Dissertations / Theses on the topic 'Ferroportin'

Ferroportin-1 (Fpn1) is a highly conserved 571-amino acid protein with the human, mouse, and rat polypeptides having 90–95% sequence identity at the amino acid level. Disruption of Fpn1 expression in zebrafish and mice results in embryonic lethality, while conditional knockout of the gene encoding Fpn1 in the intestine at the postnatal stage leads to severe iron deficiency anemia and iron accumulation in duodenal enterocytes. These studies suggest that Fpn1 is the major, if not the sole, exporter protein for iron. Export of dietary iron is thought to be facilitated by hephaestin (Hpn), a multicopper ferroxidase in the basolateral membrane of duodenal enterocytes. However, the precise mechanism of iron transport by Fpn1 and Hpn remains to be elucidated. The absence of an iron export mechanism in Saccharomyces cerevisiae was utilized to explore how Fpn1 and Hpn function in iron export. Unlike humans, S. cerevisiae transports excess iron into a storage vacuole through the vacuolar iron transporter, Ccc1p. The Δccc1 mutant fails to store excess iron; as a result, iron accumulates in the cytosol and cells die due to oxidative stress when exposed to high concentrations of extracellular iron. By expressing recombinant human Fpn1 and Hpn in S. cerevisiae, an iron export system was introduced. The functionality of rhHpn in S. cerevisiae was confirmed by both a ferrozine ferroxidase assay and a transferrin iron-loading assay. The effects of the individual or both recombinant proteins on iron sensitivity of the Δccc1 yeast with an additional fet3 (the yeast homolog of Hpn) deletion were evaluated. Recombinant human Fpn1 and Hpn suppressed the lethal phenotype of the Δccc1 mutant while co-expression of rhHpn with rhFpn1 led to a stronger rescue phenotype of the Δccc1 mutant under concentrations of high extracellular iron. The expression of rhFpn1 and / or rhHpn also relieved the copper sensitive Δfet3 mutant from copper stress. A physical interaction between rhFpn1 and rhHpn was demonstrated by cross-linking with BS³. These studies advance our knowledge of the roles Fpn1 and Hpn play in human enterocytes and suggest that these proteins are intimately involved in iron export from enterocytes into the blood.

INTRODUZIONE Le β-talassemie sono una delle malattie genetiche più frequenti in tutto il mondo con 270 milioni di portatori e 350.000 nuovi nati affetti all’anno. Questa malattia è geneticamente caratterizzata dalla perdita di produzione della catena β globinica dell'emoglobina adulta, dovuta a diverse mutazioni nel gene della β-globina. Poiché il gene beta è espresso su entrambi i cromosomi 11, possiamo avere due differenti tipi (e con differente gravità) di beta talassemia a seconda dell’assenza di entrambi o di un solo gene della beta globina: nel primo caso si ha la β talassemia MAJOR o trasfusione dipendente, nel secondo caso si ha la β talassemia MINOR o INTERMEDIA trasfusione indipendente. I nostri studi si concentrano su quest’ultima. L'assenza della catena β globina comporta diverse conseguenze per l'organismo come: - Eritropoiesi inefficace - Il sovraccarico di ferro - Il danno ossidativo Finora sono stati condotti molti studi in diversi campi (genomico, proteomico e nel metabolismo ferro) per garantire una maggiore comprensione di questa malattia. Recentemente si è scoperta una nuova proteina che potrebbe essere un eventuale regolatore o responsabile del sovraccarico di ferro nella β - talassemia; questa molecola è la ferroportina. La Ferroportina (FPN) è l'unico esportatore di ferro finora conosciuto. Essa è espressa in diversi tipi di cellule, tra cui gli enterociti duodenali, gli epatociti, i macrofagi e gli eritroblasti. Pochi anni fa, è stata segnalata l'esistenza di due trascritti alternativi della FPN con o senza le Iron Responsive Elements (IRE) sul loro promotore (FPN1A e FPN1B rispettivamente). L'espressione delle diverse isoforme della ferroportina nonché i meccanismi che la regolano nelle cellule eritroidi della β talassemia non-trasfusione dipendente (NTDT) non sono ancora noti. SCOPO Studiare il profilo di espressione delle due isoforme della ferroportina durante il differenziamento eritroide in colture controllo e di NTDT e chiarire i meccanismi che regolano la loro espressione. MATERIALI E METODI Per questi studi è stato usato un modello di eritropoiesi in vitro derivato da cellule CD34+ provenienti da sangue periferico di volontari sani (controllo) e pazienti NTDT. Il profilo dell'espressione genica delle due isoforme (FPN1A e FPN1B) è stato valutato allo stadio basale (giorno 0) e al giorno 7 e 14 della cultura (stadio di pro eritroblasti e di eritrociti ortocromatici rispettivamente) mediante la tecnica di real-time PCR (2-dCt). La percentuale relativa di ogni isoforma è stata calcolata sulla base dell’espressione della ferroportina totale (FPN1A + FPN1B). La concentrazione di ferro intra and extracellulare è stata analizzata utilizzando un kit di Ferro Assay (Biovision). In esperimenti indipendenti, colture di controllo e NTDT sono state trattate con: ferro (Ferro Ammonio Citrato [FAC] 100μM), Desferal (DFO, 4μM), protoporfirina (SNPP IX 50-20μM), eme (Emina 20-10μM) o perossido di idrogeno (H2O2 0,1mM) per indagare su un possibile ruolo di questi composti nella regolazione della ferroportina. L’espressione della FPN è stata valutata al 14esimo giorno in condizioni standard e nei trattati mediante la tecnica di real-time PCR (2-ddCt; cellule non trattate utilizzate come calibratore). RISULTATI L'espressione ferroportina aumenta durante il differenziamento eritroide, raggiungendo il livello massimo di espressione allo stadio di eritroblasti (giorno 14 di coltura) sia nel controllo sia negli NTDT. La FPN1A è l'isoforma più espressa in entrambe le condizioni. La sua espressione è più elevata negli stadi iniziali e finali dell’eritropoiesi (giorno 0 e 14), mentre l'espressione della FPN1B è maggiore nella fase intermedia di differenziamento eritroide (giorno 7). Degno di nota, l'espressione della FPN1B, anche se inferiore rispetto alla 1A, è significativamente maggiore nelle culture NTDT rispetto ai controlli, in particolare al giorno 14. La concentrazione di ferro intracellulare è diminuita in modo significativo durante il differenziamento eritroide (dal giorno 7 al giorno 14), sia nei controlli sia negli NTDT, tuttavia, al giorno 7 (stadio di eritroblasti) i livelli di ferro nelle culture NTDT sono notevolmente inferiori rispetto ai controlli. L'aggiunta di FAC, DFO, SnPP IX ed Emina nei controlli e nelle colture di NTDT non ha modificato l'espressione della ferroportina rispetto ai non trattati. L’H2O2 aggiunto ai controlli aumenta l'espressione di entrambe le isoforme della ferroportina (FPN1A: cellule non trattate: 1; H2O2: 1.33 FPN1B: cellule non trattate: 1; H2O2: 2.04). I livelli di ferro intra ed extracellulari riflettono i risultati genetici: c'è stato un aumento di ferro extracellulare causa di un aumento di espressione FPN. CONCLUSIONI L'espressione della ferroportina aumenta durante il differenziamento eritroide sia nei controlli sia nelle culture NTDT, suggerendo il suo ruolo nell’esportare il ferro intracellulare in eccesso. In entrambe le condizioni, la FPN1A è l'isoforma più espressa. Tuttavia, l'espressione dell’isoforma 1B non responsiva al ferro, anche se minore rispetto a FPN1A, è significativamente maggiore nei NTDT rispetto ai CTRL. In colture di controllo, l’espressione della FPN, ed in particolare dell’isoforma 1B, sembra essere regolata dall’aggiunta di H2O2. Questi dati suggeriscono che lo stress ossidativo, particolarmente elevato nelle NTDT, potrebbe essere uno dei principali regolatori dell’espressione dell’isoforma 1B, generando così un’importante esportazione di ferro dalle cellule NTDT.
INTRODUCTION β-Thalassemias are one of the most frequent genetic disorders worldwide with 270 million of carriers and 350.000 affected new-borns per year. This disease is genetically characterized by the loss of production of the β globin chain of the adult haemoglobin, due to several mutation within the beta globin gene. Since the beta gene is expressed on both the chromosomes 11, we can have two different type (and severity) of beta thalassemia depending on the absence of both or just one beta gene: in the first case we have the β thalassemia MAJOR transfusion dependent, in the second case we have the β thalassemia MINOR or INTERMEDIA, transfusion independent. Our studies are focused on the last one. The absence of the β globin chain implies different consequences for the organism like as: - Ineffective erythropoiesis - Iron overload - Oxidative damage Many studies have been conducted so far in different fields (genomic, protein expression and regulation, iron metabolism) in order to guarantee a major comprehension of this disease. Recently a new protein came out as a possible regulator/responsible for the iron overload in β thalassemia; this molecule is the FERROPORTIN. Ferroportin (FPN) is the only know iron exporter protein. It is expressed in different cell types including duodenal enterocytes, hepatocytes, macrophages and erythroblast cells. Few years ago it has been reported the existence of two alternative transcripts of FPN with or without an iron – responsive element (IRE) on their promoter (FPN1A and FPN1B respectively). The expression of the different ferroportin isoforms as well as the mechanisms regulating their expression in erythroid cells in non-transfusion dependent β thalassemia syndromes (NTDT) are not known yet. AIM To investigate the expression profile of ferroportin isoforms during erythroid differentiation in control and NTDT cell cultures and to elucidate the mechanisms regulating their expression. MATERIALS AND METHODS An in vitro model of erythropoiesis derived from human peripheral CD34+ cells from healthy volunteers (control) and NTDT patients was used. The expression profiling of FPN isoforms (FPN1A and FPN1B) was evaluated at baseline (day 0) and at day 7 and 14 of culture (pro erythroblasts and orthochromatic erythroblasts stage respectively) by real−time PCR (2−dCt). The relative percentage of each isoform was calculated based on total ferroportin expression (FPN1A+FPN1B). The intracellular iron concentration was analyzed by using an Iron Assay Kit (Biovision). In independent experiments, control and NTDT cultures were treated with iron (Ferric Ammonium Citrate [FAC] 100µM), Desferal (DFO, 4µM), protoporfirin (SnPP IX 50-20µM), heme (Hemin 20-10µM) or hydrogen peroxide (H2O2 0.1mM) to investigate a possible role of these compounds in ferroportin regulation; FPN expression was evaluated at day 14 in standard and treated conditions by real−time PCR (2−ddCt; untreated cells used as calibrator). RESULTS The ferroportin expression increased during erythroid differentiation; with the highest level at the end of erythroblasts stage (day 14 of cultures) both in control and NTDT cultures. The FPN1A was the more expressed isoform in both conditions. Its expression was higher at the initial and final steps of erythropoiesis (day 0 and 14), while FPN1B expression was higher at the intermediate erythroblast stages (day 7). Noteworthy, the FPN1B expression, although lower compared to FPN1A, was significantly higher in NTDT cultures than in control ones, particularly at day 14. The intracellular iron concentration decreased significantly during erythroid differentiation (from day 7 to day 14) both in control and NTDT cultures, however, at day 7 (early erythroblasts stage) the iron levels in NTDT cultures were notably lower than in controls. The addition of FAC, DFO, SnPP IX and Hemin in control and NTDT cultures did not modify the ferroportin expression compared to untreated. H2O2 added to control cells increased the expression of both ferroportin isoforms (FPN1A: untreated cells: 1; H2O2: 1.33. FPN1B: untreated cells: 1; H2O2: 2.04). The intra and extracellular iron levels reflected the genetic results: there was an increase of extracellular iron due to an increase of FPN expression. CONCLUSIONS The ferroportin expression increases during erythroid differentiation either in control than in NTDT cultures, suggesting its role in exporting the excess intracellular iron. In both conditions, the FPN1A is the more expressed isoform. However, the expression of the non−iron responsive FPN1B isoform, although lower compared to FPN1A, is significantly higher in NTDT than in control conditions. In control cultures, FPN expression, and particularly the FPN1B isoform, seems to be up regulated by H2O2 addition. These data suggest that the oxidative stress, notably higher in NTDT conditions, could be one of the major regulator of FPN1B expression, with a major iron export from NTDT erythroblast cells.

Macrophages are essential in the inflammatory response and also have a critical function in body iron homeostasis. Moreover, it is known that iron metabolism is important in the context of inflammation. Indeed, it has been demonstrated that, in line with their functions, distinct macrophage populations, such as M1 and M2 polarized cells, differ in the expression of genes involved in iron homeostasis as well as in the expression of immunoregulatory genes. The functional significance of these differencies are not completely understood: we hypotized that iron released by M2 macrophages could promote cell proliferation and extracellular matrix deposition in the resolution phase of inflammation. Moreover, in line with the increasing awareness that macrophages have an important trophic role in addition to their immunological function, macrophages may play a role of “local iron redistributors” in tissues, where they would manage iron availability for neighbouring cells. Therefore, the major aim of this project was to exploit a mouse model of impaired iron release from macrophages, caused by deletion of Ferroportin (Fpn), to understand the functions of macrophage iron in two situations of tissue repair: cutaneous wound healing and liver fibrosis. The characterization of mice with loss of macrophage Fpn showed that they are affected by transient alopecia caused by impaired hair follicle growth. The local impairment of iron distribution due to macrophage Fpn inactivation was accompanied by cellular iron deprivation and decreased proliferation in adjacent epithelial cells. By exposing mice to an iron-restricted diet we concluded that hair loss was not related to hypoferremia/anemia. Taken together, these results suggest that iron retention in resident macrophages has detrimental effects on tissue homeostasis by inhibiting the proliferation of hair follicle cells. We observed a considerable delay in the closure of excisional skin wounds of Fpnfl/flLysCre/- mice compared to controls, with defective granulation tissue formation and diminished fibroplasia. Moreover, the development of both lymphatic and blood vascular network was impaired. Conversely, inactivation of Fpn in macrophages had no impact on inflammatory processes accompanying wound healing, such as production of inflammatory molecules, content of leukocyte subsets and macrophage polarization. Altogether, these results indicate that, though it does not interfere with immune cells recruitment and local activation, Fpn deletion in macrophages impairs blood vessels formation and stromal cells proliferation, leading to delayed skin repair. Fpn inactivation in macrophages had no impact on inflammation, steatosis and fibrosis associated with exposition to the MCD diet, a model of non-alcoholic steatohepatitis. Levels of inflammatory and fibrogenic markers did not show significant differences between Fpnfl/flLysCre/- mice and controls. Interestingly, the levels of transaminases were significantly lower in mice with Fpn inactivation in macrophages, suggesting a different susceptibility to liver damage. These data suggest that, in this model, Fpn deletion in macrophages does not affect the inflammatory response to liver damage and fibrogenesis. The different susceptibility to liver damage and the different results we observed in the cutaneous wound healing and in the process of hepatic fibrosis should be further explored, perhaps using another model of fibrosis. In conclusion, the results reported in this thesis indicate that the macrophage trophic function in skin homeostasis and healing is iron and Fpn-dependent.

This dissertation focused on identifying novel chemical and genetic modulators of iron homeostasis. Iron is an essential element for human health. Disorders such as anaemia and haemochromatosis can develop when iron levels are not maintained within a normal physiological range. The findings of this program included the identification of a new iron chelating compound, demonstration of iron chelation in a haemochromatosis mouse model by a flavonol, identification of iron metabolism-related genes and variants which may assist in distinguishing suitable blood donors, and the identification of novel genes which may contribute to modulating iron homeostasis by regulating the iron exporter ferroportin.

Le fer est un oligoélément indispensable pour tout organisme vivant. Le taux de fer systémique est régulé par la fixation de l’hepcidine, hormone synthétisée majoritairement par le foie mais également par les macrophages, à la ferroportine seul exporteur du fer. L’expression de ces deux protéines est régulée par le taux de fer et les processus inflammatoires. Des mécanismes d’acquisition et de séquestration du fer sont mis en place respectivement par le pathogène et l’hôte durant l’infection et régulent en parallèle l’expression de l’hepcidine et la ferroportine. Les travaux de recherche effectués dans le cadre de ma thèse ont porté d’une part sur un aspect fondamental à améliorer nos connaissances du mécanisme de régulation de l’axe hepcidine - ferroportine en condition inflammatoire et analyser l’influence du fer sur la réponse immune au niveau des macrophages; d’autre part une deuxième partie de mes recherches s’est orientée vers une étude plus appliquée du rôle du fer dans la réponse immune induite par une infection mycobactérienne. Nous montrons que l’expression de l’hepcidine et de la ferroportine est différentiellement régulée en corrélation avec la polarisation des macrophages via les voies de signalisation intracellulaires PI3K et autres kinases. Le fer influence la polarisation des macrophages et module ainsi la réponse inflammatoire, et représente aussi un signal de danger capable de stimuler une voie MyD88-dépendante. Enfin, la réponse à l’infection Mycobacterium. bovis BCG est modulée par un régime modérément enrichi en fer, réduisant la charge bactérienne et l’inflammation
Iron is an essential trace element for all organisms. In mammals, systemic iron homeostasis relies on hepcidin, a peptide hormone synthesized by liver but also macrophages with defensing properties, and its target, the cell iron exporter ferroportin. Iron content and inflammation regulate hepcidin and ferroportin expression in mammals. During infection, pathogens develop sophisticated mechanisms for iron acquisition and sequestration. In response, host regulates the bioavailability of iron through hepcidin and ferroportin expression. First, this work contributes to improve our fundamental knowledge on hepcidin and ferroportin regulation during inflammation and analyzes the influence of iron in macrophages immune response. Second, the role of iron in response to mycobacterial infection was investigated. We show that hepcidin and ferroportin expression was regulated differentially in correlation with macrophages polarization through intracellular signaling pathways involving PI3K and others kinases. In addition, iron influenced macrophages polarization leading to a decrease of inflammatory response with a potent effect on MyD88 pathway stimulation. Finally, we showed that moderate iron-rich diet modulated Mycobacterium bovis BCG response reducing the bacterial burden and inflammation

Anemia of chronic inflammation (ACI) is a condition that develops in a setting of chronic immune activation. ACI is characterized and triggered by inflammatory cytokines and the disruption of iron homeostasis. Hepcidin, a small peptide hormone, is the master regulator of iron homeostasis, and rapidly responds to iron supply and demand. Under conditions of chronic inflammation, hepcidin is elevated, and alters the way that iron is absorbed and disrupted throughout the body, resulting in disrupted iron homeostasis through inhibition of the iron exporter protein ferroportin. Anemia arises when insufficient erythropoietic activity combined with inadequate iron supply abrogates the healthy development of mature red blood cells (RBCs). The proprotein convertase (PC) known as furin is a serine protease capable of cleaving peptide precursors into their active state. Furin is critical for normal activation of proteins and enzymes but recently, furin has been implicated in critical roles within cancers, viral and pathogenic infections, and arthritis through activating precursors novel to the disease type. Furin has previously been identified as being the sole PC responsible for generating active hepcidin. Hepcidin is initially synthesized as a larger precursor protein, before undergoing furin cleavage. Furin is known to form mature, bioactive hepcidin, with the removal of this pro-region. Our discovery of a novel regulatory site on Furin has led to potent inhibition using small molecules. This inhibition is shown with the use of in vitro fluorogenic assays, in vivo cell tissue cultures, and within an animal model of ACI. Our results demonstrate that in using these small molecules we can decrease hepcidin levels even in the presence of potent inflammatory cytokines. The inhibition of hepcidin by these small molecules causes an increase in stably expressed ferroportin on cell surfaces and the restoration of the ability to mobilize iron from storage tissues and absorption from the diet. The ability to "fine-tune" inhibition of furin in targeting its allosteric site along with its catalytic domain designates these small-molecule inhibitors as promising therapeutics for treatment of diseases ranging from Alzheimer's and cancer to anthrax and Ebola fever.

Active learning strategies are important for facilitating deep learning that may be carried throughout life, but which is still finding its way into the college setting. Educators are not often trained in effective learning practices, which reduces the cognitive and proficiency gains of their students. By providing such guidance in the formative years of a teacher’s training, we hypothesize that the learning environment will be greatly enriched and enhanced. On the opposite end of the spectrum of life and cognition, the plague of dementia also warrants examination. Alzheimer’s disease (AD), an incurable neurodegenerative disorder progressing from the medial temporal lobe, is the most common form of dementia diagnosed in people over age 65, afflicting 30-40% of those 85 years and older. Despite its prevalence, effective treatments are limited because the principal causes and triggers of AD are not entirely understood. Growing evidence demonstrates that oxidative stress (OS) is an important factor contributing to the initiation and progression of AD. A key player contributing to this OS is iron, an essential trace mineral which is required for proper neuronal function, but which generates reactive oxygen species during redox transitions. Intracellular labile iron pool (LIP) levels are strictly regulated by proteins such as transferrin (import), ferroportin (export), and ferritin (storage). However, when these proteins become dysregulated, excess iron associates with other proteins such as amyloid beta (Aβ) and tau, aggregations of which are hallmarks of AD. In our hypothetical model, under extensive or prolonged OS, as occurs in AD, much larger Aβ plaques form because the stress does not abate. Hyperphosphorylated tau is the last resort to protect the cell against free iron, and aggregates when the LIP is elevated because neither iron storage in ferritin nor iron export through ferroportin can relieve the neurons of the free iron.

Background Intrauterine Growth Restriction (IUGR) and Pre-eclampsia (PE) are pregnancy pathologies associated with deficient placental function, leading to decreased nutrient and oxygen availability to the fetus. Iron (Fe) deficiency in pregnancy is associated with low birth weight and premature delivery. Nevertheless, Fe oversupply promotes the generation of free radicals and causes oxidative damage in the cells. A previous study performed in the lab where this thesis has been carried out demonstrated a significant decrease of the Fe cell-importer Transferrin Receptor (TfR1), located in the trophoblast cell (TC) microvillar membrane, in human Intrauterine Growth Restricted (IUGR) vs normal (N) placentas (Mandò C. et al. 2011). Ferroportin (FPN) is a trans-membrane protein located in the TC basal membrane that exports Fe towards the fetal circulation. Aim We hypothesized that TfR1 downregulation in IUGR placentas may be due to Fe intracellular accumulation. Thus, we measured FPN gene and protein expression in human IUGR vs N placentas. Then, we extended the Fe transporters investigation to PE placentas, by measuring TfR1 and FPN gene expression in human PE and PE associated with IUGR vs N placentas. Furthermore, we evaluated the relationship between Fe supplementation, food intake, haematologic parameters and pregnancy outcome in a cohort of Italian pregnant and healthy women. Materials and Methods Placentas were sampled at the time of elective cesarean section; villi were selected, washed and immediately frozen for following analysis. Umbilical venous and arterial blood was sampled for pH, oxygen measurements, Hb and lactate measurements, from a doubly clamped segment of the cord immediately after fetal extraction. All samples were collected in hepa- rinized syringes and kept on ice until the end of analysis. All the parameters were measured on a GEM Premier 3000 (Instrumentation Laboratory). FPN mRNA was quantified in a total of 50 N, 41 IUGR, 10 PE and 15 PE+IUGR placentas by Real Time PCR and FPN protein expression was quantified in 26 N and 14 IUGR by Enzyme-Linked ImmunoSorbent Assay. TfR1 mRNA was quantified in 28 N, 10 PE and 15 PE+IUGR placentas. For the study of Fe supplementation, 55 healthy Italian singleton pregnant women were randomized in 4 groups in relation to different doses and types of Fe supplementation (Controls, Fe Sulphate 30 mg, Fe liposomial 14 mg and Fe liposomial 28 mg). At 28-30 gestational weeks data about eating behavior were collected by food frequency questionnaires and hematologic parameters of Fe content (hemoglobin, ferritin, transferrin, serum Fe, folate, vitamin B12, homocystein) in maternal blood by biochemical analysis. Results and Discussion Fetuses from IUGR pregnancies, both with and without PE, have lower pO2 in umbilical vein, while there were no significant differences in fetal emoglobin (Hb) among controls, IUGR, PE and PE+IUGR groups. Both FPN mRNA and protein expression were not statistically different in IUGR compared to N placentas. FPN and TfR1 mRNA levels were not statistically different in PE and in PE+IUGR compared to N placentas. Our results showed no differences in FPN mRNA and protein placental levels between IUGR and N. This suggests that the Fe reaching IUGR fetuses may be decreased compared to normal pregnancies, as a consequence of TfR1 downregulation in the microvillar membranes. This could impair many cellular processes, since Fe is a very important element for enzyme functions and for a correct oxidative status in the cell. TfR1 and FPN mRNA levels were not statistically different in PE and in PE+IUGR vs. N placentas, suggesting that Fe transport is not affected in Preeclampsia. However, we aim at enlarging our analysis to reach definitive conclusions on the Fe transport system in PE placentas. No significant differences were found in the maternal hematologic parameters between the 4 groups of randomized pregnant women at 28-31 weeks, with the exception of hemoglobin, which was significantly higher in women supplemented with 28 mg of Fe liposomial compared to controls (p<0.01). The groups were homogeneous in relation to pregnancy outcomes: no differences were found in neonatal and placental weights, as well as in gestational age at delivery and in umbilical artery pH. The food frequency questionnaires analysis revealed that the control group assumed higher quantity of bioavailable iron (meat) compared to other groups. This may explain the absence of significant differences in the maternal iron status in the control group compared to the supplemented groups.

La surcharge en fer est un facteur de risque démontré de carcinome hépatocellulaire (CHC). Les mutations du gène SLC40A1, codant pour la ferroportine (FPN), conduisent à des surcharges martiales majeures. Des cas isolés de sujets atteints de maladie ferroportine et de CHC ont été rapportés. Le but de cette étude est de préciser la fréquence des mutations du gène SLC40A1 chez les malades ayant une surcharge en fer hépatique majeure et un CHC. Tous les malades ayant un CHC, pris en charge consécutivement entre janvier 2009 et septembre 2010 (234 malades) ont été étudiés dans ce travail prospectif. Douze malades ayant un génotype HFE C282Y homozygote ont été exclus. 109 malades avaient des perturbations du bilan martial sérique (élévation de la ferritinémie ou du coefficient de saturation de la transferrine). Ces patients ont eu une détermination de la concentration hépatique en fer (CHF) par IRM ou méthode biochimique. Chez les 13 malades ayant une CHF > 100 μmol/g un séquençage a été réalisé des 8 exons, des leurs jonctions et de la région 5'UTR du gène SLC40A1 par la méthode Single Condition Amplification. Aucune altération de séquence majeure n'a été observée. Le polymorphisme c.44-24 G>C (IVS1) dont l'effet pathogène est controversé, est retrouvé chez tous ces patients à l'état homozygote ou hétérozygote. Il apparaît que les mutations du gène SLC40A1, ne sont qu'une cause marginale de CHC mais le rôle du polymorphisme c.44-24 G>C (IVS1) reste à préciser.

Ferroportin is a polytopic membrane protein of 62.5 kDa that exports ferrous iron from specialized body cells into the bloodstream. To date, it is the only known iron exporter in mammals. At the systemic level, ferroportin is regulated with a negative post-translational mechanism operated by hepcidin, a peptide secreted by the liver in response to increased levels of body iron, which binds ferroportin causing its internalization and lysosomal degradation (Nemeth et al., 2004). Ferroportin is therefore an important regulator of intracellular and systemic iron. Mutations in the gene encoding ferroportin (SLC40A1) cause the iron overload disease type IV hereditary hemochromatosis (HH) or "ferroportin disease" characterized by autosomal dominant inheritance. Ferroportin disease usually presents as one of two different phenotypes. If the mutation results in a loss-of-function, patients display macrophage iron loading, high serum ferritin levels and normal to low transferrin iron saturation. In contrast to this classical phenotype, there are some gain-of-function mutations that do not affect the iron export ability of ferroportin, but result in a partial to complete resistance to hepcidin. Patients with mutations in this second category have a phenotype with features similar to those of the classical type of hemochromatosis. Specifically, the transferrin saturation is expected to be markedly elevated and iron accumulate mostly in parenchymal cells (Pietrangelo, 2006). The PhD work was focused on the study of ferroportin. To date, the ferroportin crystal structure has not yet been resolved and there is no information on the iron export mechanism. For the analysis of this complex membrane protein two different approaches were chosen: first, using bioinformatics techniques, a structural model of human ferroportin has been built and, on this basis, a mechanism of iron transport was hypothesized. The significance of the model was experimentally tested through iron export measurements in cells transfected with recombinant wild type and mutant ferroportin. In addition, a system for heterologous expression of ferroportin has been developed. The purpose was to produce enough protein, in a quantity higher than those so far obtained (Rice et al., 2009), which would provide the basis for the biochemical and structural characterization of ferroportin. Obviously, any attempt to disclose the three dimensional structure of ferroportin or the molecular mechanism of iron export, may be useful not only to explain the different pathological phenotypes associated to alterations of the transporter, but also to develop or improve the actual treatment of the ferroportin disease.
La ferroportina è una proteina politopica di membrana di 62,5 kDa che media l’esporto del ferro ferroso da cellule specializzate dell’organismo alla circolazione sanguigna. Ad oggi, è l’unica proteina deputata a svolgere questa funzione ad essere stata identificata nei mammiferi. A livello sistemico, la ferroportina è soggetta ad un meccanismo di regolazione negativa post-traduzionale operato dall’epcidina, peptide secreto dal fegato in risposta ad innalzamento dei livelli di ferro nell’organismo, che legandosi ad essa innesca un meccanismo di internalizzazione e degradazione proteica lisosomiale (Nemeth et al., 2004). La ferroportina rappresenta quindi un importante regolatore dei quantitativi di ferro intracellulari e sistemici dell’organismo. Mutazioni a carico del gene codificante per ferroportina (SLC40A1) sono causative di una sindrome da sovraccarico di ferro, denominata emocromatosi (HH) di tipo IV o “malattia da ferroportina”, caratterizzata da trasmissione autosomica dominante. L’HH di tipo IV si esplicita in due possibili fenotipi sulla base della alterazione funzionale che la proteina subisce. Se la mutazione produce una perdita di funzione si assiste ad accumulo di ferro a livello macrofagico, incremento dei livelli di ferritina sierica e si riscontrano normali valori di saturazione della transferrina. In contrasto a questa manifestazione fenotipica classica, ci sono alcune mutazioni a guadagno di funzione che non influenzano l’esporto del metallo attraverso il canale proteico, ma che determinano la produzione di ferroportina con parziale, o in alcuni casi completa, resistenza all’epcidina. I pazienti con mutazioni di questa seconda tipologia presentano un fenotipo patologico con tratti simili a quelli dell’emocromatosi di tipo classico. Nello specifico, si assiste a incremento dei livelli di saturazione della transferrina sierica e accumulo di ferro per lo più a livello parenchimale (Pietrangelo, 2006). Il lavoro di dottorato è stato incentrato sullo studio della ferroportina. Ad oggi infatti, la struttura cristallografica di questo trasportatore non è stata ancora risolta e mancano informazioni sul meccanismo messo in atto per l’esporto del ferro. Per l’analisi di questa complessa proteina di membrana sono stati scelti due differenti approcci: da un lato, è stato sviluppato, mediante tecniche di bioinformatica, un modello strutturale sulla base del quale è stato ipotizzato un meccanismo di trasporto del ferro, verificato attraverso la produzione e lo studio della funzionalità di mutanti ad hoc di ferroportina. Dall’altro lato, è stato messo a punto un sistema eterologo di espressione per ferroportina, indirizzato alla produzione di quantitativi proteici, maggiori di quelli osservati finora (Rice et al., 2009), che fornissero le basi per procedere alla sua caratterizzazione biochimica e strutturale. È evidente come qualsiasi studio volto all’identificazione della struttura proteica o del meccanismo di funzionamento di ferroportina, possa risultare utile anche alla comprensione dei fenotipi patologici associati alle alterazioni del trasportatore e allo sviluppo o al miglioramento delle attuali tipologie di trattamento della malattia da ferroportina.

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. In mammals since no controlled means of eliminating unwanted iron has evolved, body iron balance is maintained by alterations in dietary iron intake. This occurs in the duodenum where most dietary iron is absorbed. Absorption involves at least two steps, uptake of iron from the intestinal lumen and then its transport into the body, processes that occur at the apical and basal membranes of enterocytes, respectively. In chapter one of this thesis the background information relevant to iron absorption is described. Despite numerous studies, the role of these proteins in iron absorption remains unclear, partly because many studies have reported them in non-enterocyte cell lines where the expression of the proteins involved in iron absorption is unlikely and therefore the physiological significance of the findings uncertain. Therefore, the study of iron absorption would value from additional cell lines of intestinal origin being used, preferably derived from a species used to comprehensively study this process in vivo, namely the rat. Validation of such a model would enable comparisons to be made from a molecular level to its relevance in the whole organism. In chapter 3 of this thesis, the rat intestinal cell line 6 (IEC-6) was examined as a model of intestinal iron transport. IEC-6 cells expressed many of the proteins involved in iron absorption, but not the ferrireductase Dcytb, sucrase or αvβ3 integrin. In addition, in IEC-6 cells the expression of the apical transporter divalent metal transporter 1 (DMT1), the iron storage protein ferritin, the uptake of Fe(II) and Fe(III) were regulated by cellular iron stores as is seen in vivo. This suggests that IEC-6 cells are of a lower villus enterocyte phenotype. Presented in chapter 4 is the study of the uptake of iron from Fe(II):ascorbate and Fe(III):citrate by IEC-6 cells in the presence of a blocking antibody to the putative basolateral transporter ferroportin1 and of colchicine and vinblastine, different pHs, and over-expression of DMT1. It was shown that optimal Fe(II) uptake required a low extracellular pH and was dependent on DMT1. Uptake of Fe(III) functioned optimally at a neutral pH, did not require surface ferrireduction, and was increased during over-expression of DMT1. These observations suggest that intravesicular ferrireduction takes place before transport of Fe(II) to the cytoplasm by DMT1. This pathway was not blocked by a functional antibody against αvβ3 integrin but was inhibited by competition with unlabeled iron citrate or citrate alone. Surprisingly, a functional antibody against ferroportin1 had no effect on efflux but significantly reduced (p<0.05) uptake of Fe(II) by 40-50% and Fe(III) by 90%, indicating two separate pathways for the uptake of iron from Fe(II)-ascorbate and from Fe(III)-citrate in IEC-6 cells. Presented in chapter 5 is the development and validation of a technique for the removal of freshly isolated enterocytes from the rat duodenum and their use to study iron transport processes that enabled comparisons to be made between these cells, IEC-6 cells and the human enterocyte cell line Caco-2 cells. In chapter 6 a blocking antibody to ferroportin1 was shown to inhibit uptake of Fe(II) but not release of iron in freshly isolated duodenal enterocytes from rats and Caco-2 cells supporting the findings obtained with IEC-6 cells described in chapter 4. Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane indicating its expression at the microvillus membrane. Confirming this, ferroportin1 was shown along the microvillus membrane of Caco-2 cells, in enriched microvillus membrane preparations and in enterocytes of duodenum tissue of rats where it co-localised with lactase. The significant findings to emerge from this thesis are that the IEC-6 cell is a valid model to study iron absorption producing results consistent with those found in freshly isolated enterocytes and in human enterocyte-like cells. In particular, ferroportin1 functions in the uptake of iron at the apical membrane possibly by modulating surface binding of Fe(II) to DMT1 or the activity of DMT1. In addition to this in Fe(II) uptake from Fe(III) ferroportin1 may also affect the number of Fe(III): citrate binding sites. Preliminary studies further characterizing the function of ferroportin1 at the apical membrane and at intracellular sites of IEC-6 cells along with integration of these data are discussed in chapter 7.

Mestrado em Biomedicina Molecular
Iron is found in almost all living organisms, playing a central role in host-pathogen interactions and being crucial for both host and pathogens. In the host, iron is a crucial element, since it plays a key role in biological processes such as oxygen transport, biosynthesis of DNA, energy production and regulation of gene expression. However, high concentrations of iron can also be toxic to cells due to the ability to generate hydroxyl radicals. Thus, vertebrates developed proteins to transport and store iron: transferrin and ferritin, respectivetly. Hepcidin is a key protein of iron metabolism, since it binds to ferroportin, the iron exporter, regulating the release of iron to the serum. On the other hand, iron is also fundamental for pathogens that required it to its growth and proliferation, to the expression of virulence factors and to metabolic processes. Thereby, during infection, the host and the pathogen compete by this metal. Pathogens developed multiple strategies to acquire iron from the host during infection. Thus, making iron unavailable for microorganisms is a central mechanism in host defense. In this work, we investigated the regulation of iron metabolism in host during infection with Listeria monocytogenes, a gram-positive bacterium and Salmonella Typhimurium, a gram-negative bacterium in order to verify whether there are alterations in host iron metabolism depending of infection type and if hepcidin have a central role in these alterations. C57BL6 male mice were infected with 104 CFU of L. monocytogenes, S. Typhimurium, or an equivalent volume of vehicle and sacrificed at different time points. Bacterial load quantification, non-heme iron determination in liver, evaluation of iron distribution in tissue, histopathologic analyses and the expression of genes related with iron metabolism were analyzed. Our results show that in both infections with L. monocytogenes and S. Typhimurium the host immune system are not able to irradiate the infection and, thus, the bacterial load increases during the experiment. Regarding the hematological and serological parameters, a reduction of red blood cells and hematocrit is observed, as well as, of serum iron levels. The levels of interleukin-6 and hepcidin increase at different time points in each infection. Additionally, non-heme iron concentration increases in liver during infection with both pathogens. Histopathological alterations were also detected during infection with L monocytogenes and S. Typhimurium. Our data suggests that both infections induce alterations in host iron metabolism. However, the infection with S. Typhimurium appears to have earlier and more severe effects in the host than infection with L. monocytogenes.
O ferro é encontrado em quase todos os seres vivos, desempenhando um papel central nas interacções entre o hospedeiro e o patógeno e sendo essencial para ambos. Para o hospedeiro, o ferro é um elemento crucial, uma vez que desempenha um papel chave em processos biológicos como o transporte de oxigénio, a biossíntese de DNA, produção de energia e regulação da expressão génica. No entanto, elevadas concentrações de ferro também podem ser tóxicas para as células devido à capacidade de gerarem radicais hidroxilo. Assim, os vertebrados possuem proteínas para transportar e armazenar o ferro, a transferrina e a ferritina respetivamente. A hepcidina é uma proteína chave do metabolismo do ferro, uma vez que se liga à ferroportina, o exportador do ferro, regulando a libertação de ferro para o soro. Por outro lado, o ferro é também fundamental para os patógenos, que o requerem para o seu crescimento e proliferação, para a expressão de factores de virulência e para vários processos metabólicos. Assim, durante a infecção, o hospedeiro e o patógeno competem por este metal. Os patógenos desenvolveram múltiplas estratégias para adquirir o ferro a partir do hospedeiro durante a infeção. Deste modo, tornar o ferro indisponível para os microrganismos é um mecanismo central na defesa do hospedeiro. Neste trabalho, investigámos a regulação do metabolismo do ferro no hospedeiro durante a infecção com Listeria monocytogenes, uma bactéria gram-positiva e com Salmonella Typhimurium, uma bactéria gram-negativa, de modo a verificar se existem alterações no metabolismo do ferro do hospedeiro dependendo do tipo de infeção e se a hepcidina tem um papel preponderante nestas alterações. Murganhos machos C57BL6 foram infectados com 104 CFU de L. monocytogenes, S. Typhimurium, ou um volume equivalente de veículo e sacrificados a diferentes tempos experimentais. A quantificação da carga bacteriana, determinação do ferro não hémico no fígado, avaliação da distribuição de ferro no tecido, análise histopatológica e a expressão de genes relacionados com o metabolismo do ferro foram analisados. Os nossos resultados mostram que tanto na infeção com L. monocytogenes como na infeção com S. Typhimurium, o sistema imunitário do hospedeiro não é capaz de irradiar a infeção e, assim, a carga bacteriana aumenta durante a experiência. Em relação aos parâmetros hematológicos e serológicos, é observada a redução da quantidade de eritrócitos e do hematócrito, bem como dos níveis de ferro no soro. Os níveis de interleucina-6 e de hepcidina aumentam em diferentes tempos experimentais em cada infeção. Adicionalmente, a concentração de ferro não hémico aumenta no fígado durante a infeção com ambos os patógenos. Foram também detetadas alterações histopatológicas aquando da infeção com L monocytogenes e S. Typhimurium. Os nossos dados sugerem que ambas as infeções induzem alterações no metabolismo do ferro do hospedeiro. Contudo, a infeção com S. Typhimurium parece ter efeitos mais precoces e mais severos no hospedeiro do que a infeção com L. monocytogenes.

La ferroportine transporte du fer hors des cellules sources (entérocytes et macrophages) vers le plasma. L’hepcidine est une protéine soluble, secrétée par le foie et régulateur négatif de la ferroportine. Une expression altérée de l’hepcidine est responsable de pathologies du métabolisme du fer. Nous avons mis en évidence, lors de la bêta-thalassémie, une corrélation entre une diminution de l’expression de l’hepcidine et l’augmentation de l’activité érythropoïétique, de manière indépendante du niveau d’ARNm des gènes régulateurs du métabolisme du fer. La diminution de la biodisponibilité plasmatique du fer, lorsque l’expression de l’hepcidine était augmentée par la surcharge en fer chez des souris, soulève la question de l’intérêt potentiel de la mesure de l’hepcidine chez des patients avec une surcharge en fer secondaire pour lesquels un traitement par phlébotomies est envisagé. Nos résultats chez la souris soulèvent des questions sur le rôle de la ferroportine dans la régulation du contenu en fer du foie. La régulation de la biodisponibilité du fer plasmatique par la ferroportine hépatique, par rapport à celle des autres organes sources de fer, reste à définir. En parallèle, l’impact de l’hepcidine au niveau du foie nécessite aussi d’être précisé
Erroportin export iron from providing cells (enterocytes and macrophages) to plamsa. Hepcidin is a soluble protein secreted by the liver and negative regulator of ferroportin. An altered expression of hepcidin is responsible for iron metabolism pathologies. We highlighted a correlation between a decrease of this expression and an increase of erythropoietic activity, independently of mRNA levels of iron metabolism genes. Decrease of plasmatic iron bioavailability when hepcidin expression was increased by iron overload in mice raises the question of the potential interest of hepcidin measurement in patients with secondary iron overload and for who a venesection treatment is considered. Our results in mouse raise questions on ferroportin role in liver iron content regulation. Plasmatic iron bioavailability regulation by hepatic ferroportin compared to ferroportin from other iron providing organs needs to be defined. In parallel impact of hepcidin at the liver level needs to be specified

INTRODUCTION: Iron homeostasis is maintained in humans trough a meticulous control of intestinal iron absorption, effective utilization of iron by erythropoiesis, efficient recycling of iron from senescente erythrocytes and controlled storage of iron by hepatocytes and macrophages. Little is known about the regulation of iron metabolism in pathological conditions, in particular transfusion independent beta-thalassemia intermedia (TI) transfusion independent and thalassemia major (TM). The clinical manifestations of TI result from three key factors: ineffective erythropoiesis, chronic anemia and iron overload. In patients with thalassemia major (TM), in whom iron loading occurs mainly as a result of transfusion therapy, while in patients with TI accumulate iron primarily due to increased intestinal iron absorption and ineffective erythropoiesis. This leads to an increase in the plasma of iron from tissue and taken up by transferrin, which is saturated with the accumulation of excess free iron. This causes toxicity and tissue damage. The main regulator of iron homeostasis regulation is hepcidin, an hepatic peptide that negatively regulates iron egress from intestinal cells and macrophages by altering the expression of the cellular iron exporter ferroportin. Ferroportin (FPN) is the only mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated. In doing so, hepcidin can control both the total body iron by modulating intestinal iron absorption as well as promote iron available for erythropoiesis by affecting the efficiency of macrophages in recycling iron from effete red blood cells. Despite the key role attributed to hepcidin in the regulation of the iron, the mechanisms that regulate its expression are unknown, in particular it is not known how the increased erythropoietic activity present in TI reduces the expression of hepcidin. A candidate gene involved in this regulation is the GDF15 (growth differentiation factor 15), which is secreted by erythroblasts during erythropoiesis. The only information known about this gene is derived from studies of TM subjects and in vitro studies on cell lines, where it has been observed that in patients with TM, it was observed that the GDF15 is present at high levels in serum and its role may be to inhibit the expression of hepcidin in the liver. It is still unknown why GDF15 is more expressed in beta thalassemia patients than in healthy subjects and how GDF15 can negatively regulates the expression of hepcidin is still unknown. AIM: To determine the genes expression profile of GDF15, hepcidin and ferroportin isoforms during normal and thalassemic erythroid differentiation in standard cultures and in situations that simulate the iron depletion (deferoxamine) or saturated iron (ferric ammonium citrate), from CD34+ and macrophages of normal and thalassemia intermedia and major subjects. METHODS: After informed consent, the CD34+ cells and macrophages cells were obtained from peripheral blood of healthy volunteers and from patients with TI and TM by positive and negative respectively selection using anti-CD34-tagged magnetic beads. The CD34+ cells were cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The macrophages cells were cultured for 6 days with a medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) to induce macrophages differentiation. Each culture of CD34+ cells and macrophages was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4M) as iron chelating agent and ferric ammonium citrate (FAC 100 M) at day 0 of culture. The expression profiling of GDF15, hepcidin and ferroportin genes were evaluated at baseline, day 7 and day 14 by real-time PCR (2^-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit. RESULTS IN CD34+ CELLS: GDF15 expression and secretion increased significantly during erythroid differentiation either in normal, in TI and TM cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were significantly higher compared to control and to TM which had lower values. On the contrary, hepcidin is significantly expressed only in the TM. At day 14 in control cultures GDF15 expression was up-regulated by DFO and down-regulated by FAC addition. In TI GDF15 expression was down-regulated both by DFO and by FAC. In TM GDF15 expression was down-regulated by DFO and up-regulated by FAC addition. There was the same trend for the secretion of the GDF15 protein. In control cultures, FPN total expression increased significantly during erythroid differentiation, while in TI and TM cultures FPN total was highly expressed at erythroid progenitors stage (day 0 of culture) and decreased at early erythroblasts stage (day 7) and late erythroblasts stage (day 14). In control cultures, FPN1A/FPNTOT was highly expressed at day 0 of culture, decreased significantly at day 7 and increased significantly at day 14. In TM cultures it was expressed at day 0 and decreased both at day 7 and at day 14. In TI cultures, the FPN1A/FPNTOT was highly expressed only day 7. In control cultures the FPN1B/FPNTOT was significantly expressed only at early erythroblasts stage, whereas in TI and TM cultures it was highly expressed at baseline although decreased during differentiation. At day 14 in thalassemia intermedia cultures FPN1B/FPNTOT expression were higher compared to control and to TM. At day 14 in control cultures FPN1A/FPNTOT and FPN1B/FPNTOT expression were not modificated by addition DFO and FAC. In TI and TM cultures, the addition of FAC was not modificated the expression of FPN1A/FPNTOT. In TI it expression was down-regulated by DFO addition, in TM it was up-regulated by DFO. In TM cultures FPN1B/FPNTOT was up-regulated by DFO while it was down-regulated by FAC. In TI FPN1B/FPNOT expression was up-regulated both by DFO and by FAC. RESULTS IN MACROPHAGES CELLS: In untreated control cultures both FPN1A/FPNTOT and FPN1B/FPNTOT were highly expressed, while they were down-regulated both by DFO and by FAC. In TI and TM cultures FPN1A/FPNTOT and FN1B/FPNTOT were not expressed both in untreated macrophages and in treated macrophages. In control and TI cultures GDF15 expression was up-regulated by DFO and down-regulated by FAC addition. In TM cultures GDF15 expression was not modificated by addition DFO and FAC. DISCUSSION: In TI and TM cultures total FPN was highly expressed at erythroid progenitors stage and could contribute to iron overload typical of thalassemia. Conversely, in control cultures the ferroportin was expressed at late erythroblasts stage maybe because the now mature cells does not need iron and made it available to other parts of the body. This correlated with the low levels of hepcidin and with the positive expression of GDF15. In TM cultures the absence of GDF15, the presence of hepcidin and the lack feroportina was due to low concentration of intracellular iron. Instead in TI cultures was much GDF15, a marker of ineffective erythropoiesis, little hepcidin and the good levels of FPN. In TM the ineffective erythropoiesis was suppressed by transfusions. The GDF15 increased during the normal differentiation and thus may play a role in these stages and its expression was modulated by iron levels. In TI and TM the GDF15 was essential for growth but in TI there was no modulation by the iron concentrations. The FPN1A seemed to be important at the beginning and end of erythroid differentiation, while in mid favored retention of iron in erythroblasts for to make hemoglobin. Similar to the situation of the TM but also at the end of erythroid differentiation did not express FPN1A because in reality it had not completed erythropoiesis. In TI the 1A isoform was high at day 7 of culture and low at 14° may be due ineffective erythropoiesis and delayed differentiation. In the control 1B isoform was high at day 7 to escape the repression due to the system IRE / IRP involves the isoform 1A so that, if the body was in conditions of iron deficiency, erythroid cells can export it to ensure the flow to other organs. In TI and TM cells the FPN1B isoform was highly expressed in the early stages of erythroid differentiation, possibly contributing to iron overload in both forms of thalassemia. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expression. Control cells treated with an iron chelator or iron did not show changes in the expression of isoform 1A and 1B. The TM cells under conditions of iron depletion increased the expression of FPN1A as the chelator, by subtracting the extracellular iron, created an imbalance of the ion and the cell expressed FPN1A to export it outside. TI cells however, in contact with DFO, decreased the expression of FPN1A because the chelator removed directly to intracellular iron and therefore the cell did not need a transporter. The isoform FPN1B was mostly expressed in TI and TM cells treated with DFO compared to untreated cells, as the decrease caused by the iron chelator, increased the ineffective erythropoiesis and as the 1B represented the ineffective erythropoiesis, it does not could only increase. The untreated control macrophages expressed both isoforms of ferroportin because the recycled iron from macrophages it happened in was two possible ways: it may be stored with the ferritin molecules and used later or exported out of the plasma and therefore needed precisely ferroportin. Conditions of iron depletion or iron saturation, however, strongly down regulated the expression of both isoforms, assuming a more importance of regulation by heme. In TI and TM cells ferroportin was not expressed because the macrophages, affected by extracellular iron overload, repressed the expression of FPN not to be exported more iron potentially toxic. The expression of GDF15 in control macrophages was the same pattern of CD34: conditions of iron depletion increased the expression of GDF15 and conditions of iron saturation decreased its presence. In fact, increased levels of GDF15 caused an increase of iron efflux from macrophages to make it available to others tissues. Decreased levels of GDF15 however, increase the activity of macrophages and helped to retain iron in macrophages for limiting a accumulation of toxic iron. Macrophages TI was the same trend as control macrophages, therefore the regulation of GDF15 was again influenced by the levels of iron. These data suggest that in TI cultures existed two different systems of regulation of GDF15 depending on the type of cell involved: in fact in CD34 cells was an regulation insensitive to variations in iron while in macrophages was an iron-dependent regulation, as in controls cells.

Le fer est un élément vital mais en excès il peut devenir toxique pour l’organisme. Une régulation fine de son adsorption (au niveau du duodénum) et de son recyclage (au niveau des macrophages tissulaires) est donc indispensable. Cette régulation implique l’hepcidine, un peptide produit majoritairement par les hépatocytes, et qui induit l’endocytose et la dégradation du seul exportateur de fer connu chez les mammifères, la ferroportine. Nous avons étudié, dans les macrophages, la localisation subcellulaire de la ferroportine, sa régulation et nous avons recherché des partenaires protéiques fonctionnels ou régulateurs potentiels de cette protéine. Nous avons montré que ce transporteur est localisé dans des radeaux lipidiques (rafts) à la surface des macrophages. De plus, la désorganisation des rafts induit une diminution de la dégradation de la ferroportine par l’hepcidine. Le transport de fer au niveau cellulaire semble impliquer une ferroxidase, la céruloplasmine. Nos macrophages en culture expriment une céruloplasmine cytosolique et une céruloplasmine membranaire liée à la membrane par une ancre GPI (glycosylphosphatidylinositol). Après un traitement au fer, la ferroportine et les deux formes de céruloplasmine sont surexprimées avec une céruloplasmine membranaire localisée dans des rafts de même densité que ceux de la ferroportine. Ensemble, nos observations soulignent l’importance fonctionnelle des rafts dans le recyclage du fer macrophagique. Une approche protéomique des fractions rafts contenant la ferroportine nous suggèrent déjà certains acteurs moléculaires impliqués dans l’activité et la régulation de la ferroportine macrophagique
Iron is an essential element for the organism but can become toxic when present in excess. A proper regulation of iron absorption (in the duodenum) and iron recycling (in tissues macrophages) is therefore necessary. Hepcidin, a small peptide produced by hepatocytes, is involved in this systemic regulation. In fact, hepcidin induces the endocytoses and the degradation of ferroportin, the only known iron exporter in mammals. In this thesis, we studied the subcellular localization and the regulation of macrophage ferroportin and we looked forward potentials functional or regulatory partners of this exporter. We showed that ferroportin is localized in lipid rafts at the cell surface of macrophages. Furthermore, disruption of lipid rafts causes a decrease in the hepcidine induced ferroportin degradation. Cellular iron export seems to involve a ferroxidase named ceruloplasmin. We observed that cultured mouse macrophages produce a cytosolic and a GPI (glycosylphosphatidylinositol) anchored membrane ceruloplasmin. The expression of both forms of ceruloplasmin and ferroportin is strongly stimulated after iron treatment. Moreover, after iron treatment, the GPI-ceruloplasmin and ferroportin are co-expressed into lipid rafts. All together, our observations emphasize the role of lipid rafts in macrophage iron recycling. Finally, a proteomic approach of the ferroportin containing lipid rafts fractions pointed out some proteins which could be involved in the regulation and/or the activity of the macrophage ferroportin

Mammalian iron homeostasis is maintained by an intricate network of diverse proteins that constantly survey systemic iron levels and carefully regulate the uptake of iron from the diet. Control of this uptake is critically important because once iron is absorbed, mammals have no regulated mechanism for its removal. The portal through which iron enters the body is ferroportin, a multipass membrane protein expressed on the basolateral membrane of epithelial cells in the duodenum. The iron export function of ferroportin is primarily regulated by the serum peptide hormone hepcidin, which is secreted from the liver when systemic iron levels are high. Hepcidin acts as a negative regulator of iron uptake by binding to ferroportin at the cell surface and inducing its internalization and degradation. Genetic defects in ferroportin, hepcidin, or the proteins involved with sensing systemic iron levels lead to iron overload diseases known as hereditary hemochromatosis. Using the tools of biophysics and cell biology, we sought to study ferroportin and its interaction with hepcidin in order to better understand this critical bottleneck in iron uptake and how genetic defects within ferroportin might lead to disease. We developed the first protocols for the overexpression, detergent-solubilization, and purification of recombinant ferroportin. We determined that detergent-solubilized ferroportin is a monomer capable of binding hepcidin in vitro. We characterized the expression and subcellular localization of ferroportin in mammalian tissue culture and determined that both the amino- and carboxy-termini of ferroportin are cytosolic. We developed cell-based assays for the hepcidin-induced internalization of ferroportin and used these to characterize the route of internalization from the plasma membrane through early endosomes to degradative lysosomal compartments. Using live-cell imaging techniques, we showed that this internalization depended on intact microtubules. We expanded this cell-biological study to include sixteen disease-related ferroportin mutants and reported that each mutant was expressed on the plasma membrane like wild-type ferroportin, but that only a subset of the mutants were capable of being internalized by hepcidin. These studies form a foundation for future biophysical and cell-biological studies of ferroportin function.

Iron is an essential element for human organism, because it cooperates as a cofactor of enzymes in many metabolic pathways. Iron is a component of hemoglobin, and thus it is indispensable for the oxygen transport to tissues. It can exist as a ferrous or ferric form. However, ferrous iron paticipates in reactions in which highly reactive hydroxyl group can be formed. This product is harmful for the organism. Non-heme iron is taken up to the circulation through duodenal enterocyte. Iron excretion is carried out only by desquamation of the enterocytes or by bleeding. Therefore, iron intake must be strictly regulated. Iron overloading is observed in some chronic diseases (hereditary hemochromatosis, alcohol liver disease). In contrary, iron depletion can be a case of iron deficiency anemia. The aim of this master thesis is to determine the expression of iron transport molecules in duodenum in chronic diseases which originate due to disturbances of iron intake regulation. We determine the expression of molecules of iron transport (DMT1, Dcytb, ferroportin, hephaestin) on mRNA level by qPCR and on protein level by western blot. The level of serum hepcidin was determined by ELISA. Our results show an increased expression of mRNA of transporters DMT1 and ferroportin as well as ferrireductase Dcytb and ferroxidase...