Macrophages are the largest cells in the immune system and they regulate inflammatory signalling and inform cell fate decisions. Many signals, including those mediated by Tumor Necrosis Factor alpha (TNFα) converge on a few key intracellular signalling pathways, including the Nuclear Factor kappa B (NFκB) network. The NFκB signalling pathway plays a vital role in the regulation of many different cellular responses, including the production of TNFα itself, which is required to sustain and propagate immune responses to, for example, infection or tissue damage. In this thesis we report on studies-both experimental and theoretical-of the NFκB signalling pathway in macrophages. Our collaborators stimulated these cells with various doses of Lipopolysaccharide (LPS), a molecule that forms the major component of the outer membrane of Gram-negative bacteria: in these experiments it serves as a proxy for bacterial infection. The macrophages, studied in vitro, respond as they are believed to do in tissues, by secreting certain signalling molecules called cytokines: the level of secretion proved to depend on the strength of the LPS stimulus. Further, heterogeneity of macrophage signalling was observed in response to a range of LPS doses. Within individual macrophages LPS stimulation results in oscillatory behaviour of NFκB localisation-NFκB shuttles in and out of the nucleus-with an amplitude (peak nuclear concentration) that also depends on the LPS dose. Heterogeneity was also observed in cells that were stimulated with the same dose intensity. This raises an important question about how immune cells coordinate inflammatory activity in the presence of this variability. In this thesis we aim to achieve an understanding of the system through the qualitative analysis of mathematical models of it. This work explores both the parametric sensitivity and bifurcation analyses for two mathematical models of NFκB in macrophages. Parametric sensitivity analysis is used to investigate the role of parameters on the model's output, especially on certain features of the signal-peak amplitudes, inter-peak intervals and areas beneath curves-that are commonly measured in single-cell experiments. Local bifurcation analysis is conducted in order to show all the possible behaviours produced when varying parameters.

Optimal CD4+ T cell activation is key to the generation of a productive immune response. Naïve circulating CD4+ T cells are quiescent under normal conditions and undergo activation only upon encounter of the T cell receptor (TCR) with Major Histocompatibility Complex (MHC)-encoded class II molecules on antigen presenting cells (APCs). Processed antigens (derived from pathogens, tumors or self tissue during autoimmunity) in complex with MHC class II are recognized by specific TCRs on CD4+ T cells. After this encounter, the highly complex and regulated process of CD4+ T cell activation results in the differentiation of naïve T cells into effectors and their clonal expansion. Apart from binding to its cognate peptide-MHC-II complex, several other factors define the extent and magnitude of T cell activation. This context is an important determinant of the nature of the subsequent T cell response. One of the factors involved is the strength of the signal (SOS) which is delivered to the cell upon ligation of the TCR to the MHC-peptide complex. The SOS, which can vary from weak to strong, is determined by the affinity/avidity of the TCR for the MHC-peptide complex, antigen concentrations, the duration of engagement, etc. Extreme weak or strong signals can lead to non-productive T cell responses with the former resulting mostly in anergy and the latter in cell death. Signals of optimal strength are the ones that translate into functional T cell responses. However, mechanisms by which signal strength information is translated into distinct T cell responses are still not very well understood. Binding of the TCR to the MHC-peptide complex triggers several signaling cascades and leads to generation of intracellular signaling intermediates, including Ca2+. Rise in intracellular Ca2+ levels is one of the first events to occur upon initiation of T cell activation. The initial increase is brought about due to release of Ca2+ from intracellular smooth endoplasmic reticulum stores. Once intracellular stores have been emptied, the increase is sustained by a process termed as capacitative Ca2+ entry, involving opening of Ca2+ channels in the plasma membrane known as Ca2+ release activated channels (CRACs). Consequently, Ca2+ flows from the extracellular milieu into the cell. A sustained Ca2+ increase is essential for activation of the transcription factor, NF-AT whose primary job is to initiate transcription of IL-2, a cytokine crucial for CD4+ T cell proliferation. The other intracellular signaling intermediates which are the focus of work presented in this study are reactive oxygen species (ROS). TCR signaling leads to generation of ROS, which may be either mitogenic or detrimental to T cell activation. Low levels of ROS, especially H2O2, inactivate phosphatases leading to activation of kinases and signaling pathways resulting in increased proliferation. However, high levels of ROS cause oxidative stress leading to reduced T cell activation, hyporesponsiveness and death. The experimental system used for this study consists of purified mouse lymph node CD4+ T cells. These cells were activated with varying strengths of the primary signal to better understand the roles of Ca2+ and ROS in modulating T cell activation and function. The signal strength was either varied by addition of different concentrations of ionomycin or thapsigargin, pharmacological agents that increase intracellular Ca2+ concentrations. Alternatively, signal through the surface TCR-CD3 complex was initiated using anti-CD3 in two modes: soluble (weak signal) or plate immobilized (strong signal). Increasing concentrations of ionomycin or thapsigargin or changing the mode of anti-CD3 from soluble to plate bound enhances IL-2 amounts, thereby converting a weak signal to a strong one. The work presented has been divided into three parts, each dealing with a distinct aspect of T cell activation. I. SOS and CTLA4-CD80/CD86 interactions: The binding of the TCR to its cognate MHC-peptide complex delivers the primary signal. However, this alone is not sufficient to drive T cell activation and an additional costimulatory signal emanating from the binding of CD28, a constitutively expressed receptor on T cells, to its ligands CD80 and CD86 is required. Another receptor that binds to CD80 and CD86 is CTLA-4 although it does so with a ~100 fold higher affinity. CTLA-4, unlike CD28, is expressed upon T cell activation and is considered to downregulate T cell activation. Its role as a negative regulator is highlighted by the phenotype of Ctla4 -/-mice which die of massive lymphoproliferation. However, there have also been reports of some plasticity in the effects mediated by CTLA-4. Previous work from our laboratory showed that CTLA-4-CD80/CD86 interactions could either inhibit or stimulate T cell activation depending on the SOS. To identify the molecular mediators of the differential effects of CTLA-4, the role of Ca2+ and ROS was evaluated. During activation with phorbol myristate acetate (PMA) and low amounts of ionomycin, intracellular amounts of Ca2+ were greatly reduced upon blockade of CTLA-4-CD80/CD86 interactions. Further experiments demonstrated that CTLA4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of ionomycin or thapsigargin (strong SOS) but the opposite occurred with PMA and low amounts of ionomycin or thapsigargin (weak SOS). These results were confirmed by activating cells with anti-CD3 either in the soluble or plate bound form. Considerably higher amounts of intracellular Ca2+ were present in cells activated with plate bound anti-CD3 compared to those activated with soluble anti-CD3. These amounts, further augmented by CTLA-4-CD80/CD86 interactions, probably became toxic to cells as increased proliferation was observed, using reagents that blocked these interactions. The opposite, however, was seen in cells activated with soluble anti-CD3. Also, CTLA4-CD80/CD86 interactions enhanced the generation of ROS. Studies with catalase revealed that H2O2 is required for IL-2 production and cell cycle progression during activation with a weak SOS. However, the high amounts of ROS produced during activation with a strong SOS reduced cell cycle progression. Together, this study identifies intracellular Ca2+ and ROS to play important roles in the modulation of T cell responses by CTLA4-CD80/CD86 interactions. II. SOS and CD4 downregulation: This study was initiated to identify early T cell functional responses that would help predict the strength of the primary signal. Using the in vitro culture system of varying signal strengths, it was found that CD4 surface expression was controlled by signal strength. CD4 is a surface glycoprotein expressed on the TH subset along with the TCR. It performs two main functions: First, binding to MHC class II and strengthening the TCR-MHC interaction, i.e. functioning as a coreceptor. Second, due to its association with p56lck a src family tyrosine kinase, the presence of CD4 along with the TCR enhances signal transduction. Also, CD4 acts as a receptor for entry for the AIDS virus. It is known that CD4 is downregulated from the surface and degraded upon T cell activation by a protein kinase-C dependent process in human and mouse T cells. Experiments presented in this study showed increased CD4 downregulation with a strong signal. The roles of intracellular mediators were assessed and high intracellular Ca2+ amounts, but not PMA activation, was required for sustained CD4 downregulation. Also, increased H2O2 amounts in cells activated with a strong SOS inhibited CD4 downregulation. Most interestingly, the pattern of CD4 downregulation was different between peripheral T cells and thymocytes, suggesting a correlation with CD4+ T cell development. III. Modulation of CD4+ T cell activation by small molecule plant growth regulators: An important area of investigation in T cell biology is the identification of molecules that modulate T cell activation. Towards this end, the mechanisms by which small molecule plant growth regulators, naphthalene acetic acid (NAA), 2,4 dichlorophenoxyacetic acid (2,4D) and indole acetic acid (IAA), influence CD4+ T cell activation was studied. It is useful to recall that IAA is the natural auxin present in plants, NAA is a synthetic auxin and 2,4D is a herbicide. These compounds, but not structurally similar control molecules, increased the activation and IL-2 production in CD4+ T cells activated with either soluble anti-CD3 or a combination of PMA and ionomycin. An investigation into the mechanisms of action by these compounds revealed increased early generation of intracellular ROS and Ca2+. Interestingly, the nature of their effects was found to rely on the strength of the primary signal: IL-2 and proliferation were increased in cells activated with a weak signal, but lowered proliferation was observed in cells activated with a strong signal. Cells activated with strong signal posses high amounts of ROS and Ca2+ and further increase in their amounts by IAA, NAA and 2,4D resulted in growth suppression. However, augmentation of Ca2+ and ROS amounts in cells activated with a weak signal was mitogenic. The role of these compounds during in vivo T cell responses needs to be addressed. Taken together, results presented in this study emphasize the importance of the role of SOS in determining T cell responses. In addition, the roles of Ca2+ and ROS downstream of the primary signal in modulating CD4+ T cell activation were demonstrated.

The mechanisms that regulate the differentiation of stem cells (SCs) into cardiomyocytes are still unclear and the role of endogenous molecules on this process remains unexplored. One such molecule is the bioactive phospholipid lysophosphatidic acid (LPA) which accumulates in the myocardium following acute infarction and exerts multiple biological functions, including the regulation of cell growth and differentiation as well as cell survival (Tigyi et al., 2003; Sengupta, et al., 2004). Experiments were therefore carried out in this thesis to reveal whether LPA can induce the differentiation of stem cells into cardiomyocytes and to identify the signalling mechanisms that mediate this effect. All experiments were carried out in the mouse P19 carcinoma stem cell line. Treatments with LPA in the absence and presence of various pharmacological compounds were conducted in embryoid bodies (EBs) formed from the P19 cells in sterile Petri dishes over 4 days. The EBs were subsequently transferred into 6-well cell culture plates and cultured for specific time points. Lysates were generated and subjected to western blotting for expression of cardiac- specific myosin light chain -1v (MLC-1v). To look at the expression of LPA receptors (LPAR1-LPAR5) experiments were carried out by RT-PCR using specific primers for each LPA receptor and the role of the latter in mediated responses to LPA were examined in the presence of the LPAR 1/3 antagonist, Ki16425, or the LPAR 4 receptor blocker suramin. In addition, experiments were carried out investigating the role of Gαi and specific signalling pathways that may be involved in the differentiation of P19 cells. These were carried out using potent inhibitors/antagonists of Gαi inhibitor (Pertussis toxin), PI3K inhibitor (LY294002), Akt inhibitor (Akt inhibitor XIII), PKC inhibitor (Bisindolylmaleimide I BIM-I), ROCK inhibitor (Y-27632), p38-MAPK inhibitor (SB203580) and ERK1/2 inhibitor (PD98059). Further experiments were carried out to establish whether the presence of LPA results in the phosphorylation of the targeted kinases. These studies were however limited to Akt, p38 MAPK and ERK1/2. Incubation of cells with LPA resulted in the differentiation of P19 cells into cardiomyocytes as reflected by the induction of MLC-1v. The latter increased significantly above basal in a time-dependent manner, reaching a maximum 10 days after plating EBs in 6-well plates. The induction of MLC-1v was more pronounced in cells incubated with 5 μM LPA at 6 days but showed little concentration differences at day 12. RT-PCR analysis confirmed the expression of LPA receptors 1 to 4 but not 5. Pre-incubating cells with suramin and Ki16425 concentration-dependently inhibited MLC-1v expression with 0.05 mg/ml and 10 μM respectively, virtually abolishing the expression of MLC-1v. Additionally, inhibitors of LPAR1/3 and LPAR4 receptors and all the signalling inhibitors except SB203580 abolished the phosphorylation of ERK1/2. Similarly, p38 MAPK activation was completely abolished by LPAR1/3 and LPAR4 receptor antagonists, Interestingly, only LY294002 (5 μM) and Y27632 (10 μM) abolished the LPA induced activation of p38 MAPK while SB203580, BIM-I, Akt inhibitor XIII and PD95080 caused no significant changes to the phosphorylation of p38 MAPK. In conclusion, the studies carried out in this thesis have shown that LPA can induce P19 stem cells to differentiate into cardiomyocytes and they are linked to the well characterised LPA receptors (LPAR1/3 and 4). These receptors are coupled to downstream signalling pathways of which those involving the ROCK, PI3K, PKC and/or Akt may be critical, and may converge on ERK1/2. Inhibition of any of these pathways has the potential to suppress differentiation. In contrast, signalling leading to p38 activation may potentially suppress differentiation but this needs further clarification.

En combinant un nouveau système de culture cellulaire à partir d'hépatocytes différenciés avec du virus de l’hépatite C (VHC) purifié, nous pouvons induire un profil transcriptomique caractéristique des patients à risque élevé de développer un carcinome hépatocellulaire (CHC). En utilisant ce modèle, nous avons découvert le rôle fonctionnel de l'EGFR comme élément moteur de la signature du risque de développement d'un CHC. De plus, nous avons identifié des gènes candidats impliqués dans le développement du CHC. Pour étudier les maladies du foie in vivo, nous avons caractérisé l'expression des protéines phosphatases dans des biopsies hépatiques de patients infectés par le VHC. Nous avons observé une régulation négative de PTPRD, un suppresseur de tumeur, causé par une augmentation de miR-135a-5p qui cible l'ARNm de PTPRD. Par ailleurs, l'analyse in silico montre que l'expression de PTPRD dans le tissu hépatique est corrélée à la survie chez les patients atteints de CHC. / By combining a cell culture system of hepatocyte-like cells with purified hepatitis C virus (HCV), we effectively simulated chronic infection in vitro. We found this infection model induces a transcriptomic profile of chronic HCV patients at high risk of developing hepatocellular carcinoma (HCC). Using this model, we have uncovered the functional role of EGFR as a driver of the HCC risk signature and revealed candidate drivers of the molecular recalibration of hepatocytes leading to liver cancer. In an approach to study liver disease in vivo, we opted to screen for protein phosphatase expression in liver biopsies of chronic HCV patients. We observed a downregulation of PTPRD, a well-known tumor suppressor. We demonstrated that this effect is mediated by an increase in miR-135a-5p which targets PTPRD mRNA. Moreover, in silico analysis shows that PTPRD expression in adjacent liver tissue of HCC patients correlates with survival and reduced tumor recurrence after surgical resection.

L'ozone troposphérique est un polluant secondaire majeur. Outre son rôle de gaz à effet de serre direct, l'ozone fait partie des polluants atmosphériques les plus toxiques et la pollution qu’elle engendre, affecte aussi bien la santé humaine que la productivité végétale. Les travaux présentés dans cette thèse porte sur l’étude du rôle des canaux ioniques de la membrane plasmique en réponse à une forte exposition à l’ozone ainsi que leurs interactions avec les évènements de signalisation mis en place lors du processus de PCD induit par ce stress sur des cellules en culture d’A. thaliana. Nous avons montré que cette mort cellulaire génétiquement contrôlée est caractérisée par une plasmolyse semblable à « l’Apoptosis Volume Decrease » (AVD) décrit en animal. Ce processus est promu par des cascades de signalisation où, dans un premier temps, les canaux anioniques sont très précocement activés potentiellement par l’acide oxalique issu de la dégradation de l’ascorbate par l’O3. Les données suggèrent une interconnexion entre les courants anioniques, l’influx cacique et une génération de ROS dépendante de la NADPH oxydase. Dans un deuxième temps, des canaux K+ rectifiants sortants sont activés de manière retardée et participent à la PCD. Cette activation retardée pourrait être due à une régulation post-transcriptionnelle des canaux GORK induite par l’O2•-. Enfin, nous avons également mis en évidence des activités enzymatiques de type caspase, au niveau cytoplasmique et nucléaire. Ces activités enzymatiques semblent être corrélées à la baisse de la teneur vacuolaire en ions K+, mais des données complémentaires sont nécessaires afin de comprendre les mécanismes sous-jacents. Ce travail souligne l’importance et la complexité de la régulation des canaux anioniques et potassiques et ce, dans les processus de signalisation et la mécanistique menant à la mort cellulaire programmée chez les plantes. / Tropospheric ozone is a major secondary pollutant. In addition to its role in greenhouse effect gas, ozone is one of the most toxic air pollutants, and its pollution affects both human health and crop productivity. The work presented in this thesis concerns the role of ion channels in the plasma membrane in response to acute exposure to ozone and their interactions with signaling events leading to O3-induced PCD in A. Thaliana cultured cells. We have shown that cell death was genetically controlled and characterized by cell shrinkage similar to the mechanism of "Apoptosis Volume Decrease" (AVD) described in animal. This process is initially promoted by an early activation of a plasma membrane anion channel, amongst which ascorbate-derived oxalic acid production potentially participates to this activation. Our data further suggests an interplay between anion channel with well known plant responses to O3, Ca2+ influx and NADPH-oxidase generating reactive oxygen species (ROS) in mediating the oxidative cell death. In a second step, K+ outwards rectifying currents are activated in a delayed manner and participate to PCD. This delayed activation could be due to O2•- post-transcriptional regulation of GORK channels. Finally, we also demonstrated caspase-like activities in the cytoplasm and the nucleus. These enzyme activities appear to be correlated with the decrease in vacuolar K+ ion content, but require additional data to understand the underlying mechanisms. This work highlights the importance and the complexity of ion channels regulation in the signaling pathway and the mechanistic processes leading to programmed cell death in plants.

Neurogenesis continues postnatally in two major neural stem cell (NSC) niches: The subventricular zone (SVZ) and dentate gurus of the hippocampus. SVZ NSCs self-renew and produce transit amplifying progenitor cells that, in turn, divide and give rise to neuroblasts. These neuroblasts migrate to the olfactory bulbs, via the rostral migratory stream (RMS), where they terminally differentiate into mature neurons. The postnatal SVZ (pSVZ) is more gliogenic than its adult counterpart (aSVZ), contributing to robust postnatal astrocytogenesis and oligodendrogenesis in the surrounding brain parenchyma. Studies examining Galectin-3 (Gal-3) in the aSVZ showed it has functions in regulating neuroblast migration, microglial activation, oligodendrocytic differentiation, and angiogenesis. However, the role of Gal-3 in pSVZ lineage progression is unknown. This thesis aims to unravel the roles of Gal-3 in regulating pSVZ lineage progression, fate choices, and NSC activation. In doing so, the thesis tackles the molecular pathways possibly involved in mediating the effects of Gal-3. I found through co-immunoprecipitation that Gal-3 was bound to β-catenin and both proteins were co-expressed in the aSVZ. In addition, expression of Gal-3 and Wnt/β-catenin signalling were downregulated as SVZ cells progressed through the lineage and became migratory. I hypothesised that Gal-3 may regulate lineage progression through regulation of Wnt/β-catenin signalling. To explore this hypothesis, Gal-3 overexpression, knockdown or control plasmids were co-electroporated with a Wnt/β-catenin reporter into the SVZ of postnatal day two mice. I found lineage progression was not altered by Gal-3 overexpression. Surprisingly, contrary to evidence described in the cancer literature, Gal-3 overexpression reduced Wnt/β-catenin signalling. This was accompanied by an acute reduction in proliferation. Also, more cells expressed p27/Kip1 in the SVZ, and more cells migrated into the RMS, suggesting increased cell cycle exit. However, NSC proliferation and clonal neurosphere forming capacity were not altered by Gal-3 overexpression, indicating that NSC activation was not influenced by Gal-3. While olfactory neuronogenesis was not altered by Gal-3 overexpression, striatal astrocytogenesis was increased while oligodendrogenesis was dampened. Further experiments revealed phosphorylation of Smad proteins 1/5/8 was increased in vivo and in vitro after Gal-3 overexpression. These findings indicate that Gal-3 positively regulated BMP signalling in the SVZ, possibly contributing to Gal-3's pro-gliogenic effects. Taken together, this thesis supports a model whereby a subpopulation of Gal-3-responsive pSVZ cells reacted to Gal-3 overexpression by acutely exiting the cell cycle, and possibly through the same mechanisms, switched from oligodendrocytic to astrocytic fate. These cellular responses might have been brought about, at least partially, by acute suppression of Wnt/β-catenin and activation of BMP signalling. These novel findings emphasise the regulatory actions of Gal-3 on pSVZ lineage progression through Wnt/β- catenin and BMP signalling.

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover. SLK activity is regulated by the LIM domain-binding proteins Ldb1/2. In addition to playing role in tumor initiation and progression, these proteins have been demonstrated to interact with LMO4. Therefore, this project assessed the ability of LMO4 to interact and regulate SLK activity. Results show that LMO4 can directly bind to SLK and activate its kinase activity. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding. Cre deletion of LMO4 inhibits cell migration and SLK activation, and impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Src/Yes/Fyn-deficient cells (SYF) express very low levels of LMO4 and do not recruit SLK to the leading edge. Src-family kinase inhibition impairs SLK recruitment to the leading edge, suggesting that both expression of LMO4 and the recruitment of SLK to the leading edge require c-Src activity. In conclusion, cell migration and activation of SLK requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. This study also investigated whether LMO4 deletion through MMTV-Cre-driven excision would impair mammary tumorigenesis in a PyMT mouse model of breast cancer. No difference in Overall Survival was observed between animals with and without LMO4 expression. Western blot analysis and IHC showed that tumors expressed LMO4 protein in animals genotyped as Cre-positive. This result suggests that expression of LMO4 is required for tumor initiation in the PyMT model of murine mammary carcinoma. This project has established a novel cytosolic role for the transcriptional co-activator LMO4 and validated it’s involvement in the regulation of SLK and cell migration. This pathway may provide a novel therapeutic strategy as LMO4 appears to be critical to the initiation and progression of breast cancer.

No description available.

In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.

Les cellules souches embryonnaires (ES) sont porteuses de grands espoirs en recherche biomédicale dans le but d’apporter un traitement définitif à l’ostéoarthrose. Parce que certaines articulations des chevaux sont similaires à celles des humains, cet animal représente un modèle important dans l’évaluation de stratégies de régénération du cartilage. Cependant, pour expérimenter un traitement par les cellules ES chez le cheval, des cellules ES équines (eES) n’ont toujours pas pu être dérivées. Dans ce contexte, l’objectif principal de cette étude est de dériver des lignées de cellules eES. Le premier objectif de notre étude consiste à optimiser la technique de dérivation des cellules eES. Nous démontrons que la lignée de cellules nourricières et le stade de développement des embryons influencent l’efficacité de la technique de dérivation tandis que l’inhibition de voies de signalisation menant à la différenciation des cellules ES ne l’influence pas sous nos conditions. Le deuxième objectif de notre étude est de caractériser de façon plus approfondie les lignées de cellules eES obtenues. Nous démontrons que les cellules eES dérivées expriment autant des marqueurs associés aux cellules pluripotentes qu’aux cellules différenciées et que l’inhibition de voies de signalisation menant à la différenciation n’influence pas l’expression de ces marqueurs. Pour conclure, nous confirmons avoir dérivé des lignées de cellules semblables au cellules eES (eES-like) ne correspondant pas complètement aux critères des cellules ES. / Embryonic stem (ES) cells carry high hopes for biomedical research in order to provide definitive treatment for osteoarthritis. The horse is considered to be an important animal model for examining osteoarthritis treatments. However, despite almost thirty years of research, authentic equine ES (eES) cells have not yet been derived. In this context, the main objective of this study was to derive eES cell lines. The first objective of our study was to optimize the technique for deriving eES cells. We show that different feeder cell lines and embryo development stages influence the effectiveness of this technique while the use of cell signalling inhibitors does not influence eES cell derivation. The second objective was to characterize markers of pluripotency and differentiation in eES cell lines by RT-PCR. We demonstrate that the eES cells express both markers associated with pluripotent cells and differentiated cells and that the presence of cell signalling inhibitors in the culture medium does not influence the expression of these markers. In conclusion, we confirm having derived eES-like cells but these do not meet all the molecular criteria of authentic ES cells.