Pseudomonas aeruginosa is resistant to many clinically-relevant antibiotics and this is partly attributable to multidrug efflux systems in this organism. One of these, MexEF-OprN, exports chloramphenicol, fluoroquinolones and trimethoprim and is positively regulated by MexT, encoded by the mexT gene that is located upstream of the efflux genes. MexT also positively regulates the mexS gene that encodes an oxidoreductase of unknown function and whose loss increases expression of mexEF-oprN. A null mutation in the mexS gene of P. aeruginosa increased expression of two 3-gene operons, PA4354-PA4355-PA4356 and PA2813-2812-2811. This increased expression, which paralleled an increase in mexEF-oprN expression in the same mutant, was, like mexEF-oprN, MexT-dependent. The PA4356 (xenB) gene product is homologous to a xenobiotic reductase in P. fluorescens shown to remove nitro groups from trinitrotoluene and nitroglycerin. As nitration is a well-known result of nitrosative stress, a role for xenB (and the co-regulated mexEF-oprN and PA2813-2812-2811) in a nitrosative stress response was hypothesized and tested. Using S-nitrosoglutathione (GSNO) as a source of nitrosative stress, expression of mexEF-oprN, xenB and PA2811 was shown to be GSNO-inducible, though in the case of xenB and PA2811 this was only seen in a mutant lacking MexEF-OprN and only for xenB, this GSNO-inducible expression was dependent upon MexT. Consistent with MexT being required for mexEF-oprN and PA4354-PA4355-PA4356 expression, MexT bound to their promoter regions. Chloramphenicol, a nitroaromatic antimicrobial that is a substrate for MexEF-OprN and resembles a nitrosated nitrosative stress product accommodated by this efflux system induced expression of mexEF-oprN, but not xenB, or PA2811, and this was dependent upon the MexT regulator. In addition to MexT positive-regulating activity of genes in response to nitrosative stress, MexT also negatively regulates the expression of mexAB-oprM through direct binding to its promoter region and the oprD gene, encoding an outer membrane porin that provides a portal of entry for basic amino acids and carbapenem, whose expression was reduced only by GSNO. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2011-05-15 20:59:57.018

In the midst of debate over the treatment of culture in foreign language education and the increased exposure to culture in the global economy, the knowledge of other cultures is more significant than ever. In order to improve teaching culture, materials have been examined and revised. The purpose of this exploratory study is to describe how culture is addressed in the fourteen high school EFL reading textbooks in Japan, that were approved by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) at the time of this study. In previous studies on EFL textbooks in Japan by Kitao (1979, 1988) and Iwata, Ogawa, Wen, Sakamoto, Takarada, Horio, Muto, and Mogi (2001) among others, the findings included the cultural elements categorized by various criteria, defining topics and types of reading texts and illustrations, classifying the student activities regarding culture, the descriptions of societies represented, the unnatural use of the English language that are edited for grammatical correctness, and the lack of elements that are usually present in the natural course of conversations or in the authentic passages. This study of the reading passages and pre- and post-reading activities was guided by several theories and studies: First, the authentic use of a language reflects the writer's culture (Brown, 1987; Byrnes, 1990; Gilmore, 2004; Honeyfield, 1977; Kramsch, 1993; Lamie, 1998; McKay, 1982; Shrum & Glisan, 2005; Swaffar, 1985), and the culture is reflected in the vocabulary (Sapir, 1949). Second, the readers' own cultural knowledge (C1) should be activated in understanding the culture of others (C2), comparing and contrasting C1 and C2 so that the differences and similarities will be more evident (Byrnes, 1990; Kolb, 1984; Kramsch, 1993; National Standards in Foreign Language Education Project [NSFLEP], 2005). The results show 1) that the reading passages were edited mostly by deleting and altering portions of the originals, and thus the culture was not present as in the original passages, and 2) pre-reading and post-reading activities/questions were literal and not designed to foster cultural understanding. / Ed. D.

Pseudomonas aeruginosa est un pathogène opportuniste à Gram-négatif, responsable d’infections nosocomiales chez des patients immunodéprimés et principale cause de morbidité et de mortalité chez les patients atteints de mucoviscidose. Les traitements utilisés contre P. aeruginosa peuvent être mis en échec en raison des nombreux mécanismes de résistance développés par la bactérie tels que les systèmes d’efflux RND, capables d’exporter les antibiotiques à l’extérieur de la cellule. Parmi ces systèmes, MexEF-OprN est très peu produit dans les souches sauvages mais il est surproduit chez les mutants appelés nfxC et conduit à une résistance aux fluoroquinolones, au chloramphénicol et au triméthoprime. Ces mutants ont également la particularité de résister de façon concomitante aux carbapénèmes et d’être peu virulents. Notons enfin que la pompe MexEF-OprN est codée par un opéron à trois gènes, mexEF-oprN, dont la transcription est activée par MexT, un régulateur appartenant à la famille LysR.Les mutants nfxC étant peu décrits dans le contexte clinique, nous avons évalué leur prévalence et caractérisé les événements génétiques conduisant à la surexpression de mexEF-oprN. A partir d’une collection de 221 souches cliniques isolées au CHRU de Besançon, et sélectionnées en raison de leur sensibilité diminuée à la ciprofloxacine et à l’imipénème, 19.5% surexprimaient mexEF-oprN. Nous avons par la suite caractérisé 22 souches non-redondantes et montré que seulement 13.6% d’entre elles possédaient des mutations inactivatrices dans le gène mexS alors que 40.9% avaient des mutations conduisant à la substitution d’un seul acide-aminé. Il est apparu que ces dernières mutations avaient des effets modérés sur les profils de résistance et de virulence alors que les mutations inactivatrices donnaient des hauts niveaux de résistance mais aucune virulence. Enfin, nous n’avons pas pu identifier de mutations génétiques pouvant expliquer la surexpression de mexEF-oprN des 45.5% de souches restantes, suggérant l’existence des mécanismes de régulation encore inconnus de cet opéron.Nous avons donc étudié des mutants résistants au chloramphénicol, sélectionnés in vitro à partir de la souche de référence PA14. Leur caractérisation nous a permis de découvrir un nouveau type de mutants surproducteurs de MexEF-OprN que nous avons appelé nfxC2. Tous possédaient des mutations gain-de-fonction sur le gène PA14_38040 (nommé cmrA) codant pour un régulateur de la famille AraC, jamais étudié auparavant. Chez les mutants nfxC2, l’expression de cmrA est augmentée, ainsi que celle de l’opéron mexEF-oprN et ceci, d’une façon MexS- et MexT-dépendante. De façon intéressante, ces mutations dans cmrA font apparaître un phénotype résistant sans toutefois altérer la virulence de la souche. Une analyse transcriptomique a montré que CmrA pouvait activer l’expression de 11 gènes parmi lesquels PA14_38020 apparaît comme étant nécessaire pour l’activation indirecte de mexEF-oprN. Ce gène code pour une quinol monooxygenase partageant des domaines conservés avec YgiN, une enzyme d’Escherichia coli qui participe à la réponse contre les électrophiles. D’ailleurs, l’exposition de la souche PA14 à des concentrations sub-inhibitrices d’électrophiles toxiques (glyoxal, méthylglyoxal et cinnamaldéhyde) active suffisamment la pompe MexEF-OprN pour générer un phénotype de résistance et ce, de façon CmrA-dépendante. Enfin, cette même exposition aux électrophiles active également deux autres pompes RND, à savoir MexAB-OprM et MexXY/OprM. Les voies de régulation conduisant à l’activation de ces deux opérons d’efflux seront étudiées prochainement au laboratoire. / Pseudomonas aeruginosa is a Gram negative opportunistic pathogen, responsible for several nosocomial infections in immunocompromised patients, and the main cause of mortality and morbidity of patients suffering from cystic fibrosis. Treatment of P. aeruginosa infections turns to be difficult due to its natural resistance to antibiotics, increased in part by the overproduction of RND efflux pumps capable to export antibiotics out of the cell. Amongst these systems, MexEF-OprN exports several antibiotics such as fluoroquinolones, chloramphenicol and trimethoprim. This efflux pump is quiescent in wild-type strains but it is highly produced in nfxC mutants, making them resistant to MexEF-OprN substrates. In addition, these mutants are characterized by their concomitant resistance to carbapenems and their low-virulence profile. MexEF-OprN is encoded by a three-gene operon, mexEF-oprN, whose transcription is activated by MexT, a member of the LysR family of transcriptional regulators. In the clinical context, nfxC mutants being poorly described, we evaluated their prevalence and characterized the genetic events responsible for mexEF-oprN overexpression. A collection of 221 clinical isolates from the University Hospital of Besançon exhibiting a reduced susceptibility to ciprofloxacin and imipenem was screened. We found that 19.5% of these strains overexpressed mexEF-oprN and further characterization of the 22 non-redundant mutants showed that only 13.6% of these mutants harbored a disrupted mexS gene. Moreover, 40.9% of nfxC clinical strains harbored missense mutations in mexS conducing to the substitution of a single amino-acid residue in the encoding protein. Interestingly, these mutations were associated to moderate effects on resistance and virulence factor production while disruptive mutations produced highly resistant but completely non-virulent strains. For the 45.5% of remaining strains, we failed to identify genetic mutations, which could explain mexEF-oprN overexpression; this indirectly suggested that there might be additional regulatory loci controlling the expression of this operon.We thus studied chloramphenicol resistant mutants selected in vitro derived from reference strain PA14 and found a new class of MexEF-OprN overproducers, which we called nfxC2, harboring gain-of-function mutations in a so-far uncharacterized gene, PA14_38040 (hereafter called cmrA) coding for an AraC transcriptional regulator. In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the expression of mexEF-oprN through MexS and MexT, resulting in a multi-drug resistant phenotype without altering virulence factor production. Transcriptomic experiments showed that CmrA positively regulates the expression of 11 genes, including PA14_38020, which is required for the MexS/MexT-dependent activation of mexEF-oprN. Gene PA14_38020 is predicted to code a quinol monooxygenase sharing conserved domains with YgiN of Escherichia coli, which was reported to be involved in the response of the bacterium to electrophiles. Interestingly, exposure of strain PA14 to sub-inhibitory concentrations of toxic electrophiles (glyoxal, methylglyoxal or cinnamaldehyde) strongly activates the CmrA-pathway and upregulates mexEF-oprN sufficiently to provoke the resistance to the pump substrates. Finally, we found that the same exposure to electrophiles is capable to activate two other RND pumps, MexAB-OprM and MexXY/OprM. The regulatory pathways conducing to activation of these two efflux operons will be elucidated at the laboratory.

The opportunistic pathogen P. aeruginosa is a leading cause of hospital-accquired infections, and is also the primary cause of morbidity and mortality in patients with cystic fibrosis (CF). In this thesis, I describe the identification and characterization of a novel LysR-type transcription regulator (LTTR) of P. aeruginosa named BexR. I show that BexR exhibits reversible ON/OFF bistable expression, which leads to the bistable expression of several genes including one encoding a virulence factor. I present results suggesting that this bistable expression depends on positive feedback of BexR. This work illuminates the simplicity with which a transcription regulatory network can exhibit a complex behavior and generate phenotypic diversity in a clonal population.