The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO) enzyme is increased post-stroke and contributes to the development of post-stroke depression (PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production. The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for depression severity using the Center for Epidemiological Studies Depression Scale (CES-D). Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared. Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within 28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an etiological role in PSD.

The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO) enzyme is increased post-stroke and contributes to the development of post-stroke depression (PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production. The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for depression severity using the Center for Epidemiological Studies Depression Scale (CES-D). Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared. Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within 28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an etiological role in PSD.

P. aeruginosa est un pathogène opportuniste capable d'infecter un large spectre d'hôtes. Elle possède un vaste arsenal de facteurs de virulence. Le système de sécrétion de type III (SSTT) est un facteur de virulence majeur dont la régulation est complexe pour permettre une adaptation la plus précise possible de la bactérie au cours de l'infection. Nous nous sommes intéressés à déterminer le rôle potentiel de nouveaux acteurs de l'adaptation de P.aeruginosa au cours de l'infection. La porine OprF qui représente la protéine la plus abondante de la membrane externe de P. aeruginosa lui permettrait d'évaluer l'état d'activation du système immunitaire de son hôte afin d'adapter sa virulence. Chez P. aeruginosa, le tryptophane est le précurseur des kynurenines qui sont également produites par l'hôte à partir du tryptophane et qui, dans ce dernier contexte, sont des immunomodulateurs. Peu ou pas d'études ont été réalisées pour mettre en œuvre un éventuel rôle d'immunomodulation ou dans la virulence des kynurénines bactériennes. Dans un premier temps, nous nous sommes intéressés à un signal anciennement découvert au laboratoire et qui réprime l'expression du SSTT à haute densité bactérienne. Nous avons montré que ce signal exerce une régulation post-transcriptionnelle en plus d'une inhibition de la transcription des gènes du SSTT. Le métabolisme du tryptophane et de l'anthranilate semble être au cœur de ce processus de régulation. En inactivant des voies du catabolisme du tryptophane, nous avons montré que la production de ce signal dépend partiellement de la voie des kynurénines mais ne dépend pas ni des voies classiques du quorum sensing ni de l'opéron phnAB, impliqué dans la synthèse de l'anthranilate. Cependant, la voie des phénazines pourrait être impliquée dans la production de ce signal. Par CLHP couplée à la spectrométrie de masse, nous avons pu séparer des espèces moléculaires réprimant le SSTT et qui sont contenues dans ce signal, mais l'identification précise nécessite plus d'investigations. Dans un second temps, nous nous sommes intéressés aux kynurénines produites par la bactérie. Nous avons confirmé que P. aeruginosa produit des kynurénines et le gène kynA est le gène clé de la voie de synthèse de ces métabolites. En utilisant des fusions transcriptionnnelles, nous avons montré que le tryptophane et la kynurénine régulent positivement la production des kynurénines en agissant sur l'expression des gènes clés. D'autres parts, nous avons remarqué que la bactérie module l'activité de la voie métabolique des kynurénines issue du tryptophane en fonction de son état de croissance. Nous avons montré qu'au cours du dialogue interrègne bactérie/hôte, la voie des kynurénines de P. aeruginosa est stimulée par certains composants du système immunitaire. Grâce à un modèle d'infection pulmonaire aiguë, nous avons prouvé que les kynurénines produites par la bactérie sont importantes pour sa virulence. Selon notre hypothèse les kynurénines pourraient avoir une action sur la réponse immune, mais cela reste à déterminer. Dans un troisième temps, nous nous somme focalisés sur la porine OprF. Nous avons montré que la mutation ∆oprF est à l'origine d'une altération de la production mais vraisemblablement pas de la sécrétion des exotoxines du SSTT. Un ligand connu d'OprF, l'interféron gamma, module la voie des kynurénines. OprF pourrait donc avoir un rôle central dans les différents aspects de la régulation de la virulence. Nous avons donc produit des anticorps monoclonaux anti-OprF. Ces derniers se sont révélés capables de reconnaître spécifiquement la protéine OprF. Afin de vérifier l'efficacité de ces anticorps, des expériences de neutralisation de la bactérie in vitro puis in vivo seront réalisées. Mots clés : Pseudomonas aeruginosa, Système de Sécrétion de Type III, régulation, catabolisme du tryptophane, kynurénines, OprF. / P. aeruginosa is an opportunistic pathogen capable of infecting a wide host range. It possesses a large arsenal of virulence factors. The type III secretion system (TTSS) is a major virulence factor whose regulation is complex to allow the most accurate adaptation of the bacteria during infection. We were interested to determine the potential role of new actors in the adaptation of P. aeruginosa during infection. OprF represents the most abundant protein of the outer membrane of P. aeruginosa. This protein allows bacteria to assess the activation status of the host's immune system to adapt its virulence. In P. aeruginosa, tryptophan is the precursor of kynurenines that are also produced by the host from tryptophan and in the latter context, are immunomodulators. Little or no studies have been done to determine a possible role of bacterial kynurenines in immune modulation or virulence. Initially, we were interested in a signal previously discovered in the laboratory and which suppresses the expression of TTSS at high bacterial density. We have shown that this signal exerts a post-transcriptional regulation in addition to inhibition of TTSS genes transcription. The metabolism of tryptophan and anthranilate appears to be at the heart of this regulatory process. By inactivating pathways of tryptophan catabolism, we showed that production of this signal depends partly on the kynurenines pathway but does not depend neither classical ways of quorum sensing or phnAB operon involved in the synthesis of anthranilate. However, the phenazines pathway could be involved in the production of this signal. By HPLC coupled with mass spectrometry, we were able to separate molecular species suppressing the TTSS and which are contained in this signal, but accurate identification requires further investigation. In a second time, we were interested to kynurenines produced by the bacterium. We confirmed that P. aeruginosa produces kynurenines and KynA is the key gene in the synthesis of these metabolites. We showed that tryptophan and kynurenine upregulate the production of kynurenines by acting on the expression of key genes. Other shares, we found that the bacterium modulates the activity of the kynurenines pathway depending on its state of growth. We showed that during the dialogue bacteria / host, the pathway of kynurenines in P. aeruginosa is stimulated by certain immune system components. With an acute lung infection model, we proved that kynurenines produced by the bacterium are important to its virulence. We hypothesized that the kynurenines could have an effect on the immune response, but this remains to be determined. In a third time, we focused on the protein OprF. We showed that mutation ΔoprF is causing an alteration in production but probably not the secretion of TTSS exotoxins. One known ligand of OprF is the gamma interferon. It modulates the pathway of kynurenines. OprF could therefore have a central role in various aspects of the regulation of virulence. So, we produced monoclonal anti-OprF which recognizes specifically the protein OprF. To verify the effectiveness of these antibodies, neutralization experiments of the bacteria in vitro and in vivo will be realized.

Monitoring the impact of anthropogenic activities, particularly in industrial regions, requires ecological screening tools and frameworks that provide a comprehensive understanding of ecosystem responses to environmental changes. Biological indicators, organisms like algae, insects, fish, and sentinel mammals, are critical for assessing ecosystem health, particularly in areas of high industrial activity. The aim of this thesis was to identify a cross-species biomarker that can assess organismal health and environmental stress across various species, organs, and biological matrices. A range of biological systems and signaling pathways related to xenobiotic metabolism, energy homeostasis, immune responses, and stress adaptation were explored, leading to the identification of the Tryptophan-Kynurenine Pathway, which consumes 60-90% of tryptophan in vertebrates. Tryptophan and its metabolites play key roles in diverse physiological processes, including cell growth and maintenance, immunity, disease states, and the coordination of adaptive responses to environmental and dietary cues. This adaptive response suggests that kynurenine-tryptophan ratio (KTR) may serve as a marker for exposure to a variety of environmental stress conditions, including toxicants, nutrient scarcity, predatory stress, and habitat loss—stressors that are prevalent in areas of high industrial activity. In recent years, the KTR is increasingly recognized as a sensitive biomarker in human diseases induced or exacerbated by stress; however, its role in environmental exposure and wildlife health remains unexplored. This thesis explores the question of whether KTR can be utilized as a cross-species biomarker for environmental stress or environmental exposure to toxicants, particularly focusing on the Athabasca Oil Sands Region (AOSR). In vitro studies with mammalian hepatocytes exposed to polycyclic aromatic compounds (PACs): benzo[a]pyrene (BaP), and a Bitumen Water Accommodated Fraction (BitWAF) demonstrated that KTR increases were driven by elevated kynurenine levels, indicating disruption of tryptophan metabolism via the aryl hydrocarbon receptor (AhR). Further studies using acid extractable organics from Oil Sands Process-Affected Water (OSPW), Naphthenic Acid Fraction Components (NAFCs) showed metabolic reprogramming, including altered glucose and fatty acid uptake and mitochondrial dysfunction, mediated through PPARα activation and upregulation of Tdo2, the enzyme responsible for kynurenine production. In vivo studies of longnose and white suckers from the AOSR were conducted to assess the relationship between KTR and CYP1 enzyme activity (EROD). These studies revealed species-specific responses, with an inverse correlation between KTR and EROD in longnose suckers and a direct correlation in white suckers. These findings validate KTR as a biomarker for environmental exposure in wildlife, with significant implications for monitoring ecosystem health. Collectively, this work demonstrates the potential of KTR as a novel biomarker for environmental toxicology, offering a valuable tool for assessing organismal stress across species in response to environmental contaminants. / Thesis / Doctor of Philosophy (PhD) / Human activities, especially industrial operations, can significantly impact the environment. To monitor these effects, scientists use various tools and organisms to assess ecosystem health. This research introduces a new approach to measuring environmental stress in wildlife by focusing on two key molecules: tryptophan and kynurenine. These molecules are part of a conserved biological pathway that helps all organisms manage stress, repair cells, adapt to their environment, and maintain overall health. Tryptophan, an essential amino acid, is broken down into kynurenine, and the balance between them— known as the kynurenine-tryptophan ratio (KTR)—can indicate the level of stress an organism is experiencing. This thesis investigates whether KTR can detect environmental stress caused by industrial activity, particularly from petroleum-derived chemicals in the Athabasca Oil Sands Region (AOSR). In laboratory experiments, mammalian liver cells were exposed to oil sands compounds and complex mixtures from oil sands wastewater. These compounds changed KTR, showing that the liver’s stress response was activated, and tryptophan metabolism was disrupted. The study also found that these chemicals affected cellular energy use and the way cells process fats and sugars. Furthermore, we examined fish species in the AOSR: longnose and white suckers. Results showed that KTR varied depending on the species and the location of exposure. In white suckers, KTR increased in response to stress, while in longnose suckers, it decreased, indicating species-specific responses to environmental changes. Overall, our findings suggest that KTR could serve as a useful tool for measuring environmental stress in different species and ecosystems, especially in areas affected by anthropogenic or industrial activity. Understanding how KTR changes in response to pollution can help scientists better monitor and protect wildlife and ecosystem health.