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L’anémie est un symptôme fréquent chez les patients atteints de cancer. La libération de la cytokine pro-inflammatoire TNFα, un inhibiteur connu de l’érythropoïèse, en est l’une des causes. L’érythropoïèse est un processus nécessitant l’arrêt de la prolifération et l’autophagie. Les résultats précédents ont montré que le TNFα inhibe l’expression des marqueurs érythroïdes et module l’expression de facteurs de transcription (FT) hématopoïétiques. Notre objectif est d’étudier l’implication de la voie TNFα/sphingomyélinase (SMase)/céramide dans l’inhibition de l’érythropoïèse en utilisant des cellules souches hématopoïétiques CD34+ induites à se différencier par l’érythropoïétine recombinante (Epo). Par l’utilisation de céramides exogènes, de SMase bactérienne et d’inhibiteurs de SMases, nous montrons l’implication de la voie SMase/céramide dans l’inhibition de l’expression des marqueurs érythroïdes mais également dans l’induction de la différenciation myéloïde avec une augmentation de l’expression du CD11b. Cet effet sur la différenciation est corrélé à la modulation du réseau FT/miR impliquant GATA-1, GATA-2 et PU.1 et les miR-144, 451, 155, 146a et 223. De plus, l’analyse par microscopie électronique à transmission, l’absence de formation de punctae GFP-LC3 et l’accumulation de SQSTM1/p62 montrent que le TNFα et les céramides inhibent l’autophagie induite par l’Epo. L’analyse des protéines impliquées dans la régulation de l’autophagie montre que le TNFα et les céramides activent mTOR. Son implication est confirmée par l’utilisation de rapamycine et l’inhibition de ULK1 et Atg13. De plus, le TNFα et les céramides inhibent l’expression de bécline 1 et de la formation du complexe Atg5-Atg12. Ces résultats démontrent que la voie TNFα/SMase/céramide joue un rôle dans l’homéostasie hématopoïétique par l’inhibition de l’érythropoïèse au profit de la myélopoïèse, en impactant les réseaux de régulation FT/miR et le processus d’autophagie / Anemia is a common symptom in cancer patients. It can be caused by the release of pro-inflammatory cytokines such as TNFα, a known inhibitor of erythropoiesis. Erythropoiesis involves proliferation arrest and autophagy. Our previous studies showed that TNFα inhibits the expression of erythroid markers as well as hematopoietic transcription factors (TF) expression. The aim is to study the involvement of TNFα/sphingomyelinase (SMase)/ceramide pathway in erythropoiesis inhibition using recombinant erythropoietin (Epo)-induced CD34+ hematopoietic stem cells. Using exogenous ceramides, a bacterial SMase and sphingomyelinase inhibitors, we show the involvement of SMase/ceramide pathway in the inhibition of erythroid markers as well as the induction of myeloid differentiation as shown by the increase in CD11b expression. This effect is correlated to the modulation of the TF/miR network involving GATA-1, GATA-2 and PU.1 as well as miR-144, 451, 155, 146a and 223. We show that TNFα and ceramides inhibit Epo-induced autophagy through transmission electron microscopy analysis, the absence of GFP-LC3 punctae formation and SQSTM1/p62 accumulation. Analysis of proteins involved in autophagy regulation showed that TNFα and ceramides activate mTOR, which is confirmed using rapamycin as well as the inhibition of ULK1 and Atg13. Moreover, TNFα and ceramides inhibit Beclin 1 expression and Atg5-Atg12 complex formation. These results demonstrate the role of TNFα/SMase/ceramide pathway in hematopoietic homeostasis through an erythropoiesis-myelopoiesis switch resulting from perturbation of TF/miR network and autophagy

Lors de variations de PO2 et PCO2, différents mécanismes se mettent en place afin demaintenir l’oxygénation des tissus, notamment au niveau du métabolisme et de la ventilation. En casde stimulation hypoxique ou hypercapnique, on observe alors une réponse ventilatoire, caractériséepar une augmentation progressive de la ventilation. Parmi les facteurs qui influencent la réponse àl’hypoxie, on trouve l’érythropoïétine (Epo) qui, en plus de son rôle dans l’érythropoïèse, possèded’autres rôles, notamment au sein du système nerveux central. Cette thèse présente l’étude del’implication de l’Epo et de différents systèmes de neurotransmission dans les réponses ventilatoiresà l’hypoxie (RVH) et à l’hypercapnie (RVHc).Nous avons alors pu mettre en évidence l’implication du NO, du glutamate et de la sérotonine dansla RVH et dans l’acclimatation ventilatoire à une hypoxie prolongée (VAH) chez un modèle de sourisanémique déficient en Epo (Epo-TAgh) et un animal adapté à la vie en altitude, la plateau Pika. Nousavons ensuite étudié l’impact de la déficience en Epo sur la RVHc, et nous avons confirmé que l’Epon’était pas nécessaire à l’obtention de la RVHc, tout en mettant en évidence un rôle de l’Epo sur lepatron ventilatoire et sur l’implication de certaines structures du système nerveux central dans lamise en place de cette réponse. Une étude en parallèle sur les femelles a permis de mettre enévidence que le cycle oestral n’était pas impliqué dans les réponses ventilatoires mais qu’il semble yavoir une interaction entre l’Epo et les hormones sexuelles femelles dans la RVH et la RVHc. Enfin,différentes expériences réalisées lors de collaborations (Chili, Canada) ont permis d’étudier les effetsde l’Epo sur les chémorécepteurs centraux et périphériques dans la mise en place des réponsesventilatoires.In fine, ces différentes expériences ont permis de préciser les différents facteurs impliqués dans lamise en place des réponses ventilatoires à l’hypoxie et à l’hypercapnie, ce qui pourrait aider par lasuite à mieux comprendre les modifications respiratoires induites par des pathologiques liées àl’anémie ou l’exposition prolongée à l’altitude. / When PO2 and PCO2 are modified, various mechanisms are being established to maintaintissue oxygenation, such as ventilation and metabolism adaptations. In case of hypoxia orhypercapnia stimulation, we observed a ventilatory response, characterized by an increase in minuteventilation. Among the factors involved in the hypoxic response, Epo plays a key role. In addition toits role in erythropoiesis, Epo has other functions, especially in the central nervous system. Thisthesis presents the study of Epo involvement in the ventilatory responses to hypoxia (HVR) andhypercapnia (HcVR).We demonstrate the involvement of NO, glutamate and serotonin in the HVR and in acclimatizationto sustained hypoxia (VAH) in Epo deficient mice (Epo-TAgh) and in an animal adapted to highaltitude, the plateau Pika. Then we studied the impact of Epo-deficiency on HcVR and confirmed thatEpo is not mandatory to obtained HcVR but we demonstrate that Epo can modulate the ventilatorypattern and central nervous system structures involvement in this response. During this study, wealso demonstrate that in female mice, estrous cycle is not involved in HVR or HcVR but it seems thatthere is an interaction between Epo and female sexual hormones in these responses. Finally, someexperiments in collaboration with different countries (Chile, Canada) allowed us to study the effectsof Epo on peripheral and central chemoreceptors during HVR and HcVR.In fine, these experiments allows us to specify the factors involved in ventilatory responses tohypoxia and hypercapnia, which could be helpful to better understand respiratory pathologies suchas anemia or pathologies associated with high altitude.

Anemia of prematurity is characterized by a progressive decline in hemoglobin level during the first month of life. Unlike term newborns, preterm infants become anemic and often require red blood cell transfusions. Various factors contribute to the development of this anemia. These include short infant red blood cell (RBC) lifespan, decline in erythropoiesis rate after birth, and blood losses caused by repeated phlebotomies. The objectives of this work were to develop novel models to evaluate fetal and neonatal erythropoiesis, and to study in vivo adult and neonatal RBC survival in low birth weight preterm anemic infants. The model developed to evaluate fetal erythropoiesis was based on the in utero growth of the fetus over time. Neonatal erythropoiesis rate was estimated using a hemoglobin (Hb) mass-balance based method that has the advantage of not relying on specific structural pharmacodynamic model assumptions to describe the Hb production, but instead utilizes simple mass balance principles and nonparametric regression analysis to quantify the amount of Hb produced and the Hb production rate during the first month of life. To study RBC survival, two separate models, one describing the elimination of neonatal RBCs produced under non-steady state conditions, and the second describing the elimination of adult RBCs produced under steady state conditions were developed and applied to the RBC survival data obtained from low birth weight anemic preterm infants. The proposed mathematical models and its implementation provides a flexible framework to study both in utero non-steady state (non-SS) fetal erythropoiesis and neonatal erythropoiesis in newborn infants.

Abstract SWAP-70 originally identified as a signaling protein exclusively expressed in B-cells has been recently described in other cells of the hematopoietic system, such as mast cells and dendritic cells. Here we describe a novel role of SWAP-70 in hematopoiesis, specifically in regulation of erythropoiesis. SWAP-70 protein expression is detected at the stage of the hematopoietic stem cell (HSC). Its expression persists throughout several stages of multipotent and myeloid progenitors. In erythroid development SWAP-70 is found from early committed to erythroid lineage precursors, burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E); however its expression declines with erythroid maturation and it is lastly detectable at the basophilic erythroblast stage. The protein’s deficiency leads to 3-fold increase in HSC numbers in the bone marrow (BM). The lack of SWAP-70 does not affect intermediate myeloid progenitors and the first erythroid committed progenitor, BFU-E. Hematopoietic tissues (BM and spleen) of Swap-70-/- mice carry 2-times less CFU-Es, thus SWAP-70 appears to be important at this stage. Swap-70-/- mice have the same frequencies of later erythroid progenitors, Ter-119+ erythroblasts, in the BM but fewer in the spleen. BM and splenic Ter-119+ erythroid Swap-70-/- compartment (basophilic, polychromatic and orthochromatic erythroblasts) exhibit an altered profile that is characterized by the delayed maturation of cells at the polychromatic stage. SWAP-70 deficiency is not critical for steady state erythropoiesis and does not influence blood homeostasis. Yet SWAP-70 is essential for proper stress response in conditions of anemia. Swap-70-/- mice have normal steady state hematocrite level but fail to restore it after induced anemia, thus showing sluggish blunted response to erythropoietic stress. In resting conditions Swap-70-/- early erythroid progenitors (CFU-Es) exhibit aberrant preactivation of the integrin VLA-4, which supports homotypic and heterotypic interaction within the erythroid niche, and are hyperadhesive to fibronectin. Similarly, Swap-70-/- basophilic erythroblasts are hyperadhesive to splenic tissue. Based on our data and our initial observations we propose a novel function of SWAP-70 in the c-kit signaling pathway and integrin-mediated, i.e. VLA-4, interactions that are important for HSC and erythroid progenitor maintanence and differentiation. Better understanding of mechanisms governing red blood cell development and homeostasis is of high relevance in the context of treatment of anemia, a very common blood disorder, which leads to a wide range of clinical complications and is the most common cancer-associated morbidity.

Severe anemia is a major life-threatening complication of malaria. Inappropriately low reticulocytosis in malaria patients with anemia suggests insufficient erythropoiesis, of which the mechanisms and implications are not clear. The principle growth factor that promotes erythropoiesis is erythropoietin (Epo). Studies determining the serum level of Epo in malaria infected patients have been inconclusive. Furthermore, the role of Epo and the erythropoietic response to Epo stimulation during malaria have never been examined. The purpose of the experiments performed in this thesis was, thus, to investigate the role of Epo and erythropoiesis in relation to anemia during blood-stage malaria using the murine model of Plasmodium chabaudi AS. A murine Epo specific ELISA, which was determined to be less biased by the presence of other cytokines in the samples as compared to the conventional Epo bioassay, was first developed to facilitate the research. The kinetics of Epo production in the kidney and the levels in the serum were characterized. It was demonstrated that Epo production during blood-stage malaria is mainly regulated by the degree of anemia and that renal cytokines may have only a minor effect on this response. Next, the roles of Epo and erythropoiesis during blood-stage malaria were investigated by neutralization of endogenous Epo or by administration of exogenous Epo. Timely onset of Epo-induced reticulocytosis was shown to be important for the alleviation of malarial anemia and survival. However, reticulocytosis in response to Epo stimulation is severely suppressed by infection with malaria. Dissection of the upstream events of erythropoiesis demonstrated that blood-stage malaria compromises the generation of reticulocytes by suppressing the proliferation, differentiation, and maturation of erythroid-lineage cells at various stages of erythroid development. Taken together, our data provide important insights for understanding the patho

Hemogen (Hemgn) is reported as a tissue specific transcriptional regulator in testis as well as hematopoietic tissues. It is known that Hemgn positively regulates erythroid differentiation; however,the underlying molecular mechanism is not well understood. In the current study, using proteomic approach in combination with other molecular biology tools,we have attempted to decipher the role of Hemgn in differentiating Murine erythroblast leukemia (MEL) cells as a model system. Our study reveals that Hemgn predominantly interacts with transcriptional regulators, chromatin modifiers and histones. Furthermore, using Chromatin Immunoprecipitation and knockdown approach, we have demonstrated that Hemgn is recruited to the b-globin locus, which is known to be activated during erythroid differentiation. Based on the results,we speculate that Hemgn acts as a tissue specific histone chaperone that regulates transcription during erythroid differentiation.

Adult haematopoiesis occurs in the context of a supportive stromal cell niche. The bone marrow, spleen and thymus all contain specific, but relatively poorly defined, stromal cells, which are important for maintenance of quiescence and directed differentiation. Even less is known about the haematopoietic niche during haematopoietic development. The formation of red blood cells (erythropoiesis) occurs during haematopoiesis, and is also controlled by a variety of stromal cells. This thesis examined the visceral endoderm, a group of cells that surrounds the developing epiblast and is required for primitive erythropoiesis (early blood production). We attempted to determine which factors in the visceral endoderm were responsible for induction of primitive erythropoiesis, and whether they would be useful as blood induction factors in embryonic stem cell differentiation. Thus, I attempted to immortalise the visceral endoderm using an immortalising agent (SV40Tag), driven off of a previously identified visceral endoderm gene, Indian hedgehog. We modified a bacterial artificial chromosome so that SV40Tag was driven off of the Indian hedgehog gene. The modified bacterial artificial chromosome was used in both pronuclear injections of mouse blastocysts and the electroporation of embryonic stem cells. After neither attempt produced a visceral endoderm cell line, we examined a visceral endoderm-like cell line, END2, for the presence of the blood inducing factors. We demonstrated the ability of END2 conditioned media to apparently increase expression of blood transcripts in differentiating embryonic cells indicating the presence of blood inducing factors. Expression profiles of END2 cells were compared to a previously completed embryonic stem cell differentiation profile to identify enriched genes. Two genes, angiopoietin-like 7 and Bc064033, were tested for an ability to induce blood in differentiating embryonic stem cells. When neither protein was capable of inducing blood, the END2 cells were examined for the presence of other known blood inducing factors and similarity to in vivo visceral endoderm. The END2 cells were found to produce bone morphogenetic protein 4, a potent inducer of blood in embryonic stem cell differentiation, which complicated the search for additional factors. Examination of END2 cells also indicated a lack of visceral endoderm markers such as alpha fetoprotein, indicating that the END2 cells may not be as visceral endoderm-like as expected from the current literature. The previously identified Indian hedgehog gene was also examined for its blood induction abilities in vivo. Indian hedgehog knockout mice were examined for the effect gene removal had on both primitive and definitive erythropoiesis. Levels of primitive erythrocytes were unaffected in the Indian hedgehog knockout mice, but levels of definitive erythrocytes were found to be significantly decreased. Further examination of Indian hedgehog knockout fetal livers also showed that they had decreased numbers of haematopoietic stem cells. The haematopoietic stem cells were fully capable when cultured and generated appropriate numbers of progenitor cells, indicating a non-intrinsic cause for this defect. Levels of hedgehog target genes that are usually highest in the stromal compartment were also found to be most significantly decreased in Indian hedgehog knockout fetal livers. Another hedgehog gene, desert hedgehog, was also shown to be expressed in the fetal liver and may act with Indian hedgehog to regulate stromal production in the fetal liver.

Thesis (Ph.D. )--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on October 7, 2008 ). Research advisor: Paul A. Ney, M.D. Document formatted into pages (xi, 122 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 78-110).