As síndromes talassêmicas (?- e ?-talassemia) são as desordens mais comuns e frequentes associadas com eritropoese ineficaz. O desbalanço na produção das cadeias ?- e ?-globinas resulta no comprometimento da produção de eritrócitos, em anemia e aumento de progenitores eritroides no sangue periférico. Enquanto os pacientes homozigóticos afetados por essas desordens demonstram alterações características dos parâmetros relacionados a eritropoese, a relação entre grau de anemia, eritropoese alterada e disfunção do metabolismo de ferro ainda não foram investigados nos indivíduos com ?+-talassemia heterozigótica ou ?+-talassêmia. Duzentos e vinte seis indivíduos (75 do gênero feminino e 151 do gênero masculino) foram recrutados e divididos em 5 grupos: Controle (n=28), doadores de sangue regulares (DSR, n=23), ?+-talassemia heterozigótica (TAT, n=14), ?+-thalassemia (traço ?-talassêmico, TBT, n=20) e ?0-talassemia, (?-talassemia maior, BTM, n=27). As amostras foram analisadas para parâmetros hematológicos (Micros ABX 60); ferro sérico, capacidade total de ligação ao ferro e saturação de transferrina por método colorimétrico (Pointe Scientific, Inc., Canton, MI, USA), ferritina e proteína C-reativa ultra sensível por imunoensaio (Immulite 1000); receptor solúvel de transferrina, eritropoetina, fator de diferenciação do crescimento 15 (R&D Systems) e hepcidina (Intrinsic LifeSciences, La Jolla, CA) por ELISA. As razões sTfR/log ferritina e (hepcidina/ferritina)/sTfR foram calculadas para avaliar o metabolismo do ferro. sTfR/log ferritina pode distinguir depleção dos estoques de ferro de eritropoese deficiente de ferro, enquanto (hepcidina/ferritina)/sTfR pode avaliar os estímulos contrários (disponibilidade de ferro e atividade eritropoética) que controlam a síntese de hepcidina e a absorção de ferro, na ausência de estímulos inflamatórios. Foi demonstrado que TAT teve significativa redução da hepcidina e aumento do receptor solúvel de transferrina, com parâmetros hematológicos relativamente normais. Em contraste, todos os parâmetros hematológicos de TBT foram significativamente diferentes do Controle, incluindo aumento dos níveis do receptor solúvel de transferrina, ferritina, eritropoetina e fator de diferenciação do crescimento 15. Essas alterações em ambos os grupos sugerem um balanço alterado entre eritropoese e metabolismo de ferro. Os índices sTfR/log ferritina e (hepcidina/ferritina)/sTfR estão, respectivamente, aumentado e reduzido comparados ao Controle, proporcional a severidade de cada grupo talassêmico. Em conclusão, destacamos que, pela primeira vez, foram descritas alterações no metabolismo de ferro em indivíduos com ?+-talassemia heterozigótica. Esses dados demonstram que, no contexto da saúde pública, são necessários identificação e acompanhamento dos portadores de ?+-talassemia. / The thalassemia syndromes (?- and ?-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of ?- or ?-globin chain production results in impaired red blood cell synthesis, anemia and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis and dysfunctional iron metabolism have not been investigated in both carriers of ?-thalassemia and ?-thalassemia. 226 subjects (75 females and 151 males) were recruited to this study and divided in 5 groups: Control (n=28), repeat blood donors (DSR, n=23), ?+-thalassemia heterozygous carriers (TAT, n=14), ?+-thalassemia (?-thalassemia trait, TBT, n=20) and ?0-thalassemia, (?-thalassemia major, BTM, n=27). Samples were tested for hematological parameters (Micros ABX 60); serum iron, total iron binding capacity, and transferrin saturation by the colorimetric method (Pointe Scientific, Inc., Canton, MI, USA), ferritin and high sensitive C-reactive protein by immunoassay (Immulite 1000); soluble transferrin receptor, erythropoietin and growth differentiation factor 15 (R&D Systems) and hepcidin (Intrinsic LifeSciences, La Jolla, CA) by ELISA. Were calculated the ratios sTfR/log ferritin and (hepcidin/ferritin)/sTfR to evaluate iron metabolism. sTfR/log ferritin can distinguish storage iron depletion from iron-deficient erythropoiesis, while (hepcidin/ferritin)/sTfR can be utilized to explore and quantify the opposing forces (i.e. iron availability and erythropoietic activity) regulating hepcidin synthesis and iron absorption in absence of inflammatory stimuli. We demonstrate that TAT have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, TBT have all hematological parameters significantly different from controls, including increased soluble transferrin receptor, ferritin, erythropoietin and growth differentiation factor 15 levels. These changings in both groups suggest an altered balance between erythropoiesis and iron metabolism. The indexes sTfR/log ferritin and (hepcidin/ferritin)/sTfR are respectively increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we emphasize that, for the first time in the literature, subjects with heterozygous ?+-thalassemia have altered iron metabolism. Our data demonstrate that within the context of public health, identification and monitoring of patients with ?+-thalassemia are needed.

Chan Suk-mei. / "October 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. [171]-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

As síndromes talassêmicas (?- e ?-talassemia) são as desordens mais comuns e frequentes associadas com eritropoese ineficaz. O desbalanço na produção das cadeias ?- e ?-globinas resulta no comprometimento da produção de eritrócitos, em anemia e aumento de progenitores eritroides no sangue periférico. Enquanto os pacientes homozigóticos afetados por essas desordens demonstram alterações características dos parâmetros relacionados a eritropoese, a relação entre grau de anemia, eritropoese alterada e disfunção do metabolismo de ferro ainda não foram investigados nos indivíduos com ?+-talassemia heterozigótica ou ?+-talassêmia. Duzentos e vinte seis indivíduos (75 do gênero feminino e 151 do gênero masculino) foram recrutados e divididos em 5 grupos: Controle (n=28), doadores de sangue regulares (DSR, n=23), ?+-talassemia heterozigótica (TAT, n=14), ?+-thalassemia (traço ?-talassêmico, TBT, n=20) e ?0-talassemia, (?-talassemia maior, BTM, n=27). As amostras foram analisadas para parâmetros hematológicos (Micros ABX 60); ferro sérico, capacidade total de ligação ao ferro e saturação de transferrina por método colorimétrico (Pointe Scientific, Inc., Canton, MI, USA), ferritina e proteína C-reativa ultra sensível por imunoensaio (Immulite 1000); receptor solúvel de transferrina, eritropoetina, fator de diferenciação do crescimento 15 (R&D Systems) e hepcidina (Intrinsic LifeSciences, La Jolla, CA) por ELISA. As razões sTfR/log ferritina e (hepcidina/ferritina)/sTfR foram calculadas para avaliar o metabolismo do ferro. sTfR/log ferritina pode distinguir depleção dos estoques de ferro de eritropoese deficiente de ferro, enquanto (hepcidina/ferritina)/sTfR pode avaliar os estímulos contrários (disponibilidade de ferro e atividade eritropoética) que controlam a síntese de hepcidina e a absorção de ferro, na ausência de estímulos inflamatórios. Foi demonstrado que TAT teve significativa redução da hepcidina e aumento do receptor solúvel de transferrina, com parâmetros hematológicos relativamente normais. Em contraste, todos os parâmetros hematológicos de TBT foram significativamente diferentes do Controle, incluindo aumento dos níveis do receptor solúvel de transferrina, ferritina, eritropoetina e fator de diferenciação do crescimento 15. Essas alterações em ambos os grupos sugerem um balanço alterado entre eritropoese e metabolismo de ferro. Os índices sTfR/log ferritina e (hepcidina/ferritina)/sTfR estão, respectivamente, aumentado e reduzido comparados ao Controle, proporcional a severidade de cada grupo talassêmico. Em conclusão, destacamos que, pela primeira vez, foram descritas alterações no metabolismo de ferro em indivíduos com ?+-talassemia heterozigótica. Esses dados demonstram que, no contexto da saúde pública, são necessários identificação e acompanhamento dos portadores de ?+-talassemia. / The thalassemia syndromes (?- and ?-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of ?- or ?-globin chain production results in impaired red blood cell synthesis, anemia and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis and dysfunctional iron metabolism have not been investigated in both carriers of ?-thalassemia and ?-thalassemia. 226 subjects (75 females and 151 males) were recruited to this study and divided in 5 groups: Control (n=28), repeat blood donors (DSR, n=23), ?+-thalassemia heterozygous carriers (TAT, n=14), ?+-thalassemia (?-thalassemia trait, TBT, n=20) and ?0-thalassemia, (?-thalassemia major, BTM, n=27). Samples were tested for hematological parameters (Micros ABX 60); serum iron, total iron binding capacity, and transferrin saturation by the colorimetric method (Pointe Scientific, Inc., Canton, MI, USA), ferritin and high sensitive C-reactive protein by immunoassay (Immulite 1000); soluble transferrin receptor, erythropoietin and growth differentiation factor 15 (R&D Systems) and hepcidin (Intrinsic LifeSciences, La Jolla, CA) by ELISA. Were calculated the ratios sTfR/log ferritin and (hepcidin/ferritin)/sTfR to evaluate iron metabolism. sTfR/log ferritin can distinguish storage iron depletion from iron-deficient erythropoiesis, while (hepcidin/ferritin)/sTfR can be utilized to explore and quantify the opposing forces (i.e. iron availability and erythropoietic activity) regulating hepcidin synthesis and iron absorption in absence of inflammatory stimuli. We demonstrate that TAT have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, TBT have all hematological parameters significantly different from controls, including increased soluble transferrin receptor, ferritin, erythropoietin and growth differentiation factor 15 levels. These changings in both groups suggest an altered balance between erythropoiesis and iron metabolism. The indexes sTfR/log ferritin and (hepcidin/ferritin)/sTfR are respectively increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we emphasize that, for the first time in the literature, subjects with heterozygous ?+-thalassemia have altered iron metabolism. Our data demonstrate that within the context of public health, identification and monitoring of patients with ?+-thalassemia are needed.

(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....

Iron storage disease is a significant cause of liver disease and mortality in captive Egyptian fruit bats (Rousettus aegyptiacus). The nature of the susceptibility in this and other captive exotic species to iron storage disease is not clear. Hepcidin, a key iron regulatory hormone, is involved in the regulation of iron absorption in humans and other mammalian species and a deficiency in hepcidin has been associated with a number of genetic mutations resulting in hemochromatosis in humans. The objectives of this thesis were to identify whether there is a functional mutation in the hepcidin gene in the Egyptian fruit bat that may increase the susceptibility of this species to iron storage disease, and whether there is a functional deficiency in hepcidin gene expression in the Egyptian fruit bat in response to iron challenge. We compared the coding region of the hepcidin gene amongst several species of bats and investigated hepcidin response to intramuscular injection of iron dextran amongst three species of bats with variable susceptibility to iron storage disease; the Egyptian fruit bat, the straw-colored fruit bat (Eidolon helvum), and the common vampire bat (Desmodus rotundus). While a number of genetic differences were identified amongst species, a functional mutation that could result in decreased hepcidin activity was not identified in the Egyptian fruit bat. Bats exhibited marked variation in hepcidin gene expression, with the highest level of hepcidin response to iron challenge in the common vampire bat. While the Egyptian fruit bat exhibited significant hepcidin response to iron challenge, the magnitude of response was lower than that in the common vampire bat and lower than expected based on findings in healthy humans. The straw-colored fruit bat did not exhibit any hepcidin response despite a significant increase in iron stores, which suggests this species may have evolved an alternate mechanism for coping with excessive iron or may be more susceptible to iron overload than previously recognized. / Toronto Zoo Scholarship Fund

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), has remained the largest killer among infectious diseases for over a century. The increasing emergence of drug resistant varieties such as the multidrug resistant (MDR) and extremely drug resistant (XDR) strains are only increasing the global burden of the disease. Available statistics indicate that nearly one-third of the world’s population is infected, where the bacteria remains in the latent state but can reactivate into an actively growing stage to cause disease when the individual is immunocompromised. It is thus immensely important to rethink newer strategies for containing and combating the spread of this disease. Extraction of iron from the host cell is one of the many factors that enable the bacterium to survive in the harsh environments of the host macrophages and promote tuberculosis. Host–pathogen interactions can be interpreted as the battle of two systems, each aiming to overcome the other. From the host’s perspective, iron is essential for diverse processes such as oxygen transport, repression, detoxification and DNA synthesis. Infact, during infection, both the host and the pathogen are known to fight for the available iron, thereby influencing the outcome of the infection. It is of no surprise therefore, that many studies have investigated several components of the iron regulatory machinery of M.tb and the host. However, very few attempts have been made to study the interactions between these components and how such interactions lead to a better adapted phenotype. Such studies require exploration at multiple levels of structural and functional complexity, thereby necessitating the use of a multiscale approach. Systems biology adopts an integrated approach to study and understand the function of biological systems. It involves building large scale models based on individual biochemical interactions, followed by model validation and predictions of the system’s response to perturbations, such as a gene knock-out or exposure to drug. In multiscale modeling, an approach employed in this thesis, a particular biological phenomenon is studied at different spatiotemporal levels. Studying responses at multiple scales provides a broader picture of the communications that occur between a host and pathogen. Moreover, such an analysis also provides valuable insights into how perturbation at a particular level can elicit responses at another level and help in the identification of crucial inter-level communications that can possibly be hindered or activated for a desired physiological outcome. The broad objectives of this thesis was to obtain a comprehensive in silico understanding of mycobacterial iron homeostasis and metabolism, the influence of iron on host-pathogen interactions, identification of key players that mediate such interactions, determination of the molecular consequences of inhibiting the key players and finally the global response of M.tb to altered iron concentration. Perturbation of iron homeostasis holds a strong therapeutic potential, given its essentiality in both the host and the pathogen. Understanding the workings of iron metabolism and regulation in M.tb has been a main objective, so as to ultimately obtain insights about specific therapeutic strategies that capitalize on the criticality of iron concentration. An in-depth study of iron metabolism and regulation is performed at different levels of temporal and spatial scales using diverse methods, each appropriate to investigate biological events associated with the different scales. The specific investigations carried out in the thesis are as follows, a) Reconstruction of a host-pathogen interaction (HPI) model, with focus on iron homeostasis. This study represented the inter-cellular level analysis and was crucial for the identification of key players that mediate communication between the host and pathogen. Additionally, the model also provided a mathematical framework to study the effect of perturbations and gene knock-outs. b) Understanding the influence of iron on IdeR, an iron-responsive transcription factor, also identified as a key player in the HPI model. The study was carried out at the molecular level to identify atomistic details of how IdeR senses iron and the resulting structural modifications, which finally enables IdeR-DNA interaction. The study enabled identification of residues for the functioning of IdeR. c) Genome scale identification of genes that are regulated by IdeR to obtain an overview of the various biological processes affected by changing iron concentrations and IdeR mutation in M.tb. d) To understand the direct and indirect influences of iron and IdeR on the M.tb proteome using large scale protein-protein interaction network. The study enabled identification of highest differentially regulated genes and altered activity of the different biological processes under differing iron concentrations and regulation. e) Systems level analysis of the M.tb metabolome to investigate the metabolic re-adjustments undertaken by M.tb to adapt to altered iron concentration and regulation. The conceptual details and the background of each of the methods used to study the specific aims are provided in the Methodology chapter (Chapter 2). Construction of the host-pathogen interaction (HPI) model and the insights obtained from this study are presented in Chapter 3. A rule based HPI model was built with a focus on the iron regulatory mechanisms in both the host and pathogen. The model consisted of 194 rules, of which 4 rules represented interactions between the host and pathogen. The model not only represented an overview of iron metabolism but also allowed prediction of critical interaction that had the potential to form bottleneck in the system so as to control bacterial proliferation. Infact, model simulation led to the identification of 5 bottlenecks or chokepoints in the system, which if perturbed, could successfully interfere with the host-pathogen dynamics in favour of the host. The model also provided a framework to test perturbation strategies based on the bottlenecks. The study also established the importance of an iron responsive transcription factor, IdeR for regulating iron concentration in the pathogen and mediating host-pathogen interactions. Additionally, the importance of mycobactin and transferrin as key molecular players, involved in host-pathogen dynamics was also determined. The model provided a mathematical framework to test TB pathogenesis and provided significant insights about key molecular players and perturbation strategies that can be used to enhance therapeutic strategies. Given the importance of IdeR in HPI, its molecular mechanism of activation and dimerization was explored in Chapter 4. The main objective of the study was to explore the structural details of IdeR and its iron sensing capacity at the molecular level. A combination of molecular dynamics and protein structure network (PSN) were used to analyse IdeR monomers and dimers in the presence and absence of iron. PSNs used in this thesis are based on non-covalent interactions between sidechain atoms and are quite efficient in identifying iron induced subtle conformational variations. The study distinctly indicated the role of iron in IdeR stability. Further, it was observed that IdeR monomers can take up two major conformations, the ‘open’ and ‘close’ conformation with the iron bound structure preferring the ‘close’ conformation. Major structural changes, such as the N-terminal folding and increased propensity for dimerization were observed upon iron binding. Interestingly, careful analysis of structure suggests a role of these structural modifications towards DNA binding and has been tested in the next chapter. Overall, the results clearly highlight the influence of iron on IdeR activation and dimerization. The predisposition of IdeR to bind to DNA in the presence of metal is clearly visible even when the simulations are performed solely on protein molecules. However, to confirm the conjectures proposed in this chapter and to obtain the atomistic details of IdeR-DNA interactions, the IdeR-DNA complex was investigated. Chapter 5 focuses on the mechanistic details of IdeR-DNA interactions and the influence of iron on the same. IdeR is known to bind to a specific stretch of DNA, known as the ‘iron-box’ motif to form a dimer-of-dimer complex. Molecular dynamics followed by protein-DNA bipartite network analysis was performed on a set of four IdeR-DNA complexes to obtain a molecular level understanding of IdeR-DNA interactions. A striking observation was the dissociation of IdeR-DNA complex in the absence of iron, undoubtedly establishing the importance of iron for IdeR-DNA binding. At the residue level, hydrogen bond and non-covalent interactions clearly established the importance of N-terminal residues for DNA binding, thereby confirming the conjecture put forth in the previous chapter. An important aspect studied in this chapter is the allosteric nature of IdeR-DNA binding. Recent years have witnessed a paradigm shift in the understanding of allostery. Unlike the classical definition of allostery that was based on static structures, the newer definition is based on the conformational ensemble as represented by the shift in the energy landscape of the protein. The allosteric nature of IdeR-DNA complex was probed using simulated trajectories and indeed they suggest iron to be an allosteric regulator of the protein. Finally, based on the known experimental data and observations presented in Chapters 4 and 5, a multi-step model of IdeR activation and DNA binding has been proposed. In chapter 6, a global perspective of IdeR regulation in M.tb was obtained. This was important to gain insights about the influences of iron and its regulation at the M.tb cellular level. A genome scale identification of all possible IdeR targets based on the presence of ‘iron-box’ motif in the promoter region of the genes was carried out. An interesting aspect of this study was the use of energetic information from previous molecular dynamics study as an input for generation of the motif. A total of 255 such IdeR targets were identified and converted into an IdeR target network (IdeRnet). Along with IdeRnet, an unbiased systems level protein-protein interaction network was also generated. To study the response of the pathogen to external perturbations, iron-specific gene expression data was integrated into the network as node weights and edge weights. Analysis of IdeRnet provides interesting associations between fatty acid metabolism and IdeR regulations. Specific genes such as fadD32, DesA3 or lppW have been found to be affected by IdeR mutation. While IdeRnet discusses the direct associations, the global level responses are monitored by analysing pathways for the flow of information in the protein-protein interaction network (PPInet). Comparisons of the PPInets under conditions such as altering iron concentrations and lack of iron homeostasis led to the identification of the ‘top-most’ active paths under the different conditions. The study clearly suggests a halt in the protein synthesis machinery and decreased energy consumption under iron scarcity and an uninhibited consumption of energy when iron homeostasis is perturbed. In the final chapter (Chapter 7), flux balance analyses has been used to investigate the influence of iron on M.tb metabolism. The importance of iron for metabolic enzymes has already been established in the previous chapter. Additionally, M.tb is known to produce siderophores, an important metabolite that requires amino acids as its precursors, for iron extraction. All this, together highlighted the importance of iron and its regulation of M.tb metabolism. Flux balance analysis has been used previously to study the metabolic alterations that occur in an organism under different conditions. For this study, iron specific gene expression data was also incorporated into the model as reaction bounds and the flux values so obtained were compared in different environmental conditions. The study provided valuable insights into the metabolic adjustments taken up by M.tb under iron stress conditions and correlates well with the responses observed from the interactome as well as experimental observations. Most significantly, changes were observed in the energy preferences of the cell. For instance, it was noted that while the wild type strain of M.tb prefers synthesis of ATP via glycolysis, the IdeR mutant strain preferred oxidative phosphorylation. The picture becomes clearer when one accounts for the uncontrolled utilization of energy and rapid activation of protein synthesis machinery in the IdeR mutant strain. Biological systems are inherently multiscale in nature and therefore for a successful drug target regime, analysis of the genome to the phenome, which captures interactions at multiple levels, is essential. In this thesis, a detailed understanding of iron homeostasis and regulation in M.tb at multiple levels has been attempted. More importantly, insights obtained from one level, formed questions in the next level. The study was initiated at the inter-cellular level, where the influence of iron on HPI was modeled and analysed. From this study, IdeR, an iron-responsive transcription factor was identified as a key player that had the potential to alter host-pathogen interactions in the favour of the host. For a complete understanding of how IdeR regulates iron homeostasis, it was imperative to obtain a molecular level insight of its mechanism of action. Finally, the various aspects of IdeR regulation were investigated at the cellular level by analysing direct and indirect influences of IdeR on M.tb proteome and metabolome. The study suggests certain therapeutic interventions, such as 1) reduction in the concentration of free transferrin various, 2) mutations at the N-terminal sites of IdeR, 3) regulation of proteins involved in production of mycolic acids by iron and 4) perturbation of altering energy sources, which capitalize on iron and should be investigated in detail. In summary, the consequences of iron on TB infection were studied by threading different levels. This is based on the belief that most biological functions involve multiple spatio-temporal levels with frequent cross talks between the different levels, thereby making such multiscale approaches very useful.

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.

Adaptation to chronic hypoxia activates endogenous signaling cascades, which lead to cardiac protection against acute ischemia/reperfusion (I/R) injury. The molecular mechanism of this phenomenon has not been fully clarified yet. However, it was proved that reactive oxygen species (ROS) take part in cardioprotective signaling pathway inducted by chronic hypoxia. The high level of ROS must be precisely regulated by antioxidative system of a cell. The aim of diploma thesis was to examine the effect of intermittent hypobaric hypoxia (IHH, 7 000 m) on relative amount of antioxidative enzymes (peroxiredoxin 6 - PRX6, thioredoxin 1 and 2 - TRX1 and TRX2, thioredoxin reductase 1 - TRXR1) and also enzymes of iron metabolism (heme oxygenase 1 and 2 - HO1 and HO2, aconitase 1 and 2 - ACO1 and ACO2), which participate in regulation of cell redox state. Moreover, we studied the effect of adaptation to IHH and an antioxidant tempol on relative amount of calcium-independent phospholipase A2 (iPLA2). iPLA2 can remove peroxidized fatty acids from membrane phospholipids. On the other hand, iPLA2 can damage cell in I/R conditions. All enzymes were studied in homogenates from normoxic and IHH adapted rat left ventricular myocardium by Western blot. Adaptation to IHH caused a decrease of PRX6 and on the opposite an increase of...

Background SARS-CoV-2 infection causes acute respiratory distress, which may progress to multiorgan failure and death. Severe COVID-19 disease is accompanied by reduced erythrocyte turnover, low hemoglobin levels along with increased total bilirubin and ferritin serum concentrations. Moreover, expansion of erythroid progenitors in peripheral blood together with hypoxia, anemia, and coagulopathies highly correlates with severity and mortality. We demonstrate that SARS-CoV-2 directly infects erythroid precursor cells, impairs hemoglobin homeostasis and aggravates COVID-19 disease. Methods Erythroid precursor cells derived from peripheral CD34+ blood stem cells of healthy donors were infected in vitro with SARS-CoV-2 alpha variant and differentiated into red blood cells (RBCs). Hemoglobin and iron metabolism in hospitalized COVID-19 patients and controls were analyzed in plasma-depleted whole blood samples. Raman trapping spectroscopy rapidly identified diseased cells. Results RBC precursors express ACE2 receptor and CD147 at day 5 of differentiation, which makes them susceptible to SARS-CoV-2 infection. qPCR analysis of differentiated RBCs revealed increased HAMP mRNA expression levels, encoding for hepcidin, which inhibits iron uptake. COVID-19 patients showed impaired hemoglobin biosynthesis, enhanced formation of zinc-protoporphyrine IX, heme-CO2, and CO-hemoglobin as well as degradation of Fe-heme. Moreover, significant iron dysmetablolism with high serum ferritin and low serum iron and transferrin levels occurred, explaining disturbances of oxygen-binding capacity in severely ill COVID-19 patients. Conclusions Our data identify RBC precursors as a direct target of SARS-CoV-2 and suggest that SARS-CoV-2 induced dysregulation in hemoglobin- and iron-metabolism contributes to the severe systemic course of COVID-19. This opens the door for new diagnostic and therapeutic strategies.