technologie possible pour la production à grande échelle durable de H2 nécessaire pour l'économie future de l'hydrogène. Cependant, l'obstacle majeur à l'élaboration d'un processus pratique a été la faiblesse des rendements qui sont obtenus, généralement autour de 25%, bien en sous des rendements pouvant être atteints pour la production de biocarburants à partir d'autres processus. L'objectif de cette thèse était de tenter d'améliorer la production d'H2 par la manipulation physiologique et le génie métabolique. Une hypothèse qui a été étudiée était que la production d'H2 pourrait être améliorée et rendue plus économique en utilisant un procédé de fermentation microaérobie sombre car cela pourrait fournir la puissance supplémentaire nécessaire pour une conversion plus complète du substrat et donc une production plus grande d'H2 sans l'aide de l'énergie lumineuse. Les concentrations optimales d’O2 pour la production de H2 microaérobie ont été examinées ainsi que l'impact des sources de carbone et d'azote sur le processus. La recherche présentée ici a démontré la capacité de Rhodobacter capsulatus JP91 hup- (un mutant déficient d’absorption-hydrogénase) de produire de l'H2 sous condition microaérobie sombre avec une limitation dans des quantités d’O2 et d'azote fixé. D'autres travaux devraient être entrepris pour augmenter les rendements d'H2 en utilisant cette technologie. De plus, un processus de photofermentation a été créé pour améliorer le rendement d’H2 à partir du glucose à l'aide de R. capsulatus JP91 hup- soit en mode non renouvelé (batch) et / ou en conditions de culture en continu. Certains défis techniques ont été surmontés en mettant en place des conditions adéquates de fonctionnement pour un rendement accru d'H2. Un rendement maximal de 3,3 mols de H2/ mol de glucose a été trouvé pour les cultures en batch tandis que pour les cultures en continu, il était de 10,3 mols H2/ mol de glucose, beaucoup plus élevé que celui rapporté antérieurement et proche de la valeur maximale théorique de 12 mols H2/ mol de glucose. Dans les cultures en batch l'efficacité maximale de conversion d’énergie lumineuse était de 0,7% alors qu'elle était de 1,34% dans les cultures en continu avec un rendement de conversion maximum de la valeur de chauffage du glucose de 91,14%. Diverses autres approches pour l'augmentation des rendements des processus de photofermentation sont proposées. Les résultats globaux indiquent qu'un processus photofermentatif efficace de production d'H2 à partir du glucose en une seule étape avec des cultures en continu dans des photobioréacteurs pourrait être développé ce qui serait un processus beaucoup plus prometteur que les processus en deux étapes ou avec les co-cultures étudiés antérieurément. En outre, l'expression hétérologue d’hydrogenase a été utilisée comme une stratégie d'ingénierie métabolique afin d'améliorer la production d'H2 par fermentation. La capacité d'exprimer une hydrogénase d'une espèce avec des gènes de maturation d'une autre espèce a été examinée. Une stratégie a démontré que la protéine HydA orpheline de R. rubrum est fonctionnelle et active lorsque co-exprimée chez Escherichia coli avec HydE, HydF et HydG provenant d'organisme différent. La co-expression des gènes [FeFe]-hydrogénase structurels et de maturation dans des micro-organismes qui n'ont pas une [FeFe]-hydrogénase indigène peut entraîner le succès dans l'assemblage et la biosynthèse d'hydrogénase active. Toutefois, d'autres facteurs peuvent être nécessaires pour obtenir des rendements considérablement augmentés en protéines ainsi que l'activité spécifique des hydrogénases recombinantes. Une autre stratégie a consisté à surexprimer une [FeFe]-hydrogénase très active dans une souche hôte de E. coli. L'expression d'une hydrogénase qui peut interagir directement avec le NADPH est souhaitable car cela, plutôt que de la ferrédoxine réduite, est naturellement produit par le métabolisme. Toutefois, la maturation de ce type d'hydrogénase chez E. coli n'a pas été rapportée auparavant. L'opéron hnd (hndA, B, C, D) de Desulfovibrio fructosovorans code pour une [FeFe]-hydrogénase NADP-dépendante, a été exprimé dans différentes souches d’E. coli avec les gènes de maturation hydE, hydF et hydG de Clostridium acetobutylicum. L'activité de l'hydrogénase a été détectée in vitro, donc une NADP-dépendante [FeFe]-hydrogénase multimérique active a été exprimée avec succès chez E. coli pour la première fois. Les recherches futures pourraient conduire à l'expression de cette enzyme chez les souches de E. coli qui produisent plus de NADPH, ouvrant la voie à une augmentation des rendements d'hydrogène via la voie des pentoses phosphates. / Biological hydrogen (H2) production represents a possible technology for the large scale sustainable production of H2 needed for a future hydrogen economy. However, the major obstacle to developing a practical process has been the low yields that are obtained, typically around 25%, well below those achievable for the production of other biofuels from the same feedstock. The goal of this thesis was to improve H2 production through physiological manipulation and metabolic engineering. One investigated hypothesis was that H2 production could be improved and made more economical by using a microaerobic dark fermentation process since this could provide the extra reducing power required for driving substrate conversion to completion and hence more H2 production might be obtained without using light energy. The optimal O2 concentrations for microaerobic H2 production were examined as well as the impact of carbon and nitrogen sources on the process. The research reported here proved the capability of Rhodobacter capsulatus JP91 hup- (an uptake-hydrogenase deficient mutant) to produce H2 under microaerobic dark conditions with limiting amounts of O2 and fixed nitrogen. Further work should be undertaken to increase H2 yields using this technology. In addition, a photofermentation process was established to improve H2 yield from glucose using R. capsulatus JP91 hup- strain either in batch and/or continuous culture conditions. Some technical challenges in establishing the proper operational conditions for increased H2 yield were overcome. A maximum yield of 3.3 mols of H2/ mol of glucose was found for batch cultures whereas in continous cultures it was 10.3 mols H2/ mol glucose, much higher than previously reported and close to the theoretical maximum value of 12 mols H2/ mol glucose. In batch cultures the maximum light conversion efficiency was 0.7% whereas it was 1.34% in continuous cultures with a maximum conversion efficiency of the heating value of glucose of 91.14%. Various approaches to further increasing yields in photofermentation processes are proposed. The overall results suggest that an efficient single stage photofermentative H2 production process from glucose using continuous cultures in photobioreactors could be developed which would be a much more promising alternative process to the previously studied two stage photofermentation or co-culture approaches. Furthermore, the heterologous expression of hydrogenases was used as a metabolic engineering strategy to improve fermentative H2 production. The capability of expressing a hydrogenase from one species with the maturation genes from another was examined. One strategy demonstrated that the orphan hydA of R. rubrum is functional and active when co-expressed in E. coli with hydE, hydF and hydG from different organisms. Co-expression of the [FeFe]-hydrogenase structural and maturation genes in microorganisms that lack a native [FeFe]-hydrogenase can successfully result in the assembly and biosynthesis of active hydrogenases. However, other factors may be required for significantly increased protein yields and hence the specific activity of the recombinant hydrogenases. Another strategy was to overexpress one of the highly active [FeFe]-hydrogenases in a suitable E. coli host strain. Expression of a hydrogenase that can directly interact with NADPH is desirable as this, rather than reduced ferredoxin, is naturally produced by its metabolism. However, the successful maturation of this type of hydrogenase in E. coli had not been previously reported. The Desulfovibrio fructosovorans hnd operon (hndA, B, C, and D genes), encoding a NADP-dependent [FeFe]-hydrogenase, was expressed in various E. coli strains with the maturation genes hydE, hydF and hydG of Clostridium acetobutylicum. Hydrogenase activities were detected in vitro, thus a multi-subunit NADP-dependent [FeFe]-active hydrogenase was successfully expressed and matured in E. coli for the first time. Future research could lead to the expression of this hydrogenase in E. coli host strains that overproduce NADPH, setting the stage for increased hydrogen yields via the pentose phosphate pathway.

Made available in DSpace on 2015-05-14T12:59:29Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1529227 bytes, checksum: de14b6706c9f0d50230f124a34766410 (MD5) Previous issue date: 2011-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Among the natural products with experimentally proven analgesic properties, the monoterpernes are of great importance, recognized as the main chemical constituents of essential oils from aromatic plants. ( )-Carvone is an example of monoterpene with antinociceptive properties, founded as the main active constituent of oils from some species of the genus Mentha. Since the peripheral antinociceptive mechanism of ( )-carvone is not well established, this study aimed to better characterize it. Initially, it was performed a structure-activity study by which was possible to demonstrate the importance of the carbonyl group into the molecule of ( )-carvone, during its blocking effect in the rat peripheral nervous excitability, as also that the replacement of that group for a hydroxyl enhanceed this effect. After shown that ( )-carvone had low cytotoxicity, we investigated the effects of this monoterpene in the voltage-gated sodium channels of (Nav) and the transient receptor potential vanilloid 1 (TRPV1), involved in peripheral nociception, using neurons from the dorsal root ganglion (DRG) of rats. With the technique of Whole-cell Patch-clamp it was demonstrated that ( )-carvone (1 mM) was able to reduce Na+ influx from 8.7±1.6 nA (control) to 5.3±1.1 nA (p <0.05). Later, by fluorescence microscopy assays, we observed that ( )-carvone increased the Ca2+ levels in DRG neurons, possibly via TRPV1 channels. The involvement of these channels was further confirmed by specific antagonism and heterologous expression in HEK 293 by transfecting these cells with TRPV1-cDNA of Rattus novergicus. Then we demonstrated that ( )-carvone acts in the TRPV1 channel in a concentration-dependent manner, promoting its desensitization. Additionally, it was discarded the participation of another TRP channel involved in pain, the TRPM2, during the effect of ( )-carvone. Therefore, this study showed that the replacement of a carbonyl by a hydroxyl group in the molecule of ( )-carvone could increase the efficiency of this monoterpene in reducing the peripheral nerve excitability. Such effect was demonstrated as being a result of the Nav channel blockade, as well as the activation and subsequent desensitization of TRPV1 channels, indicating the great potential of this monoterpene as a peripheral antinociceptive molecule or prototype of a novel analgesic drug. / Dentre os produtos de origem natural com propriedades analgésicas comprovadas experimentalmente, destacam-se os monoterpernos, tidos como os principais constituintes químicos dos óleos essenciais de plantas aromáticas. A ( )-carvona é um exemplo de monoterpeno com propriedades antinociceptivas, encontrada como o principal constituinte ativo dos óleos de algumas espécies do gênero Mentha. Uma vez que o mecanismo antinociceptivo periférico de ( )-carvona não está estabelecido, o presente trabalho se propõe a melhor caracterizá-lo. Inicialmente realizou-se um estudo estrutura-atividade, onde foi possível demonstrar a importância do grupo carbonila ligado à molécula de ( )-carvona, durante o seu efeito bloqueador da excitabilidade nervosa periférica de rato, e ainda, que a substituição desse grupo por uma hidroxila potencializou esse efeito. Após demonstrarmos que ( )-carvona possuía baixa citotoxicidade, investigou-se os efeitos desse monoterpeno sobre o canais de sódio dependentes de voltagem (Nav) e nos canais receptores de potencial transiente vanilóides 1 (TRPV1), envolvidos na nocicepção periférica, utilizando-se neurônios do gânglio da raiz dorsal (DRG) de rato. Por meio da técnica de Whole-cell Patch-clamp foi possível demonstrar que ( )-carvona (1 mM) foi capaz de reduzir o influxo de Na+ de 8,7±1,6 nA (controle) para 5,3±1,1 nA (p< 0,05). Posteriormente, através de ensaios de microscopia de fluorescência, observou-se que ( )-carvona aumentou os níveis de Ca2+ em neurônios DRG, possivelmente via canais TRPV1. O envolvimento desses canais foi posteriormente confirmado por antagonismo específico e por expressão heteróloga em HEK 293, por transfecção dessas células com cDNA-TRPV1 de Rattus novergicus. Em seguida, demonstrou-se que ( )-carvona atua sobre o canal TRPV1, de modo dependente de concentração, promovendo a sua dessensibilização. Adicionalmente, foi descartada a participação de outro canal TRP envolvido na dor, como o TRPM2, durante o efeito de ( )-carvona. Portanto, o presente estudo evidenciou que a substituição do grupamento carbonila por uma hidroxila na molécula de ( )-carvona poderia aumentar a eficiência desse monoterpeno na redução da excitabilidade nervosa periférica. Este efeito foi demonstrado como sendo resultante do bloqueio de canais Nav, bem como pela ativação e posterior dessensibilização de canais TRPV1, indicando a grande potencialidade deste monoterpeno como uma molécula antinociceptiva periférica ou protótipo de nova droga analgésica.