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To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux. / Master of Science

A imobilização produz efeito deletério sobre a musculatura estriada. A avaliação das propriedades mecânicas do músculo esquelético é um parâmetro de mensuração para elucidar se o exercício físico pode constituir importante forma de prevenção ou reversão dos défices decorrentes da imobilização. Seis grupos (GC1, GI, GC2, GIL, GIE, GE), com dez animais cada, foram utilizados com o objetivo de avaliar a interferência do exercício físico por esteira em algumas propriedades mecânicas de modelos animais sujeitos previamente à imobilização do membro posterior. Os animais dos grupos GI, GIL e GIE foram submetidos a vinte e um dias de imobilização gessada do membro posterior direito. Os animais dos grupos GIE e GE foram submetidos a protocolo de treino em esteira por vinte e um dias. Os grupos GC1 e GC2 serviram como controle. Após alcançarem à idade ideal, os animais sofreram eutanásia e os gastrocnêmios das patas direitas foram submetidos a ensaios de tração para análise da carga máxima suportada, deformação na carga máxima e tensão. Os resultados obtidos mostraram que a imobilização exerce papel deletério sobre as propriedades mecânicas do músculo esquelético, a tensão não apresenta necessariamente correlação com a carga máxima e que o protocolo de exercícios adotado neste estudo não foi capaz de restaurar todas as propriedades mecânicas, principalmente a carga máxima. / Immobilization has a deleterious effect on skeletal muscles. The evaluation of the mechanical properties of the skeletal muscle is a parameter used to elucidate if physical activity may prevent or revert the deficits caused by immobilization. Six groups (GC1, GI, GC2, GIL, GIE, GE), with ten animals each, were used to assess the interference of treadmill running on mechanical properties of animal models submitted to plaster cast immobilization of the right hind limb. The animals in groups GI, GIL and GE were submitted to twenty-one days of right hind limb immobilization. Animals in groups GIE and GE were submitted to a treadmill running protocol for twenty-one days. The groups GC1 and GC2 served as controls. The animals underwent euthanasia after reaching the age limit and the gastrocnemius of the right hind limb was submitted to a traction test to analyze the ultimate load, ultimate load deformation and tension. The results showed that immobilization caused a deleterious effect on the muscle\'s mechanical properties and the tension is not necessarily correlated with maximum load. Furthermore the running protocol as used in this study was not able to restore all the normal muscle mechanical properties, mainly the maximum load.

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Pseudomonas aeruginosa (PA) est la troisième bactérie impliquée dans les maladies nosocomiales et est la principale cause de mortalité des patients atteints de la mucoviscidose. PA est résistante à la plupart des traitements antibiotiques. Trouver de nouvelles stratégies thérapeutiques est devenu un enjeu majeur de santé publique, l’une d’entre elles est l’inhibition de facteurs de virulence. Parmi ceux-ci, les lectines sont des protéines impliquées dans l’adhésion et la formation de biofilm via des interactions avec des sucres (PA-IL, PA- IIL, FliC, FliD, PilA, PilY1 et CupB6).Le but de ce travail est donc de trouver des leurres moléculaires ayant une forte affinité pour ces lectines. Ceux-ci sont des motifs saccharidiques présentés de façon multivalente : glycoclusters. De part leur grande diversité structurale et leur faible quantité, un outil de criblage innovant a été développé qui consiste en une lame de verre microstructurée : le glycocluster-microarray. Les glycoclusters sont immobilisés de manière ordonnée par DNA Directed Immobilization (DDI). Deux méthodes de criblage ont été développées grâce à cet outils : 1) le criblage en solution et par compétition d’une bibliothèque de motifs saccharidiques et 2) le criblage d’une bibliothèque de glycoclusters immobilisés sur le microarray. Avec cet outil, des protocoles de mesures d’IC50 et de Kd ont aussi été fiabilisés pour caractériser les meilleurs candidats inhibiteurs des lectines. Le glycocluster- microarray présente l’avantage de n’utiliser qu’une très faible quantité de matériel (quelques picomoles) et permet de réaliser diverses analyses en parallèle.Afin de valider cet outil, une étude sur l’impact de la densité de surface en glycocluster a été menée. Le criblage de plus de 150 motifs saccharidiques a permis de sélectionner ceux ayant une forte affinité pour les lectines. L’analyse sur microarray complétée par de la modélisation moléculaire d’une bibliothèque de glycoclusters, possédant ces motifs et différentes topologies, valences et propriétés (aromaticité, charge,…), a permis d’identifier les paramètres clés dirigeant les relations structure-affinité. Une activité anti-biofilm chez PA a été démontrée avec les meilleurs glycoclusters ciblant PA-IL.Tester l’activité in vivo, chez l’animal, des meilleurs candidats est une voie à explorer. Cibler d’autres lectines comme celles présentes sur le flagelle et les pili de PA et notamment impliquées dans son adhésion précoce est aussi une voie à développer. Pour cela, des tests préliminaires ont été présentés et d’autres sont en cours faisant appel à l’utilisation de bactéries entières ainsi qu’à une détection sans marquage des lectines. / Summary: Pseudomonas aeruginosa (PA) is the third pathogen involved in nosocomial diseases and the major cause of mortality of cystic fibrosis patients. PA develops resistance to antibiotics treatments. And so, developing new therapeutic strategies is a public health issue. One of the promising strategies is to inhibit virulence factors involved in the adhesion and the biofilm formation of PA. Some of these virulence factors are lectins which interact with sugars (PA-IL, PA-IIL, FliC, FliD, PilA, PilY1 and CupB6).The goal of this work is to find molecular decoys which have a strong affinity for these lectins. These are saccharidic units with a multivalent display: glycoclusters. An innovative screening tool has been developed: the glycocluster-microarray, to study lectin/glycocluster interactions. It is a microstructured glass slide where glycoclusters are immobilized by DNA Directed Immobilization (DDI). Two screening methods have been developed with this microarray: 1) the screening in solution and by competition of a saccharidic units library and2) the screening of a glycoclusters library immobilized on the microarray. Protocols of IC50 and Kd measurements have also been developed with this tool to characterize the best lectins inhibitors. This tool allows to use few amount of material (few picomoles) and to do parallel analysis.To validate the microarray, a study of the impact of glycoclusters surface density has been done. The screening of more than 150 saccharidic units allowed the selection of the ones that display the best affinity forlectins. The analysis, on microarray and molecular simulations, of the glycoclusters library displaying thesesaccharidic units and several topologies, valences and properties (aromaticity, charge,…) enable to identify key parameters of structure-affinity relationships. An anti-biofilm activity has been observed for the best glycoclusters targeting PA-IL.Testing in vivo activity of these best candidates will be explored. Targeting others lectins such as the ones on the flagella and pili of PA and involved in the early adhesion needs also to be developed. To this end, preliminary tests have been showed and some are in progress.

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1). A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.    The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104. Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering. For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.