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Pulmonary hypertension and right ventricular (RV) dysfunction are major prognostic determinants in patients with heart failure with preserved ejection fraction (HFpEF). The underlying pathomechanisms remain unknown. In this context, we sought to study the pathogenesis of pulmonary hypertension and RV dysfunction in a rat model of obesity-associated HFpEF., info:eu-repo/semantics/published

The nucleus accumbens (NAc) is a key component of the brain reward circuitry. Due to its extensive connectivity with regions involved in limbic but also motor processes, its main function is to integrate reward-related information, and to generate motivation to sustain action in goal-directed behaviors. As drugs of abuse hijack the brain reward system to drive compulsion to seek and consume drugs, characterizing NAc neuronal activity is particularly important to improve our understanding of the neuronal mechanisms of addiction. Here, we focused our research on a cocaine conditioned place preference paradigm, which relies on the association between drug and environmental cues to drive place preference. Indeed, the formation and maintenance of cue-reward associations drive strong craving and relapse after re-exposure of addicted individuals to a context previously paired with drug use. In this thesis, we first described the activity of the two main neuronal populations of the NAc, the medium spiny neurons expressing the dopamine receptor D1R or D2R (D1- and D2-MSNs, respectively), during this behavior by using in vivo endoscopic calcium imaging. We showed that both neuronal populations contain the same level of information about the place preference, and consistently encode the least preferred location, both before and after conditioning. As this location/preference-based encoding is likely shaped by presynaptic inputs, we developed a pipeline to identify and quantify NAc D1- and D2-MSNs brain-wide afferents, by using a retrograde tracing approach combined with optimized immunolabeling and clearing of whole brains. Finally, we demonstrated the functional involvement of the projections between the paraventricular thalamus and the NAc in the maintenance of these cocaine-associated memories, using chemo- and opto-genetic techniques. Altogether, the work realized during this thesis contributes to a better understanding of the circuits involved in addiction, and more parti, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

In the nervous system, glial cells coexist and collaborate with neurons to process information and generate adaptive responses. However, how neurons and glia in the nervous system communicate with each other to properly perform their functions is an underexplored research topic. The survival of neurons depends on glia making it difficult to analyze intercellular communication. Here, I used the nematode Caenorhabditis elegans to circumvent these obstacles and study neuron-glial communication. Taking advantage of the invariant anatomy of C. elegans, I selected as a model a sensory organ composed of 12 ciliated neurons and two glial cells. This organ serves as a model to explore how neurons and glia communicate in the context of tissues within a living animal.Cilia are sensory projections present on several types of cells and used to detect external stimuli. The ciliated sensory neurons of C. elegans have been widely used as a model to understand cilia biology. Ciliary traffic homeostasis is key in cilia biology, and several diseases, collectively referred to as ciliopathies, are caused by dysfunctional cilia. In C. elegans, cilia dysfunction disrupts sensory responses to odors, food, repellent molecules, and temperature changes. In addition to their sensory function, cilia are believed to aid in the communication between cells and individuals by excreting extracellular vesicles larger than 100 nm, called ectosomes.First, I show how ectosomes bud from the ciliary membrane of ciliated neurons of hermaphroditic animals, a mechanism thought to be exclusive to the male ciliated neurons of C. elegans. To describe how and which ciliated sensory neurons produce ectosomes, I selected potential cargoes of ciliated ectosomes that were marked by transgenesis or knock-in. These new ectosome markers include tetraspanins TSP-6, 7 and 4, as well as other membrane receptors or sensory machinery proteins. Ciliary distribution and entry into ectosomes of these markers have been studied, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

During starvation, animals utilize the liver as lipid storage organ. For instance, patients suffering from anorexia nervosa, overnight fasted mice and starved zebrafish larvae develop fatty liver. However, the mechanisms underlying starvation-induced liver damage remain unknown. Further, it remains unclear if the resolution of hepatic steatosis is beneficial to the liver and the animal. Here, we investigated the role and regulation of starvation-induced hepatic steatosis using zebrafish (Danio rerio) as model system. By in vivo imaging of calcium and lipid droplets, we published a work showing negative correlation between the two. Using a genetically encoded calcium chelator (SpiCee), we showed that buffering of calcium signaling reduces the turnover of lipid droplets. On the other hand, we increased calcium transients in the liver by mobilization of endo-lysosomal Ca2+ stores, using selective two-pore complex 2 (TPC2) agonists. This led to a resolution of hepatic lipids. Thus, we established methods to manipulate starvation-induced hepatic steatosis by modulating Ca2+ signaling. Using this system, we investigated the impact of hepatic lipid droplet accumulation, and their turnover on the health of the organ and organism. Increasing Ca2+ flux by pharmacological activation of TPC2 channels efficiently reduced fat in the zebrafish liver. However, this process enhanced macrophage infiltration, hepatocyte phagocytosis and starvation-induced mortality. On the other hand, buffering cytoplasmic Ca2+ reduced lipid droplet turnover during starvation, leading to reduced liver inflammation and increased the lifespan of starved animals. With this, we propose that fatty liver helps with starvation resistance, while hepatic lipid flux (turnover) damages the liver. To understand how animals with enhanced starvation resistance deal with hepatic steatosis and liver damage, we investigated the starvation response in cavefish (Astyanax mexicanus) in collaboration with Dr. Nicolas Rohn, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

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Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. In this study, we evaluated the impact of CDK4/6 inhibition by palbociclib in a panel of 28 MPM cell lines, including 19 patient-derived cell lines, using a variety of approaches including RNA-sequencing. Palbociclib used alone sufficed to strongly and durably inhibit the proliferation of 23 MPM cell lines, indicating a unique sensitivity of MPM to CDK4/6 inhibition. Importantly, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4. This was associated with the high p16INK4A (CDKN2A) levels that accompany RB1 defects or inactivation, and also (unexpectedly) cyclin E1 over-expression in the presence of wild-type RB1. Prolonged treatment with palbociclib irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype including various potentially immunogenic components was also irreversibly induced. Phosphorylated CDK4 was detected in 80% of 47 MPM tumors indicating their intrinsic sensitivity to CDK4/6 inhibitors. The absence of this phosphorylation in some highly proliferative MPM tumors was linked to partial deletions of RB1, leading to very high p16 (CDKN2A) expression. Our study strongly supports the clinical evaluation of CDK4/6 inhibitory drugs for MPM treatment, in monotherapy or combination therapy., info:eu-repo/semantics/published, article accepted for publication in the peer-reviewed journal Molecular Oncology 2022-11-17.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. We evaluated the impact of CDK4/6 inhibition by palbociclib in 28 MPM cell lines including 19 patient-derived ones, using various approaches including RNA-sequencing. Palbociclib strongly and durably inhibited the proliferation of 23 cell lines, indicating a unique sensitivity of MPM to CDK4/6 inhibition. When observed, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4. This was associated with high p16INK4A (CDKN2A) levels that accompany RB1 defects or inactivation, or (unexpectedly) CCNE1 overexpression in the presence of wild-type RB1. Prolonged palbociclib treatment irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype including various potentially immunogenic components was irreversibly induced. Phosphorylated CDK4 was detected in 80% of 47 MPMs indicating their sensitivity to CDK4/6 inhibitors. Its absence in some highly proliferative MPMs was linked to very high p16 (CDKN2A) expression, which was also observed in public datasets in tumours from short-survival patients. Our study supports the evaluation of CDK4/6 inhibitors for MPM treatment, in monotherapy or combination therapy., info:eu-repo/semantics/published

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Friedreich Ataxia (FRDA) is an autosomal recessive multisystem disorder with prominent neurological manifestations, which include the early and severe loss of large primary proprioceptive neurons (PPNs) of the Dorsal Root Ganglia (DRGs) and, at a later stage, the degeneration of cerebellar and pyramidal systems. The disease is caused by the hyperexpansion of a GAA triplet repeat in the first intron of the FXN gene, encoding the mitochondrial protein frataxin (FXN), which is involved in iron-sulfur cluster biogenesis. The expanded GAA tract promotes chromatin condensation and disrupts FXN mRNA transcription, leading to reduced FXN levels, impaired mitochondrial function and altered iron metabolism. Despite notable progress in the identification of the underlying causes of FRDA, neither the details of these pathogenic processes nor the reason for the specific vulnerability of the proprioceptive system and a limited number of other cell types are yet fully understood. Human induced pluripotent stem cells (iPSCs) are used to generate models of human diseases that recapitulate the pathogenic process as it occurs in affected cell types. Studies conducted so far in FRDA have mainly utilized iPSC-derived cortical neurons and cardiomyocytes, which have shown to recapitulate essential aspects of FRDA pathogenesis that are at least partially corrected by restoring FXN expression. However, those studies can only provide a limited view of the effects that FXN silencing has in vivo, because the neuronal type utilized so far is not affected in FRDA patients. On the other side, efforts to differentiate iPSCs into primary sensory neurons have only succeeded in generating an unselected mixed neuronal population with a small minority of proprioceptors.Having an in vitro source of PPNs would enable the study of the alterations which are specifically induced and the pathways which are impaired by frataxin deficiency in these cells, helping in the understanding of their unique neuronal p, L'ataxie de Friedreich (FRDA) est une pathologie héréditaire autosomale récessive caractérisée par des manifestations neurologiques importantes, qui comprennent la perte précoce et sévère des neurones proprioceptifs (PPN) des ganglions de la racine dorsale (DRG) et, à un stade ultérieur, l’atteinte des systèmes cérébelleux et pyramidaux.La maladie est causée par une expansion de triplets nucléotidiques GAA au sein du premier intron du gène FXN, qui code pour la protéine mitochondriale frataxine (FXN), impliquée dans la biogenèse des centres fer-soufre (Fe-S). Cette expansion GAA induit la formation d’hétérochromatine et perturbe la transcription du gène FXN, entraînant une réduction des niveaux de frataxine, une altération de la fonction mitochondriale et une altération du métabolisme du fer. Malgré des progrès notables dans l'identification des causes de la maladie, ni les détails de ces processus pathogènes ni la raison de la vulnérabilité spécifique du système proprioceptif et d'un nombre limité d'autres types de cellules ne sont encore entièrement élucidés.Les cellules souches pluripotentes induites (iPSC) sont utilisées pour générer des modèles de maladies humaines qui récapitulent le processus pathogénique tel qu'il se manifeste dans les cellules affectées. Les études menées jusqu'à présent dans l’ataxie de Friedreich ont principalement utilisé des neurones corticaux et des cardiomyocytes dérivés des iPSC FRDA, qui présentent les aspects essentiels de la pathogenèse de FRDA, qui sont partiellement corrigés après la restauration de l’expression du gène FXN. Cependant, ces études ne peuvent fournir qu'une vision limitée des effets de la diminution de la transcription du gène FXN, car le type neuronal utilisé n'est pas affecté dans la maladie. D'autre part, les efforts pour différencier les iPSC en neurones sensoriels primaires n'ont réussi qu'à générer une population neuronale mixte avec une faible proportion de neurones de type proprioceptif. Disposer d'une sou, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Chemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value., SCOPUS: ar.j, info:eu-repo/semantics/published

Glandular epithelia, including the mammary and prostate glands, are generally composed by basal (BCs) and luminal cells (LCs) organised in a bilayered structure. Lineage tracing studies demonstrate that many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced in adult life by distinct pools of unipotent basal and luminal stem cells. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression. Particularly, transplantation of mammary gland (MG) BCs alone that are unipotent in lineage tracing give rise to MG outgrowth composed of BCs and LCs, promoting the multipotent potential of BCs when transplanted without LCs. In contrast, when BCs are transplanted together with LCs, BCs maintain their unipotent basal fate, suggesting that LCs may actively restrict BCs multilineage differentiation potential in physiological conditions. The nature of this mechanism is unknown.During my thesis I studied the mechanisms involved in the activation of BSC multipotency in different conditions to identify whether a common multipotent program is activated in the different multipotent conditions, as well as, in different glandular epithelia. I first studied the activation of BSC multipotency upon LC ablation in multiple epithelia both in vivo in mice and in vitro in organoids. Performing bulk and single-cell RNA sequencing after LC ablation we identified that BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs—reminiscent of the genetic program that regulates multipotency during embryonic development. Also, using mammary gland derived organoids, we could demonstrate that the communication between LCs and BCs is essential to maintain BC unipotency. By predicting ligand–receptor pairs from single-cell data, we found that TNF—which is secreted by LCs—restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

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We questioned whether EMT is required for metastasis, and we have analyzed metastasis formation in two distinct SCC models, different in terms of SCC ability to undergo EMT. We demonstrated that in spontaneous mouse model in which tumors presented a wide range of EMT (from well differentiated to fully EMT-like) presented high metastatic incidence: 49% (20/70) of mice presented LN metastasis and 28% (34/70) of mice presented lung metastases. Importantly, metastatic incidence was always associated with EMT-like tumors. However, in the second mouse model, that develops mostly well-differentiated tumors, the metastatic incidence was very low: 12% (5/42) of mice developed lung metastases and 10% (4/42) developed lymph node metastases. Importantly, the rare metastases were associated with tumors presenting some degree of EMT, supporting that EMT is needed for metastases arising from skin SCC.To understand whether EMT can occur at the intermediate step between primary tumor and distant metastasis, we analyzed blood from mice based on YFP reporter. We uncovered that most CTCs were EMT-like: 80% of CTCs were EpCAM-negative. This finding suggests that EpCAM marker, a gold standard used to detect CTCs, is not optimal as it doesn’t detect CTCs of EMT phenotype thus underestimating the real number of CTCs. Importantly, we noticed that the presence of CTCs was always associated with lung metastases and indeed can be used to predict prognosis of oncologic disease. Our immunofluorescent analysis of lung and lymph nodes showed that the majority of metastases were of differentiated phenotype. For the lung metastases, 71% (36/51) of them presented epithelial phenotype YFP+/K14+/Vim-, 14% (7/51) presented differentiated epithelial features but did not express K14 or Vimentin, 12% (6/51) presented an EMT hybrid epithelial phenotype YFP+/K14+/Vim+, whereas only 4% (2/51) were purely mesenchymal YFP+/K14-/Vim+. Histological analyses were confirmed by FACS analysis showing that lung metast, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Microglia, the resident macrophage population of the central nervous system (CNS), contribute to the development and homeostasis of the brain and spinal cord, and to the maintenance of parenchymal integrity in the event of injury or pathogenic aggression. Recent studies have also demonstrated the involvement of these cells in a wide range of severe pathological conditions, for which the replacement of "defective" microglia by in vitro reprogrammed cells is now considered a promising therapeutic avenue. Consequently, in recent years, the mechanisms controlling the origin and development of microglia have been the subject of intense research. Although numerous fate mapping studies in mice have revealed that microglia originate from embryonic hematopoietic progenitors with self-renewal properties within the CNS parenchyma, the precise nature of these progenitors is, however, still debated.In this work, we set out to answer this important question by using the zebrafish, a model of choice for the study of vertebrate developmental hematopoiesis, to better understand the dynamics of microglia development. Because the early steps of microglia ontogeny take place early during embryogenesis, the optical transparency of the zebrafish embryo is an unprecedented advantage for studying these processes non-invasively. By combining the use of hematopoietic mutants with novel cell fate tracking approaches, we identified primitive macrophages as the unique precursors of embryonic microglia in the zebrafish embryo. Surprisingly, our results also demonstrate that this initial wave of microglial cells is subsequently replaced at the larval stage by a distinct population that maintains through adulthood, and originates from embryonic hematopoietic stem cells.These observations led us to investigate the signaling pathways that control the development of each of the two microglial waves in vivo. We focused on the role of Colony-stimulating factor 1 receptor (CSF1-1R), a fundamental regula, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished