La caractérisation d’échantillons biologiques s’effectue très souvent au microscope optique. Or, il est techniquement difficile d’isoler quelques cellules rares parmi une culture hétérogène en se basant strictement sur des caractéristiques observables au microscope, comme la localisation, la morphologie ou le déplacement, car il n’existe pas nécessairement de marqueur moléculaire unique qui leur sont associés. Afin de répondre à cet enjeux, le laboratoire dans lequel j’ai effectué mon stage de maîtrise a récemment développé la Single Cell Magneto Optical Capture (scMOCa), qui utilise des réactifs communs et un laser de faible puissance pour attacher des billes ferromagnétiques à la membrane plasmique cellulaire et permet d’isoler magnétiquement les cellules d’intérêt. Le présent ouvrage rapporte l’application de la scMOCa à la migration de cellules métastatiques ainsi que les adaptations apportées à la technique nécessaires à la réalisation du projet. Notamment, le volume de cellules uniques capturé a été augmenté d’un facteur d’environ 250 grâce à l’automatisation de la technique et à l’étude du photoblanchiement de la fluorescéine, phénomène à la base de la scMOCa. Brièvement, l’expérience consiste à capturer les cellules uniques présentant les phénotypes migratoires les plus importants, définis par l’analyse de leur trajectoire, parmi une culture hétérogène de cellules métastatique. Les résultats de l’expérience démontrent une conservation des phénotypes migratoires après plusieurs mitoses. Aussi, l’expression génétique relative fait ressortir des gènes et groupes de gènes propres à la migration cellulaire. / The characterization of biological samples depends heavily on the optical microscope. However, it is technically challenging to isolate rare cells among a heterogeneous culture solely based on visual inspection at the microscope. Indeed, characteristics like location, morphology or displacement do not necessairly have specific related molecular markers. In order to solve this issue, the laboratory where I accomplished my master internship developped the Single Cell Magneto Optical Capture (scMOCa) wich uses commun reagents and a low powered laser to attach ferromagnetic beads on the cell plasma membrane and isolate the cells of interest with magnetic tools. The present work reports the application of scMOCa to the metastatic cell migration and the implemented adaptations to the technique in order to carry out the project, especially by increasing the number of single cells being isolated by a factor of 250. This adaptation requiered the study of photobleaching, phenomenon at the foundation of scMOCa. Briefly, the experiment consists to capture the cells presenting the most important migratory phenotypes, defined by their track analysis, among a heterogeneous metastatic cell culture. The experimental results show that the migratory phenotypes are preserved after several cell divisions. Also, the relative gene expression highlights some genes and gene groups owned to cellular migration.

Disulfide bridges play a significant role in protein-folding as well as en- zyme activity and thus regulate many intra- and extracellular processes. Sulfhydryl oxidase QSOX1 forms S-S bridges de novo, modulating the activity of its substrates and thus directly or indirectly influences vital cel- lular processes. The first part of this thesis focuses on characterization of the role of QSOX1 in cancerogenesis, using breast cancer cell lines (MCF7, MDA-MB-231) and pancreatic cancer cell line (Panc-1), while the second part emphasizes the regulation of QSOX1 expression by different oxygen concentrations. To study the effect of QSOX1 on proliferation of triple-negative cancer cells MDA-MB-231, two genetically modified cell lines - QSOX1-overexpressing and QSOX1 knockout cell lines - were constructed. While increased QSOX1 protein levels do not have a significant effect, the absence of QSOX1 leads to a decreased cellular growth. Lack of QSOX1 also results in visible change in cellular morphology. QSOX1 knockout cells can be mostly characterized as more round-shaped with less noticeable or completely missing lamellipo- dia. This finding is with agreement with to-date literature suggesting that QSOX1 is important not only for cellular proliferation but also for migration and invasiveness. While authenticating the theory of...

Establishment and maintenance of the blood system relies on the cellular and spatial organization of bone marrow. In the BM niche, sinusoidal endothelial cells (SECs) are mainly located in the trabecular zone of the metaphysis area of long bones. SECs present a fenestrated structure characterized by high permeability, low shear rates, and oxygen pressure. The hypoxic environment surrounding SECs is needed for the movement and engraftment of hematopoietic cells, but it might also facilitate the homing of malignant cells. SECs’ adaptive response to hypoxia depends on the Hypoxia Inducible Factors (HIFs) promoting angiogenesis, hematopoiesis, and other processes. The upstream regulator of HIFs, the prolyl hydroxylase domain-2 (PHD2) is considered the key cellular oxygen sensor. Our group has shown the crucial regulatory effects that PHD2 has on different physiological and pathological settings. During steady state, PHD2 modulates proliferation and mobilization of hematopoietic progenitor cells (HSCPs), as well as bone metabolism. On the other hand, PHD2 is crucial for neutrophil motility affecting their extravasation during arthritis. We have also demonstrated that loss of PHD2 in different humans and mouse tumor cell lines, as well as in myeloid cells and T-lymphocytes where it impairs tumor development. Others have shown that heterozygous deletion of PHD2 in tumor ECs can reduce distant metastasis. Due to the intricate interplay between the different players of the PHD2/HIFs axis, as well as their effect on other signaling pathways, the positive or negative impact of the hypoxia pathways has been shown to be cell type and context dependent. In the present study, we analyzed the role of hypoxia pathway proteins, mainly PHD2, in ECs in the bone/BM niche, as well as its consequent influence on the environment during physiological and pathological scenarios. We have demonstrated that endothelial PHD2 has a profound intrinsic effect on vessel morphology and functionality, affecting the crucial communication between ECs and the bone/BM niche. Further, using different transgenic mouse lines, we have identified the BM endothelial cells (BM-ECs) PHD2/HIF-2α axis directly increasing leukocytosis via vascular cell adhesion protein 1 (VCAM-1) protein downregulation. Moreover, during steady state conditions we have discovered a novel regulatory effect that PHD2 exerts on VCAM-1 by increasing miR-126-3p transcription, a well-known inhibitor of VCAM-1 expression. Lastly, the PHD2/HIF-2/miR-126-3p/VCAM-1 axis not only influenced the myeloid cell intravasation towards circulation but also increased the extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone. BM-ECs PHD2 offers a protective role against tumor homing cells in the bone/BM while serving as an important regulator in the communication between endothelium and BM niche. Concluding Remarks:  Loss of PHD2 in ECs generates profound changes in vessel function and in the hematopoietic compartment leading to increase myelopoiesis resulting in leukocytosis. The above-mentioned effect occurs in a HIF-2α-dependent manner.  PHD2/HIF-2α also transcribed in an increase of miR-126-3p expression leading to VCAM-1 downregulation. Process that resulted in increased leukocytes in circulation due to hematopoiesis dysregulation. The novel PHD2/HIF-2/miR-126-3p/VCAM-1 axis enhanced extravasation and homing of metastatic breast carcinoma cells into the bone/BM tissue, increasing tumor burden throughout the bone.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119 / Die Organisation und Aufrechterhaltung des Blutsystems hängt von der zellulären und räumlichen Organisation des Knochenmarks ab. In der BM-Nische befinden sich, hauptsächlich in der Trabekelzone des Metaphysenbereichs langer Knochen, die sinusoidale Endothelzellen (SECs). SECs weisen eine gefensterte Struktur auf, die von hoher Durchlässigkeit, geringer Scherrate und Sauerstoffdruck geprägt ist. Das hypoxische Milieu der SECs ist notwendig für Bewegung und Einwanderung der hämatopoetischen Zellen und könnte dies ebenso für bösartige Zellen begünstigen. Die Anpassung der SECs an Hypoxie hängt von den Hypoxia Inducible Factors (HIFs) ab. HIFs fördern die Angiogenese, die Hämatopoese und andere Prozesse. Die prolyl hydorxylase domain-2 (PHD2) ist der vorgeschaltete Regulator der HIFs und gilt als wichtigster zellulärer Sauerstoffsensor. Unsere Gruppe konnte zeigen welche zentralen regulatorischen Effekte die PHD2 auf verschiedene physiologische und pathophysiologische Mechanismen ausübt. Im Gleichgewichtszustand moduliert PHD2 Proliferation und Mobilisation der hämatopoetischen Vorläuferzellen (HSCPs) sowie den Knochenmetabolismus. Auf der einen Seite spielt PHD2 eine entscheidende Rolle bei der Motilität der Neutrophilen und beeinflusst daher die Extravasation bei Arthritis. Wir konnten außerdem zeigen, dass der Verlust von PHD2 in verschiedenen humanen und murinen Tumorzelllinien, sowie in myeloischen Zellen und T-Lymphozyten die Tumorentwicklung beeinträchtigt. In anderen Arbeiten wurde gezeigt, dass eine heterozygote PHD2-Deletion in Tumor-ECs eine Fernmetastasierung reduziert. In Anbetracht der komplizierten Wechselwirkung zwischen den verschiedenen Komponenten der PHD2/HIF-Signalkaskade sowie deren Effekte auf andere Signalkaskaden, übt sich in Abhängigkeit des Zelltyps und des Kontexts ein positiver oder negativer Einfluss auf die Hypoxie-Signalwege aus. In der vorliegenden Studie haben wir die Rolle von Proteinen des Hypoxiewegs, hauptsächlich PHD2, in den ECs der Knochen-/BM-Nische sowie deren daraus resultierenden Einfluss auf die Umwelt in physiologischen und pathologischen Szenarien analysiert. Wir konnten zeigen, dass das endotheliale PHD2 einen tiefgreifenden intrinsischen Effekt auf die Gefäßmorphologie und Funktionalität besitzt und damit entscheidend die Kommunikation zwischen ECs und der Knochen-/BM-Nische beeinflusst. Weiterhin konnte unter Nutzung verschiedener transgener Muslinien identifiziert werden, dass die Knochenmarksendothelzellen (BM-ECs) PHD2/HIF-2α-Achse direkt die Myelopoese, durch eine Herabsetzung der vascular cell adhesion protein 1 (VCAM-1) -Expression, steigert. Darüber hinaus haben wir einen neuartigen regulatorischen Effekt der PHD2 auf das VCAM-1 entdeckt. Hierbei wird die Expression des VCAM-1 Inhibitors miR-126-3p gesteigert. Des Weiteren beeinflusst die PHD2/HIF-2/miR-126-3p/VCAM-1 Achse nicht nur die Intravasation der Myloidzellen in Richtung des Kreislaufs, sondern auch eine Steigerung der Extravastion und Einwanderung von metastasierenden Brustkarzinomzellen in die Knochen/BM-Gewebe und steigert somit die Tumorlast in den Knochen. In Anbetracht klinischer Versuche der Krebsbehandlung mit PHD2-Inhibitoren, bietet BM-ECs PHD2 einen schützenden Effekt gegen Tumorzelleinwanderung in die Knochen/BM, während es gleichzeitig als ein wichtiger Regulator in der Kommunikation zwischen Endothelium und der BM-Nische dient. Abschließende Bemerkungen: Der Verlust von PHD2 in ECs führt zu tiefgreifenden Veränderungen in der Gefäßfunktion und im hämatopoetischen Kompartiment, was zu einer verstärkten Myelopoese und damit zu Leukozytose führt. Die oben erwähnte Wirkung tritt in einer HIF-2α-abhängigen Weise auf. PHD2/HIF-2α führte auch zu einem Anstieg der miR-126-3p-Expression, was zu einer Herunterregulierung von VCAM-1 führte. Dieser Prozess führte zu einer erhöhten Anzahl von Leukozyten im Blutkreislauf aufgrund einer Dysregulation der Hämatopoese. Die neuartige PHD2/HIF-2/miR-126-3p/VCAM-1-Achse förderte die Extravasation und Ansiedlung von metastatischen Brustkrebszellen im Knochen/BM-Gewebe, wodurch die Tumorlast im gesamten Knochen erhöht wurde.:Introduction 8 The Endothelium 9 Endothelial barrier. 9 Leukocyte transendothelial migration. 11 Intracellular mechanism for activation of adhesion molecules. 12 Cellular migration in pathological conditions. 13 Tumor Dissemination: Metastasis. 14 Metastatic cascade. 15 The bone is a preferential site for malignant cells arrival. 16 Bone/BM physiology influences metastasis. 17 The endosteal niche. 18 The perivascular niche. 19 Hypoxia pathway proteins impact on metastasis. 20 Thesis Aims 23 Aim 1: Characterize the role of the hypoxia pathway proteins in bone vasculature and the impact on the niche. 24 Aim 2: Analyze the genetic changes that resulted from PHD2 deletion in BM-ECs and their impact on the cross-talk communication with the different BM-niches. 24 Aim 3: Investigate the impact of PHD2 and downstream regulators (HIF-1/2α) on vessel functionality. 25 Materials and Methods 26 Mice. 27 Histology: tissue processing and immunofluorescence staining 28 Bone tissue processing. 28 Staining of Bone cryosections. 28 Induced skin inflammatory model. 29 Staining of ears treated with PMA. 29 Vascular morphology quantification. 29 Microscopy 31 Antibodies used for immunofluorescence. 31 Bone analysis. 31 Bone µCT measurements and analysis 31 Tartrate-resistant acid phosphatase (TRAP) staining 32 Blood and BM analysis. 32 Sysmex. 32 Flow cytometry. 32 BM cell extraction from bones. 32 BM cells staining. 33 Meso Scale Discovery (MSD) 35 Evans Blue assay. 35 BM soup ELISA. 35 RNAseq of CD31+ EMCN+ BM-ECs 36 BM-EC cell sorting for RNAseq. 36 RNA extraction and qPCRs 36 Tumor model. 36 Tumor breast carcinoma cells. 36 Tumor homing model. 37 Statistical analyses. 37 Results 38 PHD2 Conditional Knockout from Endothelial Cell Compartment. 39 Further P2EC mice characterization. 41 Transgenic deletion of PHD2 showed slight developmental retardation. 41 Spleen size showed not to be affected by deletion endothelial PHD2. 41 P2EC mice displayed increased vessel leakiness. 42 Endothelial PHD2 deletion does not affect lung endothelial cells. 43 BM-ECs PHD2-HIF-2α axis modulates leukocytosis and vessel morphology. 44 HIF-2α modulates P2EC leukocytosis and thrombocytopenia. 44 BM-ECs PHD2 deficient mice hinder vessel morphology in a HIF-2α dependent manner. 44 Endothelial PHD2-deficient mice exhibit perturbed hematopoiesis. 45 P2EC mice early progenitor displayed reduced total cell number, but frequency remained unchanged. 46 P2EC mice favor differentiation of committed progenitors with a myeloid bias. 48 P2EC mice significantly reduced the numbers and frequency of megakaryocyte/erythroid progenitor’s linage. 48 PHD2-HIF-2α deletion restored normal hematopoiesis. 50 P2EC vascular functionality during pathological conditions. 53 Endothelial PHD2 modulates leukocyte migration during localized inflammation. 53 Endothelial PHD2 shapes bone/BM tumor homing. 55 Tumor homing in the bone: generation of an early metastatic model. 56 Early metastasis limitation. 58 Endothelial PHD2 modulates tumor colonization to the bone/BM. 59 Simultaneous deletion of PHD2 and HIF-1 in BM-ECs worsen tumor metastasis to bone. 61 Simultaneous deletion of PHD2 and HIF-2 in BM-ECs showed no differences in tumor homing. 62 Deep sequencing of PHD2 deficient BM-ECs. 63 BM EC from P2EC mice display enriched leukocyte migration gene signatures. 63 P2EC mice presented genetic dysfunction in the integrin-binding system. 64 P2EC steady-state VCAM-1 expression is HIF-2α dependent. 66 BM-ECs VCAM-1 + is regulated by PHD2 through HIF-2α. 66 PHD2-dependent downregulation of VCAM-1 does not affect VE-cadherin expression. 68 BM pro-inflammatory cytokines do not contribute to VCAM-1 lower expression. 69 During steady-state, loss of VCAM-1 increased frequency BM resident mature cells. 69 BM-ECs VCAM-1 deficient mice 71 VCAM1EC mice developed leukocytosis. 71 VCAM1EC does not exhibit significant changes in hematopoiesis. 73 P2EC vessel morphology is independent of downregulation of VCAM-1 74 VCAMEC mice showed increased tumor homing in the diaphysis. 75 PHD2-HIF-2 regulatory effect on VCAM-1 is modulated by mir-126-3p. 76 HIF-2α regulates mir-126 expression in PHD2 deficient BM-ECs. 77 BM-ECs PHD2 influence bone homeostasis. 78 Loss of BM-ECs PHD2 lead to increase Osteoclast numbers and activity. 79 Osteoclast differentiation and activity could be independent of OBs. 79 Loss of PHD2 in BM-ECs leads to osteoclastogenesis. 80 BM resident Tcell CD8+ could be Increasing Osteoclast Activation. 82 Discussion. 83 Mouse Model: Conditional Deletion of Endothelial PHD2. 85 Endothelial PHD2 Modulates Myelopoiesis. 86 BM-EC PHD2 regulates vessel morphology and functionality under steady-state independent of VCAM-1. 88 Endothelial VCAM-1 downregulation does not impaired neutrophil migration during inflammation. 88 BM-ECs PHD2 is a Gatekeeper of Tumor Homing in the Bone. 89 HIF-2α dependent Mir-126 activation leads to VCAM-1 downregulation 91 Endothelial PHD2 controls Osteoclastogenesis independent of BM RANKL. 94 References 96 List of Abbreviations 107 Summary 109 Zusammenfassung 111 Acknowledgements 113 Deklaration 114 Appendix 118 List of Figures. 118 List of tables 119

ERK3 est une protéine de la famille des MAP kinase (MAPK) classifiée comme atypique car elle présente des différences notables comparées aux propriétés redondantes des MAPK dites classiques. ERK3 est notamment une protéine très instable dégradée constitutivement par le système ubiquitine protéasome. Par conséquence, son activité biologique est principalement contrôlée par la régulation de sa dégradation. Pourtant, les facteurs impliqués dans la régulation de la stabilité de ERK3 restent mal compris. Ce travail de thèse vise ainsi à affiner notre compréhension des mécanismes de régulation de la stabilité de ERK3. De manière intéressante, nous avons montré dans une première étude qu’un pH acide stabilise fortement ERK3 alors qu’à l’inverse, un pH basique induit sa rapide dégradation par le protéasome. De plus, la déplétion génétique de NBCn1, un transporteur de bicarbonate impliqué dans la régulation du pH intracellulaire, augmente également la stabilité de ERK3. Ainsi, des variations de pH intracellulaire régulent finement la dégradation de ERK3. Nous avons également montré dans une deuxième étude l’importance de ERK3 dans la progression tumorale dans le cancer du sein. La surexpression de ERK3 au niveau transcriptionnel ou protéique est associée à un mauvais pronostic dans le cancer du sein, que ce soit au niveau de la survie globale ou de la survie sans métastase. Ainsi, la déplétion de ERK3 entraîne une diminution drastique du nombre de métastases au foie et aux poumons. ERK3 est également impliquée dans la migration cellulaire in vitro. Nous avons montré pour la première fois que la stabilité d’une kinase peut être modulée par le pH. Or, le pH est impliqué dans de nombreux processus biologiques comme, entre autres, la prolifération cellulaire, la migration, l’invasion et la mort cellulaire. Les résultats obtenus pendant ce doctorat ouvrent donc de nouveaux champs d’exploration pour étudier l’activité biologique de ERK3 dans des contextes dépendants du pH. / ERK3 is an atypical member of the MAP kinase (MAPK) family because its regulation differs from the canonical module of classical MAPK. ERK3 is also an unstable protein constitutively degraded by the ubiquitin proteasome system (UPS). Therefore, ERK3 stability regulation is an essential element in the control of its biological activity. However, the components implied in the regulation of its stability by the UPS are mainly unknown. This thesis aims to understand the regulation mechanisms controlling ERK3 degradation to better explore its biological function. In a first study, we showed that an acidic extracellular pH strongly stabilizes ERK3. At the opposite, a basic pH triggers its rapid degradation by the proteasome. Moreover, genetic depletion of NBCn1, a bicarbonate transporter involved in the regulation of the intracellular pH (pHi), also impacts ERK3 stability. We demonstrated that pHi variation finely regulates ERK3 degradation. We also explored the role of ERK3 in breast cancer progression in a second study. In breast cancer, high ERK3 expression correlates with a poor overall survival as well as a higher risk to develop metastases. ERK3 depletion triggers a severe decrease in the number of liver and lungs metastasis in a in vivo metastasis model. We also demonstrated that ERK3 is involved in cell migration in vitro. We showed for the first time that a kinase stability is modulated by pH variation. pH homeostasis is finely regulated in cells to assure important cellular functions such as proliferation, invasion, and survival. Therefore, ERK3 protein levels regulation by the pH raises new potential functions to explore for this kinase in a context pH dependent.