A desnutrição protéico-energética (DPE) afeta mais de 1 bilhão de pessoas no mundo, principalmente crianças, idosos e pacientes hospitalizados. Ela provoca alterações metabólicas e hormonais, além de afetar o tecido hemopoético. O comprometimento da hemopoese provoca anemia e leucopenia, modificando a resposta imune inata e adquirida do organismo. Dessa forma, é comum a associação entre desnutrição e infecção, levando ao comprometimento do tratamento e aumento da morbidade e mortalidade de indivíduos hospitalizados. Após a recuperação nutricional, é relatada a reversão das alterações bioquímicas e hormonais, bem como das alterações na hemopoese e na resposta imune. Porém, muitos resultados são controversos, existindo dúvidas quanto à reversibilidade das alterações. Assim, nos propusemos a avaliar os efeitos da recuperação nutricional nos parâmetros bioquímicos, hormonais, hematológicos e imunológicos em modelo murino de desnutrição. Os animais desnutridos apresentaram perda de peso significativa, redução de proteínas totais, albumina, glicose, insulina e IGF-1, bem como aumento de glutamina plasmática, glutamina sintetase muscular e corticosterona. Houve redução dos parâmetros hepáticos e musculares, bem como alteração na sensibilidade à insulina, evidenciada pelos testes de OGTT e ITT. Todas as alterações descritas caracterizam o quadro de desnutrição. Após a recuperação nutricional, alguns parâmetros foram normalizados, mas as concentrações de glicose, insulina e IGF-1 permaneceram reduzidas. Da mesma forma, as alterações na concentração de DNA hepático e na sensibilidade à insulina permaneceram nos animais renutridos. A pancitopenia periférica e hipocelularidade da medula óssea e do baço observadas nos animais desnutridos foram revertidas após a renutrição. A avaliação de macrófagos peritoniais mostrou reversão parcial do comprometimento da capacidade e adesão e espraiamento, bem como da atividade fungicida nos animais renutridos. A produção de peróxido de hidrogênio continuou baixa após a recuperação nutricional, enquanto a produção de óxido nítrico voltou a aumentar. O comprometimento da produção de citocinas pró-inflamatórias decorrente da desnutrição não foi completamente revertido, visto que, em camundongos Swiss Webster, somente a produção de TNF-α retornou ao normal, enquanto em camundongos C56BL/6J a produção de nenhuma citocina foi restabelecida. A avaliação da via de sinalização do fator de transcrição NFkB mostrou alteração na expressão de MyD88, TRAF-6, IkKβ e IkBα em animais desnutridos. Após a recuperação nutricional, algumas dessas proteínas não retornaram ao normal. Os animais desnutridos também apresentaram comprometimento da ativação de NFkB, que não foi normalizada após a recuperação nutricional. Sendo assim, é possível afirmar que o retorno a uma dieta normoprotéica não é suficiente para reverter todas as alterações causadas pela desnutrição. / Protein-energy malnutrition (PEM) affects more than 1 billion people worldwide, mainly children, elderly and hospitalized patients. It causes metabolic and hormonal changes, besides affecting hematopoietic tissue. Impaired hematopoiesis causes anemia and leukopenia, modifying innate and acquired immune response of the organism. Thus, it is common the association between malnutrition and infection, leading to impaired treatment and increasing morbidity and mortality in hospitalized patients. After nutritional recovery, it is reported reversal of biochemical and hormonal changes, as well as, reversal of changes in hematopoiesis and immune response. However, many results are controversial, and there are doubts about the reversibility of the changes. Thus, we proposed to evaluate the effects of nutritional recovery biochemical, hormonal, haematological and immunological parameters in a murine model of malnutrition. The malnourished animals showed significant weight loss, reduction in total protein, albumin, glucose, insulin and IGF-1, as well as increased plasma glutamine, corticosterone and muscle glutamine synthetase. There was a reduction in muscle and liver parameters as well as change in insulin sensitivity, evidenced by the tests of OGTT and ITT. All modifications described characterize the malnutrition. After nutritional recovery, there was normalization of some parameters, but the concentrations of glucose, insulin and IGF-1 remained low. Likewise, changes in hepatic DNA concentration and insulin sensitivity remained in renourished animals. Peripheral pancytopenia and hypocellularity in bone marrow and spleen observed in malnourished animals were reversed after refeeding. The evaluation of peritoneal macrophages showed partial reversal of impairment of adhesion and spreading ability, as well as fungicidal activity in animals renourished. The hydrogen peroxide production remained low after nutritional recovery, while nitric oxide production increased again. Impaired production of proinflammatory cytokines due to malnutrition was not completely reversed, whereas in Swiss Webster mice, only the production of TNF-α returned to normal, whereas in C56BL/6J mice no cytokine production was restored. The assessment of the signalling pathway of transcription factor NFkB showed alterations in the expression of MyD88, TRAF-6 IkKβ and IkBα in malnourished animals. After nutritional recovery, some of these proteins didn\'t return to normal. Malnourished animals also showed impaired activation of NFkB, which wasn\'t normalized after nutritional recovery. Therefore, it is possible to say that the return to a normal diet is not enough to reverse all the changes caused by malnutrition.

Des études récentes ont mis en évidence qu’au moins 70% du génome humain est transcrit et produit une myriade d’ARN non codants. Au début de ma thèse j’ai utilisé des données de RNA-Seq sens-spécifique pour identifier les transcrits divergents dans les tissus primaires de souris. J’ai utilisé aussi des données ChIP-Seq afin d’analyser leurs caractéristiques épigénétiques. Nous avons trouvé que la transcription divergente est associée de manière significative à des gènes liés à la régulation de la transcription et le développement.Dans un deuxième temps, je me suis intéressé à l'identification et la caractérisation des lncRNA chez l'homme. J’ai appliqué des approches statistiques pour quantifier leur expression et identifier ceux qui sont (dé)régulés dans un contexte normal ou leucémique Dans un troisième temps. Au cours de ma thèse, je me suis attaché à étudier le mécanisme moléculaire épigénomique ainsi qu'à développer un pipeline bioinformatique permettant d'identifier les gènes (codant ou non codant) associés à des profils H3K4me2/3 étendus. Ainsi, j’ai mis en évidences que ces profils étendus étaient directement dépendants d'un processus transcriptionelle impliquant des nouveaux mécanismes de régulation. Cette étude a donné aussi lieu à une publication dont je suis cosignataire en premier auteur. (Zacarias, Belhocine et al. Journal of Immunology 2015). Cette nouvelle approche devrait s'avérer très utile dans d'autres modèles développementaux et/ou pathologiques et peuvent être utilisé comme outil de prioritisation des candidats les plus relevant dans des approches plus globale. / Recent studies indicate that at least 70% of the human genome is transcribed into a myriad of both coding and non-coding RNAs. at the beginning of my thesis I used RNA-Seq data to identify divergent transcripts in mouse primary tissues. I also used the ChIP-Seq data to analyze their epigenetic characteristics. The results demonstrated that divergent transcription was significantly associated with genes related to transcription regulation and development. In a second phase, I was interested in the LncRNAs identification and characterization during the development of human T lymphocytes and in T acute lymphoblastic leukemia (T-ALL). I applied statistical approaches to quantify their expression and identify those that are regulated in a normal or leukemic contextSubsequently, I determined the most appropriate approach to prioritize the functional role of LncRNAs. Indeed, I focused on studying the molecular epigenomic mechanism marking and developed a bioinformatics pipeline to identify genes (coding or non-coding) associated with the extended profiles of H3K4me2/3. Evidence generated through the pipeline demonstrated that these extended profiles were directly dependent on specific transcriptional process involving new regulatory mechanisms.In conclusion, this body of work has resulted in a more comprehensive approach to determining the true functional role of LncRNAs in both normal biological context and in human disease.

L'hématopoïèse est le processus développemental qui permet la formation des cellules qui composent le sang. Au niveau moléculaire, de nombreux facteurs de transcription permettent une régulation fine de ce processus et la dérégulation de leur activité, en affectant la différenciation ou la prolifération des cellules sanguines, peut conduire à l'apparition d'hémopathies telles que les leucémies. De manière intéressante, de nombreux gènes contrôlant l'hématopoïèse sont conservés entre la Drosophile et l'homme. Ces dernières années, cet insecte a donc émergé en tant que modèle pour l'étude du développement normal et pathologique des cellules hématopoïétiques. En tirant profit de cette conservation, mon travail de thèse a visé à caractériser, chez la Drosophile, le rôle et le mode d'action des protéines de la famille " Myeloid Leukemia Factor " (MLF). En effet, bien que le membre fondateur de cette famille soit impliqué dans le développement de Leucémies Aigües Myéloïdes chez l'homme, ces protéines restent très peu caractérisés. Les travaux réalisés dans l'équipe montrent que MLF contrôle l'homéostasie du système sanguin de la Drosophile, et qu'un aspect conservé de la fonction des protéines MLF est de réguler l'activité de facteurs de transcription de type RUNX dont Lozenge (LZ). Dans ce contexte, j'ai cherché à déterminer plus précisément la fonction de MLF dans l'hématopoïèse et à comprendre comment MLF régule les facteurs RUNX. In vivo, j'ai montré que MLF contrôle non seulement le nombre de cellules sanguines RUNX+/LZ+ mais aussi leur différenciation en "cellules à cristaux". L'établissement par RNAseq du transcriptome de ces cellules en contexte sauvage ou mutant pour mlf m'a permis d'identifier de nouveaux marqueurs de ce lignage et de montrer que mlf régule l'expression de nombre d'entre eux. De plus j'ai mis en évidence que ces deux aspects de la fonction de mlf passent par la régulation de LZ. Ainsi, bien que lz soit nécessaire au développement des cellules à cristaux, une diminution de son expression s'accompagne d'une augmentation du nombre de ces cellules qui présentent alors des caractéristiques " hyper-différenciées " et une sur-activation de la voie de signalisation Notch. Ces données mettent en exergue l'importance de la régulation du niveau du facteur RUNX LZ par MLF au cours de l'hématopoïèse. D'autre part, en utilisant une lignée de cellules sanguines de Drosophile en culture (cellules Kc167), j'ai pu montrer que MLF régule post-traductionnellement le niveau de LZ et que MLF et LZ interagissent physiquement. Pour ouvrir de nouvelles pistes quant aux mécanismes moléculaires d'action de MLF, j'ai également cherché ses partenaires par spectrométrie de masse. J'ai ainsi identifié la protéine chaperonne DNAJ1/HSP40 et j'ai pu mettre en évidence que ce partenaire de MLF est aussi impliqué dans la régulation de l'activité et du niveau d'expression de LZ en culture cellulaire. J'ai ensuite généré un mutant de ce gène chez la Drosophile par CRISPR et j'ai pu montrer qu'il contrôle lui aussi le développement des cellules sanguines LZ+, probablement en interaction avec MLF. Mes résultats suggèrent donc que MLF pourrait faire partie d'un complexe chaperon impliqué dans le contrôle de l'activité de LZ et dans l'hématopoïèse. / Haematopoiesis is the developmental process responsible for the formation of all blood cell types. At the molecular level, many transcription factors allow tight regulation of this process and deregulation of their activity, by affecting blood cell proliferation or differentiation, can lead to the appearance of various diseases including leukaemia. Interestingly, many genes implicated in haematopoiesis are conserved between Drosophila and human. Consequently, this insect has emerged as a potent model to study normal and pathological blood cell development. Taking advantage of this conservation, my thesis aimed at characterizing the role and mode of action of the "Myeloid Leukemia Factor" (MLF) family in Drosophila. Indeed, although the founding member of this family is involved in the development of Acute Myeloid Leukaemia in humans, this family remains poorly characterised. Previous work showed that MLF controls Drosophila blood cell homeostasis and that one conserved aspect of MLF function is to regulate the activity of RUNX transcription factor activity, including that of the Drosophila hematopoietic factor LOZENGE (LZ). Further to these results, I sought to determine more precisely MLF function in haematopoiesis and its molecular mechanism of action on RUNX factors. In vivo, I showed that mlf controls not only the number of LZ + blood cells but also their differentiation into "crystal cells. Notably, the establishment of wild type or mlf-/- LZ+ cells transcriptome by RNAseq allowed me to identify new markers for this lineage and revealed that mlf regulates the expression of a large number of them. Interestingly, I found that mlf controls both LZ+ cell number and their differentiation by regulating LZ level. Indeed, although lz is required for crystal cell development, a decrease in lz level is associated with increased LZ+ cell number and these cells exhibit "hyper-differentiated" phenotypes as well as Notch signalling pathway over-activation. These data underline the crucial role of RUNX level regulation by MLF for normal blood cell development. In parallel, using a Drosophila blood cell line (Kc167 cells), I showed that MLF physically interacts with LZ and post-translationally regulates its level. To open new leads concerning the molecular mode of action of MLF, I undertook a proteomic approach to identify its partners. Thereby, I found that the chaperon protein DNAJ1/HSP40 binds to MLF and I demonstrated that DNAJ1 is also implicated in the regulation of LZ level and activity in Kc cells. Using a CRISPR approach, I then generated a null dnaj1 allele in Drosophila and its phenotypic characterisation allowed me to show that DNAJ1 also controls the development of LZ+ blood cells, probably in interaction with MLF. All together, my results suggest that MLF could be part of a " chaperon " complex involved in controlling RUNX activity and haematopoiesis.

Bone marrow (BM) transfer from flagellin-treated mice has been reported to improve the survival of lethally-irradiated mice. Although the mechanism for flagellin’s antiviral and antibacterial effects have been elucidated, there remains a gap in knowledge regarding its radioprotective effects. Here, we report that flagellin treatment results in a 5-fold increase in the proliferation of Lin-Sca-1+C-Kit+(LSK) cells, a heterogeneous stem and multipotent cell population in BM, with the most striking increase within the ST-HSC, MPP2 and MPP3 subpopulations. Furthermore, the presence of TLR5 but not NLRC4 on radiosensitive, non-LSK cells in BM was both sufficient and necessary for the observed phenomenon. Finally, adoptive transfer of MPP3 cells along with an insufficient amount of whole bone marrow cells (WBM) to lethally-irradiated mice significantly improved their survival, recapitulating the effects of Whole bone marrow from flagellin-treated mice.

Blood cells are continuously produced throughout our lifetime by a rare pluripotent cell that primarily resides in the adult bone marrow. This hematopoietic stem cell (HSC) must maintain a careful balance between self-renewal, differentiation and apoptosis in order to support hematopoiesis for such a long duration. Understanding the mechanism of balance between these fates is crucial to our understanding and clinical application of these cells. From previous studies, we know Src homology 2 domain containing 5' inositol phosphatase 1 (SHIP) plays an important role in HSC homeostasis and function. Most interestingly SHIP impacts HSC homing to the bone marrow niche. An ideal location and environment is essential for HSC to fulfill their physiological roles. Here we present evidence that SHIP is expressed by cells of the HSC niche. Furthermore, SHIP deficiency severely alters this environment and thus damages HSC function. In addition to the extrinsic effects of a SHIP-deficient microenvironment on HSC, there is an intrinsic requirement for SHIP expression in confining HSC to the bone marrow niche. We previously demonstrated that lack of SHIP leads to an increase in peripheral HSC. Here we demonstrate that SHIP-deficient HSC from the spleen can provide radioprotection and sustained multi-lineage repopulation in lethally irradiated hosts. This indicates extramedullary HSC can function outside the traditional bone marrow niche in SHIP-deficient mice. Combined, these studies indicate both extrinsic and intrinsic factors contribute to HSC homeostasis and function. In order to better understand the signaling pathways involved in self-renewal and differentiation, we applied an array-based technology to hematopoietic cells at various levels of differentiation. Comparing the phosphorylation signature, or 'kinome', of these cell types can help pinpoint signaling mechanisms important for HSC self-renewal and lineage commitment.

No description available.

Imatinib mesylate (imatinib) represents a potent molecularly targeted therapy against the oncogenic tyrosine kinase, BCR-ABL. Although imatinib has shown considerable efficacy against chronic myeloid leukemia (CML), displaying high rates of complete hematological and complete cytogenetic responses, treatment with imatinib is not curative and overtime advanced-stage CML patients often become refractory to further treatment. Acquired resistance to imatinib has been associated with mutations within the kinase domain of BCR-ABL, BCR-ABL gene amplification, leukemic stem cell quiescence as well as over-expression of the multidrug resistance (MDR1) gene. However, in vitro resistance models often fail to consider the role of the tumor microenvironment in the emergence of the imatinib-resistant phenotype. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrixes, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib sensitivity, we utilized an in vitro co-culture bone marrow stroma model. Using a transwell system, we demonstrated that soluble factors secreted by the human bone marrow stroma cell line, HS-5, were sufficient to cause resistance to apoptosis induced by imatinib in CML cell lines. We subsequently determined that culturing CML cells in HS-5-derived conditioned media (CM) inhibits apoptosis induced by imatinib and other second generation BCR-ABL inhibitors. These data suggest that more potent BCR-ABL inhibitors will not overcome resistance associated with the bone marrow microenvironment. Additionally, we determined that CM increases the clonogenic survival of CML cells following treatment with imatinib. HS-5 cells are reported to express several cytokines and growth factors known to activate signal transducer and activator of transcription 3 (STAT3). Given its crucial role in the survival of hematopoietic cells, we asked whether, 1) CM derived from HS-5 cells can activate STAT3 in CML cells and 2) does activation of STAT3 confer resistance to BCR-ABL inhibitors. We demonstrated that exposure of the CML cell lines, K562 and KU812, to CM caused an increase in phospho-Tyr STAT3, while no increases in phospho-Tyr STAT5 were noted. Moreover, resistance was associated with increased levels of the STAT3 target genes, Bcl-xl, Mcl-1 and survivin. Furthermore, reducing STAT3 levels with siRNA sensitized K562 cells cultured in CM to imatinib-induced cell death (p<0.05, Student’s t-test). Importantly, STAT3 dependency was specific for cells grown in CM, as reducing STAT3 levels in regular growth conditions had no effect on imatinib sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib resistance and provide preclinical rationale for using STAT3 inhibitors to increase the efficacy of imatinib within the context of the bone marrow microenvironment.

Reproductive mode has been remarkably labile among squamate reptiles and the evolutionary transition from oviparity to viviparity commonly has been accompanied by a shift in the pattern of embryonic nutrition. Structural specializations for placental transfer of nutrients during intrauterine gestation are highly diverse and many features of the extraembryonic membranes of viviparous species differ markedly from those of oviparous species. However, because of a high degree of evolutionary divergence between the species used for comparisons it is likely that the observed differences arose secondarily to the evolution of viviparity. We studied development of the extraembryonic membranes and placentation in the reproductively bimodal lizard Lacerta vivipara because the influence of reproductive mode on the structural/functional relationship between mothers and embryos can best be understood by studying the most recent evolutionary events. Lecithotrophic viviparity has evolved recently within this species and, although populations with different reproductive modes are allopatric, oviparous and viviparous forms interbreed in the laboratory and share many life history characteristics. In contrast to prior comparisons between oviparous and viviparous species, we found no differences in ontogeny or structure of the extraembryonic membranes between populations with different reproductive modes within L. vivipara. However, we did confirm conclusions from previous studies that the tertiary envelope of the egg, the eggshell, is much reduced in the viviparous population. These conclusions support a widely accepted model for the evolution of squamate placentation. We also found support for work published nearly 80 years ago that the pattern of development of the yolk sac of L. vivipara is unusual and that a function of a unique structure of squamate development, the yolk cleft, is hematopoiesis. The structure of the yolk sac splanchnopleure of L. vivipara is inconsistent with a commonly accepted model for amniote yolk sac function and we suggest that a long standing hypothesis that cells from the yolk cleft participate in yolk digestion requires further study.

Indiana University-Purdue University Indianapolis (IUPUI) / Mitochondria are the major ATP producing source within cells. There is increasing data supporting a direct involvement of mitochondria and mitochondrial function in regulating stem cell pluripotency. Mitochondria have also been shown to be important for hematopoietic stem and progenitor cell function. Hematopoietic stem cells have lower numbers of mitochondria (mass), lower mitochondrial membrane potential, and lower ATP levels as compared to other blood cell types. Mitochondria play an important role in hematopoietic stem and progenitor cells, thus we investigated the role of the chemokine, SDF-1/CXCL12, in mitochondrial function in hematopoietic stem and progenitor cells using an SDF-1/CXCL12 transgenic mouse model. We found increased mitochondrial mass is linked to CD34 surface expression in hematopoietic stem and progenitor cells, suggesting that mitochondrial biogenesis is linked to loss of pluripotency. Interestingly these hematopoietic progenitor cells have low mitochondrial membrane potential and these mitochondrial become active prior to leaving the progenitor cell compartment. We also tested the ability of SDF-1/CXL12 to modulate mitochondrial function in vitro by treating the human leukemia cell line, HL-60, and primary mouse lineage- bone marrow cells with SDF-1/CXCL12. We found significantly reduced mitochondrial function at two hours while mitochondrial function was significantly increased at 24 hours. This suggests that SDF1/CXCL12 regulates mitochondrial function in a biphasic manner in a model of hematopoietic progenitors and immature blood cells. This suggests SDF1/CXCL12 may play a role in regulating mitochondrial function in hematopoiesis. We also investigated STAT3 in hematopoietic stem and progenitor cells. Mitochondrial STAT3 plays an essential role in regulating mitochondrial function. By using a knockout (Stat3-/-) mouse model we found that Stat3-/- hematopoietic progenitor cells had reduced colony forming ability, slower cell cycling status, and loss of proliferation in response to multi-cytokine synergy. We also found mitochondrial dysfunction in Stat3-/- hematopoietic stem and progenitor cells. Our results suggest an essential role for mitochondria in HSC function and a novel role for SDF-1/CXCL12 and STAT3 in regulating mitochondrial function in hematopoietic stem and progenitor cells.

Final publication is available at http://www.sciencedirect.com/science/article/pii/S0006291X15310664 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19598号 / 医博第4105号 / 新制||医||1014(附属図書館) / 32634 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM