Neste projeto foram abordados procedimentos analíticos para extração, separação e identificação de espécies de arsênio encontradas normalmente em quantidades de traços e ultra-traços em amostras biológicas e ambientais. Entre as amostras alvo deste estudo, teve-se alimentos de origem marinha como por exemplo camarão. Foi avaliada a estabilidade das espécies de arsênio nas soluções geradas pelos processos de extração. Para separação das espécies inorgânicas (arsenito e arsenato), metiladas (ácidos mono e dimetil arsênio) e orgânicas (arsenobetaina) empregaram-se as técnicas eletroforese capilar (CE) e cromatografia líquida (LC). Os sistemas de separação para a determinação das espécies de arsênio foram acoplados com os espectrômetros massas (ICP-MS), e de fluorescência atômica (AFS). Os sistemas acoplados apresentaram resolução e sensibilidade na determinação das espécies de arsênio nas amostras estudadas neste trabalho. A extração com água de espécies de As utilizando-se banho de ultra-som apresentou eficiência acima de 78%. A estabilidade das espécies nas soluções padrão e nos extratos das amostras foi mantida por um período de até uma semana quando armazenadas em geladeira (+4°C). Visando uma política de química limpa, foi desenvolvida uma metodologia para evitar desperdiço preparando micro-volumes de amostras e soluções padrão, para especiação de arsênio por eletroforese capilar. Para tal se empregou um injetor seqüencial, também foi desenvolvido um dispositivo de injeção hidrodinâmica para eletroforese capilar. Além disto, as soluções residuais geradas durante a pesquisa analítica foram tratadas para a recuperação de arsênio e boro. A extração no ponto nuvem foi empregada para recuperar As entanto que a precipitação por mineralização hidrotérmica foi aplicada para a recuperação de B . A aplicação deste procedimento resultou na extração de 80% de arsênio e 75% de boro. Isto, permitiu converter grandes volumes de resíduos líquidos perigosos em um pequeno volume de resíduos sólidos / Analytical procedures for extraction, separation and identification of arsenic species at ultra-trace levels in biological and environmental samples were investigated. Stability studies of arsenic species in sample extracts and standard solutions were carried out. Separation of arsenite, arsenate, monomethylarsonic and dimethylarsinic acids and also arsenobetaine were carried out by capillary electrophoresis or liquid chromatography. Determination of the separated arsenic species was accomplished by coupling the separation techniques to inductively coupled plasma mass spectrometry (ICP-MS) or atomic fluorescence spectrometry (AFS). Satisfactory resolution and sensibility were achieved by the coupled systems described. Extraction efficiency better than78% were achieved with water in an ultrasonic bath at the laboratory temperature (23-27°C). Whereas, stability of species in solutions stored in refrigerator at +4°C was efficient for aperiod up to one week. Micro-volumes of standards and sample solutions mixed in a sequential injector were prepared for arsenic speciation by capillary electrophoresis, aiming green chemistry. A time controlled micro-volume injector device for hydrodinamic injections was also developed. Finally, waste solutions from arsenic speciation analysis in a system with hydride generation were treated for arsenic and boron removal. Cloud point extraction for recovery of arsenic and hydrothermal mineralization of boron by calcium hydroxide were employed. Satisfactory removal of 80% of arsenic and 75% of boron from waste solution was achieved. These procedures allowed to reduce volumes of hazardous waste solutions

La présente thèse traite, au travers d’une analyse fondamentale et expérimentale, l’effet de texturation par détente instantanée contrôlée (DIC) sur les phénomènes de transfert au cours des processus de séchage, d’extraction mécanique et de comportement rhéologique. Les produits concernés ont été les graines de caroube (gomme et germe) et deux variétés de graines de tournesol (linoléique et oléique). La texturation DIC permet d’agir sur la cinétique de séchage par flux d’air ce qui conduit à une intensification des processus de séchage par flux d’air tout en assurant une bonne préservation de la qualité nutritionnelle du produit fini. L’étude expérimentale a été couplée à une analyse phénoménologique de la cinétique à travers une modélisation de type CWD (coupled Washing/Diffusion). Cette dernière étude conduit à une évaluation de l’impact de la texturation sur la diffusivité effective Deff et l’accessibilité initiale δWs. D’autre part, l’étude expérimentale en vue de l’optimisation de l’opération a été réalisée à travers la méthode des plans d’expériences. La modification des paramètres opératoires fonctionnels de la DIC, à savoir la pression absolue de vapeur d’eau saturée sèche (P), et le temps de traitement thermique (t), ainsi que le nombre des cycles (c) sur l’ensemble des performances de l’opération a été établie sur le comportement rhéologique de la gomme de caroube. Une deuxième partie de la thèse a été consacrée à l’étude fondamentale et expérimentale des procédés d’extraction d’huile de tournesol des deux variétés de graines linoléiques et oléiques. Le traitement thermomécanique par détente instantanée contrôlée (DIC) permet, à travers une modification contrôlée de la structure de la matière première, d’agir sur l’extraction mécanique par presse à froid des huiles de tournesol, ainsi que sur l’extraction par solvant de l’huile à partir du tourteau résiduel. Un complément à cette opération a consisté en l’extraction par solvant (n-hexane) opérée sur l’ensemble des graines de tournesol. La texturation DIC a conduit à une grande augmentation du rendement en huile pour les variétés linoléique et oléique. La préservation de la qualité de l’huile et du tourteau prouve l’adéquation de la DIC avec l’absence pratique de la dégradation chimique. Notre étude a également concerné la définition d’un nouveau procédé de désodorisation. Il s’agit de l’utilisation de l’Autovaporisation Multi-Flash MFA sur le raffinage de l’huile de tournesol. Cette technologie innovante de désodorisation se distingue par son faible niveau de température assurant ainsi une sévérité nettement inférieure à celle des méthodes conventionnelles à haute température. / This thesis deals with a fundamental and experimental analysis of the effect of instant controlled pressure drop (DIC) on the transfer phenomena and rheological behavior, which occurs during the drying process and mechanical and solvent extraction. The products concerned were carob seeds (gum and germ) and two varieties of sunflower seeds (linoleic and oleic). DIC texturing makes it possible to greatly modify the drying kinetics, leading to an effective intensification of the drying processes while maintaining a good preservation of the nutritional quality of the finished product. The experimental study was coupled with a phenomenological kinetic model using the CWD (coupled Washing/Diffusion). This study leads to evaluate the impact of texturing on the effective diffusivity Deff and the initial accessibility δWs. On the other hand, the experimental study for the optimization of the operation was carried out through an adequate experimental design method. The impact of the DIC processing parameters, namely the absolute pressure of saturated dry water vapor (P), and the heat treatment time (t), as well as the number of cycles (c) was established on the rheological behavior of the carob bean gum. A second part of our work was devoted to the theoretical and experimental study of sunflower oil extraction processes of the two types of linoleic and oleic seeds. By acting as a controlled modification of the raw material, (DIC) texturing allowed increasing the yield of sunflower oil by both cold pressing and solvent extraction from the residual cake. DIC texturing has led to a large increase in oil yield for both linoleic and oleic varieties. The preservation of the quality of oil demonstrates that DIC was perfectly adequate with a perfect absence of chemical degradation. Our study also involved the definition of a new deodorization process. This is the use of the Multi-Flash Autovaporization MFA on the refining of sunflower oil. This innovative deodorization technology is characterized by its low temperature level, thus ensuring a much lower severity than conventional high temperature methods.

Cette thèse aborde deux sujets de recherches, le premier est sur l’existence et l’unicité des solutions des Équations Différentielles Doublement Stochastiques Rétrogrades (EDDSRs) et les Équations aux Dérivées partielles Stochastiques (EDPSs) multidimensionnelles à croissance surlinéaire. Le deuxième établit l’existence d’un contrôle optimal strict pour un système controlé dirigé par des équations différentielles stochastiques progressives rétrogrades (EDSPRs) couplées dans deux cas de diffusions dégénérée et non dégénérée.• Existence et unicité des solutions des EDDSRs multidimensionnels :Nous considérons EDDSR avec un générateur de croissance surlinéaire et une donnée terminale de carré intégrable. Nous introduisons une nouvelle condition locale sur le générateur et nous montrons qu’elle assure l’existence, l’unicité et la stabilité des solutions. Même si notre intérêt porte sur le cas multidimensionnel, notre résultat est également nouveau en dimension un. Comme application, nous établissons l’existence et l’unicité des solutions des EDPS semi-linéaires.• Contrôle des EDSPR couplées :Nous étudions un problème de contrôle avec une fonctionnelle coût non linéaire dont le système contrôlé est dirigé par une EDSPR couplée. L’objective de ce travail est d’établir l’existence d’un contrôle optimal dans la classe des contrôle stricts, donc on montre que ce contrôle vérifie notre équation et qu’il minimise la fonctionnelle coût. La méthode consiste à approcher notre système par une suite de systèmes réguliers et on montre la convergence. En passant à la limite, sous des hypothèses de convexité, on obtient l’existence d’un contrôle optimal strict. on suit cette méthode théorique pour deux cas différents de diffusions dégénérée et non dégénérée. / In this Phd thesis, we considers two parts. The first one establish the existence and the uniquness of the solutions of multidimensional backward doubly stochastic differential equations (BDSDEs in short) and the stochastic partial differential equations (SPDEs in short) in the superlinear growth generators. In the second part, we study the stochastic controls problems driven by a coupled Forward-Backward stochastic differentialequations (FBSDEs in short).• BDSDEs and SPDEs with a superlinear growth generators :We deal with multidimensional BDSDE with a superlinear growth generator and a square integrable terminal datum. We introduce new local conditions on the generator then we show that they ensure the existence and uniqueness as well as the stability of solutions. Our work go beyond the previous results on the subject. Although we are focused on multidimensional case, the uniqueness result we establish is new in one dimensional too. As application, we establish the existence and uniqueness of probabilistic solutions tosome semilinear SPDEs with superlinear growth generator. By probabilistic solution, we mean a solution which is representable throughout a BDSDEs.• Controlled coupled FBSDEs :We establish the existence of an optimal control for a system driven by a coupled FBDSE. The cost functional is defined as the initial value of the backward component of the solution. We construct a sequence of approximating controlled systems, for which we show the existence of a sequence of feedback optimal controls. By passing to the limit, we get the existence of a feedback optimal control. The convexity condition is used to ensure that the optimal control is strict. In this part, we study two cases of diffusions : degenerate and non-degenerate.

A tese enfatiza o desenvolvimento e aplicação de amostradores difusionais para pré-concentração de traços de poluentes gasosos e sua conjugação com técnicas de separação e determinação, especialmente, a eletroforese capilar. Foram implementadas novas aplicações para um amostrador com múltiplos filamentos microporosos de polipropileno, previamente desenvolvido pelo grupo de pesquisa, e para o novo dispositivo de coleta de dimensões reduzidas, composto por monofilamento microporoso de polipropileno (CMDS) com volume interno de 30 µL. Para o estabelecimento da vazão estável em 1,0 µL min-1 de fase aceptora pelo capilar poroso, recorreu-se à pressurização do reservatório com uma bomba pneumática de aquário e regulagem de vazão por um capilar de sílica fundida e uma válvula de agulha. Outra bomba pneumática foi utilizada para aspirar o fluxo de ar amostrado. As amostras foram coletadas seqüencialmente em frascos de 200 µL, mantidos sob temperatura reduzida em unidade de refrigeração do tipo Peltier acoplado ao amostrador. O CMDS monofilamentar, mais compacto e robusto, apresentou alta eficiência de coleta para a pré-concentração de formaldeído (CH2O), ácidos fórmico e acético e amônia da fase gasosa da atmosfera. Para as espécies com elevada constante de Henry, água deionizada serviu como fase aceptora, enquanto que para amônia, recorreu-se ao deslocamento do equilíbrio por um aceptor ácido, de modo a reter o analito na forma de NH4 +. A concentração das espécies em fase líquida foi determinada posteriormente por eletroforese capilar com detecção condutométrica sem contato (CE-C4D). Os limites de detecção, em fase líquida, para formiato, acetato, formaldeído (determinado na forma do aduto hidroximetanosulfonato HMS) e amônio foram estimados em 1,0, 1,5, 1,2 e 1,2 µmol L-1, respectivamente (equivalentes à 0,9, 3,0, 1,0 e 0,7 µg m-3 dessas espécies na atmosfera, respectivamente). Para a determinação de CH2O por injeção em fluxo (FIA) acoplada à detecção amperométrica em eletrodo de ouro platinizado, fez-se uso do coletor multifilamentar, por atender melhor à necessidade de volume de amostra. A interferência de espécies comumente presentes na atmosfera, como H2O2 e SO2, pôde ser contornada realizando coletas em presença de peróxido de hidrogênio para promover a oxidação do S(IV) à S(VI) e, posteriormente, destruindo o oxidante em reator contendo enzima catalase imobilizada. A determinação de CH2O foi implementada com sucesso por FIA com detecção por amperometria em eletrodo de ouro platinizado. Um CMDS portátil que funciona à pilha, próprio para amostragens em campo, foi desenvolvido utilizando, para o deslocamento do ar amostrado, uma bomba de pistão retirada de aparelho automático para medida de pressão arterial. O amostrador foi empregado na coleta de H2S, SO2 e alquil-mercaptanas em fase gasosa da atmosfera, utilizando fase aceptora alcalina para promover a desprotonação e conseqüente fixação das espécies em meio líquido. Os analitos foram determinados por CE-C4D ou por microextração em fase sólida (SPME) seguida de determinação por GC. O uso conjunto do antioxidante ascorbato para conservação das amostras e de etanol para fixação dos compostos voláteis em meio aquoso permitiu o estabelecimento de um protocolo completo para coleta e detecção dessas espécies reduzidas de enxofre, com sensibilidade suficiente para monitorar emissões de origem biogênica, o que foi exemplificado na prática coletando amostras próximo a um córrego contaminado com esgoto. A perfeita combinação do diminuto volume de fase aceptora do CMDS com a demanda de nanolitros de amostra da CE-C4D culminou com o desenvolvimento de um sistema de análise total (TAS) com gerenciamento de fluidos baseado em propulsão por bombas de aquário (de baixo custo e alta durabilidade) e válvulas solenóide de estrangulamento, controladas por computador. Como exemplo de aplicação inovador e bem sucedido do TAS desenvolveu-se a análise concomitante e em tempo quase real de ácidos fórmico e acético no ar, com freqüência de 10 pares de dados por hora. As vantagens do sistema CMDS-CE-C4D incluem simplicidade, versatilidade, consumo de reagentes e amostra e geração de resíduos minimizada, robustez e rapidez enquanto uma amostra é coletada, o eletroferograma da anterior é adquirido sem necessidade de bombas de alta pressão ou colunas dispendiosas como as requeridas para HPLC / The development and application of porous membrane diffusion samplers for fast and efficient pre-concentration of an array of trace air pollutants was emphasized in this thesis, in conjunction with compatible separation and determination techniques, especially capillary electrophoresis. New applications were found for a formerly developed diffusion scrubber based on a bundle of microporous hollow polypropylene capillary membranes and for a scaled down version with a single core capillary membrane diffusion scrubber (CMDS) comprising an internal volume of 30 µL, were used for sampling of the trace level pollutants to an adequate liquid acceptor. The low-flow of acceptor solution, 1.0 µL min-1, required by the CMDS was satisfied by pressurization of the reservoir with an aquarium pump combined with flow regulation by a silica capillary as hydrodynamic resistor and a needle valve. Another aquarium pump was used for the aspiration of the sampled air through the sampler. The low volumes collected in the CMDS were stored in 200 µL vials inserted in a cooling plate of a Peltier device. The robust and compact system was used for sampling of formaldehyde (CH2O), formic acid, acetic acid and ammonia in the gaseous phase of the atmosphere. For chemical species with high Henry´s constant, deionized water suffices as acceptor phase. Otherwise, equilibria displacement to a non-volatile ion, like NH4 + for NH3 sampling, promoted quantitative retention in the acceptor phase. The concentrations of the analytes in the liquid phase were determined by capillary electrophoresis with capacitively coupled contactless conductometric detection (CEC4D). The detection limits obtained in the liquid phase for formate, acetate, formaldehyde (in the form of the adduct hydroxymethanesulfonate HMS) and ammonium were 1,0, 1,5, 1,2 e 1,2 µmol L-1 respectively (what corresponds to 0,9, 3,0, 1,0 e 0,7 µg m-3 of the respective gaseous species in the air). The higher volume of acceptor phase provided by the sampler with a bundle of microporous membrane capillaries (~600 µL) are in tune with the needs of flow injection analysis (FIA), as demonstrated for amperometric detection of CH2O on a platinized gold electrode. The interferences from SO2 and H2O2 were overcome by adding H2O2 to the acceptor solution to promote the oxidation of S(IV) to S(VI) and destruction of the oxidant afterwards in a column with immobilized catalase enzyme. The aquarium pump used for gas aspiration was substituted to a piston pump, taken from arterial blood pressure meter fed with 8 V D.C., to turn possible field collections with the CMDS device. The system was used for the sampling of H2S, SO2 e alkyl-mercaptans in the gaseous phase of the atmosphere. To succeed on that, the samplings were performed in alkaline media to promote deprotonation of the species (and stabilization in non-volatile forms). The collected analytes were determined by CE-C4D or by solid phase microextraction (SPME) followed by GC analysis. The joint use of the antioxidant ascorbate for sample preservation and ethanol for fixation of the volatile compounds allowed the establishment of a complete protocol for sampling, storage and detection of the sulfur reduced species with enough sensitivity for monitoring biogenic emissions from waste discharge. Perfect matching of the low-volume characteristics of the CMDS device and the CE-C4D equipment led to the conception of a low-cost automatic CMDS-CE-C4D total analysis system (TAS) and its innovative and successful application to near-realtime simultaneous analysis of formic acid and acetic acid in air. During the evaluation of one sample, the TAS collects a new one, with valves, pumps and high voltage delivery under software control. Advantages include rapidity (10 data points per hour for each analyte), high preconcentration efficiency, simplicity and versatility, minimum sample and reagent consumption and residue generation (green analytical method), no need of costly high pressure pumps and separation columns like those used in HPLC

A presente dissertação trata da implementação e otimização de um sistema de detecção condutométrica sem contato capacitivamente acoplada (C4D) para Eletroforese Capilar (CE). O sistema é caracterizado pela compactação do sistema de detecção, versatilidade e flexibilidade de instalação em diferentes equipamentos comerciais de eletroforese capilar e home made. Desde a década de 80, a eletroforese capilar vem se consolidando como uma das técnicas de separação mais relevantes. Normalmente, os instrumentos comerciais são disponibilizados com detectores ópticos e detectores eletroquímicos. A C4D é utilizada em eletroforese capilar posicionando-se dois eletrodos tubulares envoltos ao capilar. A aplicação de sinais de alta frequência entre os eletrodos permite monitorar variações de condutividade da solução dentro do capilar. Assim, a resposta do detector depende de diversos fatores como mobilidade do analito, do co-íon do eletrólito, da frequência e amplitude do sinal aplicado entre os eletrodos e da geometria dos mesmos. A ausência de componentes móveis torna o presente detector compacto (6,5 cm3) e robusto. O presente C4D é constituído de um oscilador local funcionando a 1,1 MHz, um circuito capaz de converter corrente em tensão, retificar e filtrar os sinais analógcos, além de apresentar um conversor de sinais analógicos em digitais (Conversor A/D) de alta resolução (21 bits efetivos). Outra vantagem é a possibilidade de determinar o diâmetro interno do capilar através da leitura do conversor A/D quando se faz passar um fluxo de ar e, posteriormente água através do capilar. A diferença de leitura de conversor A/D pode ser convertida em diâmetro interno mediante uma curva de calibração. Em uma aplicação típica, os limites de detecção baseado no critério 3s (sem filtragem inicial) foi de 0,6, 0,4, 0,3, 0,5, 0,6 e 0,8 µmol L-1 para K+, Ba2+, Ca2+, Na+, Mg2+ e Li+, respectivamente, é comparável a outras implementações de alta qualidade de um detector condutométrico sem contato capacitivamente acoplado. / This dissertation deals with the implementation and optimization of a Capacitively Coupled Contactless Conductivity Detector (C4D) applied to capillary electrophoresis (CE). The system is characterized by compaction of the detection system, installation flexibility and versatility in different commercial capillary electrophoresis equipment and home-made. Since the 80s, capillary electrophoresis has been consolidated as one of the most important separation techniques. Typically, commercial instruments are available to optical detectors and electrochemical detectors. The C4D is used in capillary electrophoresis positioning two electrodes encased in tubular capillary. The application of high frequency signals between the electrodes allows monitoring changes in conductivity of the solution within the capillary. Thus, the detector response depends on several factors such as mobility of the analyte, the co-ion electrolyte, the frequency and amplitude of the signal applied between the electrodes and the geometry of the same. The absence of moving parts makes this compact detector (6.5 cm3) and robust. This C4D is composed of a local oscillator, working at 1.1 MHz, a circuit capable of converting current into voltage, rectify and filter the analogs signals, besides presenting a high-resolution analog-to-digital converter (ADC) (21 bits effective). Another advantage is the possibility of determining the internal diameter of the capillary by reading the ADC when it is passed a stream of air and then water through the capillary. A readings different ADC can be converted into inner diameter through a curve a calibration curve. In a typical application, the detection limits based on the 3s criterion (without baseline filtering) was 0.6, 0.4, 0.3, 0.5, 0.6 and 0.8 µmol L-1 for K+, Ba2 +, Ca2 +, Na+, Mg2+ and Li+, respectively, is comparable to other high-quality implementations of a capacitively coupled contactless conductivity detector.

A slab-coupled optical fiber sensor (SCOS) falls into a narrow class of all-dielectric optical fiber electric field sensors, which makes it a perfect candidate for measurements of high electric fields in environments where presence of conductors is highly perturbing to the system under test. Its nonlinear response to high fields requires a new nonlinear calibration technique. A nonlinear calibration method is explained and demonstrated to successfully measure high electric fields, as well as high voltages with dynamic range up to 50 dB. Furthermore, a SCOS can be fitted into narrow spaces and make highly localized measurements due to its small size. This allows a SCOS to be integrated inside a standard high voltage coaxial cable, such as RG-218. Effects of partial discharge and arcing is minimized by development of a fabrication method to avoid introduction of impurities, especially air-bubbles, into the cable during SCOS insertion. Low perturbation of the measured voltage is shown by simulating the introduced voltage reflections to be on the order of –50 dB. It is also shown that a SCOS can be inserted into other cables without significant perturbation to the voltage. A complete high voltage and high electric field measurement system is built based on the high-voltage modifications of the SCOS technology. The coaxial SCOS is enhanced for robustness. Enhancements include packaging a SCOS into stronger ceramic trough, strengthening the fiber with kevlar reinforced furcation tubing and protecting the sensor with metal braces and protective shells. The interrogator is protected from electromagnetic interference with an RF-shielded box. Reduction in power losses introduced by the new PANDA-SCOS technology allows interrogator bandwidths to be increased up to 1.2 GHz. The whole measurement process is streamlined with dedicated software, developed specifically for high voltage and electric field measurements with support for the nonlinear calibration.

A slab-coupled optical fiber sensor (SCOS) falls into a narrow class of all-dielectric optical fiber electric field sensors, which makes it a perfect candidate for measurements of high electric fields in environments where presence of conductors is highly perturbing to the system under test. Its nonlinear response to high fields requires a new nonlinear calibration technique. A nonlinear calibration method is explained and demonstrated to successfully measure high electric fields, as well as high voltages with dynamic range up to 50 dB. Furthermore, a SCOS can be fitted into narrow spaces and make highly localized measurements due to its small size. This allows a SCOS to be integrated inside a standard high voltage coaxial cable, such as RG-218. Effects of partial discharge and arcing is minimized by development of a fabrication method to avoid introduction of impurities, especially air-bubbles, into the cable during SCOS insertion. Low perturbation of the measured voltage is shown by simulating the introduced voltage reflections to be on the order of −50 dB. It is also shown that a SCOS can be inserted into other cables without significant perturbation to the voltage.A complete high voltage and high electric field measurement system is built based on the high-voltage modifications of the SCOS technology. The coaxial SCOS is enhanced for robustness. Enhancements include packaging a SCOS into stronger ceramic trough, strengthening the fiber with kevlar reinforced furcation tubing and protecting the sensor with metal braces and protective shells. The interrogator is protected from electromagnetic interference with an RF-shielded box. Reduction in power losses introduced by the new PANDA-SCOS technology allows interrogator bandwidths to be increased up to 1.2 GHz. The whole measurement process is streamlined with dedicated software, developed specifically for high voltage and electric field measurements with support for the nonlinear calibration.

Unnatural amino acids (uAA) expand the structural and functional possibilities of proteins. Numerous previous studies have demonstrated uAA as a powerful tool for protein engineering, but challenges also remain. Three notable such challenges include: (1) the fitness of uAA-incorporated proteins are difficult to predict and time-consuming to screen with conventional methods, (2) uAA incorporation in difficult-to-express proteins (e.g. membrane proteins such as G-protein coupled receptors) remain challenging, and (3) the incorporation of multiple types of uAA are still limited. In response, we pose cell-free protein synthesis (CFPS), a rapid and versatile in vitro expression system, as a platform to explore solutions to these challenges. The "cell-free" nature of CFPS enables it to accelerate protein expression and tolerate extensive modifications to its translational environment. In this work, these advantages were utilized to address the aforementioned challenges by: (1) rapidly expressing and screening uAA-containing proteins, (2) incorporating uAA in functional G-protein coupled receptor in the presence of membrane-mimicking lipid additives, and (3) engineer the translational environment extensively towards multiple uAA incorporation.

No description available.

La symbiose mycorhizienne à arbuscules (MA) est une association bénéfique établie entre les membres d'un ancien sous-phylum de champignons, les Gloméromycètes, et les racines de la majorité des plantes terrestres. Les champignons MA procurent de l'eau et des minéraux (azote et phosphore principalement) à leur plante hôte et obtiennent de cette dernière des molécules carbonées sous forme d'hexoses et de lipides. Des études récentes ont montré que certaines protéines sécrétées par les champignons MA peuvent être des régulateurs importants de l'association (Kloppholz et al., 2011 ; Tsuzuki et al., 2016). Notre objectif était d'identifier de nouvelles protéines fongiques contribuant à la mise en place de la symbiose. Des protéines prédites pour être préférentiellement sécrétées par le champignon MA Rhizophagus irregularis dans les racines ont été identifiées au début de ma thèse (Kamel et al., 2017). Certaines d'entre-elles présentaient une structure ressemblant aux précurseurs de phéromones sexuelles d'Ascomycètes. Ces protéines sont connues pour être maturées dans les voies de sécrétion en petits peptides qui sont ensuite sécrétés. Leur reconnaissance par un récepteur couplé à la protéine G (GPCR) aboutit à la fusion cellulaire de deux types sexuels opposés. Dans le cas de R. irregularis, seule la reproduction clonale a été décrite, mais des données génomiques récentes remettent en question son statut d'organisme asexué (Ropars et al., 2016). Une grande partie de ma thèse a été dédiée à la caractérisation fonctionnelle de ce type de peptides chez R. irregularis. Nous avons montré que deux peptides étaient effectivement produits et sécrétés par R. irregularis. L'utilisation de peptides synthétiques nous a permis de mettre en évidence que l'un d'eux stimulait la colonisation de M. truncatula mais était également perçu par le champignon lui-même, induisant la transcription de son propre gène précurseur et d'un GPCR. Ce peptide stimulateur de la symbiose est composé de seulement trois acides aminés et il peut être produit à partir de trois précurseurs protéiques. Par des approches de génétique inverse (HIGS et VIGS), nous avons confirmé l'importance de ces précurseurs dans l'établissement de la symbiose.[...] / Arbuscular Mycorrhizal (AM) symbiosis is a beneficial association established between members of an ancient subphylum of fungi, the Glomeromycotina, and the roots of the majority of terrestrial plants. AM fungi provide water and minerals (mainly nitrogen and phosphorus) to their host plant in exchange for organic carbon in the form of hexoses and lipids. Recent studies have shown that certain proteins secreted by AM fungi are important symbiosis regulators (Kloppholz et al., 2011, Tsuzuki et al., 2016). Our aim was to identify new fungal proteins involved in the establishment of symbiosis. Proteins predicted to be preferentially secreted by the AM fungus Rhizophagus irregularis in the roots were identified at the beginning of my thesis (Kamel et al., 2017). We noticed that some of them had a structure resembling the sex pheromone precursors of Ascomycota. These proteins are known to be processed in the secretory pathway into small peptides which are then secreted. Their recognition by a G protein-coupled receptor (GPCR) leads to cell fusion of two opposite sex types. In the case of R. irregularis, only clonal reproduction has been described. However, recent genomic data question its status as an asexual organism (Ropars et al., 2016). A large part of my thesis was dedicated to the functional characterization of this type of processed peptides in R. irregularis. We show that two of them are actually produced and secreted by R. irregularis. Treatments with synthetic forms of these peptides revealed that one of them stimulated the colonization of M. truncatula but was also perceived by the fungus itself, inducing the transcription of its own precursor gene and of a GPCR gene. This symbiosis-stimulating peptide is composed of only three amino acids and can be produced from three different protein precursors. Using reverse genetics (HIGS and VIGS), we confirmed the importance of these precursors in the symbiosis establishment. [...]