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Made available in DSpace on 2014-06-11T19:28:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-02Bitstream added on 2014-06-13T18:35:10Z : No. of bitstreams: 1 000739913.pdf: 3500532 bytes, checksum: d1f5204e156e6108ea196d3ebbee589a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A perda do bulbo ocular compromete não só a estética, mas também a tonicidade muscular da região facial do paciente, uma vez que com a ausência do globo ocular os músculos orbiculares dos olhos podem sofrer atrofia. Desse modo, o objetivo do presente estudo foi verificar a atividade elétrica dos músculos orbiculares, antes e após a instalação de próteses oculares em pacientes que foram submetidos à enucleação unilateral do bulbo ocular. Foram selecionados, por meio de anamnese e exame clínico, 12 pacientes voluntários com indicação de prótese. O sinal eletromiográfico foi realizado com o auxílio do eletromiógrafo, em quatro situações clínicas: Repouso (R), Abertura e Fechamento Normal das Pálpebras (AFN), Abertura e Fechamento Rápido das Pálpebras (AFR) e Apertamento (A). Esses registros foram realizados antes da instalação da prótese ocular, e após 7, 30 e 60 dias da instalação e uso da mesma. Os mesmos ensaios foram realizados no músculo orbicular do olho sadio do paciente, resultados que serviram como controle do estudo. Os dados obtidos foram submetidos à análise estatística pelo programa SPSS (p<0.05) e o t-teste foi utilizado para comparar os músculos superior e inferior por período de tratamento (inicial, 7, 14, 30 e 60 dias), para as quatro condições clínicas. Nas quatro condições clínicas avaliadas foi verificado diferença estatisticamente significativa em relação ao período inicial e após 7 dias da instalação da prótese. O fascículo superior do músculo orbicular do olho apresentou maiores valores de atividade elétrica em relação ao fascículo inferior em todas as situações clínicas avaliadas. Os menores valores de atividade elétrica foram observados durante o período inicial para a condição de repouso (OS 8.418 / OI 5.933) e os maiores após 60 dias na condição... / The eye loss besides affecting patient’s aesthetics, it compromises the muscle tone of the facial region owing to the atrophy of orbicular muscles. Thus, although the use of ocular prosthesis does not return patient’s vision, it fills the anophtalmic cavity restoring the cosmetic and muscle tone. The aim of this present study was to evaluate the electrical activity of orbicular muscles before and after ocular prosthesis insertion of patients who underwent unilateral enucleation of eyeball. The electrical activity of the orbicular muscles was assessed through the Myosystem BR1 electromyograph in four clinical situations: (1) rest, (2) normal opening and closing of the eyelid, (3) fast opening and closing of the eyelids, and (4) clenching. The electrodes were placed in the fascicles of upper (UO) and lower (LO) orbicular muscles. Electromyographic examinations were performed before and after 7, 14, 30 and 60 days of prosthesis insertion. T-test (p<.05) was used to compare the upper and lower orbicular muscles for each period of evaluation in all clinical conditions. A total of 12 patients of both genders were treated and they aged from 42 to 80 years. Several factors were the cause of anophthalmia and the trauma during job accident was the main reason. A statistical significant difference in the electromyographic data was observed for all four clinical conditions when comparing the baseline with the 7-day prosthesis insertion periods. The UO exhibited higher values of electrical activity than LO for all clinical situations. The lowest electrical activity was noted for the baseline period during the rest condition (UO 8.418 /LO 5.933), while the greatest one after 60 days of prosthesis insertion during clenching (UO 131.504 / LO 117.123). After ocular prosthesis insertion, a significant increase in the electrical activity values of the orbicular muscles was observed.

Orientador: Daniela Micheline dos Santos / Co-orientador: Marcelo Coelho Goiato / Banca: Eduardo Piza Pellizzer / Banca: Aldiéris Alves Pesqueira / Resumo: A perda do bulbo ocular compromete não só a estética, mas também a tonicidade muscular da região facial do paciente, uma vez que com a ausência do globo ocular os músculos orbiculares dos olhos podem sofrer atrofia. Desse modo, o objetivo do presente estudo foi verificar a atividade elétrica dos músculos orbiculares, antes e após a instalação de próteses oculares em pacientes que foram submetidos à enucleação unilateral do bulbo ocular. Foram selecionados, por meio de anamnese e exame clínico, 12 pacientes voluntários com indicação de prótese. O sinal eletromiográfico foi realizado com o auxílio do eletromiógrafo, em quatro situações clínicas: Repouso (R), Abertura e Fechamento Normal das Pálpebras (AFN), Abertura e Fechamento Rápido das Pálpebras (AFR) e Apertamento (A). Esses registros foram realizados antes da instalação da prótese ocular, e após 7, 30 e 60 dias da instalação e uso da mesma. Os mesmos ensaios foram realizados no músculo orbicular do olho sadio do paciente, resultados que serviram como controle do estudo. Os dados obtidos foram submetidos à análise estatística pelo programa SPSS (p<0.05) e o t-teste foi utilizado para comparar os músculos superior e inferior por período de tratamento (inicial, 7, 14, 30 e 60 dias), para as quatro condições clínicas. Nas quatro condições clínicas avaliadas foi verificado diferença estatisticamente significativa em relação ao período inicial e após 7 dias da instalação da prótese. O fascículo superior do músculo orbicular do olho apresentou maiores valores de atividade elétrica em relação ao fascículo inferior em todas as situações clínicas avaliadas. Os menores valores de atividade elétrica foram observados durante o período inicial para a condição de repouso (OS 8.418 / OI 5.933) e os maiores após 60 dias na condição... / Abstract: The eye loss besides affecting patient's aesthetics, it compromises the muscle tone of the facial region owing to the atrophy of orbicular muscles. Thus, although the use of ocular prosthesis does not return patient's vision, it fills the anophtalmic cavity restoring the cosmetic and muscle tone. The aim of this present study was to evaluate the electrical activity of orbicular muscles before and after ocular prosthesis insertion of patients who underwent unilateral enucleation of eyeball. The electrical activity of the orbicular muscles was assessed through the Myosystem BR1 electromyograph in four clinical situations: (1) rest, (2) normal opening and closing of the eyelid, (3) fast opening and closing of the eyelids, and (4) clenching. The electrodes were placed in the fascicles of upper (UO) and lower (LO) orbicular muscles. Electromyographic examinations were performed before and after 7, 14, 30 and 60 days of prosthesis insertion. T-test (p<.05) was used to compare the upper and lower orbicular muscles for each period of evaluation in all clinical conditions. A total of 12 patients of both genders were treated and they aged from 42 to 80 years. Several factors were the cause of anophthalmia and the trauma during job accident was the main reason. A statistical significant difference in the electromyographic data was observed for all four clinical conditions when comparing the baseline with the 7-day prosthesis insertion periods. The UO exhibited higher values of electrical activity than LO for all clinical situations. The lowest electrical activity was noted for the baseline period during the rest condition (UO 8.418 /LO 5.933), while the greatest one after 60 days of prosthesis insertion during clenching (UO 131.504 / LO 117.123). After ocular prosthesis insertion, a significant increase in the electrical activity values of the orbicular muscles was observed. / Mestre

There is a growing recognition that removal of an eye may cause a significant impact on a person's body image and her or his role in society; and may evoke a variety of emotional responses. The loss of an eye does not only signal disfigurement, it also means a loss of a body part and a vital sense; that of sight. Without vision, individuals have difficulty communicating. The emotional responses to this loss many a times, go unrecognized as the doctors and nurses who are in close contact with the patient at this time are not necessarily prepared to provide emotional care. This study seeks to address this gap by identifying the emotional impact of loss of an eye and sight on people's lives and the implication it has for health care workers, especially nurses. The eliciting of the different feelings and experiences of these patients can provide information for the formulation and design of protocols for holistic health care management. A non-experimental exploratory and descriptive design was used to conduct In-depth conversational interviews with seven purposively selected participants who had enucleation or evisceration between 2000 and 2005. Information-rich data yielded findings that clearly stressed the need for greater sensitization to the problem. All the participants expressed shock at the final diagnosis of enucleation or evisceration even if this was on their request. Patients wanted to know about the operation and its outcome, the prosthesis, how will it look like and its fit. Findings indicate that answers to these questions were not provided. Patients were not adequately emotionally prepared pre-operatively and were therefore not appropriately cared for post-operatively. Families were not satisfactorily involved and as such were not in a position to provide emotional support that the patients needed The recommendation was that a study to explore the health care team's knowledge in the psychological and emotional management of patients in crisis should be conducted as a benchmark for further training. / Health Studies / M.A. (Health Studies)

There is a growing recognition that removal of an eye may cause a significant impact on a person's body image and her or his role in society; and may evoke a variety of emotional responses. The loss of an eye does not only signal disfigurement, it also means a loss of a body part and a vital sense; that of sight. Without vision, individuals have difficulty communicating. The emotional responses to this loss many a times, go unrecognized as the doctors and nurses who are in close contact with the patient at this time are not necessarily prepared to provide emotional care. This study seeks to address this gap by identifying the emotional impact of loss of an eye and sight on people's lives and the implication it has for health care workers, especially nurses. The eliciting of the different feelings and experiences of these patients can provide information for the formulation and design of protocols for holistic health care management. A non-experimental exploratory and descriptive design was used to conduct In-depth conversational interviews with seven purposively selected participants who had enucleation or evisceration between 2000 and 2005. Information-rich data yielded findings that clearly stressed the need for greater sensitization to the problem. All the participants expressed shock at the final diagnosis of enucleation or evisceration even if this was on their request. Patients wanted to know about the operation and its outcome, the prosthesis, how will it look like and its fit. Findings indicate that answers to these questions were not provided. Patients were not adequately emotionally prepared pre-operatively and were therefore not appropriately cared for post-operatively. Families were not satisfactorily involved and as such were not in a position to provide emotional support that the patients needed The recommendation was that a study to explore the health care team's knowledge in the psychological and emotional management of patients in crisis should be conducted as a benchmark for further training. / Health Studies / M.A. (Health Studies)

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T18:46:31Z : No. of bitstreams: 1 saraiva_nz_dr_jabo.pdf: 2226785 bytes, checksum: ed58cf70cd41ad428b58b7a952966bd3 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Uma das etapas mais críticas do procedimento de transferência nuclear (TN) é a remoção da cromatina do oócito para a produção de citoplastos. O objetivo deste trabalho foi estudar o efeito de diferentes ambientes citoplasmáticos obtidos a partir de três técnicas de enucleação (convencional, assistida quimicamente e induzida quimicamente) sobre o remodelamento nuclear e desenvolvimento embrionário, avaliando-se o perfil de expressão dos genes XIST, G6PD e HSPA1A em embriões bovinos. Para isso, quatro experimentos foram delineados. No primeiro experimento, verificou-se que o processo de enucleação pode ser iniciado a partir de 1,0 h de tratamento com demecolcina nas duas técnicas de enucleação química. A dinâmica nuclear e de microtúbulos de oócitos ativados tratados com demecolcina foi avaliada em um segundo experimento, e oócitos tratados apresentaram redução da densidade dos microtúbulos, porém, essas estruturas não desapareceram completamente na maioria dos oócitos. No experimento III, a demecolcina não apresentou efeitos significativos na atividade do fator promotor de maturação (MPF) e da proteína cinase ativada por mitógeno (MAPK) quando utilizada na concentração 0,05μg/mL. No último experimento, a demecolcina não prejudicou o desenvolvimento embrionário e também não alterou o perfil de expressão dos genes XIST, G6PD e HSPA1A em embriões reconstituídos com células embrionárias; porém, quando foram avaliados os níveis de transcritos desses genes em embriões reconstituídos com células somáticas, observou-se maior expressão relativa do XIST e do G6PD em embriões oriundos da técnica de enucleação assistida quimicamente em comparação aos embriões produzidos pela técnica convencional. Portanto, conclui-se que a enucleação química não altera a reprogramação nuclear nem... / Removal of the oocyte chromatin for production of cytoplasts is one of the most critical steps of the standard nuclear transfer (NT) procedure. The aim of this work was to study the effect of different cytoplasmic environments from three enucleation techniques (conventional, chemical-assisted, and chemical-induced enucleation) on nuclear reprogramming and embryonic development, evaluating the expression patterns of XIST, G6PD and HSPA1A genes in bovine embryos. Therefore, four experiments were designed. In the first experiment, it was verified that the enucleation procedure can be initiated 1.0 h after starting demecolcine treatment on both chemical enucleation techniques. The nuclear and microtubular dynamics of activated oocytes treated with demecolcine were evaluated in a second experiment, and treated oocytes showed decreased microtubule density, but these structures did not completely disappear in most oocytes. In experiment III, demecolcine at a concentration of 0.05μg/mL had no significant effect on maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) activity. In the last experiment, demecolcine had no detrimental effects on embryonic development. Also, the expression patterns of XIST, G6PD and HSPA1A were not altered in reconstituted embryos derived from embryonic donor cells; however, evaluation of transcript levels of these genes in embryos reconstituted using somatic donor cells revealed higher relative expression of XIST and G6PD in embryos derived from chemicalassisted enucleation in comparison to embryos those produced by the conventional technique. In conclusion, chemical enucleation has no effect on nuclear reprogramming and embryonic development after nuclear transfer using embryonic donor cells. Also, chemical-assisted enucleation increases XIST and G6PD expression in nuclear transfer embryos... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-20Bitstream added on 2014-06-13T18:58:52Z : No. of bitstreams: 1 saraiva_nz_me_jabo.pdf: 431247 bytes, checksum: a83cbb84b25e1b75bfb892f8bc6cc5d0 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo desse estudo foi avaliar a ação da demecolcina na composição de microtúbulos, maturação e desenvolvimento in vitro de oócitos bovinos, submetidos ao tratamento em metáfase I (MI) e metáfase II (MII). No experimento I, observamos que a concentração 0,05æg/mL foi a mais eficaz na indução de enucleação no grupo MI (15,2%) e na formação de protrusão no grupo MII (55,1%). No experimento II, verificamos manifestações da demecolcina em apenas 0,5 h de tratamento, pelo aumento significativo de categorias de oócitos sem microtúbulos. Houve nova polimerização dessas estruturas, quando oócitos expostos à droga em MII, foram cultivados em meio livre desse agente. No experimento III, evidenciamos influência negativa da demecolcina na maturação nuclear de oócitos tratados em MI, durante 12 horas (4,9% de oócitos em MII) e um diferente comportamento quanto à distribuição de grânulos corticais (GC); enquanto no grupo MI houve tendência à antecipação na migração de GC para a periferia, após 2 horas de exposição à droga, no grupo MII, observou-se nesse momento, ação prejudicial da mesma. Ainda, verificamos incompleta expansão das células do cumulus em oócitos expostos à demecolcina por 12 horas. No experimento IV, obtivemos alta eficácia da técnica de enucleação (90,6%) e grande variação quanto ao desenvolvimento até o estádio de blastocisto (12,5 a 47%), não verificando-se ação prejudicial da droga no desenvolvimento embrionário. / The aim of this study was to evaluate demecolcine action on microtubules composition, maturation and in vitro development of bovine oocytes, submitted to the treatment in metaphase I (MI) and metaphase II (MII). In the experiment I, we observed that 0.05æg/mL was the most efficient concentration to induce the enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%). In experiment II, we verified demecolcine manifestations already after 0.5 hour of treatment, supported by the significant increase of categories of oocytes without microtubules. There was new polymerization of these structures when oocytes exposed to the drug in MII were cultured in agent-free medium. In experiment III, we evidenced negative influence of demecolcine on nuclear maturation of oocytes treated on MI for 12 hours (4.9% of oocytes in MII) and a different behavior for cortical granules (CG) distribution; while in MI group there was a tendency for the anticipation of CG migration to periphery, after 2 hours of drug exposition, in the MIII group, was observed a harmful effect of the drug. Moreover, we verified incomplete expansion of cumulus cells in oocytes exposed to demecolcine for 12 hours. In experiment IV, we got high effectiveness of enucleation technique (90.6%) and wide variation for development to the blastocyst stage (12.5 to 47%), and no harmful effects of the drug on embryonic development were observed.

Resumo: Uma das etapas mais críticas do procedimento de transferência nuclear (TN) é a remoção da cromatina do oócito para a produção de citoplastos. O objetivo deste trabalho foi estudar o efeito de diferentes ambientes citoplasmáticos obtidos a partir de três técnicas de enucleação (convencional, assistida quimicamente e induzida quimicamente) sobre o remodelamento nuclear e desenvolvimento embrionário, avaliando-se o perfil de expressão dos genes XIST, G6PD e HSPA1A em embriões bovinos. Para isso, quatro experimentos foram delineados. No primeiro experimento, verificou-se que o processo de enucleação pode ser iniciado a partir de 1,0 h de tratamento com demecolcina nas duas técnicas de enucleação química. A dinâmica nuclear e de microtúbulos de oócitos ativados tratados com demecolcina foi avaliada em um segundo experimento, e oócitos tratados apresentaram redução da densidade dos microtúbulos, porém, essas estruturas não desapareceram completamente na maioria dos oócitos. No experimento III, a demecolcina não apresentou efeitos significativos na atividade do fator promotor de maturação (MPF) e da proteína cinase ativada por mitógeno (MAPK) quando utilizada na concentração 0,05μg/mL. No último experimento, a demecolcina não prejudicou o desenvolvimento embrionário e também não alterou o perfil de expressão dos genes XIST, G6PD e HSPA1A em embriões reconstituídos com células embrionárias; porém, quando foram avaliados os níveis de transcritos desses genes em embriões reconstituídos com células somáticas, observou-se maior expressão relativa do XIST e do G6PD em embriões oriundos da técnica de enucleação assistida quimicamente em comparação aos embriões produzidos pela técnica convencional. Portanto, conclui-se que a enucleação química não altera a reprogramação nuclear nem... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Removal of the oocyte chromatin for production of cytoplasts is one of the most critical steps of the standard nuclear transfer (NT) procedure. The aim of this work was to study the effect of different cytoplasmic environments from three enucleation techniques (conventional, chemical-assisted, and chemical-induced enucleation) on nuclear reprogramming and embryonic development, evaluating the expression patterns of XIST, G6PD and HSPA1A genes in bovine embryos. Therefore, four experiments were designed. In the first experiment, it was verified that the enucleation procedure can be initiated 1.0 h after starting demecolcine treatment on both chemical enucleation techniques. The nuclear and microtubular dynamics of activated oocytes treated with demecolcine were evaluated in a second experiment, and treated oocytes showed decreased microtubule density, but these structures did not completely disappear in most oocytes. In experiment III, demecolcine at a concentration of 0.05μg/mL had no significant effect on maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) activity. In the last experiment, demecolcine had no detrimental effects on embryonic development. Also, the expression patterns of XIST, G6PD and HSPA1A were not altered in reconstituted embryos derived from embryonic donor cells; however, evaluation of transcript levels of these genes in embryos reconstituted using somatic donor cells revealed higher relative expression of XIST and G6PD in embryos derived from chemicalassisted enucleation in comparison to embryos those produced by the conventional technique. In conclusion, chemical enucleation has no effect on nuclear reprogramming and embryonic development after nuclear transfer using embryonic donor cells. Also, chemical-assisted enucleation increases XIST and G6PD expression in nuclear transfer embryos... (Complete abstract click electronic access below) / Orientador: Joaquim Mansano Garcia / Coorientadora: Simone Cristina Méo Niciura / Banca: Cláudia Lima Verde Leal / Banca: César Roberto Esper / Banca: Fernanda da Cruz Landim Alvarenga / Banca: Paulo Henrique Franceschini / Doutor

Orientador: Joaquim Mansano Garcia / Banca: Vera Fernanda Martins Hossepian de Lima / Banca: Claudia Lima Verde Leal / Resumo: O objetivo desse estudo foi avaliar a ação da demecolcina na composição de microtúbulos, maturação e desenvolvimento in vitro de oócitos bovinos, submetidos ao tratamento em metáfase I (MI) e metáfase II (MII). No experimento I, observamos que a concentração 0,05æg/mL foi a mais eficaz na indução de enucleação no grupo MI (15,2%) e na formação de protrusão no grupo MII (55,1%). No experimento II, verificamos manifestações da demecolcina em apenas 0,5 h de tratamento, pelo aumento significativo de categorias de oócitos sem microtúbulos. Houve nova polimerização dessas estruturas, quando oócitos expostos à droga em MII, foram cultivados em meio livre desse agente. No experimento III, evidenciamos influência negativa da demecolcina na maturação nuclear de oócitos tratados em MI, durante 12 horas (4,9% de oócitos em MII) e um diferente comportamento quanto à distribuição de grânulos corticais (GC); enquanto no grupo MI houve tendência à antecipação na migração de GC para a periferia, após 2 horas de exposição à droga, no grupo MII, observou-se nesse momento, ação prejudicial da mesma. Ainda, verificamos incompleta expansão das células do cumulus em oócitos expostos à demecolcina por 12 horas. No experimento IV, obtivemos alta eficácia da técnica de enucleação (90,6%) e grande variação quanto ao desenvolvimento até o estádio de blastocisto (12,5 a 47%), não verificando-se ação prejudicial da droga no desenvolvimento embrionário. / Abstract: The aim of this study was to evaluate demecolcine action on microtubules composition, maturation and in vitro development of bovine oocytes, submitted to the treatment in metaphase I (MI) and metaphase II (MII). In the experiment I, we observed that 0.05æg/mL was the most efficient concentration to induce the enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%). In experiment II, we verified demecolcine manifestations already after 0.5 hour of treatment, supported by the significant increase of categories of oocytes without microtubules. There was new polymerization of these structures when oocytes exposed to the drug in MII were cultured in agent-free medium. In experiment III, we evidenced negative influence of demecolcine on nuclear maturation of oocytes treated on MI for 12 hours (4.9% of oocytes in MII) and a different behavior for cortical granules (CG) distribution; while in MI group there was a tendency for the anticipation of CG migration to periphery, after 2 hours of drug exposition, in the MIII group, was observed a harmful effect of the drug. Moreover, we verified incomplete expansion of cumulus cells in oocytes exposed to demecolcine for 12 hours. In experiment IV, we got high effectiveness of enucleation technique (90.6%) and wide variation for development to the blastocyst stage (12.5 to 47%), and no harmful effects of the drug on embryonic development were observed. / Mestre

Le processus conduisant à la prolifération et différenciation des cellules souches hématopoïétiques (CSH) en cellules de toutes les lignées sanguines s’appelle l’hématopoïèse. Bien que l'engagement des CSH soit régi par les cytokines, les facteurs de transcription, les modificateurs épigénétiques et la niche des CSH, notre groupe a constaté que leur engagement vers la lignée érythroïde dépendait aussi du métabolisme de la glutamine. La glutaminolyse contribue à la biosynthèse des nucléotides de novo ainsi qu’à la production de l'alpha-kétoglutarate (αKG), intermédiaire métabolique du cycle TCA (Oburoglu et al. 2014). Il est cependant important de noter que la différenciation érythroïde est un processus unique, où chaque cellule fille est structurellement et fonctionnellement différente de sa cellule mère. Chaque division définit un stade de différenciation précis avec un dernier cycle de division produisant un réticulocyte énucléé. Ainsi, nous avons émis l'hypothèse que les réseaux métaboliques mobilisés dans les progéniteurs érythroïdes changent en fonction du stade de différenciation et que ces réseaux régulent la transition des progéniteurs d'un stade à l'autre.Au cours de ma thèse, j’ai caractérisé les états métaboliques associés aux différents stades de différenciation des progéniteurs érythroïdes. Nous avons ainsi montré qu'aux stades précoces de différenciation érythroïde, avant la différenciation terminale, les progéniteurs hématopoïétiques présentent une activité métabolique accrue avec un niveau de phosphorylation oxydative (OXPHOS) plus élevé. Ces données sont en corrélation avec l'augmentation de la génération de l’αKG à ces stades de différenciation. De plus, nous avons constaté une augmentation de l’OXPHOS de ces progéniteurs en présence d’αKG exogène. Cependant, la différenciation terminale des précurseurs érythroïdes, caractérisée par la perte de la masse mitochondriale et de leur potentiel membranaire, est associée à une diminution du niveau d'OXPHOS. Ainsi, l'administration exogène d’αKG, a fortement atténué la différenciation érythroïde terminale et l'énucléation, sans affecter la différenciation des pro-érythroblastes. Inversement, un antagoniste de l’αKG (diméthyloxalylglycine, DMOG) n'a pas altéré la différenciation terminale ou l'énucléation, malgré l'abrogation de l'OXPHOS dans les érythroblastes.Ces données suggèrent que la production d’αKG et sa contribution à l’OXPHOS perturbent l'énucléation des globules rouges. C'est pourquoi, dans le but de réduire les niveaux intracellulaires d’αKG, nous avons inhibé l’expression de l'isocitrate déshydrogénase I (IDH1), enzyme cytosolique catalysant la conversion de l'isocitrate en αKG. Cependant, comme IDH1 peut catalyser les réactions dans les deux sens, la diminution de son expression pourrait également augmenter les niveaux d’αKG. En effet, nous avons constaté que le knockdown d'IDH1 entraînait une forte atténuation de la différenciation terminale et de l'énucléation des précurseurs érythroïdes. Cet effet est probablement dû à un déséquilibre de la disponibilité des substrats ; ainsi l’administration ectopique de l’αKG ainsi que du citrate renforce l’altération de la différenciation terminale des précurseurs érythroïdes IDH1-/- ainsi que leur énucléation. Cette étude identifie donc un rôle crucial pour le métabolite αKG dans la régulation de la fonction mitochondriale et de l’OXPHOS, processus qui sont une condition sine qua non pour la différenciation des précurseurs érythroïdes au stade proérythroblaste. Nous montrons en outre que la suppression d’OXPHOS et la catalyse d’intermédiaires du TCA, substrats d’IDH1, sont requis pour les phases terminales de la différenciation érythroïde et l'énucléation.En conclusion, les résultats obtenus au cours de ma thèse mettent en évidence la nature dynamique des réseaux métaboliques qui régulent la progression des précurseurs érythroïdes tout au long des différents stades de la différenciation érythroïde. / Hematopoiesis is the process whereby hematopoietic stem cells (HSCs) proliferate and differentiate to all blood cell lineages. While HSC commitment is known to be regulated by cytokines, transcription factors, epigenetic modifiers and the HSC niche, our group found that specification of HSCs to the red cell lineage is dependent on glutamine metabolism. Glutaminolysis contributes to de novo nucleotide biosynthesis and to the generation of the alpha-ketoglutarate (αKG) TCA cycle metabolite (Oburoglu et al. 2014). Importantly though, erythroid differentiation is a unique process as each daughter cell is structurally and functionally different from its parent cell. Each division defines a stage of differentiation with the final division cycle resulting in the production of an enucleated reticulocyte which further matures to a biconcave erythrocyte. Thus, we hypothesized that progenitor metabolic networks change as a function of the erythroid differentiation stage and moreover, that they regulate the transition of progenitors from one stage of differentiation to the next.During my PhD, I assessed the metabolic alterations that occur as a function of the erythroid differentiation stage. We showed that at early stages of human red cell development, prior to terminal differentiation, hematopoietic progenitors exhibited an increased metabolic activity with a significantly higher level of oxidative phosphorylation (OXPHOS). This correlated with the increased generation of αKG and indeed, we found that ectopic αKG directly augmented OXPHOS in these progenitors. However, the terminal differentiation of erythroid precursors, characterized by the loss of mitochondrial mass and membrane potential, was associated with a decreased level of OXPHOS. Notably, ectopic αKG, which did not alter pro-erythroblast erythroid differentiation, severely attenuated terminal differentiation and enucleation. Conversely, an αKG antagonist (dimethyloxalyl glycine, DMOG) did not negatively impact on terminal differentiation or enucleation despite abrogating OXPHOS in erythroblasts.These data suggested that the production of αKG and its subsequent contribution to oxidative phosphorylation perturb red cell enucleation. We therefore downregulated isocitrate dehydrogenase I (IDH1), the cytosolic enzyme that catalyzes the conversion of isocitrate to αKG, by an shRNA approach in an attempt to decrease αKG levels. However, because IDH1 can catalyze both the forward and reverse reactions, its downregulation could also increase αKG levels. Indeed, we found that IDH1 knockdown resulted in a severe attenuation of terminal erythroid differentiation and enucleation. This effect was likely due to an imbalance in substrate availability––both ectopic αKG as well as citrate further decreased polychromatic to orthochromatic erythroblast differentiation and the subsequent enucleation of IDH1-knockdown erythroid precursors. Thus, the present study identifies a crucial role for the αKG metabolite in regulating mitochondrial function and oxidative phosphorylation, processes that are a sine qua non for erythroid precursors at the pro-erythroblast stage. We further show that terminal erythroid differentiation and enucleation requires OXPHOS suppression and the IDH1-mediated enzymatic catalysis of its TCA substrates.To conclude, the results generated during my PhD highlight the dynamic nature of the metabolic networks that regulate the progression of erythroid precursors through the distinct stages of erythroid differentiation.