L’érythropoïèse constitue un processus continu et ordonné au cours duquel des progéniteursérythroides commis prolifèrent et se différencient en globules rouges. Au cours d’un criblage visant à identifierdes gènes dont l’expression est régulée au cours de la différenciation terminale, nous avons identifié ISG15comme un gène dont l’expression est induite dans les stades tardifs de la différenciation érythroïde. ISG15appartient à la famille des protéines apparentées à l’ubiquitine et est conjuguée directement à des résidus Lysinede protéines cibles. La séquence des évènements moléculaires qui président à la modification de résidus Lysinedes protéines par ISG15 (ISGylation) est très semblable à ceux mis en oeuvre dans l’ubiquitination des protéines.Nous avons montré que l’expression d’ISG15 et des enzymes liées au processus d’ISGylation Ube1L, UbcM8 etHerc6 est induite au cours de l’érythropoièse in vivo et aussi dans des érythroblastes en culture induits à sedifférencier en réponse à l’Epo. Grâce à ce système de culture in vitro, nous avons montré que l’induction de cesgènes est majoritairement indépendante de la voie de signalisation IFN, et partiellement dépendante de la voie designalisation Epo. La comparaison de l’érythropoïèse dans des souris contrôles et des souris dans lesquelles legène ISG15 a été inactivé (ISG15-/-), montre une diminution du nombre de progéniteurs BFU-E et CFU-E dansla moelle osseuse avec une augmentation des BFU-E/CFU-E dans la rate des animaux ISG15-/-. Nous avonsaussi montré que les érythroblastes ISG15-/- sont inhibés dans leur différenciation terminale, à la fois in vivo et invitro. A l’inverse, la surexpression d’ISG15 dans les érythroblastes se révèle faciliter la différenciationérythroide. Au niveau moléculaire, nous avons montré que des acteurs importants de la différenciation érythroidepeuvent être ISGylés, comme par exemple STAT5, Globine, PLCγ et ERK2. En ce qui concerne plusprécisément STAT5, nous avons montré que son ISGylation est induite au cours de la différenciation et avonscherché à identifier les conséquences de son ISGylation. Dans le contexte d’une protéine de fusion, STAT5ISGylée se lie mieux à son site de liaison à l’ADN, une propriété associée avec une régulation positive de TfR etBCL-XL, deux gènes précédemment décrits comme étant régulés par STAT5 dans les érythroblastes. L’ensemblede ces résultats établit un nouveau rôle pour ISG15 au cours de la différenciation érythroide, en plus de sesfonctions anti-virales bien caractérisées / Erythropoiesis is an orderly continuous process during which committed erythroid progenitorcells proliferate and differentiate into mature red blood cells. In a search for genes that are deregulated duringthis differentiation process, we have identified ISG15 as being induced during the late stages of erythroiddifferentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins bythe enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we haveshown that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6are induced during erythroid differentiation. Moreover, using in vitro differentiating erythroblasts, we haveshown that the induction of these genes is mostly independent of IFN signaling, while it is partially dependent onEpo signaling in these cells. Our analysis of the ISG15 deficient mice have shown a decreased number of BFUE/CFU-E in the bone marrow, associated with an increased number of these cells in the spleen of these animals,a phenotype reminiscent of stress erythropoiesis. While ISG15-/- bone marrow and spleen-derived erythroblastsshowed a less differentiated phenotype both in vivo and in vitro, over-expression of ISG15 in erythroblasts wasfound to facilitate erythroid differentiation. At the molecular level, we have shown that important effectors oferythroid differentiation can be ISGylated, including STAT5, Globin, PLCγ and ERK2. Attempt to identify theconsequences of ISGylation has only been performed for STAT5. When studied in the context of a fusionprotein, ISGylated STAT5 was found endowed with a higher capacity to bind DNA, a property associated withup-regulation of TfR and Bcl-XL, two genes previously described as being regulated by STAT5 during erythroiddifferentiation. This establishes a new role for ISG15, in addition to its well-characterized anti-viral functions,during erythroid differentiation.

The primary function of erythrocytes is transport of oxygen from lungs to various tissues of the body. Red blood cell mass, due to this important role, must be controlled at precise levels. The number of erythrocytes is primarilly increased by the glycoprotein hormon erythropoietin, which expression is controlled by HIF (hypoxia inducible factor). Transcriptional factor HIF consists of the two subunits, HIFα and HIFβ. Under normoxic conditions, alfa subunit of HIF is hydroxylated by PHD protein. This hydroxylation provides a recognition motif for the VHL protein, a part of an E3 ubiquitin ligase complex that targets hydroxylated HIF for proteasomal degradation. Under hypoxic conditions, the degradation is inhibited. The alfa subunit is translocated to the nucleus, where binds the beta subunit and regulates gene expression. HIF pathway regulates a broad spectrum of cellular functions - energy metabolism, angiogenesis, apoptosis and many others. This diploma thesis is focused on HIF2α and its role in erythropoiesis. In this present study, we used CRISPR/Cas9 technology and created HEL (human erythroleukemia) cell line with knock-out of the gene for HIF2α (EPAS1). To reveal the role of HIF2α, we used specific HIF2α inhibitor in order to block its function in HEL cell line. We also tested this...

L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque. / Erythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation.

As células-tronco de pluripotência induzida (iPSC) são células geradas a partir da reprogramação de células somáticas e têm potencial para diferenciação em todos os tipos celulares do organismo adulto. A indução da diferenciação de iPSC pacienteespecífico em células hematopoéticas é uma forma de estudo da hematopoese em modelos de doenças, como a anemia falciforme, e também essencial para o desenvolvimento de terapias. O presente estudo propôs a geração de célulastronco/progenitoras hematopoéticas e progenitores eritroides in vitro a partir de iPSC derivadas de pacientes com anemia falciforme através da formação de corpos embrioides. Ao longo da diferenciação, os desenvolvimentos hematopoético e eritroide foram monitorados através de ensaios de formação de colônia e imunofenotipagem por citometria de fluxo. Neste estudo, demonstramos a presença de células com fenótipo de células endoteliais no início da diferenciação hematopoética por formação de corpos embrioides, possivelmente indicando que as células progenitoras hematopoéticas são provenientes de um endotélio hemogênico. Também verificamos a presença de células endoteliais sem potencial de endotélio hemogênico. Geramos células com características de células-tronco/progenitoras hematopoéticas, de fenótipos CD34+CD45+ e CD45+CD43+, progenitores eritroides (CD36+, CD71+ e CD235a+), bem como a formação de colônias hematopoéticas em cultura em meio semi-sólido. A linhagem de iPSC PBscd08 demonstrou maior potencial para diferenciação em células hematopoéticas e eritroide que as demais linhagens celulares avaliadas. A linhagem PBscd01, também gerada a partir de células mononucleares do sangue periférico (PBMC) de paciente com anemia falciforme, não demonstrou o mesmo potencial para a diferenciação hematopoética, gerando apenas células CD34+ e baixa porcentagem de células CD45+ e CD43+. A linhagem de iPSC PB12, gerada a partir de PBMC de indivíduo saudável, promoveu a geração de populações de células CD34+, CD45+ e CD43+, mas não duplo-positivas, e a geração de células com morfologia de células mieloides após a maturação. As linhagens celulares de iPSC demonstraram variabilidade quanto ao potencial de diferenciação hematopoética. Isto monstra a necessidade de estudos futuros para uma investigação mais detalhada. / Induced pluripotent stem cells (iPSC) are cells generated by reprogramming somatic cells, they have the potential for differentiation into all types of cells in the adult organism. The differentiation of patient-specific iPSC into hematopoietic cells is a way of studying hematopoiesis in disease models, such as sickle cell anemia, and is also essential for the development of therapies. The present study proposed the generation of hematopoietic stem/progenitor cells and erythroid progenitors from iPSC derived from patients with sickle cell anemia. Throughout the differentiation, hematopoietic and erythroid developments were monitored by colony forming cell assay and immunophenotypic analysis. In this study, we demonstrated the presence of cells with endothelial phenotype at the beginning of hematopoietic differentiation by formation of embryoid bodies, possibly showing that hematopoietic progenitor cells originate from a hemogenic endothelium. We generated cells with characteristics of hematopoietic stem/progenitor cells, of CD34+CD45+ and CD45+CD43+ phenotypes, erythroid progenitors (CD36+, CD71+ and CD235a+), as well as the formation of hematopoietic colonies in culture in semi-solid medium. The iPSC line PBscd08 demonstrated greater potential for differentiation into hematopoietic and erythroid cells than the other cell lines evaluated. The iPSC line PBscd01, also generated from peripheral blood mononuclear cells (PBMC) from patients with sickle cell anemia, did not demonstrate the same potential for hematopoietic differentiation, generating only CD34+ cells and a low percentage of CD45+ and CD43+ cells. The iPSC line PB12, generated from healthy individual PBMC, promoted the generation of CD34+, CD45+ and CD43+ cell populations, but not double-positives, and the generation of cells with myeloid cell morphology after maturation. The iPSC cell lines demonstrated variability in the potential for hematopoietic differentiation. This shows the need for future studies for a more detailed investigation.

As células-tronco de pluripotência induzida (iPSC) são células geradas a partir da reprogramação de células somáticas e têm potencial para diferenciação em todos os tipos celulares do organismo adulto. A indução da diferenciação de iPSC pacienteespecífico em células hematopoéticas é uma forma de estudo da hematopoese em modelos de doenças, como a anemia falciforme, e também essencial para o desenvolvimento de terapias. O presente estudo propôs a geração de célulastronco/progenitoras hematopoéticas e progenitores eritroides in vitro a partir de iPSC derivadas de pacientes com anemia falciforme através da formação de corpos embrioides. Ao longo da diferenciação, os desenvolvimentos hematopoético e eritroide foram monitorados através de ensaios de formação de colônia e imunofenotipagem por citometria de fluxo. Neste estudo, demonstramos a presença de células com fenótipo de células endoteliais no início da diferenciação hematopoética por formação de corpos embrioides, possivelmente indicando que as células progenitoras hematopoéticas são provenientes de um endotélio hemogênico. Também verificamos a presença de células endoteliais sem potencial de endotélio hemogênico. Geramos células com características de células-tronco/progenitoras hematopoéticas, de fenótipos CD34+CD45+ e CD45+CD43+, progenitores eritroides (CD36+, CD71+ e CD235a+), bem como a formação de colônias hematopoéticas em cultura em meio semi-sólido. A linhagem de iPSC PBscd08 demonstrou maior potencial para diferenciação em células hematopoéticas e eritroide que as demais linhagens celulares avaliadas. A linhagem PBscd01, também gerada a partir de células mononucleares do sangue periférico (PBMC) de paciente com anemia falciforme, não demonstrou o mesmo potencial para a diferenciação hematopoética, gerando apenas células CD34+ e baixa porcentagem de células CD45+ e CD43+. A linhagem de iPSC PB12, gerada a partir de PBMC de indivíduo saudável, promoveu a geração de populações de células CD34+, CD45+ e CD43+, mas não duplo-positivas, e a geração de células com morfologia de células mieloides após a maturação. As linhagens celulares de iPSC demonstraram variabilidade quanto ao potencial de diferenciação hematopoética. Isto monstra a necessidade de estudos futuros para uma investigação mais detalhada. / Induced pluripotent stem cells (iPSC) are cells generated by reprogramming somatic cells, they have the potential for differentiation into all types of cells in the adult organism. The differentiation of patient-specific iPSC into hematopoietic cells is a way of studying hematopoiesis in disease models, such as sickle cell anemia, and is also essential for the development of therapies. The present study proposed the generation of hematopoietic stem/progenitor cells and erythroid progenitors from iPSC derived from patients with sickle cell anemia. Throughout the differentiation, hematopoietic and erythroid developments were monitored by colony forming cell assay and immunophenotypic analysis. In this study, we demonstrated the presence of cells with endothelial phenotype at the beginning of hematopoietic differentiation by formation of embryoid bodies, possibly showing that hematopoietic progenitor cells originate from a hemogenic endothelium. We generated cells with characteristics of hematopoietic stem/progenitor cells, of CD34+CD45+ and CD45+CD43+ phenotypes, erythroid progenitors (CD36+, CD71+ and CD235a+), as well as the formation of hematopoietic colonies in culture in semi-solid medium. The iPSC line PBscd08 demonstrated greater potential for differentiation into hematopoietic and erythroid cells than the other cell lines evaluated. The iPSC line PBscd01, also generated from peripheral blood mononuclear cells (PBMC) from patients with sickle cell anemia, did not demonstrate the same potential for hematopoietic differentiation, generating only CD34+ cells and a low percentage of CD45+ and CD43+ cells. The iPSC line PB12, generated from healthy individual PBMC, promoted the generation of CD34+, CD45+ and CD43+ cell populations, but not double-positives, and the generation of cells with myeloid cell morphology after maturation. The iPSC cell lines demonstrated variability in the potential for hematopoietic differentiation. This shows the need for future studies for a more detailed investigation.

The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...

The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...