The uibiquitous Type II toxin – antitoxin (TA) loci encode two proteins, a toxin and an antitoxin. The antitoxin combines with and neutralizes a cognate toxin. Usually, the TA genes form an operon that is transcribed by a single promoter located upstream of the genes. In most cases, the antitoxin autoregulates the TA operon via binding to operator sites in the promoter region. In almost all such cases, the toxin act as a co-repressor of transcription as the toxin enhances the DNA binding of the antitoxin. Recently, it has been shown that toxins play an additional role in stimulating transcription, as the antitoxin and toxin ratio is important for cooperative binding of the complex to DNA. The antitoxin is rapidly degraded by cellular proteases under conditions of stress and treatment with antibiotics, which leads to activation of the toxin. The toxins of TAs belong to different gene families. The most abundant TA gene family is vapBC that, in some organisms, have expanded into cohorts of genes. For example, the major human pathogen Mycobacterium tuberculosis contains at least 88 TAs, 45 of which are vapBC loci. VapC toxins encoded by vapBC loci are PIN domain proteins (PilT N-terminal). Eukaryotic PIN domain proteins are site-specific ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. From in vitro experiments it has been postulated that VapC toxins are RNases or DNases but their exact cellular target has remained elusive. Here I show that VapC encoded by Shigella flexneri 2a virulence plasmid pMYSH6000 and the chromosome of Salmonella enterica serovar Typhimurium LT2 are site-specific endoribonucleases that specifically cleave tRNAfMet in the anticodon stem-loop in vivo and in vitro. Furthermore, I show that VapC dependent depletion of tRNAfMet leads to bacteriostatic inhibition of global translation, which surprisingly induces low-level initiation of translation at elongator codons that are correctly positioned relative to a Shine & Dalgarno sequence. I also show that VapC forms a complex with VapB and acts as a co-repressor of vapBC transcription. During steady state growth VapB is in excess of VapC. However, nutrient stress or treatment with antibiotics leads to Lon protease dependent decrease in VapB levels. Furthermore, I show that VapC in excess of VapB directly interferes with cooperative DNA binding of the VapBC complex, which is dependent on the dimerisation of the VapC toxin. 2 In conclusion, I show that enteric VapCs not only regulate global cellular translation by tRNAfMet cleavage, but also regulate vapBC transcription by conditional cooperativity.

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Organisms exposed to toxicants activate various defensive pathways such as the heat shock, xenobiotic, oxidative and metal stress responses. These sub-networks (pathways) involving groups of stress response genes act together in an integrated manner and hence constitute an overall Stress Response Network (SRN). My study aims at understanding this network of stress pathways by monitoring stress response reporter-gene outputs during exposure to single chemicals (11 heavy metals and 12 pesticides) and several mixtures, using 24 transgenic GFP reporter strains of the nematode Caenorhabditis elegans. Major stress responsive genes belonging to the heat-shock, metal-response and oxidative stress groups - plus selected genes from the xenobiotic response pathways, were selected as representative genes for this work. In addition, GFP transgenic strains for the key transcription factors that act in these stress pathways, along with the master stress regulators DAF-16 and CEP-1, were also studied. My study focused mainly on genes involved in the heat-shock stress and xenobiotic stress pathways, but responses involving other pathways are also reported, with appropriate attribution to the respective authors. Toxicant exposures of these arrays of transgenic strains were performed initially at 3 time-points on different dose ranges of single toxicants, selected on the basis of their known toxicity or widespread use (e.g. certain pesticides) in the environment. As part of a broader UKIERI project, the single toxicant exposure data were used in generating mathematical models of the stress-responsive sub-networks (performed by mathematicians Dr. Haque and Prof. King, School of Mathematical Sciences, University of Nottingham), which in turn were used to predict the likely outcome of exposures to simple toxicant mixtures. Organisms are often exposed to mixtures of toxicants in the environment and it is therefore important to understand and is possible predict the effects of toxicant mixtures. These models predicted different responses, e.g. additive effects for chemically similar (e.g. divalent) metals versus interfering effects for chemically dissimilar metals (e.g. divalent plus trivalent) in the case of the metallothionein (MTL) sub-network. By contrast, the heat-shock (HSP) sub- network model predicted only additive responses, irrespective of chemical similarity. Laboratory testing of simple binary mixtures using GFP reporter strains confirmed all of these model predictions. RNA interference was also used to knockdown key transcription factors in selected stress pathways (e.g. HSF-l, ELT-2, NHR-8 and DAF-16) in order to confirm the role of these factors in toxicant -induced gene expression. My study also investigated the effects of more complex chemical mixtures, such as soil water samples from a former mine site (P79) and two agricultural sites (P73 and P74), derived from the Spanish ECOMETRISK project. Our results identified responses to these complex mixtures that broadly agree (though only at certain time-points) with the mathematical model predictions for binary metal mixtures, in the case of soil sample P79. Interestingly, strong GFP induction was observed for several stress genes for the two agricultural soil samples and this was caused by the organic components present in these soil water samples rather than by metals. The source and nature of these organic contaminants in agricultural soils remains to be determined, though pesticide residues seem a likely culprit.

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Ce travail de thèse propose d’examiner l’intérêt de la volatolomique pour révéler dans le foie, le plasma et le tissu adipeux des composés organiques volatils (COVs) marqueurs d’exposition des animaux d’élevage aux micropolluants. Des développements méthodologiques ont d’abord été réalisés pour lever deux principaux verrous à l’application de la volatolomique. Un premier volet de ce travail méthodologique a ainsi permis de proposer des conditions d’extraction par microextraction en phase solide à la fois efficace, fidèle et reproductible pour l’analyse du volatolome de matrices biologiques complexes. Une comparaison de trois méthodes chimiométriques non-supervisées- l’Analyse en Composantes Principales (ACP), l’Analyse en Composantes Indépendantes (ACI) et l’Analyse en Composantes Communes (ACC) – a montré ensuite l’intérêt de l’ACC pour révéler des différences volatolomiques et identifier des marqueurs candidats pour chaque type d’exposition.En s’appuyant sur ces développements, un second volet de ce travail a permis d’évaluer l’intérêt de la volatolomique pour tracer l’exposition des animaux d’élevage à différents types de micropolluants. Cette recherche a confirmé la pertinence de l’analyse volatolomique du foie et a montré que le plasma pouvait permettre également un diagnostic ouvrant la voie à des contrôles in vivo de l’exposition animale. Les résultats montrent également une complémentarité informative entre les deux matrices biologiques. En revanche, le potentiel informatif de l’analyse du volatolome de tissus adipeux s’est révélé beaucoup plus limité, l’étude ne permettant pas d’identifier le moindre marqueur d’exposition. Ainsi, si la robustesse de ces marqueurs est validée par des travaux ultérieurs, ces résultats ouvrent la voie à un diagnostic de l’exposition animale aux micropolluants à l’abattoir ou sur le lieu d’élevage. / The present study proposes to examine the relevance of liver, plasma and adipose tissue volatolomics to detect volatile organic compound (VOC) markers of animal exposure to micropollutants. Methodological developments were first carried to solve two main limits to the application of volatolomics. A first part of this methodological development enabled the determination of the efficient, accurate and reproducible solid phase microextraction conditions for the analysis of the volatolome in complex biological matrices. Then, a comparison of three unsupervised chemometric methods - Principal Components Analysis (PCA), Independent Components Analysis (ICA) and Common Components Analysis (CCA) - highlighted the potential of CCA for the determination of volatolomic differences in VOC profiles and for the identification of candidate VOC markers for each contamination.Based on these methodological developments, the second part of this work studied the potential of liver, plasma and adipose tissue volatolomics to back-trace animal exposure to different types of micropolluants. These results confirmed the relevance of the volatolomic analysis of the liver and showed that the plasma could also of interest, opening the way to in vivo controls of animal exposure to micropollutants. Moreover, the results demonstrated the complementarity of the biological matrices. The informative potential of the volatolome analysis of adipose tissue is much more limited, as the study did not lead to the identification of any marker of exposure. If the robustness of these candidate markers is validated by further studies, these results pave the way for the diagnosis of animal exposure to micropollutants in slaughterhouse and during animal breeding.