The Escherichia coli (E. coli) outer membrane protease OmpT is an endopeptidase of the omptin family in gram negative bacteria. OmpT cleave preferentially between two consecutive basic residues, especially Arg-Arg, and it has been classified as an aspartyl protease based on its crystal structure although biochemical confirmation of a catalytic aspartyl residue is lacking (Vandeputte-Rutten, et al., 2001). Our lab has successfully engineered the P1 and P1’ specificity and selectivity of OmpT by employing novel strategies for the isolation of enzyme variants that cleave desired substrates from large combinatorial libraries screened by flow cytometry. However, the engineering of proteases with altered specificity beyond the P1 and P1’ residues of the substrate have not been demonstrated. By applying high throughput screening of large libraries of OmpT constructed by structure-guided saturation mutagenesis of the S2 subsite (which recognizes the P2 residue), as well as random mutagenesis by error prone viii PCR and DNA shuffling, we engineered an OmpT variant exhibiting about 56 fold change in the selectivity for the P2 position in peptide substrates. Specifically, this enzyme preferred an acidic residue (Glu) over Tyr which is preferred by the wild type OmpT. Molecular modeling was then employed to provide insights on how mutations in OmpT mediated this change in P2 specificity. A long term goal of protease engineering is to generate highly specific enzyme variants that can be used for the irreversible inactivation of disease targets. The anaphylatoxin C3a is a key mediator in inflammation and has been implicated with multiple inflammatory diseases. Since the site of anaphylatoxin C3a recognized by cellular receptors lie in its C-terminus, a protease cleaving the C-terminus of C3a could be therapeutically relevant. Using high throughput screening and directed evolution we successful isolated C3a cleaving enzyme variants and have characterized them biochemically. Finally as part of this dissertation we have employed high throughput screening methods to dissect the substrate specificity of members of the kallikrein family of mammalian proteases which are implicated in a number of physiological and disease functions. The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction and vascular cell growth. KLK6 is over-expressed in breast and ovarian cancer tissues and has been shown to cleave peptides derived from human ix myelin protein and the Aβamyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6 by substrate phage-display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at the P1 site, Ser/Arg strongly preferred at P1’ and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1’. Docking simulations of consensus peptide substrates was used to infer possible identities of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates such as ionotropic glutamate receptor (GluR) and synphilin. / text

Afin de répondre aux besoins de plus en plus importants en puissance de calcul de la part des applicationsnumériques, les supercalculateurs ont dû évoluer et sont ainsi de plus en plus compliqués àprogrammer. Ainsi, en plus de l’apparition des systèmes à mémoire partagée, des architectures ditesNUMA (Non Uniform Memory Access) sont présentes au sein de ces machines, fournissant plusieursniveaux de parallélisme. Une autre contrainte, la diminution de la mémoire disponible par coeur decalcul, doit être soulignée. C’est ainsi que des modèles parallèles tels que MPI (Message Passing Interface)ne permettent plus aux codes scientifiques haute performance de passer à l’echelle et d’exploiterefficacement les machines de calcul, et doivent donc être combinés avec d’autres modèles plus adaptésaux architectures à mémoire partagée. OpenMP, en tant que modèle standardisé, est un choix privilégiépour être combiné avec MPI. Mais mélanger deux modèles avec des paradigmes différents est unet âche compliquée et peut engendrer des goulets d’étranglement qui doivent être identifiés. Cette thèsea pour objectif d’aborder ces limitations et met en avant plusieurs contributions couvrant divers aspects.Notre première contribution permet de r éduire le surcoût des supports exécutifs OpenMP en optimisantle travail d’activation et de synchronisation des threads OpenMP pour les codes MPI+OpenMP. Dansun second temps, nous nous focalisons sur les opérations collectives MPI. Notre contribution a pourbut d’optimiser l’opération MPI Allreduce en réutilisant des unités de calcul inoccupées, et faisant intervenirdes threads OpenMP. Nous introduisons également le concept de collectives unifiées, impliquantdes tâches MPI et des threads OpenMP dans une même opération. Enfin, nous nous intéressons àl’analyse de performance et plus précisément l’instrumentation des applications MPI+OpenMP, et notredernière contribution consiste en l’implémentation et l’ évaluation de l’outil OpenMP Tools API (OMPT)dans le support exécutif OpenMP du framework MPC. Cet outil nous permet d’instrumenter des constructionsOpenMP et de conduire une analyse axée aussi bien du côté des applications que dessupports d’exécution / To provide increasing computational power for numerical simulations, supercomputers evolved and arenow more and more complex to program. Indeed, after the appearance of shared memory systemsemerged architectures such as NUMA (Non Uniform Memory Access) systems, providing several levelsof parallelism. Another constraint, the decreasing amount of memory per compute core, has to bementioned. Therefore, parallel models such as Message Passing Interface (MPI) are no more sufficientto enable scalability of High Performance applications, and have to be coupled with another modeladapted to shared memory architectures. OpenMP, as a de facto standard, is a good candidate to bemixed with MPI. The principle is to use this model to augment legacy codes already parallelized withMPI. But hybridizing scientific codes is a complex task, bottlenecks exist and need to be identified. Thisthesis tackles these limitations and proposes different contributions following various aspects. Our firstcontribution reduces the overhead of the OpenMP layer by optimizing the creation and synchronizationof threads for MPI+OpenMP codes. On a second time, we target MPI collective operations. Our contributionconsists in proposing a technique to exploit idle cores in order to help the operation, with theexample of MPI Allreduce collective. We also introduce unified Collectives involving both MPI tasks andOpenMP threads. Finally, we focus on performance analysis of hybrid MPI+OpenMP codes, and ourlast contribution consists in the implementation of OpenMP Tools API (OMPT), an instrumentation tool,inside the OpenMP runtime of MPC framework. This tool allows us to instrument and profile OpenMPconstructs and allows the analysis of both runtime and application sides

Altering the substrate specificity of proteases is a powerful process with possible applications in many areas of therapeutics as well as proteomics. Although the field is still developing, several proteases have been successfully engineered to recognize novel substrates. Previously in our laboratory, eight highly active OmpT variants were engineered with novel catalytic sites. The present study examined the roles of several residues surrounding the active site of OmpT while attempting to use rational design to modulate fine specificity enough to create a novel protease that prefers phosphotyrosine containing substrates relative to sulfotyrosine or unmodified tyrosine residues. In particular, a previously engineered sulfotyrosine-specific OmpT variant (Varadarajan et al., 2008) was the starting point for rationally designing fifteen new OmpT variants in an attempt to create a highly active protease that would selectively cleave phosphotyrosine substrates. Our design approach was to mimic the most selective phosphoryl-specific enzymes and binding proteins by increasing positive charge around the active site. Sulfonyl esters have a net overall charge of -1 near neutral pH, while phosphate monoesters have a net overall charge of -2. Selected active site residues were mutated by site-directed mutagenesis to lysine, arginine, and histidine. The catalytic activities and substrate specificities of each variant were characterized. Although several variants displayed altered substrate specificity, none preferred phosphotyrosine over sulfotyrosine containing peptides. Taken together, our results have underscored the subtle nature of protease substrate specificity and how elusive it can be to engineer fine specificity. Apparently, phosphotyrosine specific variants were not possible within the context of our starting sulfotyrosine specific OmpT derivative mutated to have single amino acid changes chosen on the basis of differential charge interactions. / text

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch. VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner. V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq. In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530. Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.

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