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Erythropoiesis is the process in which a pluripotent stem cell differentiates into a mature red blood cell (RBC). Over the last two decades understanding of factors that regulate this process has increased, which has allowed for the development of advanced in vitro culture systems that can on a small scale, generate therapeutic quantities of mature RBCs from isolated stem cell populations. Not only does this create an alternative source of RBCs for use in medical treatments, it also provides a esearch model that can be used to investigate the underlying biochemical and physiological mechanisms of human erythropoiesis.

Horseracing is a huge industry both nationally and internationally. With large financial rewards now available at some meetings the pressure to excel has grown and as a result the desire to win has led some racehorse owners to resort to the use of performance enhancing drugs. Rumours concerning the abuse of recombinant human erythropoietin (rHuEPO) in thoroughbred horseracing have been circulating since it became readily available in the late 1980s. While there are a number of commercially available EPO enzyme-linked immunosorbent assays (ELlSAs) to detect the administration of rHuEPO to horses, these kits are all restricted in their success due to the limited amount of time EPO remains in the horse's circulation. It is thought that the horse's immune system recognises the rHuEPO as a foreign protein and produces antibodies against it. This research aims to exploit this phenomenon to develop a new sensing system for the detection of rHuEPO antibodies resulting from rHuEPO administration. This research was divided into two parts. In the first section, a simple colorimetric assay based on the aggregation of gold nanoparticles for the detection of the antibodies produced as a result of rHuEPO abuse was developed. Various forms of rHuEPO have been attached to gold nanoparticles via linkage molecules to develop colorimetric assays based on the aggregation of protein-modified gold nanoparticles and its corresponding antibody. The 17 nm protein-stabilised nanoparticles are ruby red in colour. The sensing system was tested with the anti-HuEPO antibody. Upon addition of this antibody, aggregation occurred, with a subsequent change in colour from red to purple due to a red-shift in the surface plasmon absorption band, which was monitored by UV/visible spectrophotometry. For each variant of rHuEPO, the theoretical limit of detection (LOD) was established and kinetic studies (time to complete the aggregation reaction) were investigated.

L’érythropoïèse adulte est un processus complexe qui a lieu dans la moelle osseuse, il aboutit à la formation de globules rouges (GR) à partir des cellules souches hématopoïétiques. L’AMPK (AMP-activated protein kinase) est un complexe hétérotrimérique (αβγ) connu pour son rôle de régulateur du métabolisme énergétique cellulaire. L'implication de l’AMPK dans le maintien de la survie et de l’intégrité des globules rouges murins a été démontrée. En effet, les souris invalidées pour Ampk α1, β1 ou γ1 présentent une anémie hémolytique due à une anomalie de la déformabilité des globules rouges. Nous avons émis l’hypothèse que les altérations observées dans les érythrocytes déficients en AMPK pourraient se mettre en place au cours du processus de différenciation des érythroblastes. L’objectif de ce travail est donc d’étudier le rôle de l'AMPK au cours de l’érythropoïèse murine et humaine adulte. Chez la souris, nos résultats démontrent que l’absence de l’AMPK n’affecte ni la prolifération ni la survie ni la différenciation des érythroblastes Ampkα1-/-. De la même façon, l’activation de l’AMPK n’a pas d’effet sur les érythroblastes murins. Chez l’homme, nous avons montré par une approche shRNA que l’inhibition de l’expression de la sous-unité α1 de l’AMPK induit un ralentissement de la prolifération cellulaire et une anomalie de l’érythropoïèse révélée par une modification de l’expression des protéines membranaires à la surface des érythroblastes au cours de la différenciation. Nous avons également montré que l’AMPK est fortement activée dans les érythroblastes immatures (Pro-E –Baso-E) et que cette activation diminue dans les érythroblastes matures (Poly-E – Retic). Une activation de l’AMPK par des activateurs directs (GSK621 et 991) dans les érythroblastes immatures n’a pas d’effet. Par contre, le maintien de son activation par les activateurs directs dans les érythroblastes matures induit, un arrêt du cycle cellulaire en phase S, une induction de l’autophagie, une apoptose caspase dépendante conduisant à un arrêt de la différenciation au stade Baso-E. Ces résultats montrent l’importance de la diminution de l’activation de l’AMPK pour la survie et la différenciation des érythroblastes matures. L’AMPK est donc importante pour la différenciation des cellules érythroïdes humaines alors que chez la souris, elle est impliquée dans le fonctionnement du globule rouge. Notre travail illustre donc un nouveau point de divergence entre l’érythropoïèse murine et l’érythropoïèse humaine. / AMPK (AMP-activated protein kinase) is a heterotrimeric complex containing α, β, and γ subunits, known for its role in the maintenance of cellular energy homeostasis. It has been shown that Ampk is involved in maintaining integrity of murine RBCs as well as their survival. Indeed, Ampk α1-/- and Ampk γ1-/- mice present a hemolytic anemia, and their RBCs show elasticity defects in their plasma membrane. We hypothesized that the alterations observed in AMPK-deficient erythroblasts were the results of a lack of Ampk earlier during erythroid differentiation. Therefore, we aim to study the role of AMPK during human and murine erythropoiesis. We showed that the absence or activation of Ampk in mice does not affect either the survival, proliferation, or the differentiation of KO erythroblasts compared to WT ones. In human, our data show that the knockdown of the α1 subunit expression by shRNA induces a slowing down of cell proliferation and a dyserythropoiesis, indicated by the shift in pattern of cell surface markers expression during differentiation. In addition, we showed that phosphorylation of AMPK (Thr172) and its target ACC (Ser 79) are elevated in immature (Pro-E –Baso-E) erythroblasts, and then decreased conjointly with the erythroid differentiation. AMPK activation in immature erythroblasts has no effect. Conversely, in mature (Poly-E – Retic) erythroblasts, the persistence of AMPK expression induces a cell cycle arrest in S phase, followed by the induction of autophagy, and of caspase-dependent apoptosis with a differentiation arrest at basophilic erythroblast stage. Our results demonstrate the importance of finely-tuned regulation of AMPK during adult human erythropoiesis. AMPK is needed for efficient erythropoiesis in human, whereas it is involved solely in RBCs function in mice, showcasing yet another contrasting point between human and mouse erythropoiesis.

L’érythropoïèse désigne l’ensemble des phénomènes de prolifération et de maturation qui permettent d’obtenir les globules rouges à partir de cellules souches médullaires. C’est un processus de différenciation continue qui génère des cellules érythroïdes de plus en plus matures. L’érythropoïétine (EPO) est le principal régulateur de l’érythropoïèse. En effet, il est indispensable à la survie et à la prolifération des progéniteurs érythroïdes et des précurseurs érythroblastiques. Le rôle des Pims kinases dans la régulation de l'érythropoïèse n’a pas été étudié bien qu’il ait été montré que les souris invalidées pour les trois gènes de la famille Pim présentent une anémie. De plus, une forte diminution du nombre de progéniteurs érythroïdes est observée chez les souries invalidées pour le gène Pim2 et Pim1, suggérant l’importance de cette kinase dans l’érythropoïèse murine. Aucune étude n’a été réalisée pour comprendre le rôle des PIMs dans l’érythropoïèse humaine. Nous avons donc cherché à comprendre l'implication des PIMs kinases dans l'érythropoïèse normale humaine. Nous avons pu montrer que les PIMs kinases sont exprimées dans des érythroblastes humains normaux. En effet, elles sont fortement exprimées en début de différenciation et leur expression diminue au cours de la maturation terminale. Nous avons mis en évidence que l'expression des trois PIMs est régulée au niveau transcriptionnel par l'EPO. Nous avons également montré que les PIMs kinases régulent positivement la prolifération des cellules érythroïdes immatures. Nous avons aussi observé qu'elles soutiennent l'expression du récepteur de la transferrine (TFR1) à la surface des érythroblastes immatures. TFR1 est important pour les érythroblastes car il permet de faire entrer le fer dans ces cellules, où il est intégré dans l’hème permettant la synthèse de l'hémoglobine. L'hémoglobine est nécessaire à la fonction du globule rouge dans le transport de l'oxygène ainsi que pour l'oxygénation des cellules. Ensuite, nous avons voulu étudier le rôle de chacune de ces trois kinases dans les érythroblastes. Nous avons mis en évidence que la kinase PIM2 joue un rôle clef dans l'érythropoïèse normale en étant indispensable à la survie des cellules érythroïdes. Au niveau moléculaire, l’inhibition de PIM2 ne modifie pas la balance des facteurs pro et anti-apoptotiques mais entraîne des dommages à l’ADN. L'apparition de ces dommages est suivie d'une induction de l'apoptose cellulaire. Dans une pathologie comme la Maladie de Vaquez (MV), nous avons mis en évidence que l'inhibition des PIMs diminue le nombre et la taille des colonies érythroïdes, entraîne la diminution de prolifération cellulaire et aussi, pour certains patients, l'apoptose de leurs cellules. Nos résultats mettent donc en évidence que les PIMs sont importantes dans l’érythropoïèse normale humaine et que les inhibiteurs des PIMs pourraient être un traitement alternatif aux traitements classiques dans la MV. / Erythropoiesis encompasses both the proliferation and maturation of bone marrow stem cells, leading to the production of Red Blood Cells (RBCs). It is a process of continuous differentiation that generates more and more mature erythroid cells. Survival and proliferation of erythroid progenitors and erythroblastic precursors is dependent on the cytokine Erythropoietin (EPO), which is the main regulator of erythropoiesis. The PIMs kinases are proto-oncogenes involved in survival and proliferation, whose family is comprised of three members in Humans. The role of PIMs kinases in the regulation of erythropoiesis has not been studied, although it has been shown that mice knocked out for all three genes of the PIM family have anemia. In addition, a sharp decrease in the number of erythroid progenitors is observed in mice knocked out for both the Pim2 and Pim1 gene. All in all, it suggests the importance of these kinases in murine erythropoiesis, and may be also relevant in human erythropoiesis. Therefore, we sought to understand the involvement of PIMs kinases in normal human erythropoiesis. We have been able to show that PIMs kinases are expressed in normal human erythroblasts. Indeed, they are strongly expressed at the beginning of the erythroid differentiation, and their expression decreases during terminal maturation. We have shown that the expression of the three PIMs kinases is regulated at the transcriptional level by EPO. We also have shown that PIMs kinases positively regulate the proliferation of immature erythroid cells. We observed that they support the expression of the transferrin receptor 1 (TFR1) on the surface of immature erythroblasts. TFR1 is crucial for erythroblasts as it allows iron to enter these cells, where it is used for hemoglobin synthesis. Hemoglobin is absolutely required for RBCs function as oxygen transporters, responsible for the oxygenation of the whole organism. Then, we wanted to study the role of each of these three kinases in erythroblasts. We have shown that PIM2 kinase plays a key role in normal erythropoiesis by being essential for the survival of erythroid cells. At the molecular level, inhibition of PIM2 does not alter the balance of pro- and anti-apoptotic factors, but results in damages to DNA. The appearance of these damages is followed by an induction of cellular apoptosis. In a pathology such as Polycythemia Vera (PV), we have demonstrated that the inhibition of PIMs decreases both the number and the size of erythroid colonies. It also slows down cell proliferation, and for some patients, it induces cellular apoptosis. Our results highlight that PIMs are important in human normal erythropoiesis and that inhibitors of PIMs could be an alternative to conventional treatments in PV.

Orientadora : Maria Suely Soares Leonart / Co-orientadores : Aguinaldo José do Nascimento e Tomoe Nakashima / Dissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias da Saúde / Resumo: O eritrócito humano maduro, quando submetido à sobrecarga oxidativa por condições patológicas do eritrócito ou por agentes oxidantes pode apresentar depleção de glutationa reduzida (GSH), um dos responsáveis pelo sistema de defesa antioxidante do eritrócito; oxidação da molécula de hemoglobina; e agregação de complexos de ferro junto à membrana. Tais conseqüências podem ocasionar anormalidades na membrana eritrocitária e hemólise. Este trabalho tem por objetivo, estudar a ação antioxidante dos flavonóides quercetina e rutina, e de extrato hidroalcoólico de Vitis virtifera (EHV), em eritrócitos humanos normais, submetidos à sobrecarga oxidativa, in vitro, induzida por agentes oxidantes como terc-butilhidroperóxido (TBH) ou cloridrato de fenil-hidrazina (CF). Coletou-se sangue venoso de 12 indivíduos com idades entre 18 e 40 anos e considerados saudáveis, em EDTAK3. Lavou-se os eritrócitos em tampão fosfato 28 mM, NaCl 123 mM, pH 7,4 três vezes por centrifugação a 1220 x g, ressuspendendo-os até volume globular de cerca de 40 %. Submeteu-se os eritrócitos, pré incubados ou não com quercetina 100 (xM, à ação oxidante de CF 0,5 mM durante 15 minutos e; pré-incubados ou não com quercetina, rutina ou extrato hidroalcoólico de Vitis virtifera, à ação oxidante de TBH (0,5 - 5 mM), durante 0 a 30 minutos. Realizou-se a determinação da concentração de GSH [BEUTLER, Red cell metabolism, 1984], da formação de metahemoglobina (MetHb) [EVELYN; MALLOY, JJBioLChem., 126: 655, 1938], modificado por BEUTLER (1995) e realizou-se a contagem de corpos de Heinz (CH) segundo técnica preconizada por BEUTLER et ai [J.Lab.&Clin.Méd., 45: 40, 1955], modificado por CLARO (2002). Observou-se uma diminuição da concentração de GSH (4,0-0,1 nmoles/ g Hb) proporcional ao aumento da concentração de TBH (0,5 - 1,0 mM). A formação de MetHb aumentou (8 - 35 %) assim como a de CH (2 -16 %), em função da concentração de TBH (0,5 - 5 mM). A incubação com quercetina 100 pM (10 - 30 minutos) preveniu parcialmente a formação de GSH (0,25 -1,3 junoles/g Hb) induzida por TBH 1 mM. Quercetina (2 - 20 JIM) preveniu parcialmente a formação de CH (7,5 - 6,7 %) induzida por TBH 3 mM. Em amostras oxidadas por CF 0,5 mM, a quercetina 10 - 120 pM preveniu parcialmente a formação de MetHb (4 - 3,6 %). A rutina 100 (xM preveniu parcialmente a depleção de GSH (0,4 pmoles/g Hb) por TBH (0 pmoles/g Hb). A rutina (40 - 140 jiM) preveniu parcialmente a formação de CH (7,6 - 4,9 %) induzida por TBH 3 mM (9,0 %). O EHV (0,1 mg/ml) preveniu parcialmente a depleção de GSH (4,4 ±0,1 jimoles/g Hb) induzida por TBH 0,5 mM enquanto que EHV (0,5 - 1,0 mg/ml) preveniu quase que na totalidade a depleção de GSH (7,4 ± 0,4 e 7,3 ±0,1 (imoles/ g Hb, respectivamente) induzida por TBH 0,5 mM. O EHV (1,0 - 5,0 mg/ml) preveniu parcialmente a formação de CH (6,4 - 4,0 %) induzida por TBH 3 mM. A associação entre quercetina 100 pM e mesilato de desferrioxamina 4 mM, inibiu parcialmente a formação de MetHb (30 ± 1,2 %) induzida por TBH 5 mM. Os resultados sugerem que, nas concentrações testadas, a quercetina é parcialmente eficaz contra a sobrecarga oxidativa promovida por CF. O estresse oxidativo promovido por TBH foi parcialmente prevenido na presença de quercetina, rutina ou EHV. / Abstract: The mature human erythocytes, submitted to oxidative stress under pathological conditions or by oxidizer agents can present: depletion of reduced glutatione (GSH), responsible for the antioxidant defense system of the erythocytes, oxidation of the hemoglobin molecule, or aggregation of complexes of iron close to the membrane. These can cause abnormalities in the membrane and hemolysis. The aim of this work was to study the antioxidative action of the flavonoids quercetin and rutin, and of hydroalchoolic extract of Vitis vinifera (HEV), in human erythocytes submitted to the oxidative stress, induced by oxidizer agents fert-butylhydroperoxide (TBH) or phenylhydrazine hydrochloride (PH), in vitro. Venous blood was collected in EDTAK3 from 12 healthy individuals with ages between 18 and 40 years. The erythocytes was washed three times under centrifugation at 1220 x g, in 28 mM phosphate buffer, pH 7.4, and 123 mM NaCL, and ressuspended at a final globular volume of 40%. The erythocytes was then preincubated with 100 jiM quercetine and then submitted to the oxidizer action of 0.5 mM PH for 15 min and, preincubated with quercetin, rutin and hydroalchoolic extract of Vitis vinifera, to the oxidizer action of TBH (0.5 - 5 mM), for up to 30 min. The concentration of GSH was determinate [BEUTLER, Red cell metabolism, 1984], the methemoglobin (MetHb) was monitored [EVELYN; MALLOY, J.BioI.Chem., 126: 655, 1938], modified by BEUTLER et at. (1995) and tiie Heinz bodies (HB) was evaluated acording to BEUTLER et al. [J.Lab.&CIin.Med,, 45: 40, 1955], modified by CLARO (2002). A concentration decrease of GSH was observed (4.0 - 0.1 jimoles /g Hb) as a function of the increase of the concentration of TBH (0.5 - 1.0 mM). The formation of MetHb (8 - 35 %) and HB (2 -16 %), increased as a function of the TBH concentration (0.5 - 5 mM). The incubation with 100 (iM quercetin (10 - 30 min) partially prevented the GSH formation (0.25 - 1.3 jimoles/g Hb) induced by 1 mM TBH. Quercetin (2 - 20 pM) partially prevented HB formation (7.5 - 6.7 %) induced by 3 mM TBH. Erythrocyte samples oxidized by 0.5 mM PH and treated with 10 - 120 jiM quercetin had the formation of MetHb (4 - 3.6%) partially prevented. The 100 pM rutin partially prevented the depletion of GSH (0.4 junoles/g Hb) in the absence of TBH. The rutin (40 - 140 pM) partially prevented the formation of HB (7.6 - 4.9 %) induced by 3 mM TBH (9.0 %). HEV (0.1 mg/ml) partially prevented the depletion of GSH (4.4 ± 0.1 jimoles/g Hb) induced by 0.5 mM TBH while HEV (0.5 - 1.0 mg/ml) almost prevented the totally depletion of GSH (7.4 ± 0.4 and 7.3 ± 0 . 1 junoles/g Hb, respectively) induced by 0.5 mM TBH. HEV (1.0 - 5.0 mg/ml) partially prevented the formation of HB (6.4 - 4.0 %) induced by 3 mM TBH. The association of 100 JIM quercetin and 4 mM deferrioxamine mesilate, partially inhibited the formation of MetHb (30 ± 1.2 %) induced by 5 mM TBH. The results suggest that quercetin, at the used concentrations was partially effective against the oxidative stress promoted by PH. The erythrocyte oxidative stress promoted by TBH was partially prevented in the presence of quercetin, rutin and HEV.

Γενετικές παραλλαγές σε γονιδιακούς τόπους που βρίσκονται εντός (cis) αλλά και εκτός (trans) του συμπλέγματος των ανθρώπινων β-σφαιρινικών γονιδίων έχει δειχθεί πως συσχετίζονται σημαντικά με τα επίπεδα της εμβρυϊκής αιμοσφαιρίνης (HbF). Ένα από τα γονίδια αυτά είναι το γονίδιο MAP3K5 (ή ASK1), το οποίο είναι μέλος της οδού της MAP κινάσης, ενεργοποιείται από διάφορες παθολογικές καταστάσεις και από προ-φλεγμονώδεις κυτοκίνες, συμβάλλοντας στην κυτταρική απόπτωση. Πρόσφατα αποτελέσματα από το εργαστήριό μας έδειξαν ότι η παρουσία του σπάνιου αλληλομόρφου C σε μονονουκλεοτιδικούς πολυμορφισμούς (SNP) του συγκεκριμένου γονιδίου, και συγκεκριμένα στους rs9376230 και rs9483947, συσχετίζεται με μειωμένα επίπεδα της εμβρυϊκής αιμοσφαιρίνης (HbF) (Tafrali et al., 2013). Για το λόγο αυτό, υποθέσαμε ότι οι πολυμορφισμοί αυτοί συνδέονται με πιθανές αλλαγές σε ρυθμιστικές περιοχές του γονιδίου ΜΑΡ3Κ5 οι οποίες τροποποιούν τα επίπεδα έκφρασής του. Έτσι, ερευνήσαμε τον υποκινητή του γονιδίου ΜΑΡ3Κ5 ως προς τη συχνότητα παρουσίας τεσσάρων ή πέντε αντιγράφων της μικροδορυφορικής αλληλουχίας 5’ – GCGCG – 3’ (θέση -51 έως -27 από τη θέση έναρξης της μεταγραφής). Με την προσέγγιση αυτή θελήσαμε να εξακριβώσουμε εάν οι μικροδορυφορικές αυτές αλληλουχίες συνδέονται με τους μονονουκλεοτιδικούς πολυμορφισμούς και, κατά συνέπεια, με τα επίπεδα της HbF σε ασθενείς με ενδιάμεση ή μείζονα β-μεσογειακή αναιμία και σε μη-θαλασσαιμικά άτομα. Για τον σκοπό αυτό, χρησιμοποιήθηκαν 11 ασθενείς με ενδιάμεση β-μεσογειακή αναιμία, 15 ασθενείς με μείζονα β-μεσογειακή αναιμία και 60 μη-θαλασσαιμικά άτομα ελληνικής καταγωγής. Ο προσδιορισμός του γονότυπου πραγματοποιήθηκε με εκλεκτική ενίσχυση του τμήματος του υποκινητή του γονιδίου ΜΑΡ3Κ5, μεταξύ των θέσεων -273 έως και +79, και στη συνέχεια, είτε με αλληλούχιση σε αυτοματοποιημένο αναλυτή ή με ανάλυση ετεροδιμερών. Η ανάλυση έδειξε ότι υπάρχει σχεδόν απόλυτη σύνδεση των σπάνιων αλληλομόρφων C τόσο για τον rs9376230 όσο και για τον rs9483947 με τη μικροδορυφορική αλληλουχία των πέντε επαναλήψεων, σχηματίζοντας έναν απλότυπο. O συγκεκριμένος απλότυπος συσχετίζεται με χαμηλά επίπεδα της HbF και το φαινότυπο της βαριάς β-μεσογειακής αναιμίας, αφού σε ασθενείς με μείζονα β-μεσογειακή αναιμία εντοπίζεται με συχνότητα 60% και αποκλίνει στατιστικώς σημαντικά από τις αντίστοιχες συχνότητες σε ασθενείς με ενδιάμεση β-μεσογειακή αναιμία (p=0,04) και σε μη-θαλασσαιμικά άτομα (p=0,003). Τα αποτελέσματα αυτά δείχνουν ότι οι πολυμορφισμοί rs9376230 και rs9483947 συνδέονται με τη μικροδορυφορική αλληλουχία του υποκινητή, η οποία έχει πιθανό λειτουργικό ρόλο στα επίπεδα έκφρασης του γονιδίου ΜΑΡ3Κ5. / Genetic variations in loci that are in cis or trans of the human beta-globin gene cluster are shown to correlate significantly with the levels of fetal hemoglobin (HbF). One of these genes is MAP3K5 (or ASK1), a member of the MAPK pathway, which is activated by various stresses and pro-inflammatory cytokines that contribute to cellular apoptosis. Recent results from our laboratory have shown that the presence of the rare C allele in two intronic SNPs of MAP3K5, namely rs9376230 and rs9483947, is associated with reduced levels of fetal hemoglobin (HbF) (Tafrali et al., 2013). For this reason, we assumed that these polymorphisms may be associated with changes in the regulatory regions of MAP3K5 which could modify its expression levels. Thus, we investigated the frequency of a microsatellite marker at the proximal promoter region of MAP3K5, regarding the presence of four or five repeats of a 5-bp short tandem repeat (STR), am y 5’ – GCGCG – 3’ (positio -51 to -27 from transcription start site). With this approach, we wanted to ascertain whether this STR is associated with the previously studied SNPs (Tafrali et al., 2013) and consequently with th v s o bF i β-thalassemia intermediate or major patients compared to non-thalassemic individuals. For this purpose, we analyzed the DNA samples from 11 β-tha ass mia i t rm ia a 15 β-thalassemia major patients, as well as 60 non-thalassemic controls of Western Greek origin. Genotyping was performed by selectively amplifying a MAP3K5 gene promoter fragment between positions -273 to +79, followed by either sequencing or heteroduplex analysis. The analysis showed that there is an almost perfect correlation of the rare C allele for rs9376230 and the rare C allele for rs9483947 with the five STR repeats, forming a haplotype associated with low levels of HbF and with the phenotype of severe b-thalassemia. This haplotype is detected with a r qu cy o 60% i β-thalassemia major patients and it statistica y sig i ica t y viat s rom th corr spo i g r qu ci s i β-thalassemia intermedia patients (p = 0.04) and in non-thalassemic individuals (p = 0.003). These results indicate that the polymorphisms rs9376230 and rs9483947 are in linkage with the promoter STR polymorphism, which may have a functional role in MAP3K5 gene expression.

L’érythropoïèse est le processus de différentiation d’un progéniteur érythroïde multipotent en globules rouges. La différentiation érythroïde est essentiellement contrôlée par le récepteur à l’érythropoïétine (EPOR). Nous avons montré que le récepteur à la transferrine de type 2 (TFR2) est un membre important du complexe formé par l’EPOR. Le TFR2 présente, comme l’EPOR une expression restreinte qui dépend du type cellulaire. Ainsi son expression n’a pu être détectée que dans le foie, l’érythron et l’intestin grêle. Le rôle du TFR2 a été exploré dans les hépatocytes et il a été montré qu’il joue le rôle d’un senseur de fer dans cette lignée et de ce fait contribue à l’homéostasie du fer. Nous avons déterminé le rôle du TFR2 dans les érythroblastes et montré que TFR2 est une protéine escorte de l’EPOR qui contribue à l’érythropoïèse in vitro et in vivo. De plus, nos travaux montrent que le TFR2 est requis pour la production de GDF15 (Growth Differentiation Factor 15) dans les érythroblastes. D’autre part nous avons démontré que la production de GDF15 est augmentée par l’EPO, la déplétion intracellulaire en fer et l’activité transactivatrice de P53. L’inhibition de l’expression de P53, réalisée au cours de l’étude de son rôle dans la production de GDF15, a révélé son implication dans l’érythropoïèse normale. Nous avons mis en évidence l’existence de plusieurs formes du TFR2. Deux d’entre elles résultent de l’utilisation de sites distincts d’initiation de la traduction. Ces deux isoformes sont régulée différemment au cours de la maturation des érythroblastes. La troisième isoforme, appelée TFR2 soluble (sTFR2), est relargée dans le plasma suite au clivage du TFR2. Nous avons montré que la production du sTFR2 est inhibée en présence du ligand de TFR2, la transferrine saturée en fer (holoTF) alors que le TFR2 est stabilisé dans ces mêmes conditions. Les rôles spécifiques des trois formes du TFR2 doivent encore être élucidés. / Erythropoiesis is the differentiation process of a multipotent erythroid progenitor into red blood cells. Erythroid differentiation is primarily controlled by the erythropoietin receptor (EPOR). We showed that the Transferrin receptor 2 (TFR2) is an important member of the EPOR complex. TFR2 has like EPOR a lineage-restricted expression and can solely be detected in the liver, erythron and small intestine. TFR2 function has been explored in hepatocytes where it plays the role of an iron sensor and contributes to iron homeostasis. We determined the role of TFR2 in erythroblasts and showed that TFR2 is an escort protein for EPOR that contributes to optimal erythropoiesis in vitro and in vivo. Moreover we evidenced that TFR2 is absolutely required for the production of Growth differentiation factor 15 (GDF15) in erythroblasts. We further demonstrated that GDF15 production is increased by EPO levels, by intracellular iron depletion as well as by P53 trans-activation activity. The inhibition of P53 expression, realized for the study of its role in GDF15 production, revealed its implication in normal erythropoiesis. We evidenced that TFR2 is expressed under several forms, two of which result from the utilization of distinct translational initiation sites. These two isoforms are differently regulated during erythroid maturation. The third form called soluble TFR2 (sTFR2) is released in the plasma after TFR2 cleavage. We showed that sTFR2 production is inhibited in the presence of TFR2 ligand, iron loaded transferrin (holoTF) whereas cell surface TFR2 expression is stabilized by holoTF. The specific roles of the three forms of TFR2 expressed by erythroblasts remain to be elucidated.