The aging of the hematopoietic system is driven in part by defects occurring in hematopoietic stem cells (HSC). Given that HSCs provide the organism with blood and immune cells lifelong, understanding the mechanisms underlying HSC aging is vital to develop interventions that address the deterioration of the hematopoietic system at its root. Past work has indicated roles for both intrinsic and extrinsic processes in driving HSC decline during aging. Still, their roles are not fully understood, especially the relationship between different drivers, and the mechanisms by which HSCs maintain functionality in the face of age-related insults. To better understand cell-intrinsic regulation of HSC aging, we investigated nucleolar DNA damage marks stemming from replication stress in old HSCs, and connected it with nucleolar stress induction which impairs protein translation and cell cycling. Although nucleolar stress dampens old HSC activity, we reveal the cytoprotective effect of the p53-mediated nucleolar stress response to be essential for preserving the residual potential of old HSCs. Additionally, though inflammation from the niche contributes to HSC aging, the exact role of microenvironmental alterations often remains unclear. Here, we uncover an important role for IL-1 derived from endosteal stromal cells in driving both HSC and niche cell aging, and demonstrate inhibition of IL-1 signaling as a tractable strategy that counters niche deterioration to improve HSC function. These findings unveil new mechanisms of HSC aging, raise the possibility that nucleolar stress signaling could be harnessed to improve the output of old HSCs in clinical settings, and demonstrate the therapeutic viability of IL-1 blockade in improving old HSC function.

Le Strontium 90 (90Sr) est un radionucléide d’origine anthropogénique, relâché en grandes quantités dans l’environnement à la suite d’essais nucléaires aériens ou d’accidents d’installations nucléaires. Le 90Sr persiste à long terme dans l’environnement, ce qui conduit à la contamination chronique par ingestion de populations des territoires contaminés. L’induction de tumeurs osseuses liées à la fixation du 90Sr a été largement décrite. Par contre, l’occurrence d’effets non cancéreux est beaucoup moins connue. Nous avons utilisé un modèle murin avec une contamination chronique par ingestion d’eau contenant 20 kBq/l de 90Sr. Une étude de biocinétique a confirmé l’accumulation de 90Sr dans les os, avec un taux d’accumulation plus rapide durant la croissance osseuse. Cette accumulation est plus élevée dans les os des femelles que chez les males. Les doses absorbées au corps entier varient de 0.33 ± 0.06 mGy (naissance) à 10.6 ± 0.1 mGy (20 semaines). La dose au squelette peut aller jusqu’à 55 mGy. L’ingestion de 90Sr induit une modification de l’expression des gènes impliqués induisant à un déséquilibre favorisant la résorption osseuse, mais sans répercussion sur la morphologie de l’os. Aucun effet majeur n’a été observé pour le système hématopoïétique. Par contre, des modifications mineures du système immunitaire ont été observées. Afin d’évaluer la fonctionnalité du système immunitaire, un test de vaccination avec les antigènes TT et KLH a été utilisé. Les résultats montrent chez les animaux contaminés une diminution significative de la production d’immunoglobulines spécifiques, une modification de la balance Th1/Th2 dans la rate et une différenciation lymphoïde B perturbée. Ces résultats permettent de mieux comprendre certaines des conséquences non cancéreuses de l’exposition chronique à faible dose à des radionucléides à demi-vie longue pouvant être rejetés accidentellement. / Strontium 90 (90Sr) is a radionuclide of anthropogenic origin released in large quantities in the environment as a result of nuclear atmospheric tests or accidents at nuclear facilities. 90Sr persists on a long-term basis in the environment, leading to chronic contamination by ingestion of populations living on contaminated territories. The induction of bone tumours associated with the fixation of 90Sr has been widely described. However, the occurrence of non-cancer effects is much less known. We used a mouse model with chronic contamination by ingestion of water containing 20 kBq/l of 90Sr. A biokinetic study confirmed the accumulation of 90Sr in the bones, with an increased rate of accumulation during bone growth. This accumulation was higher in the bones of females than in males. The whole-body absorbed doses ranged from 0.33 ± 0.06 mGy (birth) to 10.6 ± 0.1 mGy (20 weeks). The absorbed dose for the skeleton was up to 55 mGy. Ingestion of 90Sr induced a change in the expression of genes inducing an imbalance in favour of bone resorption, but without effect on bone morphology. No significant effect was observed for the hematopoietic system. On the other hand, minor modifications were observed for the immune system. To evaluate the functionality of the immune system, a vaccination test with TT and KLH antigens was used. Results showed in contaminated animals a significant decrease in the production of specific immunoglobulins, changes in the Th1/Th2 balance in the spleen and a disrupted B lymphocyte differentiation. These results improve the understanding of some of the non-cancerous consequences of chronic exposure at low dose of radionuclides with a long half-life, which can be accidentally released.

Made available in DSpace on 2014-10-09T12:53:55Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:29Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:53:55Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:29Z (GMT). No. of bitstreams: 0 / O presente estudo teve como objetivo avaliar o efeito da inibição das cadeias inflamatórias reguladas pela ação do interferon-gama (IFN-γ) e da enzima óxido nítrico sintase indutível (iNOS) no dano radioinduzido após exposição a dose letal (8 Gy) ou moderada a severa (4 Gy) nos tecidos hematopoiéticos (baço e medula) de modelos experimentais irradiados nestas doses. Grupos de camundongos isogênicos C57Bl/6j foram expostos à radiação correspondente a 4 ou 8 Gy em exposições de corpo inteiro em fonte panorâmica de 60Co. Da mesma forma, foram irradiados camundongos cuja expressão de IFN-γ ou iNOS é ausente ou indetectável. Outros grupos receberam via oral por toda a duração do experimento um inibidor atividade de iNOS, aminoguanidina, ou via intraperitoneal uma quimiocina primordial promotora da hematopoiese, CXCL12, até o quarto dia após a exposição. Outra divisão experimental recebeu os dois agentes concomitantemente. Os animais foram sacrificados nos dias 2º, 4º e 8º após a irradiação, e fragmentos dos baços e fêmures foram preservados para histologia. Os esplenócitos e células não aderentes da medula óssea femoral foram removidos e divididos, fornecendo alíquotas para posterior RT-PCR e suspensões celulares apropriadas para ensaios de citometria de fluxo específicos para a detecção da freqüência de populações CD34+. Nestes mesmos dias de experimento, alíquotas de sangue caudal foram coletadas para contagem de hemácias e plaquetas periféricas. Os resultados mostraram que a ausência da produção de interferon-gama no local irradiado aumenta a sobrevivência e a quantidade de células progenitoras hematopoiéticas e que a ausência de iNOS ou seu bloqueio funcional diminuem a extensão do dano radioinduzido nos tecidos hematopoiéticos. Além disso, foi possível observar que a suplementação com CXCL12 sintética aumenta a freqüência do fenótipo CD34+ P.chave: radiação ionizante; medula óssea; óxido nítrico; aminoguanidina nos baços dos modelos testados, e que seu efeito parece antagonizar com a inibição da produção de NO pela aminoguanidina. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Als Schlüsselfaktor des hämatopoietischen Systems spielt PU.1 eine ent-scheidende Rolle in der Entwicklung der meisten hämatopoietischen Li-nien. Das PU.1 Expressionslevel bestimmt das Differenzierungspotential hämatopoietischer Stammzellen und Vorläufer. In den unterschiedlichen Zelltypen werden verschiedene Expressionsstärken etabliert. Wie diese zelltypischen Expressionslevel vonPU.1 generiert werden, ist bisher weit-gehend unbekannt. In der vorliegenden Doktorarbeit wurde mit Hilfe eines transgenen Maus-modells die cis-regulatorische Einheit von PU.1 definiert, um mit nachfol-genden molekularbiologischen und genomweiten Ansätzen Mechanismen der zellspezifischen Regulation von PU.1 zu aufzuzeigen. Die Definition der cis-regulatorischen Einheit von PU.1 erfolgte mit Hilfe eines transgenen Mausmodells, welches ein humanes PU.1 BAC Kons-trukt trägt. Es konnte gezeigt werden, dass humanes und murines PU.1 substituierbar sind und den gleichen Regulationsmechanismen unterlie-gen. Mit Hilfe genomweite DNaseI hypersensitivity Analysen, Methylie-rungs- und Bindungsstudien konnte ein neuer Regulationsmechanismus beschrieben werden, der eine spezifische kombinatorische Interaktion verschiedener cis-regulatorischer Elemente erfordert. Durch Reportergenassays in verschiedenen Zelltypen war es möglich, einen myeloischen Enhancer zu identifizieren. Es konnte gezeigt werden, dass PU.1 mit zelltyp-spezifischen Transkriptionsfaktoren interagiert, um unterschiedliche Bindungsmuster an seinen regulatorischen Elementen zu etablieren. Dadurch kommt es zu den spezifischen Expressionstärken von PU.1 / The transcription factor PU.1 occupies a central role in controlling myeloid and early B cell development and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis-element (URE) whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the URE alone is insufficient to confer physiological PU.1 expression, but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross-talk between different cis-elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell-type specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements.

Strontium 90 (90Sr) is a radionuclide of anthropogenic origin released in large quantities in the environment as a result of nuclear atmospheric tests or accidents at nuclear facilities. 90Sr persists on a long-term basis in the environment, leading to chronic contamination by ingestion of populations living on contaminated territories. The induction of bone tumours associated with the fixation of 90Sr has been widely described. However, the occurrence of non-cancer effects is much less known. We used a mouse model with chronic contamination by ingestion of water containing 20 kBq/l of 90Sr. A biokinetic study confirmed the accumulation of 90Sr in the bones, with an increased rate of accumulation during bone growth. This accumulation was higher in the bones of females than in males. The whole-body absorbed doses ranged from 0.33 ± 0.06 mGy (birth) to 10.6 ± 0.1 mGy (20 weeks). The absorbed dose for the skeleton was up to 55 mGy. Ingestion of 90Sr induced a change in the expression of genes inducing an imbalance in favour of bone resorption, but without effect on bone morphology. No significant effect was observed for the hematopoietic system. On the other hand, minor modifications were observed for the immune system. To evaluate the functionality of the immune system, a vaccination test with TT and KLH antigens was used. Results showed in contaminated animals a significant decrease in the production of specific immunoglobulins, changes in the Th1/Th2 balance in the spleen and a disrupted B lymphocyte differentiation. These results improve the understanding of some of the non-cancerous consequences of chronic exposure at low dose of radionuclides with a long half-life, which can be accidentally released.

When a multicellular organism suffers damages to tissues/organs it heals itself by either substituting the lost cellular matrix by scar formation or by regenerating the lost tissue. Regeneration likely occurs by a recapitulation of the developmental process that formed the organ. Many processes regulating organ development are based on epithelial-mesenchymal interactions and a strict control of organ specific stem/progenitor cells. Elucidation of the molecular basis of these processes is therefore vital in order to develop novel therapies in regenerative medicine. The LIM homebox gene Lhx2 is interesting in this context since Lhx2 has been shown to be important for the formation of several organs by regulating epithelial-mesenchymal interactions and progenitor cell function. Targeted inactivation of Lhx2 leads to a lethal anemia due to malformed liver and severe neural abnormalities such as hypoplasia of the forebrain and anophtalmia. Thus, elucidation of the mechanisms of the function of Lhx2 in different organ systems would give important insights into the molecular mechanisms regulating epithelial-mesenchymal interactions and stem/progenitor cell function. To elucidate the function of Lhx2 in the hematopoietic system Lhx2 was initially expressed in hematopoietic progenitor cells derived from ES cells differentiated in vitro using retroviral vectors. This approach led to the generation of hematopoietic stem cell (HSC)-like cell lines suggesting that Lhx2 could impact HSC function. However neither the specificity nor the efficiency of the Lhx2-induced phenotype could be determined using this approach. To be able to elucidate the function of Lhx2 in the hematopoietic system, an ES cell line with inducible Lhx2 expression was generated. Lhx2 expression induces self-renewal of a distinct hematopoietic progenitor cell from which HSC-like cell lines were established. Down-regulation of Lhx2 in these HSC-like cell lines leads to a rapid loss of stem cell character, providing a good model to study the molecular function of Lhx2 in hematopoietic stem/progenitor cells. A global gene expression analysis was performed comparing the Lhx2+ stem cell population to the Lhx2- differentiated progeny. This approach identified genes putatively linked to self-renewal/differentiation of HSCs. A considerable proportion of the genes showed an overlapping gene expression pattern with Lhx2 expression in tissue of non-hematopoietic origin suggesting that Lhx2 function in stem/progenitor cells partly overlap with Lhx2 function during organ development. In order to define other Lhx2-dependent progenitor cell populations and to generate a tool to analyze the function of Lhx2 in organ development a new transgenic mouse model was generated. By using a specific part of the Lhx2 promoter to drive expression of Cre recombinase in vivo (Lhx2-Cre mice) we have been able to define the first eye committed progenitor cells in the forebrain. By using the Lhx2-Cre mice it will be possible to distinguish the function of genes during eye development from their function in the patterning of the forebrain e.g. the eye field transcription factors. Conditional inactivation of Lhx2 in these eye specific progenitor cells causes an immediate developmental arrest. The transgene is also active in Lhx2-/- embryonic forebrain, but re-expression of Lhx2 in Lhx2-/- progenitor cells only promote formation of retinal pigment epithelium cells. Analysis of genes expressed by the Lhx2+ stem cell population allowed us to define novel genes putatively linked to Lhx2 function in eye development. Thus, we have defined the progenitor cells in the forebrain committed to eye development and the expansion and patterning of these progenitors are dependent on Lhx2. Although commitment to eye development is Lhx2-independent, Lhx2 might be important for the acquisition of the oligopotent fate of these progenitor cells.

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.

Indiana University-Purdue University Indianapolis (IUPUI) / Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.