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La lipase B de Candida antarctica (CALB) est un enzyme présentant des propriétés énantiosélectives très intéressantes pour l’obtention de molécules énantio pures par dédoublement cinétique de mélanges racémiques,molécules utilisées comme synthons dans l’industrie pharmaceutique. En effet, le principe actif de nombreux médicaments est efficace sous une forme énantio pure, l’autre forme chirale pouvant se révéler délétère pour l’organisme.Les travaux de la thèse s’intéressent à mieux comprendre l’origine de l’énantiosélectivité de la lipase B de Candida antarctica, en particulier pour la résolution d’alcools secondaires par des réactions de transestérification.Nous utilisons pour la première fois la méthode de la perturbation de l’énergie libre pour estimer la différence d’énergie libre entre les intermédiaires tétraédriques obtenus avec les formes R et S d’alcools énantiomères pour une série d’alcools secondaires, dans le but de prédire in silico l’énantiosélectivité de la CALB. Les paramètres cinétiques apparents d’une réaction avec deux alcools substrats énantiopurs sont expérimentalement déterminés et permettent de définir la contribution respective du Km et du kcat de chaque énantiomère pour la définition de l’énantiosélectivité. L’étude expérimentale de l’effet d’empreinte par des molécules co-substrats est réalisée,ainsi qu’une étude par modélisation moléculaire de l’effet d’empreinte par le premier ester substrat de la réaction qui pourrait modifier la conformation du site actif de la CALB. La troisième partie porte sur l’étude de la CALB et de trois variants (T42V, S47A et T42V/S47A) chez lesquels les acides aminés dans la poche stéréospécifiques ont mutés. T42V et S47A permettent d’obtenir une augmentation de l’énantiosélectivité. L’étude propose une étude détaillée de la conformation du site actif à partir de simulations de trajectoires de dynamique moléculaire / The lipase B from Candida antarctica is an enzyme displaying enantioselective properties which are interesting to obtain enantio pure compounds by kinetic resolution of racemic mixtures, which are used as pharmaceutical intermediates and fine chemicals. Indeed, for most of the drugs, only one of the two chiral formsis efficient as bioactive compound, whereas the other chiral form may display deleterious effects. Present work concerns the understanding of the origin of Candida antarctica lipase B enantioselectivity, and more especially in case of the resolution of secondary alcohols by transesterification. We used, for the first time, the free energy perturbation method to evaluate the free energy difference between tetrahedral intermediates with R and S alcohol enantiomers for a series of secondary alcohols in order to predict in silico enantiomeric ratio of CALB-catalyzed reactions. The apparent kinetic parameters were experimentally determined for two enantio pure substrates and allow to evalute the relative contribution of both Km and kcat for R and S enantiomers in the enantiomeric ratio of CALB-catalyzed reactions. Experimental study of imprinting effect hypothesis by co-substrate molecules was done. Molecular modeling studies of imprinting effect hypothesis were performed, in which the first substrate ester of the reaction could mould the active site. At least, the third part of this thesis concerns the study of wild-type CALB and three different variants (T42V and S47A which allow to increase enantioselectivity and T42/S47A) of CALB by molecular modeling. A detailed study of the conformation of the stereo specificity pocket in the active site is presented, based on molecular dynamics simulations.

Il a récemment été démontré que la glace de mer antarctique pouvait jouer un rôle significatif dans la dynamique des gaz à effet climatique (dont le dimethylsulfure ou DMS) dans les régions polaires. Ce travail s’est d’abord attaché à la mise au point d’une méthode de mesure fiable du diméthylsulfoxyde (DMSO) dans la glace de mer, supprimant les interférences générées par la production de DMS au sein de l’échantillon en réponse au choc osmotique subi lors de la fonte de l’échantillon de glace. Une procédure de détermination séquentielle du DMS, par broyage à sec, puis du dimethylsulfoniopropionate (DMSP) et du DMSO sur le même échantillon de glace a été développée et utilisée à large échelle dans ce travail. Les données du présent travail ont été acquises dans le cadre de deux programmes d’observation intégrés menés sur la glace de mer antarctique à des saisons différentes mais avec une méthodologie commune :1) choix de sites d’étude homogènes afin de minimiser l’impact de la variabilité spatiale sur l’interprétation des résultats dans une optique d’évolution temporelle et 2) priorité à la caractérisation du cadre physico-chimique (texture, température, salinité, couvert de neige, susceptibilité au drainage des saumures,….) avant toute autre analyse. L’étude menée dans le cadre du programme ISPOL (nov.–dec. 2004) a permis d’observer que la stratification des saumures a un impact positif sur la conversion du DMSP en DMS au sein de la glace mais ralentit les flux de DMS et DMSP vers l’océan. Le couvert de glace est caractérisé à cette période de l’année par une perte nette de DMSP et génère des flux combiné de DMS et DMSP du même ordre de grandeur que les flux de DMS atmosphériques mesurés dans le cadre d’autres études. L’étude menée dans le cadre du programme SIMBA (sept.–oct. 2007) a permis de mettre en évidence l’importance du forçage atmosphérique sur le régime thermique et la dynamique du DMS/P/O dans la glace. Les communautés d’algues de surface produisent de fortes concentrations de DMS/P/O en réponse au stress thermique, osmotique et potentiellement radiatif durant les périodes de refroidissement et la mise en place d’un régime soutenu de drainage des saumures contribue à évacuer périodiquement les hautes concentrations de DMS/P/O produites dans la glace vers l’océan sous-jacent. Le couvert de glace affichant une production nette de DMS/P/O à cette période de l’année génère des flux combinés de DMS et DMSP plus de dix fois supérieurs à ceux observés pour la glace estivale. L’étude menée sur de la glace artificielle a permis de mettre en évidence l’impact des processus physico-chimiques sur la signature en gaz de la glace en croissance constituant un premier pas vers la modélisation des transports de gaz dans la glace de mer et leurs échanges au travers des interfaces glace-océan et glace-atmosphère. <p><p><p>SUMMARY - It has recently been demonstrated that Antarctic sea ice recently demonstrated plays a potentially significant role in the dynamics of climatically significant gases (amongst which dimethylsulphide or DMS) in Polar Regions. This research work has initially focused on the development of a reliable method for the determination of dimethylsulphoxide (DMSO) within sea ice, avoiding interferences generated by DMS production within the sample in response to the osmotic shock caused by melting. A sequential determination procedure of DMS, dimethlsulphoniopropionate (DMSP) and DMSO on the same ice sample has been developed and used on a large amount of samples in the present work. Data presented in this research project have been collected in the framework of two integrated sea ice observation programs focused on Antarctic sea ice at different seasons but following a common approach: 1) choice of homogeneous study sites to minimize the impact of spatial variability on the interpretation of the results in a time series perspective and 2) priority given to the characterization of the physicochemical framework (texture, temperature, salinity, snow cover, susceptibility to brine drainage,…) prior to any other study. The study conducted in the framework of the ISPOL experiment (Nov.–Dec. 2004) demonstrated that stratification of the brine inclusions network positively influenced the conversion of DMSP into DMS but decreased fluxes of DMS and DMSP towards the ocean. The ice cover at that time of the year is characterised by a net DMSP loss and generates combined DMS and DMSP fluxes whose values fall in the range of atmospheric DMS flux from sea ice measured in the frame of other studies. The study conducted in the framework of the SIMBA experiment (sept.–oct. 2007) emphasized the importance of atmospheric thermal forcing on the sea ice thermal regime and DMS/P/O dynamics. The surface community of algae produced elevated levels of DMS/P/O in response to thermal, osmotic and potentially radiative stress during periods of atmospheric cooling while the development of an intense brine drainage regime contributed to periodically release the elevated levels of DMS/P/O produced in the sea ice towards the underlying ocean. The ice cover exhibited at that time of the year a net production of DMS/P/O and produced combined DMS and DMSP fluxes more than ten times higher than those observed for summer sea ice. The study conducted on laboratory prepared growing sea ice emphasised the impact of physicochemical processes on the gas signature of growing sea ice and represents a first step towards modelling gas exchanges within sea ice and across its interfaces with the ocean and the atmosphere.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

L-asparaginase (L-ASNase) é uma enzima com propriedades interessantes para a indústria médica, farmacêutica e de alimentos, que tem recebido atenção especial, inclusive no Brasil, por fazer parte do protocolo de tratamento de distúrbios linfoproliferativos, como a leucemia linfoblástica aguda (LLA). No mercado desde a década de 1970, as enzimas de origem bacteriana enfrentam algumas limitações por provocarem reações adversas graves em quase 80% dos pacientes em tratamento. Nesse contexto, L-ASNases provenientes de leveduras se destacam como alternativa, por serem mais próximas às congêneres humanas. A Antártica ainda é um ambiente pouco explorado, com grande diversidade de microrganismos com potencial para a produção de moléculas biológicas de interesse industrial. Nesse contexto, 150 leveduras isoladas de amostras de sedimento marinho coletadas na Península Antártica como parte do projeto MICROSFERA (PROANTAR/CNPq) foram avaliadas para a produção de L-ASNase. A triagem resultou em 9 isolados produtores, dos quais 7 pertencem ao gênero Leucosporidium. A linhagem L. muscorum CRM 1648 foi a que produziu mais enzima (540 U.L-1), com maior produtividade (5,6 U.L-1.h-1) e, por isso, foi alvo deste estudo. A análise univariada de fontes de carbono e nitrogênio indicou maior crescimento desse microrganismo e produção de L-ASNase em meio CD com extrato de levedura, prolina e sacarose. Ureia, cloreto de amônio e sulfato de amônio resultaram em baixa ou nenhuma produção da enzima, sugerindo que a metabolização de fontes de nitrogênio por essa linhagem está sob a influência do fenômeno de repressão catabólica pelo nitrogênio (RCN). Dois delineamentos experimentais do tipo fatorial completo resultaram em um aumento de 10 vezes na produção e produtividade da enzima (4582,5 U.L-1 e 63,6 U.L-1.h-1, respectivamente). A análise univariada da concentração inicial de inóculo (X0), pH inicial do meio, temperatura e adição de água do mar mostrou que a melhor condição para a produção foi: pH = 5,5 ou 6,5, cultivo a 15°C com adição de água do mar (25-50% m/v). A variável X0 não foi significativa nas concentrações avaliadas. Cultivos em biorreator (batelada) foram conduzidos em quatro diferentes níveis de oxigênio dissolvido (OD): (1) OD não controlado e abaixo de 20%, (2) OD não controlado e acima de 20%, (3) OD controlado em 80% e (4) OD controlado em 20%. Os resultados mostraram que OD é fator limitante para o crescimento de L. muscorum CRM 1648 e produção de L-ASNase por essa levedura e deve ser mantido acima de 35% para maior produção da enzima.Neste trabalho, a composição do meio e condições de cultivo foram estabelecidas para favorecer a produção de uma nova L-ASNase livre de atividade glutaminásica por levedura adaptada ao frio, abrindo espaço para novos estudos acerca de seu potencial antileucêmico e possível uso como alternativa às enzimas já existentes no mercado no tratamento de LLA. / L-asparaginase (L-ASNase) is an enzyme with interesting properties for medical, pharmaceutical and food industry, which has received special consideration, especially in Brazil, for being part of lymphoproliferative disorders treatment, such as acute lymphoblastic leukemia (ALL). Bacterial enzymes are on the market since the 1970s and face some limitations related to theirserious adverse reactions that reach almost 80% of all patients in treatment. In this context, L-ASNases from yeasts are highlighted as important alternative to bacterial enzymes, due to the closerphylogeny to human congeners. Antarctic environment has much to be explored, with a vast diversity of microorganisms with potential to produce biomolecules with industrial interest. A total of 150 yeasts isolated from Antarctic marine sediments as part of MICROSFERA project (PROANTAR/CNPq) were evaluated for L-ASNase production. The screening resulted in 9 producers, 7 species from the genus Leucosporidium. L. muscorum CRM 1648 was the strain that yielded the highest L-ASNase activity (540 U.L-1) and volumetric productivity (5.6 U.L-1.h-1). Carbon and Nitrogen sources were evaluated by a method of one-factor at a time (OFAT). From the gather results, sucrose, yeast extract and proline resulted in a maximal growth and highest enzyme production.The absence or low production of L-ASNase in medium with urea, ammonium chloride and ammonium sulfate suggests the presence of nitrogen catabolic repression (NCR). Carbon and nitrogen concentration were evaluated by full factorial design and yielded about ten times higher enzyme and volumetric productivity (4582.5 U.L-1 and 63.6 U.L-1.h-1, respectively). Initial inoculum concentration (X0), initial pH, temperature and concentration of seawater in the culture were evaluated by OFAT analysis and the best condition for L-ASNase production was: pH = 5.5 or 6.5, at 15 °C with addition of seawater (25-50 wt%). X0 was not considered a significant variable. Bioreactor assays (in batch regime) were performed in four different dissolved oxygen (DO) levels: (1) without DO control (DO remained under 20%), (2) without DO control (DO remained above 20%), (3) DO controlled at 80%, and (4) DO controlled at 20%.The results showed that DO is a key factor for growth of L. muscorum CRM 1648 and production of L-ASNase by this yeast and should be maintained above 35% for higher production of this enzyme.At this work, the medium and culture conditions were established to support the production of a novel glutaminase-free L-ASNase by a cold adapted yeast, opening a new path for further studies regarding its antileukemic potential and possible use as an alternative for ALL treatment.

VILLELA, FRANCO N. J. Análise decadal do fluxo de CO2 entre o oceano e a atmosfera na passagem de Drake, Oceano Austral. 2011. 148 f. Dissertação (mestrado) Programa de Pós-Graduação em Ciência Ambiental (PROCAM), Universidade de São Paulo, São Paulo, 2011. Para a área delimitada pelos paralelos 60ºS e 62,5ºS e pelos meridianos 60ºW e 65ºW, localizada no sul da Passagem de Drake, no Oceano Austral, próximo à Península Antártica, foram calculadas as distribuições médias de 2000 a 2009, sazonais e anual, do fluxo de CO2 na interface oceano-atmosfera e de suas variáveis associadas: a pressão parcial de CO2 na superfície marinha (PCO2sw), a pressão parcial de CO2 na atmosfera (PCO2ar), a diferença da pressão parcial de CO2 entre o oceano e a atmosfera (PCO2) e a taxa de transferência gasosa (TR), que é produto do coeficiente solubilidade do CO2 na água do mar pela velocidade de transferência gasosa. A parametrização utilizada no cálculo dos fluxos foi a de Takahashi et al. (2009) com TR dependente da velocidade do vento ao quadrado multiplicada por um fator de escala 0,26. A área de estudo tem cerca de 75 mil km2 e foi dividida em uma grade espacial de 0,5º x 0,5º, resultando em 50 quadrículas. Foram utilizados mais de 46 mil medições de PCO2sw, que na média espacial variou de 362,7 ±11,2 a 371,9 ±17,5 µatm, no verão e primavera respectivamente. A PCO2 variou de -0,4 a 5,7 µatm no outono e primavera, respectivamente. A TR variou de 0,065 ±0,04 a 0,088 ±0,002 gC.mês-1.m-2.µatm-1, no verão e inverno, respectivamente. O fluxo líquido, se tomando a concentração de gelo como negligenciável, variou de -0,039 ±0,865 a 0,456 ±1,221 gC.m-2.mês-1, no outono e inverno, respectivamente. O fluxo total anual de carbono, estimado através da média espacial por quadrícula, foi de 95 GgC.ano-1. Dessa maneira, na estimativa anual, a superfície do mar se comporta como fonte de CO2 para a atmosfera, principalmente devido à região da plataforma continental com PCO2sw consideravelmente maior que o da atmosfera. Sazonalmente sugere-se que no verão a maior disponibilidade de radiação solar, a temperatura da superfície do mar (TSM) mais elevada e os ventos mais fracos favorecem a produção de biomassa fitoplanctônica, fazendo com que a bomba biológica seja o processo dominante na diminuição da PCO2sw e na absorção de CO2 atmosférico pela superfície marinha. Já no inverno, os ventos se intensificam e, associados com o forte resfriamento da TSM, promovem a mistura com águas profundas ricas em carbono inorgânico dissolvido, levando a superfície marinha a um estado de supersaturação de CO2 em relação à atmosfera. Ventos circumpolares de oeste mais intensos e deslocados para sul tem sido apontados como a causa do aumento da PCO2sw em igual ou maior taxa do que ocorre na atmosfera. Na área de estudo foi levantada uma tendência média da intensidade do vento de 0,23 ±0,03 m.s-1.década-1 e um aumento na freqüência da componente zonal de oeste (positiva) de 1,47 ± 1,13 % .década-1. Sugere-se que estas tendências estejam relacionadas com o Modo Anular Austral (SAM). Entretanto, a tendência decadal estimada para a PCO2sw foi menor que para a atmosfera, apesar de ambas indicarem tendência de aumento. Acredita-se que a grande variabilidade e distribuição esparsa de dados tenham mascarado a magnitude da estimativa da tendência de PCO2sw. / VILLELA, FRANCO N. J. Decadal analysis of the CO2 sea-air flux in the Drake Passage, Southern Ocean 2011. 148 f. Dissertação (mestrado) Programa de Pós-Graduação em Ciência Ambiental (PROCAM), Universidade de São Paulo, São Paulo, 2011. For the area bounded by parallels 60°S and 62.5°S and meridians 60°W and 65°W, located in the southern Drake Passage in the Southern Ocean, near the Antarctic Peninsula, mean seasonal and annual distributions of CO2 flux at the ocean-atmosphere interface, from 2000 to 2009, have been computed, as well as their associated variables: the CO2 partial pressure at sea surface (PCO2sw), the CO2 partial pressure in atmosphere (PCO2ar), the CO2 pressure difference between ocean and atmosphere (PCO2), and the gas transfer rate (TR), which is the product of the CO2 solubility coefficient in sea water by the gas transfer velocity. The parameterization used to calculate fluxes was that of Takahashi et al. (2009) with TR depending on the squared wind speed multiplied by a scale factor 0.26. The study area has about 75,000 km2 and was divided into a grid of 0.5° x 0.5°, resulting in 50 area boxes. Over 46,000 PCO2sw measurements were used, which in the spatial mean varied from 362.7±11.2 to 371.9±17.5 µatm, in summer and spring, respectively. The PCO2 varied from 0.4 to 5.7 µatm in autumn and spring, respectively. TR varied from 0.065±0.04 to 0,088±0.002 gC.month-1.m-2.µatm-1, in summer and winter, respectively. The net flux, taking ice concentration as negligible, varied from 0.039±0.865 to 0.456±1.221 gC.month-1.m-2, in autumn and winter, respectively. The total annual carbon flux, estimated through the spatial mean per square, was 95 GgC.y-1. Thus, in the annual estimate the region acts as a source to the atmosphere, mainly due to the continental shelf having PCO2sw considerably greater than that of the atmosphere. Seasonally, it is suggested that in summer the greater availability of solar radiation, warmer sea surface temperature (SST), and weaker winds favor the production of phytoplanktonic mass, making the biological pump the dominating process in lowering the PCO2sw and the absorption of atmospheric CO2 by the sea surface. On the other hand, in winter winds intensify and, in association with the strong cooling of the SST, promote mixing with deep waters rich in dissolved inorganic carbon, leading the sea surface to a state of supersaturation in CO2 relative to the atmosphere. Stronger circumpolar west winds and displaced to the south have been pointed as the cause for the increase of PCO2sw at a rate equal to or greater than that occurring in the atmosphere. In the study area it has been detected a mean trend of wind intensity 0.23±0.03 m.s-1.decade-1 and an increase in the western zonal component of 1.47±1.3%.decade-1. It is suggested that these trends are related to the Southern Annular Mode (SAM). However, the decadal trend estimated for the PCO2sw was smaller than for the atmosphere, in spite of both indicating increasing tendencies. It is believed that the great variability and scatter distribution of the data have masked the magnitude of the PCO2SW trend estimate.