No description available.

Staphylococcus aureus est une bactérie pathogène d’importance pouvant causer une multitude d’infections allant d’infections cutanées à des infections systémiques mortelles. Ce pathogène infecte l’homme ainsi que les animaux comme les porcs, bovins, volailles, etc. Chez les animaux, cette bactérie cause, entre autres, la mammite bovine. Il existe des souches de S. aureus qui sont résistantes à la méthicilline (SARM). Le nombre d’infections reliées aux SARMs est en constante évolution, car en plus de leur forte présence dans les hôpitaux, ces bactéries sont aussi présentes dans la communauté et auprès des animaux d’élevage. En plus de la résistance à la classe antibiotique des ß-lactamines, dont la méthicilline fait partie, plusieurs souches de SARMs sont résistantes à d’autres classes d’antibiotiques (macrolides, fluoroquinolones, aminoglycosides). De plus, plusieurs souches de S. aureus produisent des biofilms, soit des structures qui protègent les bactéries contre plusieurs facteurs de leur environnement. La plupart des antibiotiques sont moins efficaces pour tuer les bactéries se retrouvant dans cette structure. L’inefficacité de la plupart des antibiotiques existants envers ce pathogène demande la recherche de nouvelles classes d’antibiotiques ayant de nouvelles cibles thérapeutiques ce qui permettrait de cibler ce pathogène et de l’éliminer. De récentes recherches ont permis de découvrir une nouvelle classe d’antibiotiques, soit des pyrimidines modifiées, qui sont des ligands spécifiques du riborégulateur guanine, compris dans la classe des riborégulateurs purines. Ces molécules antibiotiques ont une cible très spécifique : elles sont efficaces lorsque le riborégulateur guanine contrôle spécifiquement la transcription du gène guaA, qui code pour la GMP synthétase. La position activée du riborégulateur résulte en la pleine transcription du gène guaA et subséquemment en la synthèse du GMP. Lorsqu’il y a un excès de guanine intracellulaire, ce ligand se lie spécifiquement à l’aptamère en 5’ du riborégulateur provoquant ainsi la formation d’une tige de terminaison qui empêche la transcription de guaA. En fait, chez S. aureus, le riborégulateur guanine contrôle tout l’opéron xpt-pbuX-guaB-guaA. Il a été démontré que la molécule 2,5,6-triaminopyrimidine-4-un (PC1), une pyrimidine modifiée, favorise la conformation terminatrice du riborégulateur guanine et ainsi, l’arrêt artificiel de la transcription de guaA. Par contre, cette molécule n’est pas encore parfaite. Il y a dimérisation en un composé inactif en présence d’oxygène. La suite des efforts de recherche a consisté à produire et identifier de nouvelles molécules de type pyrimidine dérivées de PC1 ou autres analogues de guanine et qui agissent comme antibiotiques en liant le riborégulateur guanine retrouvé chez les staphylocoques. En particulier, nous avons démontré l’efficacité in vitro et in vivo de la molécule GRL-206 contre les infections causées par les SARMs et les souches bovines de S. aureus. En parallèle, l’efficacité de GRL-206 a aussi été testée contre Staphylococcus epidermidis, un staphylocoque à coagulase négative (SCN) produisant des biofilms.

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No description available.

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This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

RasGRP1 is an intracellular signaling protein expressed in lymphocytes that is responsible for activating Ras GTPases. Positive regulation of RasGRP 1 requires translocation to cellular membranes where lipid-anchored Ras can be accessed. Plasma membrane localization of RasGRP 1 in response to antigen receptors requires both the Cl domain and the plasma-membrane targeting (PT) domain. The Cl domain binds to diacylglycerol (DAG) at membranes. The PT domain binds its putative ligand at the plasma membrane and is negatively regulated by an adjacent suppressor of PT (SuPT) domain. RasGRP1 also contains a pair of EF-hands, with Ca²⁺-binding capability, but with no known regulatory role. In DT4O cells, RasGRP1 translocates to the plasma membrane and activates the Ras ERK pathway in response to B cell receptor (BCR) signaling. By introducing point mutations in the Ca²⁺-binding loops of each of the EF-hands, I found that a potential Ca²⁺- interaction loop in the first EF-hand is required for RasGRP1 translocation and the consequential activation of the Ras-ERK pathway in response to BCR signaling. However, RasGRP1 translocation is not regulated by BCR-generated Ca²⁺ flux. EF-hands were not required for Cl domain-mediated membrane localization, but were needed for PT-mediated plasma membrane targeting. EF-hands enhanced PT-domain mediated plasma membrane localization by repressing the SuPT domain. The REM and GEF domains, which co ordinately bind to and catalyze guanine nucleotide exchange on Ras GTPases, needed to be present and Ras-bound for this EF-hand mechanism to be effective. When not bound to Ras, the REM-GEF domain complex suppressed both plasma membrane and endomembrane targeting of RasGRP 1 by an EF-hand independent mechanism. Finally, membrane localization and activation of a naturally occurring splice variant of RasGRP 1, found overexpressed in systemic lupus erythematosus (SEE) patients, was examined. This splice variant lacks exon 11, which encodes the segment of RasGRP1 between the GEF domain and the first EF-hand. Removal of exon 11 resulted in a defect in plasma membrane localization that was partially overridden by deletion of SuPT, while membrane localization control via the REM-GEF complex was not affected. Therefore, exon 11 deletion via alternative splicing appears to functionally disable the first EF-hand of RasGRP1.

The formation of base pair radical anions and cations is closely related to many fascinating research fields in biology and chemistry such as genetic mutation, radiation-induced DNA damage and dynamics of charge transfer in DNA. However, the relevant knowledge so far mainly comes from studies on isolated base pair radical anions and cations, and their behavior in the DNA environment is less understood. In this study, we focus on how the nucleobase sequence affects the properties of the guanine¡Vcytosine (G:C) base pair radical anion and cation. The energetic barrier and reaction energy for the proton transfer along the N1(G)¡VH¡E¡E¡EN3(C) hydrogen bond and the stability of (G:C)¡E (i.e., electron affinity and ionization potential of G:C) embedded in different sequences of base-pair trimer were evaluated using density functional theory and two-layer ONIOM method. The computational results demonstrated that the presence of neighboring base pairs has an important influence on the behavior of (G:C)¡E in the gas phase. The excess electron and positive hole were found to be localized on the embedded G:C and the charge leakage to neighboring base pairs was very minor in all of the investigated sequences. Accordingly, the sequence behavior of the proton transfer reaction and the stability of (G:C)¡E is chiefly governed by electrostatic interactions with adjacent base pairs. However, the effect of base stacking, due to its electrostatic nature, is severely screened upon hydration, and thus, the sequence dependence of the properties of (G:C)¡E in aqueous environment becomes relatively weak and less than that observed in the gas phase. The effect of geometry relaxation associated with neighboring base pairs as well as the possibility of proton transfer along the N2(G)¡VH¡E¡E¡EO2(C) channel have also been investigated. The implications of the present findings to the electron transport and radiation damage of DNA are discussed.

RasGRP1 is an intracellular signaling protein expressed in lymphocytes that is responsible for activating Ras GTPases. Positive regulation of RasGRP 1 requires translocation to cellular membranes where lipid-anchored Ras can be accessed. Plasma membrane localization of RasGRP 1 in response to antigen receptors requires both the Cl domain and the plasma-membrane targeting (PT) domain. The Cl domain binds to diacylglycerol (DAG) at membranes. The PT domain binds its putative ligand at the plasma membrane and is negatively regulated by an adjacent suppressor of PT (SuPT) domain. RasGRP1 also contains a pair of EF-hands, with Ca²⁺-binding capability, but with no known regulatory role. In DT4O cells, RasGRP1 translocates to the plasma membrane and activates the Ras ERK pathway in response to B cell receptor (BCR) signaling. By introducing point mutations in the Ca²⁺-binding loops of each of the EF-hands, I found that a potential Ca²⁺- interaction loop in the first EF-hand is required for RasGRP1 translocation and the consequential activation of the Ras-ERK pathway in response to BCR signaling. However, RasGRP1 translocation is not regulated by BCR-generated Ca²⁺ flux. EF-hands were not required for Cl domain-mediated membrane localization, but were needed for PT-mediated plasma membrane targeting. EF-hands enhanced PT-domain mediated plasma membrane localization by repressing the SuPT domain. The REM and GEF domains, which co ordinately bind to and catalyze guanine nucleotide exchange on Ras GTPases, needed to be present and Ras-bound for this EF-hand mechanism to be effective. When not bound to Ras, the REM-GEF domain complex suppressed both plasma membrane and endomembrane targeting of RasGRP 1 by an EF-hand independent mechanism. Finally, membrane localization and activation of a naturally occurring splice variant of RasGRP 1, found overexpressed in systemic lupus erythematosus (SEE) patients, was examined. This splice variant lacks exon 11, which encodes the segment of RasGRP1 between the GEF domain and the first EF-hand. Removal of exon 11 resulted in a defect in plasma membrane localization that was partially overridden by deletion of SuPT, while membrane localization control via the REM-GEF complex was not affected. Therefore, exon 11 deletion via alternative splicing appears to functionally disable the first EF-hand of RasGRP1.