Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment‐screening strategy is ...described that uses ADE to co‐crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co‐crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid‐handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co‐crystallization experiments to be discretely positioned on a single data‐collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co‐crystallized with a small 33‐compound mini‐library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure‐based drug discovery using as little as 2.5 nl of each major component.
A method is presented for screening fragment libraries using acoustic droplet ejection to co-crystallize proteins and chemicals directly on micromeshes with as little as 2.5 nl of each component. ...This method was used to identify previously unreported fragments that bind to lysozyme, thermolysin, and trypsin. Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment-screening strategy is described that uses ADE to co-crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co-crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid-handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co-crystallization experiments to be discretely positioned on a single data-collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co-crystallized with a small 33-compound mini-library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure-based drug discovery using as little as 2.5 nl of each major component.
L'objectif de ce mémoire consiste à étudier l'influence de la localisation de l'endommagement et de son étendue sur la fiabilité d'un pont existant en béton armé. À . cette fin, le programme LRT ...(Laval Rell~ability Toolbox) est développé dans le but d'évaluer la fiabilité de structures en considérant le système, c'est-à-dire l'interaction entre les éléments structuraux. La fiabilité du pont à l'étude est évaluée en considérant .la flexion et l'effort tranchant des poutres, à partir de fonctions d'états limites définies selon le Code canadien sur le calcul des ponts routiers, CAN/CSA -S6-00. Les facteurs faisant varier les indices de fiabilité des éléments structuraux et du pont, lorsque les poutres sont endommagées par la corrosion des armatures, sont examinés en simulant des scénarios d'endommagement diminuant à différents degrés la résistance à la flexion et/ou à l'effort tranchant d'un ou plusieurs . segments de poutres. En plus de démontrer l'importance de considérer le comportement du système lors de l'évaluation de structures redondantes, les résultats démontrent que pour le pont étudié, ce n'est pas tant l'intensité de l'endommagement qui influence sa fiabilité, mais surtout le mode de défaillance dominant, les sections critiques et le nombre de poutres endommagées. Ces conclusions auraient toutefois pu être différentes dans le cas d'un pont pour lequel l'écart entre les indices de fiabilité des poutres associés à la flexion et à l'effort tranchant serait moins grand. Enfin, la configuration du système et la corrélation définie entre les composantes sont aussi d'une grande importance dans le calcul de l'indice de fiabilité de la structure, car ils influencent grandement l'indice de fiabilité obtenu.
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