La L-asparaginase est une enzyme d’intérêt thérapeutique pour le traitement des leucémies aigües lymphoblastiques participant à l’hydrolyse de son substrat naturel L-asparagine conduisant à l’apoptose des cellules cancéreuses. À ce jour, la L-asparaginase d’origine bactérienne fait partie intégrante des formulations car possédant des activités catalytiques élevées mais provoquant de nombreux effets secondaires liés à une immunogénicité. Trois enzymes avec une activité Lasparaginase produites chez l’homme ont été découvertes récemment mais possèdent des activités catalytiques qui sont 1000 à 2000 fois inférieures aux enzymes d’origine bactérienne. Augmenter l’activité catalytique de ces enzymes par évolution dirigée pourraient permettre leurs utilisations en thérapeutique en plus de potentiellement participer à la réduction de l’immunogénicité chez les patients. Ces travaux de doctorat décrivent le développement d’outils pour l’expression etla détection de l’activité L-asparaginase à l’échelle d’une cellule individuelle. La L-asparaginase d’E. coli, utilisée en thérapeutique, a servi de référence et a permis de démontrer que le test AUR est le plus adapté pour la mesure de l’activité en microfluidique. L’expression de l’enzyme à partir de différents vecteurs d’expression a montré que l’expression périplasmique semble la plus adaptée pour le ciblage permettant un bon rendement et une bonne accessibilité pour le substrat. La viabilité des cellules à l’issu des mesures a été aussi démontrée. Ces outils pourront être directement utilisés pour le criblage de banques de mutants de L-asparaginase d’origine humaine en microfluidique. Les propriétés de la L-asparaginase ont aussi été utilisées pour démontrer la potentielle utilisation de billes de silice en tant que biocatalyseurs où sont confinées des bactéries. Ces billes sont des excellents supports pour la croissance de microorganismes qui peuvent rester viables au-delà d’une semaine. L’expression d’enzymes peut être induite et l’activité catalytique être aisément contrôlée en faisant varier la concentration bactérienne au sein du matériau. La combinaison de différentes populations bactériennes offre la possibilité d’effectuer des réactions en cascade. Le recyclage de ces billes pour différents cycles de réactions a également été démontré. Ces matériaux bioactifs peuvent avoir de nombreuses applications dans le domaine des biotechnologies. / L-asparaginase is an enzyme of therapeutic value for the treatment of acute lymphoblastic leukemia. Ths enzyme catalyzes the hydrolysis of L-asparagine conducting to apoptosis of cancer cells. To date, L-asparaginase of bacterial origine is used in the treatments due to high catalytic activities but causing a number of side effects linked with an immunogenicity. The human produces three enzymes with L-asparaginase activity but their catalytic activities are 1000 to 2000 times lower than the bacterial enzymes. Increase the catalytic activity of these enzymes by directed evolution could allow their uses in therapeutic in addition to potentially reduce immunogenicity in patients. This PhD work describes the development of tools for expression and detection of L-asparaginase at the single cell level for their applications in the screening of human L-asparaginase libraries in microfluidic. E. coli L-asparaginase, used in therapy, served as a reference and allowed to demonstrate that AUR assay is most suitable for measuring activity in microfluidic. Expression of the enzyme from different expression vectors showed that the periplasmic expression seems to be the most successful for screening enabling a good yield and good accessibility for the substrate. The viability of the cells following the measures has been shown. These tools might be used for the screening of mutants libraries of human L-asparaginases in microfluidic. The properties of L-asparaginase were also used to demonstrate the potential use of silica beads as biocatalysts in which bacteria are confined. These beads are excellent supports for the growth of microorganisms which may remain viable beyond one week. The expression of the enzymes may be induced and the catalytic activity can be reliably controlled by varying the concentration of bacteria within the material. The combination of various bacterial populations provides the possibility to carry out cascades reactions. The recycling of these beads for several cycles of reactions was also demonstrated. These bioactive materials have many potential applications in the field of biotechnologies.

O protozoário Neospora caninum é considerado um dos principais causadores de abortos infecciosos na pecuária de corte e de leite no mundo. No Brasil, a presença do parasita varia de acordo com a região do país e com o tipo de rebanho, com perdas maiores na agroindústria do leite que ultrapassam US$ 50 milhões ao ano. A neosporose possui como principal manifestação clínica as perdas fetais ou abortamentos em bovinos, além de comprometimento neuronal esporádico em cães. Apesar da sua importância, a neosporose não possui atualmente tratamento eficaz e as opções de controle envolvem basicamente medidas de prevenção. As vacinas ainda são uma alternativa de desenvolvimento exploradas, sobretudo aquelas que utilizam antígenos recombinantes. Uma dificuldade ao se produzir proteínas recombinantes em sistemas de expressão heterólogos é a formação de corpúsculos de inclusão que são agregados de proteínas insolúveis. Estes agregados devem ser submetidos a processos dispendiosos para tornarem-se ativos, resultando num baixo rendimento da proteína final. Diversos recursos são utilizados para obtenção de proteínas solúveis, desde o emprego de diferentes técnicas de purificação, solubilização até o uso de tags de afinidade. Nosso grupo de pesquisa desenvolveu e patenteou o vetor pET28/NcMIC2-like1/1081 que possui uma tag rica em ácido glutâmico acoplada, conhecida como Nc1081. Esta tag foi capaz de estimular a solubilidade da proteína micronêmica tipo 2 de N. caninum (NcMIC2-like1) fazendo com que a proteína recombinante (rNcMIC2-like1/1081) fosse expressa na forma solúvel. O presente trabalho procurou dar continuidade ao estudo com a rNcMIC2-like1/1080 e com a tag através da clonagem e expressão de uma outra proteína relacionada à invasão do parasita conhecida como proteína associada à adenil ciclase (NcCAP). A sequência codificadora da proteína foi clonada no vetor contendo a tag Nc1081, a proteína recombinante (rNcCAP/1081) foi expressa com um peso de 38 kDa na forma solúvel em tampão não desnaturante Tris-Cl. No teste de ELISA, as proteínas rNcMIC2-like1 e a rNcCAP acopladas ou não a tag foram detectadas com intensidade semelhante pelos respectivos soros policlonais anti-rNcMIC2-like1 e anti-rNcCAP, demonstrando que a presença da tag não influenciou no reconhecimento das proteínas. As proteínas recombinantes solúveis rNcMIC2-like1/1081 e rNcCAP/1081 ainda foram testadas através de ensaios in vitro de invasão e adesão. No ensaio de invasão, a forma solúvel de NcMIC2-like1 (rNcMIC2-like1/1081) na concentração de 5 ?g/mL, aumentou a invasão das células VERO pelo parasita, enquanto sua forma insolúvel (rNcMIC2-like1) diminuiu a invasão. Já no teste de adesão (binding), foi possível identificar que a rNcMIC2-like1/1081 aderiu à proteína serina treonina fosfatase 2C, ao fator de alongamento alfa 1 e a proteína hipotética identificada ii como NCLIV_029860, enquanto a rNcCAP/1081 aderiu à proteína hipotética NCLIV_041330. O presente trabalho permitiu o início de estudos das propriedades funcionais das proteínas NcMIC2-like1 e NcCAP que seriam inviáveis com suas formas recombinantes insolúveis, demonstrando a aplicabilidade do vetor com a tag 1081. / Neospora caninum parasite is considered as a major cause of infectious abortion in beef and milk cattle in the world. In Brazil, the presence of the parasite varies in dairy or beef herd and according to the region with greater losses in dairy industry that exceed $ 50 million per year. Neosporosis has as main clinical manifestation fetal losses or abortion in cattle, in addition to sporadic neuronal impairment in dogs. Despite its importance, neosporosis has currently no effective treatment and control options rely primarily on preventive measures. Vaccines still represent an alternative explored by the veterinary industry, especially those employing recombinant antigens. The difficulties regarding production of recombinant proteins in heterologous expression systems are formation of inclusion bodies, which are insoluble protein aggregates. These aggregates have to undergo costly procedures to become active, resulting in low yield of final protein. Many techniques are used to obtain soluble proteins ranging from different purification or solubilizing techniques to the use of affinity tags. Our research group has developed and patented a vector pET28/NcMIC2-like1/1081 that has a rich glutamic acid tag attached, known as Nc1081. This tag is capable of stimulating the solubility of the recombinant micronemic protein type 2 like 1 of N. caninum (rNcMIC2-like1), expressingsoluble rNcMIC2-like1/1081. This study aimed to continue the study with rNcMIC2-like1/1080 and the tag by cloning and expressing another protein related to parasite invasion known as cyclase-associated protein (NcCAP). The protein coding sequence was cloned into the vector containing the Nc1081 tag, recombinant protein (rNcCAP/1081) was expressed with a molecular weight of 38 kDa in soluble form in non-denaturing Tris-Cl. In the ELISA test, rNcMIC2- like1 protein and rNcCAP coupled or not with the tag were detected with similar intensities by the respective polyclonal anti-sera rNcMIC2-like1 and antirNcCAP, demonstrating that the tag did not decrease the recognition of the proteins. Soluble recombinant proteins rNcMIC2-like1/1081 and rNcCAP/1081 were also tested in vitro in invasion and binding tests. In the invasion assay, a soluble form of NcMIC2-like1 (rNcMIC2-like1/1081) at a concentration of 5 ?g/mL increased the VERO cell invasion by the parasite, while its insoluble form (rNcMIC2-like1) decreased invasion. In the binding test, the rNcMIC2-like1/1081 bound to protein serine threonine phosphatase 2C, the elongation factor 1 alpha and the hypothetical protein identified as NCLIV_029860, while rNcCAP/1081 bound to the hypothetical protein NCLIV_041330. The current work allowed the initial functional studies of the NcMIC2-like1 and NcCAP proteins, otherwise unfeasible with their insoluble recombinant forms, demonstrating the applicability of the vector with the tag 1081.

En conséquence de l'extension de l’utilisation des nanoparticules dans différents secteurs industriels, le nombre de travailleurs potentiellement exposés ne cesse de croître, sans parfaitement connaître les propriétés toxicologiques de ces matériaux. Étant donné que les nanoparticules peuvent se trouver en suspension dans l’atmosphère professionnelle, l'inhalation représente une voie d'exposition professionnelle majeure. De ce fait, l’évaluation des risques liés à l’exposition aux nanomatériaux requiert d’entreprendre des études de toxicologie sur des modèles cellulaires des voies aériennes. Dans ce manuscrit, les réponses cellulaires et moléculaires des macrophages alvéolaires de rat (NR8383) exposés à des nanoparticules d’oxydes métalliques : ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 et TiO2-NRCWE001, ont été étudiées, en combinant des analyses toxicologiques classiques (caractérisation des nanoparticules par microscopie électronique à transmission et par diffusion dynamique de la lumière, évaluation de la cytotoxicité par tests WST-1 et libération de LDH); et de criblage moléculaire à haut débit (analyses de transcriptomique et de protéomique). Des cellules NR8383 ont été exposées aux nanoparticules ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 et TiO2-NRCWE001 pendant 24 h ce qui a permis de déterminer une dose sub-toxique pour chaque nanoparticule à laquelle les macrophages ont été exposés pour l’analyse moléculaire. Quatre heures suite à l’exposition des cellules aux nanoparticules, de nombreux gènes et protéines étaient différentiellement exprimés. Le stress oxydant était la réponse biologique adverse suite à l’exposition des cellules aux nanoparticules composées de zinc. En revanche, l’inflammation était la principale voie activée dans les cellules exposées à la forme anatase et rutile des nanoparticules de TiO2. En conclusion, cette étude expose les « empreintes biologiques » des deux groupes de nanoparticules d’intérêt. Enfin, notre étude combinée à des travaux antérieurs de la littérature pourraient aussi être profitables pour valider les biomarqueurs d’exposition et d’effets aux nanomatériaux suggérés afin de prédire les effets biologiques adverses. / As a consequence of the extension of the use of nanoparticles in different industrial sectors, the number of potentially exposed workers continues to grow, without fully knowing the toxicological properties of these materials. Since nanoparticles can be aerosolized in the occupational atmosphere, inhalation is the major occupational exposure route. For this reason, risk assessment of exposure to nanomaterials requires toxicology studies to be conducted on cellular models of the airways. In this manuscript, the cellular and molecular responses of rat alveolar macrophages (NR8383) exposed to metallic oxide nanoparticles: ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 and TiO2-NRCWE001, were studied, combining conventional toxicological analyzes (characterization of nanoparticles by transmission electron microscopy and dynamic light scattering, evaluation of cytotoxicity by WST-1 assays and LDH release); and high throughput molecular screening (transcriptomic and proteomic analyzes). NR8383 cells were exposed to the ZnO, ZnFe2O4, NiZnFe2O4, Fe2O3, TiO2-NM105 and TiO2-NRCWE001 nanoparticles for 24 h which allowed for the determination of a sub-toxic dose for each nanoparticle to which the macrophages were exposed for molecular analysis. Four hours after exposure NR8383 to nanoparticles, many genes and proteins were differentially expressed. Oxidative stress was the adverse biological response following exposure of cells to nanoparticles composed of zinc. In contrast, inflammation was the main activated pathway in cells exposed to the anatase and rutile form of TiO2 nanoparticles. In conclusion, this study exposes the "biological fingerprints" of the two groups of nanoparticles of interest. Finally, our study, combined with previous literature studies, could also be beneficial in validating biomarkers of exposure and effects of nanomaterials suggested in order to predict adverse biological effects.

Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...