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Trypanosoma cruzi, l'agent étiologique de la maladie de Chagas, est transmis aux humains par des insectes hématophages de la sous-famille des Triatominae. Son cycle de vie intravectoriel se déroule exclusivement dans le tractus digestif de ces insectes et est étroitement lié à sa physiologie. La compréhension de la physiologie digestive, ainsi que l'exploration des mécanismes régissant l'interaction entre le parasite et les triatomines, promettent de dévoiler de nouvelles cibles cruciales pour altérer le développement du parasite et réduire la compétence vectorielle. Ce travail de thèse s'est focalisé d'une part sur l'exploration de la physiologie digestive de Rhodnius prolixus, qui, malgré son statut comme organisme modèle en physiologie des insectes et vecteur majeur de la maladie de Chagas, présente plusieurs questions concernant sa biologie digestive encore méconnues. Cela a permis de disséquer la cascade multi-peptidases impliquée dans le catabolisme de l'hémoglobine et d'illuminer le rôle digestif joué par l'intestin moyen antérieur. D'autre part, ce travail a permis d'explorer l'impact de l'établissement du parasite sur le vecteur en dévoilant des perspectives fondamentales sur l'interaction moléculaire complexe entre les triatomines et le trypanosome. L'infection par T. cruzi module le protéome de l'intestin moyen de l'insecte en manipulant l'expression de protéines impliquées dans des processus cellulaires cruciaux. De plus, la présence du parasite déclenche une réponse immunitaire systémique, caractérisée par l'expression d'un ensemble de protéines immunitaires qui pourraient agir pour maîtriser la parasitémie dans le tube digestif, empêchant ainsi la propagation de l'infection dans l'hémocoele. Ce travail a mis en lumière diverses facettes de la biologie des triatomines, et présente des cibles prometteuses exploitables pour le contrôle de la transmission de la maladie de Chagas., Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Infection by the Human Immunodeficiency Virus type 1 (HIV-1) remains a major global public health issue. Despite effective antiretroviral therapy, HIV-1 persistence occurs in cellular reservoirs, necessitating continuous drug intake leading HIV-1 infection into a chronic disease. Therefore, an HIV cure requires either eliminating these viral reservoirs or achieving an deep blockade of HIV-1 transcription in the absence of treatment. Deciphering the underlying molecular mechanisms that drive HIV-1 into latency is critical for targeted HIV cure efforts.This Ph.D. thesis aims to enhance the knowledge on the molecular mechanisms of HIV-1 latency, specifically at epigenetic level. First, this work contributes to the discovery of a novel epigenetic actor of HIV-1 latency, known as UHRF1, in both infected T-lymphoid and myeloid cells. UHRF1 acts as a HIV-1 transcriptional repressor by modulating both DNA and histone methylation. In addition to DNA methylation, many evidence have highlighted DNA hydroxymethylation as a new epigenetic mark involved in cellular transcriptional regulation. This Ph.D. thesis demonstrated the presence of DNA hydroxymethylation along both the latent and the reactivated HIV-1 provirus. Finally, this work led to the discovery of another novel epigenetic actor of HIV-1 latency, known as KLF16. This cellular transcription factor regulates HIV-1 transcription by modulating both histone acetylation and methylation in both the infected T-lymphocytic cell line and the infected monocytic cell line. Altogether, our findings expand our understanding of the molecular mechanisms involved in HIV-1 transcriptional latency, specifically at epigenetic level. Moreover, the identification of novel HIV-1 regulators have unveiled new drug targets that could be critical in HIV-1 cure efforts., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The Prdm12 gene encodes the first transcriptional regulator that, once mutated, causes congenital insensitivity to pain (CIP) in humans. In recent years, epigenetic mechanisms have opened an avenue for the development of novel kind of analgesics. Work of our laboratory has shown that in mice, during development, Prdm12 is strongly expressed in somato-sensory ganglia where it is selectively transcribed in neurons from the nociceptive lineage, and that it remains expressed in mature neurons where it controls nociception. However, its mechanisms of action remain largely unknown. In my work, we have investigated the mechanisms behind Prdm12’s role in the specification of the nociceptive lineage in nervous system development. Results obtained have confirmed that Prdm12 is required for progenitor survival. They also revealed that it is required in developing somato-sensory neuron to repress an alternate viscero-sensory fate. Using a transgenic mouse line of Prdm12 overexpression in an inducible conditional manner, we showed that Prdm12 does not block Phox2b expression when ectopically expressed in visceral ganglia, revealing a context dependent repressive function. Previous work from the lab showed that the loss of Prdm12 in mature nociceptors of adult mice deregulates the expression of many nociceptive genes, and reduces their response to capsaicin. A hypersensitivity to formalin was also observed. To further approach the role of Prdm12 in nociception, we performed complementary Prdm12 gain of function experiments using AAV. The preliminary results obtained further suggest a role for Prdm12 in nociception., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

A highly evolved neocortex is associated with higher-order cognitive abilities humans possess. Thus, it is imperative to understand how the human cortex develops and its associated diseases. A complex network of transcription factors orchestrates the patterning of the cortex. Among them, Dmrt5 and Emx2 are evolutionarily conserved and are expressed early and similarly in the developing cortex. Interestingly, either of their deletions results in strikingly similar phenotypes and their compound deletion leads to an adverse phenotype, suggesting they cooperate during cortical development. During the first part of my PhD, I tried to understand the mechanisms at the basis of their cooperative function using mouse transgenics, bulk RNA sequencing, chromatin immunoprecipitation followed by sequencing and mass spectrometry-based methods. Our result suggests that their deletion perturbs a similar set of gene regulatory networks. Unexpectedly, their common direct targets are rather limited, but all encode important regulators of cortical fate. Exploration of their protein partners through mass spectrometry-based methods points to possible mechanisms contributing to their functional disparity, wherein they utilise a different set of interactors that confers their distinct functional properties. In the second part of my thesis, I also explored the unique role played by Dmrt5 in the development of the choroid plexus tissue, a brain-associated epithelial-endothelial convolute tissue that is involved in most of the cerebrospinal fluid production. The choroid plexus epithelium derives from the neuroepithelial tissue, which selectively expresses Dmrt5 as opposed to Emx2. Dmrt5 conditional ablation in the medial pallium does not result in any identifiable specification defects of the choroid plexus. Yet, mice in which Dmrt5 is conditionally deleted postnatally develop hydrocephalous, a condition often linked with defective choroid plexus function. Results obtained suggest that this p, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Plasma membrane H+-ATPases are highly conserved in yeast, other fungi and plants. Their main role is to establish the H+ gradient at the plasma membrane (PM) to maintain the cytosolic pH close to neutral values and fuel the uptake of nutrients through secondary active transport systems. In both plants and yeast, PM H+-ATPases have also been described to promote activation of TORC1, the rapamycin-sensitive kinase complex controlling cell growth. This for instance occurs in yeast upon H+ influx coupled to amino acid uptake. Fungal and plant PM H+-ATPases are self-inhibited by their carboxyterminal tails unless this domain is phosphorylated at specific residues. In the yeast PM H+-ATPase (Pma1), the self-inhibitory action of the carboxyterminal tail is neutralized when the adjacent S911 and T912 are phosphorylated, but the kinase(s) and phosphatase(s) controlling their phosphorylation remain unknown.In this thesis work, with the objective of understanding the molecular events taking place at Pma1`s level upon H+ influx and triggering the signaling cascade towards TORC1, we show that the largely redundant Ptk1 and Ptk2 kinases are responsible for phosphorylating S911-T912 at the carboxyterminal tail of Pma1. We also show that the carboxyterminal tail of Pma1 is not essential to initial TORC1 activation induced by H+-coupled amino acid uptake. However, if not properly phosphorylated by Ptk1 and Ptk2 kinases, the carboxyterminal tail of Pma1 can interfere with TORC1 activation. Additionally, we provide evidence that activated TORC1 exerts negative feedback control on Pma1 by reducing phosphorylation of its carboxyterminal tail.Overall, our results shed important new light on phosphoregulation of the yeast Pma1 H+-ATPase and on its interconnections with TORC1., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Toxin-antitoxin (TA) systems are widespread in bacterial and archaeal genomes. These TA modulesgenerally encode a toxic component and its cognate antidote in the same operon. In the case of type IITA systems, the antitoxin binds to the toxin to form a nontoxic complex via protein-protein interactionand thus, the toxin is retained in an inactive state protecting the bacterial cells. Under unfavourableconditions in the cell, the TA systems are activated, and the labile antitoxin is degraded by proteases;therefore, the toxin can find its target in the bacterial cell leading to growth inhibition or cell death. Arecent discovery reveals multiple families of small alarmone synthetases (SASs) in which they areencoded in conserved bicistronic architectures that recall toxin-antitoxin modules. ToxSAS (toxicSASs) enzymes act through translation inhibition mediated by pyrophosphorylation of tRNA CCAends, such as FaRel2, or through synthesis of the toxic alarmone (p)ppApp and ATP depletion (such asFaRel). Both enzymatic reactions use ATP as a pyrophosphate donor. In this thesis, we have studiedthe FaRel2 toxSAS effector from Coprobacillus sp. D7 (Coprobacillus) promotes growth arrest in E.coli by inhibiting protein synthesis. FaRel2 transfers a pyrophosphate moiety from ATP to the 3’CCAend of the tRNA. Indeed, this modification abrogates the aminoacylation of tRNA and the starvationsignal of cellular amino acids by the ribosome-RelA enzyme. Virtually nothing is known about theneutralisation mechanism that ATfaRel2 antitoxin exerts to counteract FaRel2 toxicity and diversitytoxicity activity in the new toxSAS subfamilies. In this study, we show that the pseudo-Zn2+ fingerdomain (pZFD) of the ATfaRel2 antitoxin prevents the access of ATP to the pyrophosphate donor siteof the FaRel2 toxin, without affecting the recruitment of the tRNA pyrophosphate acceptor. On thecontrary, toxSASs producing (p)ppApp are inhibited via Tis1 antitoxin domains by occlusion of thepyrophosphate accepto, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Le virus humain T lymphotropique de type 1 (HTLV-1) est l'agent étiologique de la leucémie aiguë T chez l'adulte. L'infection par le HTLV-1 se caractérise par une phase de latence virale, qui représente une stratégie d'évasion du système immunitaire de l'hôte. Cette latence virale se manifeste par une répression de la protéine trans-activatrice Tax codée en orientation sens à partir du 5’LTR. La protéine cellulaire BCL11b a été démontrée comme étant importante pour la maintenance et l’établissement de la latence virale du HTLV-1 par son recrutement aux promoteurs du HTLV-1 et la répression de la protéine trans-activatrice Tax. De plus, p300 a été démontrée comme permettant l’acétylation de BCL11b. La lysine K686 de BCL11b a été identifiée comme étant importante pour l’acétylation globale de la protéine BCL11b mais également pour son rôle fonctionnel et sa localisation. Ensemble, les résultats ont permis de mettre en évidence le facteur BCL11b comme une cible thérapeutique potentielle dans le domaine de l’oncogenèse liée au HTLV-1., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Prdm12 has been identified as one of the few genes found mutated in patients suffering from Congenital Insensitivity to Pain (CIP), and the only one encoding an epigenetic factor, marking it as a potential therapeutic target in pain conditions. Previous works detailed that during neurogenesis Prdm12 is highly enriched in sensory ganglia in which it is selectively expressed in nociceptors and that it is pivotal for their survival and maturation. Prdm12 expression remains expressed in mature nociceptors of adults, suggesting a potential role in their functional properties, a hypothesis that we explored during this thesis, using loss of function genetic approaches to delete Prdm12 in TGs and DRGs of adult mice. The consequences of this ablation were analyzed at the molecular, electrophysiological and behavioral levels. The results obtained indicate that Prdm12 is not required anymore for the survival nociceptors at adulthood, but that it modulates the expression of a set of ion channel and receptor genes and therefore controls their excitability.Moreover, other than some in vitro studies investigating the epigenetic properties of Prdm12, there are little to no evidence of its exact mechanism of action, its potential interacting partners and the cis-regulatory elements controlling its expression in the nociceptors. During the last half of my PhD studies, in vitro assays and transgenic mouse models were used to provide insight into the mechanisms that regulate Prdm12 expression in the DRGs and how Prdm12 interacts with master epigenetic regulators EZH2 and G9a to regulate gene expression in the nociceptors. Data presented in this thesis reveal some potential upstream regulatory elements of Prdm12 that could drive its selective expression in mouse developing nociceptors. Finally, while Prdm12 is co-express in dorsal root ganglia with EZH2 and G9a during murine development and interact with them in vitro, using a conditional knockout G9a mouse model to investigate the func, Prdm12 a été identifié comme l'un des rares gènes dont les mutations induisent chez l’homme une insensibilité congénitale à la douleur (ICD), et le seul parmi ces gènes codant pour un facteur épigénétique, ce qui en fait une cible thérapeutique potentielle dans les affections douloureuses. Les travaux précédents ont montré que durant la neurogenèse Prdm12 est enrichi dans les ganglion sensoriels où il exprimé sélectivement dans les nocicepteurs et qu’il joue un rôle essentiel dans leur survie et leur maturation. L'expression de Prdm12 reste exprimée sélectivement dans les nocicepteurs matures chez l’ adulte, suggérant un rôle potentiel dans leurs propriétés fonctionnelles, une hypothèse que nous avons explorée au début de cette thèse en utilisant des approches génétiques de perte de fonction pour supprimer Prdm12 sélectivement dans les TGs et les DRGs de souris adultes. Les conséquences de cette ablation ont été analysées aux niveaux moléculaires, électrophysiologique et comportemental. Les résultats obtenus indiquent que Prdm12 n'est plus nécessaire à la survie des nocicepteurs chez l’adulte, mais qu’il module l’expression d’une batterie de codant pur des canaux ioniques et récepteurs et contrôle ainsi leur excitabilité.De plus, à l'exception de certaines études in vitro portant sur les propriétés épigénétiques de Prdm12, il existe peu voire aucune preuve de son mécanisme d'action exact, de ses partenaires potentiels d'interaction et des éléments de régulation cis-contrôlant son expression dans les nocicepteurs. Au cours de la dernière moitié de mes études doctorales, des essais in vitro et des modèles de souris transgéniques ont été utilisés pour fournir des informations sur les mécanismes régulant l'expression de Prdm12 dans les DRGs et sur la manière dont Prdm12 interagit avec les régulateurs épigénétiques maîtres EZH2 et G9a pour réguler l'expression des gènes dans les nocicepteurs. Les données présentées dans cette thèse révèlent certains éléments de régulatio, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

L’obésité représente un défi sanitaire global et substantiel. Engendrant de nombreuses comorbidités, son incidence augmente dans toutes les régions du monde et fait peser un poids important aussi bien sur les sociétés que les individus. Comprendre ses mécanismes et développer des stratégies thérapeutiques efficaces est donc un enjeu essentiel. A l’homéostasie, le tissu adipeux présente un environnement de type 2 anti-inflammatoire essentiel à sa fonction. La production de cytokines de type 2 (i.e. IL-5, IL-13) par les cellules lymphoïdes innées de type 2 (ILC2), et potentiellement les lymphocytes Th2, a notamment été associée au maintien de l’homéostasie par différents mécanismes impliquant l’accumulation d’éosinophiles et de macrophages anti-inflammatoires. Durant l’obésité, l’infiltrat immunitaire du tissu adipeux est profondément modifié. Les populations résidentes anti-inflammatoires deviennent alors dysfonctionnelles tandis que des cellules inflammatoires s’accumulent et mènent au développement d’une inflammation systémique chronique et de bas grade responsable des nombreuses comorbidités liées à l’obésité. De plus en plus d’études suggèrent que le maintien ou la restauration des populations anti-inflammatoires du tissu adipeux permet de limiter le développement de l’obésité et des troubles métaboliques associés. Identifier les mécanismes menant à la dysfonction des cellules anti-inflammatoires du tissu adipeux durant l’obésité présente donc un intérêt thérapeutique certain. Ce travail a ainsi permis d’identifier le rôle inattendu de deux voies de costimulation dans l’inhibition de la réponse de type 2 dans le tissu adipeux durant l’obésité. Premièrement, le récepteur costimulateur CD27, préalablement connu pour favoriser le développement de réponses lymphocytaires de type 1, a été identifié comme un régulateur négatif des Th2 du tissu adipeux, limitant à la fois leur survie et leur fonction. Ainsi, les souris CD27KO affichent un nombre de Th2 fonctionnels supé, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Bovine Leukemia Virus (BLV) and Human T-Lymphotropic Virus type I (HTLV-1) are two oncogenic retroviruses characterized by viral latency, thereby allowing escape from the host immune surveillance and the development of BLV- and HTLV-1-associated diseases. In this PhD thesis, we demonstrated for the first time the role of the cellular factor UHRF1 (Ubiquitin-like with PHD and RING Finger domains 1) in the transcriptional repression of BLV and HTLV-1. We showed the in vitro binding and in vivo recruitment of UHRF1 to the BLV and HTLV-1 transcriptional promoters located in the viral 5’ Long Terminal Repeat (5’LTR). We demonstrated the repressive role of UHRF1 in BLV and HTLV-1 gene expression in both basal and transactivated conditions using transient transfection assays. Moreover, we showed that UHRF1 epigenetically repressed the HTLV-1 5’LTR using RNA interference and that UHRF1 interacted with the HTLV-1 Tax transactivator. Finally, our results using primary cells from HTLV-1-infected individuals suggested the involvement of UHRF1 in the tumoral development observed in the HTLV-1-infected individuals. In conclusion, this PhD thesis provides a better understanding of BLV and HTLV-1 transcriptional regulations and offers new perspectives for developing innovative therapeutic strategies against BLV and HTLV-1 infections and their associated diseases., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

La douleur est un problème de santé publique majeur puisqu'elle touche 20 % de la population mondiale. Les traitements actuels procurent souvent un soulagement limité et sont associés à des effets secondaires. Il y a donc un besoin urgent de meilleurs traitements contre la douleur. La douleur résulte de l'activation d'un sous-ensemble de neurones somatosensoriels appelés nocicepteurs qui sont spécialisés pour détecter les stimuli potentiellement douloureux. Prdm12 est un régulateur épigénétique sélectivement exprimé dans les nocicepteurs dans le système nerveux en développement dont la mutation provoque une insensibilité congénitale à la douleur, une maladie génétique rare caractérisée par l'incapacité à ressentir la douleur. Au cours des années précédentes, notre laboratoire a montré que Prdm12 est nécessaire durant la neurogenèse sensorielle pour la génération du lignage nociceptif. L’expression de Prdm12 est maintenue dans les nocicepteurs matures suggérant un rôle fonctionnel potentiel chez l'adulte. Nos résultats ont montré que la perte de Prdm12 dans les nocicepteurs matures provoquait une dérégulation des gènes codant pour des récepteurs et des canaux ioniques impliqués dans la nociception, modifie l'excitabilité neuronale ainsi que les réponses comportementales à la formaline et à la capsaïcine. En conclusion, nos données ont montré que Prdm12 exerce également une influence sur le bon fonctionnement des nocicepteurs chez la souris adulte.Pain is a major health burden as it affects 20% of the worldwide population. Current therapies often provide limited relief and are associated with side effects. There is thus an urgent need for better analgesics. Pain results from the activation of a subset of somatosensory neurons called nociceptors that are specialized to detect potentially painful stimuli. Prdm12 is an epigenetic regulator selectively expressed in nociceptors in the developing peripheral nervous system whose mutation causes congenital insensitivity to pa, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The cuticle of C. elegans is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier. Here, we identify the lipid transfer protein GMAP-1 as a critical element setting the permeability of the C. elegans cuticle. A gmap-1 deletion mutant increases cuticular permeability to sodium azide, levamisole, Hoechst, and DiI. Expressing GMAP-1 in the hypodermis or transiently in the adults is sufficient to rescue this gmap-1 permeability phenotype. GMAP-1 protein is secreted from the hypodermis to the aqueous fluid filling the space between collagen fibers of the cuticle. In vitro, GMAP-1 protein binds phosphatidylserine and phosphatidylcholine while in vivo, GMAP-1 sets the surface lipid composition and organization. Altogether, our results suggest GMAP-1 secreted by hypodermis shuttles lipids to the surface to form the permeability barrier of C. elegans., SCOPUS: ar.j, info:eu-repo/semantics/published

This work investigated the blind spots of the mechanisms of action of fluoroquinolones (F-QNL) on the Gram-negative bacterium Escherichia coli from two perspectives (1) how cells die and (2) how certain cells manage to survive: the ‘persister cells’. F-QNLs are frequently prescribed against bacterial infections, though the lethal mode of action is still elusive. As a frequent treatment failure is associated with antibiotic resistance, it is critical to better understand the antibiotic action. In this light, the principal aim was to find out more about the putative role of Reactive Oxygen Species (ROS) in F-QNL-dependent killing. Using a single-cell approach, this work enabled to pinpoint the source, timing and level of ROS contribution during F-QNL treatment. Characteristic physiological aspects of persister cells were uncovered. Finally, new aspects of the more-kills-less phenomenon in F-QNLs were revealed., Cette étude porte sur les éléments manquants des connaissances des mécanismes d'action des fluoroquinolones (F-QNL) sur la bactérie Gram-négative Escherichia coli sous deux angles (1) comment les bactéries meurent et (2) comment certaines parviennent à survivre, connu sous le terme de « Persistance ». Les F-QNLs sont fréquemment prescrits contre les infections bactériennes, bien que l'activité létale soit méconnue. Étant donné que le fréquent échec thérapeutique est associé à la résistance aux antibiotiques, il est essentiel de mieux comprendre leur mode d’action. Dans cette optique, l'objectif principal était d'en savoir plus sur l'implication présumée des espèces réactives de l'oxygène (ROS). Avec une approche au niveau de la cellule unique, ces travaux ont permis d'identifier la source, le moment et le degré de contribution des ROS lors du traitement par les F-QNLs. Des aspects physiologiques des cellules persistantes ont été découverts. Enfin, l'énigme du phénomène ‘more-kills-less’ des F-QNLs a été éclairé., Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Onchocerciasis is a Neglected Tropical Disease that has a significant socioeconomic im-pact, especially in Sub-Saharan Africa. Numerous reports indicate that the Expanded Special Project for the Elimination of Neglected Tropical Diseases needs novel diagnostic tools before achieving its goal of successful elimination of onchocerciasis in Africa. The current diagnostic tests are either invasive, insensitive, or not applicable in the field and about 25% of persons infected cannot mount immune responses against the single antigen used in the only approved Ov-16 serological test. In the quest to identify novel biomarkers that can be used to certify that a patient is free from the disease, evaluate the progress of elimination programmes, and conduct post elimination surveil-lances, mass spectrometric analysis of Onchocerca volvulus crude extract revealed that 1392 proteins are expressed in the adult and microfilariae stages of the parasite. Computational analysis predicted six of the proteins as O. volvulus potential diagnostic targets. Linear B-epitopes were predicted from the six proteins and used to construct a multiepitope antigen (OvMCBL02). Serological analysis revealed that the OvMCBL02 test significantly differentiated between serum samples of onchocer-ciasis patients from the Kombone Health Area in the South West Region of Cameroon (n = 63) and control serum samples from Rwanda (n = 29) and Europe (n = 26) as well as between serum samples from the onchocerciasis hyperendemic region of Kombone Health Area (n = 63) and the hypoen-demic region of Bandjoun Health District (n = 54). Interestingly, the test did not cross-react with serum samples from patients suffering from related nematode infections, thereby suggesting that further characterization of the OvMCBL02 multiepitope antigen will render it an additional mem-ber of the diagnostic toolbox for the elimination of onchocerciasis., SCOPUS: ar.j, info:eu-repo/semantics/published

In vivo, dans les conditions physiologiques, la capacité de minéralisation est exclusivement portée par les ostéoblastes et les ondotoblastes. La grande majorité des cellules composant un vertébré ne peut induire la formation de cristaux d’hydroxyapatite. Toutefois, certaines cellules non osseuses comme les cellules stromales mésenchymateuses (MSCs) et les cellules musculaires lisses vasculaires (VSMCs) ont la capacité d’adopter un phénotype ostéoblastogénique dans certains contextes pathologiques et font exception à cette règle. Le processus de minéralisation résulte d’une balance entre des facteurs pro et anti-ostéoblastogéniques. Il existe donc des mécanismes permettant de réguler négativement l’ostéoblastogenèse/la minéralisation dans les tissus « mous ». Ce projet de thèse est porté sur la compréhension de ces mécanismes et plus particulièrement sur le rôle des macrophages dans cette régulation. En effet, il a été précédemment démontré au niveau de la moelle osseuse qu’une population de macrophages, appelés OsteoMacs, facilite la différenciation des cellules stromales mésenchymateuses médulaires. De façon étonnante, au laboratoire, des résultats préliminaires nous ont menés à penser que les macrophages non médullaires participeraient à la régulation négative de la différenciation ostéoblastogénique des MSCs issues d’autres tissus représentés ici par les MSCs du tissu adipeux (ATMSCs) et les VSMCs.La première partie de ce travail de recherche a consisté en la caractérisation de l’effet inhibiteur des macrophages sur la différenciation ostéoblastogénique des ATMSCs et des VSMCs, ainsi qu’à l’identification des mécanismes moléculaires gouvernant cet effet inhibiteur des macrophages. Les résultats obtenus suggèrent que les macrophages exercent cet effet sur la différenciation de façon tardive. En effet, les macrophages produisent du TGFß stocké dans le milieu extracellulaire sous forme latente. Dans les étapes tardives de la différenciation ostéoblastogénique, les, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Les troubles neurocognitifs représentent une complication post-opératoire majeure dontl’étiologie est restée longtemps spéculative. Ce n’est que récemment qu'une cascadeinflammatoire, menant à l’activation des microglies, a été identifiée et proposée. Dans cetravail, nous avons voulu éclaircir les mécanismes responsables des troubles cognitifs postopératoires à travers deux axes, l’un clinique, l’autre pré-clinique sur des modèles murins.Dans l’étude clinique, l’influence des habitudes de vie sur la prévalence des troublesneurocognitifs péri-opératoires ainsi que sur les marqueurs inflammatoires périphériques (IL6; HMGB1) classiquement associés aux troubles neurocognitifs péri-opératoires a été évaluée.Cette étude suggère que la sédentarité pré-opératoire n’est pas un facteur de risque alors que lemultilinguisme et l’absence de tendance dépressive seraient des facteurs protecteurs. De plus,cette étude révèle que le taux d’IL-6 sérique, contrairement à celui de l’HMGB1, varie enfonction du type de chirurgie et de l’âge du patient. Cette étude a aussi permis de dégager unerelation préliminaire basée sur les taux d’IL-6 et d’HMGB1 sanguins du premier jouropératoire et prédictive de l’incidence d’un déclin cognitif six semaines après l’interventionchirurgicale. Dans l’étude pré-clinique sur des modèles murins, il a été pris en considérationque plusieurs canaux K+(KCa3.1; Kv1.3; Kvir) identifiés à la surface cellulaire étaientessentiels à l’activation microgliale et l’acquisition d’un phénotype neuroinflammatoire.Atténuer l'activation microgliale via l’inhibition de ces canaux s’avère une approche rationnellepour prévenir le développement de la neuro-inflammation et du déclin cognitif aprèsintervention chirurgicale (ici résolution d’une fracture tibiale expérimentale). Nous avons ainsimontré que l’inhibition pharmacologique et génétique du canal Kv1.3 réduit laneuroinflammation et le déclin cognitif post-opératoires dans un modèle murin de fracture detibia. D’autre part, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

RelA/SpoT Homologs (RSH) enzymes control the bacterial stringent response through the synthesis and hydrolysis of the alarmone (p)ppGpp. Current biochemical and structural data is not sufficient to correctly understand RSH enzymatic activity as well as their regulation and interaction with molecular partners. This doctoral thesis has demonstrated the implication of Rel ACT domain in the regulation of its enzymatic activity and its interaction with the EIIANtr protein in Caulobacter crescentus. In addition, it has also confirmed the presence of a dedicated (p)ppGpp allosteric binding site in the catalytic domain of Rel in Bacillus subtilis and characterized the regulation of the latter’s activity during potassium starvation, notably by the resolution of the crystallographic structure of Rel complexed with the DarB protein. Through the discovery of these novel results, this thesis deepens the fundamental workings of RSH activity regulation., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The transporters involved in amino acid excretion are poorly investigated. In the frame of the current thesis, it has been shown that excretion of homoserine and threonine involves the Aqr1, Qdr2, and Qdr3 proteins of the DHA1 family. Aqr1 has been further investigated as a representative amino acid exporter. In addition, crossfeeding of the LAB (Lactobacillus fermentum) by yeast involves excretion of amino acids via the three mentioned exporters. The second chapter is focused on the detrimental problem of contamination of yeast fermentation tanks by LAB, proving a genetic solution to this by the deletion of several members of the DHA1 family. Finally, using a new proxy that was set up to detect amino acid excretion, it is suggested that excreted amino acids at stationary phase of growth could be used by different yeast populations, as a carbon source for cell survival under carbon starvation conditions., Les transporteurs impliqués dans l'excrétion des acides aminés sont peu étudiés. Dans le cadre de la présente thèse, il a été montré que l'excrétion de l'homosérine et de la thréonine implique les protéines Aqr1, Qdr2 et Qdr3 de la famille DHA1. Aqr1 a fait l'objet d'une enquête plus approfondie en tant qu'exportateur représentatif d'acides aminés. De plus, l'alimentation croisée des BL (Lactobacillus fermentum) par la levure implique l'excrétion d'acides aminés via les trois exportateurs mentionnés. Le deuxième chapitre se concentre sur le problème préjudiciable de la contamination des cuves de fermentation de levure par LAB, prouvant une solution génétique à cela par la suppression de plusieurs membres de la famille DHA1. Enfin, en utilisant un nouveau proxy qui a été mis en place pour détecter l'excrétion d'acides aminés, il est suggéré que les acides aminés excrétés en phase stationnaire de croissance pourraient être utilisés par différentes populations de levures, comme source de carbone pour la survie des cellules dans des conditions de carence en carbone., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Three different signals are necessary for the activation of T cells and therefore the development of an adaptive immune response. The first signal consists on the antigen presentation by dendritic cells (DCs) in the lymphoid organs in a Major Histocompatibility Complex (MHC) context to the antigen-specific TCR. The second one, via costimulatory molecules present on the surface of APCs and T cell receptors. Signal 3 is produced by the secretion of cytokines by APCs, which signal via cytokine receptors on T cells to polarize them toward an effector phenotype.CD70 and CD27, expressed mainly on antigen presenting cells and T lymphocytes, respectively, are members of the TNF/TNFR family. The CD27/CD70 pathway has been shown to trigger the differentiation of Th1- type CD4+ T lymphocytes. Of note, the sole expression of CD70 on immature DCs have been shown to break tolerance and induce immunity, underlying the critical role of CD70 in the regulation of immune responses in vivo.It has been shown that CD27, expressed at higher levels on Tregs, is also involved in the inhibition of inflammatory responses. Indeed, in our group Dhainaut and Moser have reported that regulatory T cells control Th1 priming by selectively inhibiting the expression of CD70 by DCs in a CD27-dependent manner. Intriguingly, these observations suggest that CD27 displays opposite functions, either inducing pro-inflammatory Th1 responses (when expressed on conventional T cells (Tconvs)) or inhibiting the inflammatory responses (when expressed on Tregs). The impact of CD27 engagement on Tconvs has been widely described but remains unknown for Tregs.The objective of this project was therefore to investigate the role of CD27 inthe function and homeostasis of Tregs in vivo, as compared to Tconvs.First, we described Tregs subsets expressing or lacking-CD27, and demonstrated that Tregs expressing CD27 exhibited an activated phenotype, suggesting that CD27 could be an activator marker for Tregs. Accordingly, we, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Toxin-antitoxin (TA) systems are small genetic modules that are widespread in prokaryotes. While initially discovered on plasmids, which they stabilize, TA systems are also abundantly found on bacterial chromosomes. Their functions are highly debated and have been examined in this thesis. First, we show that TA systems do not seem to play a role in stress response or antibiotic tolerance, as previously reported in the literature. Secondly, our single cell experiments demonstrate that the activation of TA systems promotes cell death in cells when a TA-encoding plasmid is lost, indicating that TA activation leads to death and not to a state of stress tolerance. We therefore conclude that TA systems are addictive genes that promote their own retention and that of the genetic elements that encode them., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

EN-Bovine leukemia virus (BLV) is a retrovirus naturally infecting cattle and causing enzootic bovine leukemia (EBL), a form of leukemia affecting B-lymphocytes. The main feature of BLV infection is viral latency due to genetic and epigenetic repressions of viral gene expression from the main viral promoter, the 5’LTR, thereby allowing the virus to escape the host immune system and induce tumor development. Recent data have demonstrated that the regulation of BLV transcription is not solely dependent on the 5’LTR. Indeed, two new active promoters have been identified despite the transcriptional repression of the 5’LTR: a cluster of 5 independent RNAPIII promoters is responsible for the expression of 10 viral miRNAs and a RNAPII promoter located in the 3’LTR is responsible for the expression of antisense transcripts.In the main part of our PhD thesis, we studied the involvement of the cellular factor CTCF in the epigenetic and transcriptional regulation of BLV gene expression. By conducting transient transfection experiments, we demonstrated its repressive or activating role on the promoter activity of the 5’LTR or 3’LTR, respectively, which could be related to the ability of CTCF to delimit epigenetic boundaries. Furthermore, by 4C-seq and ChIP-seq experiments in model BLV-infected cell lines, we showed the formation of chromatin loops between the BLV provirus and cellular genes associated with the presence of CTCF and of the cellular cohesin complex. Finally, by studying the phenomenon of transcriptional interference as a potential new regulatory mechanism of the activity of the different BLV promoter units, we demonstrated the interdependence between the promoter activities of the 5’LTR and 3’LTR, through CTCF. Together, these results identify CTCF as a new player in the transcriptional regulation of BLV, and potentially in the tumor development induced by this retrovirus.In the other part of our PhD thesis, we initiated the study of the expression of circular RNA, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Long RSHs are enzymes that synthetize and hydrolyse a secondary messenger molecule, (p)ppGpp. This molecule acts as a global regulator targeting metabolic processes to ensure bacterial survival upon environmental challenges. Because (p)ppGpp is linked with bacterial virulence and resistance to antibiotics, a fundamental understanding of its regulation is key to develop novel antimicrobial treatments. Long RSH have two N-terminal domains, one for hydrolysis of (p)ppGpp (HD-domain) and one for synthesis of (p)ppGpp (SYNTH-domain), and four regulatory domains at the C-terminus that control catalysis by the N-terminal domains. A combination of structure determination and biochemical assays were used to study the long RSHs at the molecular level and determine the bases of the allosteric mechanisms that control the activities of the enzymes. The data demonstrate an allosteric conformational switch between the N-terminal catalytic domains of these enzymes that prevent futile catalytic cycles. Indeed, the data shows binding of substrates to one catalytic domain allosterically inhibits substrate-binding and catalysis by the opposing catalytic domain. Furthermore, feedback activation of (p)ppGpp-synthesis, the molecular basis of which was unknown, in context of amino-acid starvation was studied. The allosteric positive feedback regulation of (p)ppGpp-synthesis was shown to exploit the conformational switch between the two catalytic states to modulate both activities. An allosteric binding-pocket was located at the linker-region between the two catalytic domains at which (p)ppGpp-synthesis is auto-induced allosterically while (p)ppGpp-hydrolysis is inhibited. Lastly, the conformational landscape involved in the regulation of these enzymes was explored using Nanobodies to discover a previously undetected regulatory hotspot. Based on the conformational switch as well, this regulatory hotspot modulates catalytic activity allosterically. The newly discovered hotspot might be impor, Doctorat en Sciences, info:eu-repo/semantics/nonPublished