Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.

Background: Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. Methods and results: We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1(-/-)mice and Pim1(-/-)2(-/-)3(-/-) triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 (-/-) and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit S-35 methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Conclusions: Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that a disintegrin and metalloproteinase 10 (ADAM10) is a critical regulator of Notch and that its deletion is embryonically lethal, we generated transgenic mice that overexpress ADAM10 at early stages of lymphoid and myeloid development (A10Tg). ADAM10 transgene expression alters hematopoiesis post-hematopoietic Lineage-Sca-1+c-kit+ (LSK) subset differentiation but prior to lineage commitment of progenitor populations. This results in delayed T cell development, abrogated B2 cell development, and dramatic expansion of functionally active myeloid derived suppressor cells (MDSCs) in A10Tg mice. Given ADAM10’s role in Notch signaling, we hypothesized that the observed hematopoietic alterations may be a consequence of perturbed Notch signaling. In fact, blockade of ADAM10 (S2) rescues B cell development and reduces myeloid cells in A10Tg LSKs. Inhibition of γ-secretase (S3) in wild type (WT) LSKs results in enhanced myelopoiesis, mimicking the phenotype of A10Tg mice. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical for hematopoietic cell-fate determination. Albeit arising in a tumor-free host, A10Tg MDSCs are functionally and phenotypically analogous to tumor-derived MDSCs. A10Tg MDSCs inhibit T cell activation in vitro, and inhibit adoptive immunotherapy (AIT) of metastatic melanoma in vivo, which can be reversed with MDSC depletion. Intriguingly, A10Tg mice are resistant to parasitic infection upon inoculation of Nippostrongylus brasiliensis. However, depletion of MDSCs abrogates this response, while adoptive transfer (AT) of MDSCs into WT mice increases their resistance. This polarized activity of MDSCs is heavily dependent upon interaction with mast cells (MCs). In fact, B16 melanoma cells metastasize more rapidly in WT mice infused with MDSCs when compared to MC-deficient mice (Kit Wsh/Wsh), with or without MDSC AT. Parallel to B16 progression, the ability of MDSCs to promote anti-Nb immunity is significantly diminished in MC-deficient (Kit Wsh/Wsh) mice even with MDSC AT. This augmentation of MDSC activity in the presence of MCs is further corroborated by in vitro co-culture assays that demonstrate a synergistic increase in cytokine production. Furthermore, MDSCs preferentially migrate to the liver in a MC-dependent manner. This interaction is mediated by MC-released histamine. In fact, MDSCs express histamine receptors (HR) and histamine induces MDSC survival, proliferation, and activation. We demonstrate that MDSC activity is abrogated with histamine blockade. Moreover, in humans, allergic patients present with an increase in MDSC population, and MDSCs purified from a stage I breast cancer patient exhibit increased survival in the presence of histamine. Taken together, our studies indicate that MCs and MC-released histamine are critical for the observed functional duality of MDSCs, ranging from immunosuppressive to immunosupportive, depending on the disease state.

Krüppel-like factors (KLFs) are a family of transcription factors with 3 Cys2/His2 zinc fingers that regulate cell differentiation and developmental processes. EKLF is involved in primitive and definitive erythropoiesis; KLF2 is implicated in the development of primitive erythroid and endothelial cells of the vasculature. Using light and electron microscopy, the yolk sacs and dorsal aortae from EKLF/KLF2 double knockout (KO) E9.5 (embryonic day 9.5) were examined to determine whether these KLFs have compensatory functions in morphology of blood cells and vessels. EKLF/KLF2 double KO E9.5 erythroid, endothelial, and mesothelial cells had more severely abnormal morphology than WT and KLF2-/-. Flow cytometry and cytospins were used to determine maturational effects of single and EKLF/KLF2 double KO primitive erythroid cells double-labeled with anti-TER119 and anti-CD71. EKLF KO and EKLF/KLF2 double KO erythroid cells display defective erythroid maturation. EKLF and KLF2 have overlapping roles in the development of embryonic erythroid and endothelial cells.

A malária é uma infecção causada por plasmódios, cujo controle depende do baço, o responsável pelo clareamento dos eritrócitos parasitos. O aumento da parasitemia induz uma ampliação do baço para resolver a infecção, onde participam células precursoras que apresentam CCD34+ na sua superfície. Estudamos a distribuição e a quantidade de células CD34+ em baços de roedores durante malárias de roedores, para compreender sua participação na ampliação do baço e no controle da infecção. Camundongos C57Bl/6j infectados com as cepas AJ e CR de Plasmodium chabaudi, e com a cepa ANKA de Plasmodium berghei, tiveram seus baços removidos e encaminhados para histologia e citometria de fluxo. A distribuição das células CD34+ mostrou-se mais intensa no 4º dia p.i. e menos intensa no 8º dia p.i.. As células CD34+ livres, por citometria de fluxo, surgem com uma onda no 4º dia p.i.. Sua quantidade é similar entre os modelos de P. chabaudi, mas diferente no P. berghei. Neste trabalho, o influxo de células CD34+ no baço não se relaciona com o controle da infecção. / Malaria is caused by Plasmodium sp., which control depends on the spleen, responsible for parasite clearing. The increase of parasitemia implies in spleen amplification to control the infection, with participation of CD34+ cells. We studied the distribution and amount of CD34+ cells in spleen during rodent malaria, to define the role of those cells in spleen amplification and infection control. C57Bl/6j mice were infected with strains CR and AJ of Plasmodium chabaudi, and ANKA strain of Plasmodium berghei. The spleen was removed and processed for histology and flow cytometry. Spleen CD34+ cells was increased in 4th day, p.i., and decreases in 8th day p.i. in all models. By flow cytometry, free CD34+ cells appears as a wave in the 4th day p.i.. P. chabaudi models presented the same level of those cells, which was larger in the P. berghei mice. In this work, increase of spleen CD34+ cells do not correlate with infection control.

O treinamento físico (TF) promove adaptações no sistema hematopoiético e o sítio NH2-terminal da enzima conversora de angiotensina I (ECA) hidrolisa um tetrapeptídeo hemorregulador negativo, o Acetil-Seril-Aspartil-Lisil-Prolina (Ac-SDKP). O objetivo do presente estudo foi investigar se o sítio NH2-terminal da ECA participaria nas adaptações hematopoiéticas induzidas pelo TF. Realizamos duas séries de experimentos. A primeira para determinarmos qual protocolo de TF seria o mais adequado para estudar as adaptações eritropoiéticas e a segunda para estudar o papel do sítio NH2-terminal da ECA nessas adaptações. Série de experimentos 1: ratas Wistar foram divididas em 3 grupos: controle (C), que realizaram TF (60 min/d, 5dias/sem) de 10 semanas uma vez ao dia (T1) e que realizaram o mesmo TF por 8 semanas, seguido de uma semana 2 vezes ao dia e uma semana 3 vezes ao dia (T2). Série de experimentos 2: ratas Wistar foram divididas em 4 grupos: controle (C), controle tratadas com captopril (10 mg.kg-1.dia-1) (C-Cap), treinadas sob o protocolo T2 (T2) e treinadas sob o protocolo T2 tratadas com captopril (T2-Cap). Foram medidos: 1) a pressão arterial (PA) e a freqüência cardíaca; 2) a hipertrofia cardíaca e atividade da citrato sintase; 3) o consumo máximo de oxigênio, o tempo de exercício e a distância percorrida em teste máximo; 4) a atividade catalítica dos terminais da ECA; 5) a concentração plasmática e na fração extracelular da medula óssea de Ac-SDKP; 6) o número e a proliferação de células tronco hematopoiéticas (CTH) no sangue e medula; 8) a reticulocitose e meia-vida das hemácias. As diferenças observadas apresentaram p<0,05. Série de experimentos 1: o protocolo T2 induziu maiores adaptações fisiológicas, morfológicas e funcionais em comparação ao T1. O protocolo T2 foi eficaz em promover adaptações no sistema eritropoiético como aumento no número e na capacidade proliferativa de CTH, no percentual de reticulócitos e redução na meia-vida das hemácias. O protocolo T2 aumentou a atividade catalítica do sítio NH2-terminal da ECA e reduziu a concentração plasmática e na fração extracelular da medula óssea de Ac-SDKP, o que não foi observado em T1. Série de experimentos 2: o grupo T2 apresentou aumento na atividade NH2-terminal da ECA, que foi inibido no grupo T2-Cap. A inibição do sítio NH2-terminal da ECA não influenciou a PA nem afetou as respostas ao treinamento. O grupo T2 apresentou redução na concentração plasmática e na fração extracelular da medula óssea de Ac-SDKP, enquanto no grupo T2-Cap não houve redução do Ac-SDKP no plasma e houve atenuação da redução na fração extracelular da medula óssea. Houve aumento no número e na proliferação de CTH na medula óssea e no sangue no grupo T2 e este aumento foi parcialmente inibido no grupo T2-Cap. Houve aumento na reticulocitose no grupo T2 e inibição parcial deste aumento no grupo T2-Cap. A meia-vida das hemácias foi reduzida em 50% no grupo T2, enquanto no grupo T2-Cap houve atenuação da redução. Concluímos que o protocolo de treino T2 estimula a hematopoiese pelo aumento na atividade do sítio NH2-terminal da ECA, aumento este que inativa o tetrapeptídeo Ac-SDKP. / Physical training (PT) promotes changes in the hematopoietic system and the NH2-terminal active site of angiotensin-converting enzyme I (ACE) hydrolyzes a tetrapeptide, negative hemoregulador, the acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). The purpose of this study was to investigate whether the NH2-terminal active site of ACE plays a role in the hematopoietic changes induced by PT. We conducted two series of experiments. The first in prder to determine which PT protocol would be more appropriate to study the erythropoietic adaptations and the second to study the role of the NH2-terminal site of ACE in these adaptations. Experiment series 1: female Wistar rats were divided into 3 groups: control (C), submitted to PT (60 min/d, 5d/week) for 10 weeks once a day (T1) and submitted to the same TF for 8 weeks, followed by a week 2 times a day and a week 3 times a day (T2). Experiment series 2: female Wistar rats were divided into four groups: control (C), control treated with captopril (10 mg.kg-1.day-1) (C-Cap), trained under the protocol T2 (T2) and trained under protocol T2 treated with captopril (Cap-T2). We measured: 1) blood pressure (BP) and heart rate, 2) cardiac hypertrophy and citrate synthase activity, 3) the maximum oxygen consumption, exercise time and distance in maximal test, 4) catalytic activity ACE terminals, 5) plasma and bone marrow extracellular fraction concentration of Ac-SDKP, 6) the number and proliferation of hematopoietic stem cells (HSC) in the blood and marrow, 8) reticulocytosis and erythrocyte life-spam. The observed differences presented p <0.05. Experiment series 1: T2 protocol induced greater physiological, morphological and functional compared to T1. T2 protocol was effective in causing changes in the erythropoietic system such as increase the number and proliferative capacity of HSC, the percentage of reticulocytes and reduced erythrocyte life-spam. The protocol T2 increased the catalytic activity of the NH2-terminal site of ACE and decreased plasma and bone marrow extracellular fraction concentration of Ac-SDKP, which was not observed in T1. Experiment series 2: the T2 group showed an increase in the activity of the NH2-terminal ACE, which was inhibited in group T2-Cap. Inhibition of the NH2-terminal site of ACE did not influence or affect the BP responses to training. The T2 group showed a reduction in plasma and bone marrow extracellular fraction of Ac-SDKP, whereas in the T2-Cap there was no reduction of Ac-SDKP in plasma and there was attenuation of the reduction in the extracellular fraction of bone marrow. There was an increase in the number and proliferation of HSC in bone marrow and blood in the T2 group and this increase was partially inhibited in group T2-Cap. There was an increase in reticulocytosis in group T2 and partial inhibition of the increase in T2-Cap group. The erythrocyte life-spam was reduced by 50% in T2, while in the T2-Cap group there was attenuation of the reduction. We conclude that the training protocol T2 stimulates hematopoiesis by increasing the activity of the NH2-terminal site of ACE, an increase that inactivates the tetrapeptide Ac-SDKP.

AA é uma doença grave caracterizada por pancitopenia e hipocelularidade medular, que pode levar a morte. Em casos severos os tratamentos são restritos ao transplante alogênico de MO e/ou uso de imunossupressores. Para isso, deve ser tipado o HLA entre doador e beneficiário. Com isso, as CTM são fontes celulares candidatas para este propósito, pois são praticamente não imunogênicas devido à sua ação parácrina de imunomodulação. Além disso, as CTM possuem um importante papel na hematopoiese. Para induzir AA utilizamos inicialmente ciclofosfamida (n=10) e radiação ionizante a 6 Gy (n=40), que foi o modelo experimental adotado. Após a radiação ocorreram três transplantes de CTIPDh com intervalo de 15 dias via IP. A resposta clínica do transplante foi monitorada pela avaliação da massa corpórea, hemograma e histopatologia da MO. O enxerto das CTIPDh na MO e seu efeito na hematopoiese, também foi verificado. Os resultados demonstraram que os animais irradiados e tratados com CTIPDh apresentaram benefícios clínicos em relação aos animais não tratados. Observamos enxertia das CTIPDh na MO e baço dos camundongos e recuperação dos componentes medulares em comparação aos animais não tratados. Assim, o transplante das CTIPDh são uma fonte terapêutica em potencial. / AA is a several disease characterized by peripheral pancytopenia and bone marrow hypoplasia, which can lead to death. In severe cases, treatments are limited to allogeneic BM transplant and/or immunosuppressants administration. For this purpose, the donor blood must be typed to identify their HLA. Thereby, MSC are appropriate candidates for therapeutic use, because they present low immunogenicity and provide immunomodulation effect in tissue repair. Furthermore, MSC have important role in supporting hematopoiesis. Thus, AA was induced using cyclophosphamide (n = 10) and 6 Gy ionizing radiation (n = 40), which caused BM ablation in mice. Thus ionizing radiation of AA model was selected. After radiation, the mice received three equal doses of hDPSC each 15-day via intraperitoneal injection. Clinical response was monitored by body mass, blood cells counting and histopathology of BM. The influence of hDPSC infusion on hematopoiesis and engraftment capacity of this cell was also investigated. Our data show the hDPSC transplantation in irradiated mice improves clinical conditions and the transplant intraperitoneal showed hDPSC grafting in BM and recovery of medullary components when compared to untreated group. The results indicate that hDPSC transplantation is a potential tool for cell-based therapeutics.

Protein malnutrition (PM) causes anemia and leukopenia by reduction of hematopoietic precursors and impaired production of mediators that induce hematopoiesis, as well as structural and ultrastructural changes in the bone marrow (BM) extracellular matrix. Hematopoiesis occurs in the bone marrow (BM) in distinct regions called niches, which modulate the processes of differentiation, proliferation and self-renewal of the hematopoietic stem cell (HSC). The perivascular niche, composed mainly by mesenchymal stem cells (MSC) and endothelial cells (EC), is the major modulator of HSC and its function extends to the migration of mature hematopoietic cells into the peripheral blood through the production of cytokines and growth factors. Thus, our hypothesis is that PM changes the perivascular niche and our objective is to evaluate whether PM affects the modulatory capacity of MSC and EC on hematopoiesis. C57BL/6 male mice were divided into Control and Malnourished groups, which received for 5 weeks, respectively, a normal protein diet (12% casein) and a low protein diet (2% casein). After this period, animals were euthanized, nutritional and hematological evaluations were performed, featuring the PM. We performed leukemic myelo-monoblasts cells transplantation and observed that these cells have a lower proliferation rate and are rather in the cell cycle G0/G1 phases in malnourished mice, indicating that the BM microenvironment is compromised in PM. MSC were isolated, characterized and differentiated in vitro into EC cells, which were evidenced by CD31 and CD144 markers. We performed the quantification of HSC and hematopoietic progenitors, as well as some regulators of proliferation and differentiation, ex vivo and after cultures with MSC or EC. We observed that PM reduces HSC and hematopoietic progenitors ex vivo. In PM, MSC promote increase in HSC and suppress hematopoietic differentiation, whereas ECs induce cell cycle arrest. Additionally, we verified that PM affects granulopoesis by decreasing the expression of G-CSFr in granule-monocytic progenitors. Thus, we conclude that PD compromises hematopoiesis due to intrinsic alterations in HSC, as well as alterations in the medullary perivascular niche. / A desnutrição proteica (DP) provoca anemia e leucopenia decorrente da redução de precursores hematopoéticos e comprometimento da produção de mediadores indutores da hematopoese. A hematopoese ocorre na medula óssea (MO) em regiões distintas chamadas de nichos, que modulam os processos de diferenciação, proliferação e auto renovação da célula tronco hematopoiética (CTH). O microambiente perivascular, composto principalmente por células tronco mesenquimais (CTM) e células endoteliais (CE), é o principal modulador das CTH e sua função se estende até a migração das células hematopoiéticas maduras para o sangue periférico, através da produção de citocinas e fatores de crescimento. Dessa forma, nossa hipótese é que a DP altera o microambiente perivascular e objetivamos avaliar se a DP afeta a capacidade modulatória das CTM e CE sobre a hematopoese. Utilizamos camundongos C57BL/6 machos, divididos em grupos Controle e Desnutrido, sendo que o grupo Controle recebeu ração normoproteica (12% caseína) e o grupo Desnutrido recebeu ração hipoproteica (2% caseína), ambos durante 5 semanas. Após este período, os animais foram eutanasiados, foi realizada a avaliação nutricional e hematológica, caracterizando a DP. Realizamos transplantes de mielomonoblastos leucêmicos e observamos que estas células apresentam menor taxa de proliferação e se encontram em maior quantidade nas fases G0/G1 do ciclo celular em camundongos desnutridos, indicando que o microambiente medular está comprometido. Isolamos CTM, que foram caracterizadas e diferenciadas in vitro em CE, o que foi evidenciado pelos marcadores CD31 e CD144. Quantificamos CTH e progenitores hematopoéticos, bem como reguladores de proliferação e diferenciação, ex vivo e após culturas com CTM ou CE. Observamos que a DP reduz CTH e progenitores hematopoéticos ex vivo. Na DP, as CTM promovem incremento de CTH e suprimem a diferenciação hematopoética, enquanto que as CE induzem parada no ciclo celular. Adicionalmente, observamos que a DP afeta a granulopoese por diminuição da expressão de G-CSFr nos progenitores grânulo-monocíticos. Dessa forma, concluímos que a DP compromete a hematopoese por alterações intrínsecas na CTH, como também por alterações ocasionadas no microambiente perivascular medular.

O treinamento físico (TF) promove adaptações no sistema hematopoiético e o sítio NH2-terminal da enzima conversora de angiotensina I (ECA) hidrolisa um tetrapeptídeo hemorregulador negativo, o Acetil-Seril-Aspartil-Lisil-Prolina (Ac-SDKP). O objetivo do presente estudo foi investigar se o sítio NH2-terminal da ECA participaria nas adaptações hematopoiéticas induzidas pelo TF. Realizamos duas séries de experimentos. A primeira para determinarmos qual protocolo de TF seria o mais adequado para estudar as adaptações eritropoiéticas e a segunda para estudar o papel do sítio NH2-terminal da ECA nessas adaptações. Série de experimentos 1: ratas Wistar foram divididas em 3 grupos: controle (C), que realizaram TF (60 min/d, 5dias/sem) de 10 semanas uma vez ao dia (T1) e que realizaram o mesmo TF por 8 semanas, seguido de uma semana 2 vezes ao dia e uma semana 3 vezes ao dia (T2). Série de experimentos 2: ratas Wistar foram divididas em 4 grupos: controle (C), controle tratadas com captopril (10 mg.kg-1.dia-1) (C-Cap), treinadas sob o protocolo T2 (T2) e treinadas sob o protocolo T2 tratadas com captopril (T2-Cap). Foram medidos: 1) a pressão arterial (PA) e a freqüência cardíaca; 2) a hipertrofia cardíaca e atividade da citrato sintase; 3) o consumo máximo de oxigênio, o tempo de exercício e a distância percorrida em teste máximo; 4) a atividade catalítica dos terminais da ECA; 5) a concentração plasmática e na fração extracelular da medula óssea de Ac-SDKP; 6) o número e a proliferação de células tronco hematopoiéticas (CTH) no sangue e medula; 8) a reticulocitose e meia-vida das hemácias. As diferenças observadas apresentaram p<0,05. Série de experimentos 1: o protocolo T2 induziu maiores adaptações fisiológicas, morfológicas e funcionais em comparação ao T1. O protocolo T2 foi eficaz em promover adaptações no sistema eritropoiético como aumento no número e na capacidade proliferativa de CTH, no percentual de reticulócitos e redução na meia-vida das hemácias. O protocolo T2 aumentou a atividade catalítica do sítio NH2-terminal da ECA e reduziu a concentração plasmática e na fração extracelular da medula óssea de Ac-SDKP, o que não foi observado em T1. Série de experimentos 2: o grupo T2 apresentou aumento na atividade NH2-terminal da ECA, que foi inibido no grupo T2-Cap. A inibição do sítio NH2-terminal da ECA não influenciou a PA nem afetou as respostas ao treinamento. O grupo T2 apresentou redução na concentração plasmática e na fração extracelular da medula óssea de Ac-SDKP, enquanto no grupo T2-Cap não houve redução do Ac-SDKP no plasma e houve atenuação da redução na fração extracelular da medula óssea. Houve aumento no número e na proliferação de CTH na medula óssea e no sangue no grupo T2 e este aumento foi parcialmente inibido no grupo T2-Cap. Houve aumento na reticulocitose no grupo T2 e inibição parcial deste aumento no grupo T2-Cap. A meia-vida das hemácias foi reduzida em 50% no grupo T2, enquanto no grupo T2-Cap houve atenuação da redução. Concluímos que o protocolo de treino T2 estimula a hematopoiese pelo aumento na atividade do sítio NH2-terminal da ECA, aumento este que inativa o tetrapeptídeo Ac-SDKP. / Physical training (PT) promotes changes in the hematopoietic system and the NH2-terminal active site of angiotensin-converting enzyme I (ACE) hydrolyzes a tetrapeptide, negative hemoregulador, the acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). The purpose of this study was to investigate whether the NH2-terminal active site of ACE plays a role in the hematopoietic changes induced by PT. We conducted two series of experiments. The first in prder to determine which PT protocol would be more appropriate to study the erythropoietic adaptations and the second to study the role of the NH2-terminal site of ACE in these adaptations. Experiment series 1: female Wistar rats were divided into 3 groups: control (C), submitted to PT (60 min/d, 5d/week) for 10 weeks once a day (T1) and submitted to the same TF for 8 weeks, followed by a week 2 times a day and a week 3 times a day (T2). Experiment series 2: female Wistar rats were divided into four groups: control (C), control treated with captopril (10 mg.kg-1.day-1) (C-Cap), trained under the protocol T2 (T2) and trained under protocol T2 treated with captopril (Cap-T2). We measured: 1) blood pressure (BP) and heart rate, 2) cardiac hypertrophy and citrate synthase activity, 3) the maximum oxygen consumption, exercise time and distance in maximal test, 4) catalytic activity ACE terminals, 5) plasma and bone marrow extracellular fraction concentration of Ac-SDKP, 6) the number and proliferation of hematopoietic stem cells (HSC) in the blood and marrow, 8) reticulocytosis and erythrocyte life-spam. The observed differences presented p <0.05. Experiment series 1: T2 protocol induced greater physiological, morphological and functional compared to T1. T2 protocol was effective in causing changes in the erythropoietic system such as increase the number and proliferative capacity of HSC, the percentage of reticulocytes and reduced erythrocyte life-spam. The protocol T2 increased the catalytic activity of the NH2-terminal site of ACE and decreased plasma and bone marrow extracellular fraction concentration of Ac-SDKP, which was not observed in T1. Experiment series 2: the T2 group showed an increase in the activity of the NH2-terminal ACE, which was inhibited in group T2-Cap. Inhibition of the NH2-terminal site of ACE did not influence or affect the BP responses to training. The T2 group showed a reduction in plasma and bone marrow extracellular fraction of Ac-SDKP, whereas in the T2-Cap there was no reduction of Ac-SDKP in plasma and there was attenuation of the reduction in the extracellular fraction of bone marrow. There was an increase in the number and proliferation of HSC in bone marrow and blood in the T2 group and this increase was partially inhibited in group T2-Cap. There was an increase in reticulocytosis in group T2 and partial inhibition of the increase in T2-Cap group. The erythrocyte life-spam was reduced by 50% in T2, while in the T2-Cap group there was attenuation of the reduction. We conclude that the training protocol T2 stimulates hematopoiesis by increasing the activity of the NH2-terminal site of ACE, an increase that inactivates the tetrapeptide Ac-SDKP.

Cette thèse est consacrée au développement de modèles mathématiques de l'hématopoïèse et de maladies du sang. Elle traite du développement de modèles hybrides discrets continus et de leurs applications à la production de cellules sanguines (l'hématopoïèse) et de maladies sanguines telles que le lymphome et le myélome. La première partie de ce travail est consacrée à la formation de cellules sanguines à partir des cellules souches de la moelle osseuse. Nous allons principalement étudier la production des globules rouges, les érythrocytes. Chez les mammifères, l'érythropoïèse se produit dans des structures particulières, les îlots érythroblastiques. Leur fonctionnement est régi par de complexes régulations intra et extracellulaire mettant en jeux différents types de cellules, d'hormones et de facteurs de croissance. Les résultats ainsi obtenus sont comparés avec des données expérimentales biologiques ou médicales chez l'humain et la souris. Le propos de la deuxième partie de cette thèse est de modéliser deux maladies du sang, le lymphome lymphoblastique à cellules T (T-LBL) et le myélome multiple (MM), ainsi que leur traitement. Le T-LBL se développe dans le thymus et affecte la production des cellules du système immunitaire. Dans le MM, les cellules malignes envahissent la moelle osseuse et détruisent les îlots érythroblastiques empêchant l'érythropoïèse. Nous développons des modèles multi-échelles de ces maladies prenant en compte la régulation intracellulaire, le niveau cellulaire et la régulation extracellulaire. La réponse au traitement dépend des caractéristiques propres à chaque patient. Plusieurs scénarios de traitements efficaces, de rechutes et une résistance au traitement sont considérés. La dernière partie porte sur un modèle d'équation de réaction diffusion qui peut être utilisé pour décrire l'évolution darwinienne des cellules cancéreuses. L'existence de “pulse solutions”, pouvant décrire localement les populations de cellules et leurs évolutions, est prouvée / The thesis is devoted to mathematical modeling of hematopoiesis and blood diseases. It is based on the development of hybrid discrete continuous models and to their applications to investigate production of blood cell (hematopoiesis) and blood diseases such as lymphoma and myeloma. The first part of the thesis concerns production of blood cells in the bone marrow. We will mainly study production of red blood cells, erythropoiesis. In mammals erythropoiesis occurs in special structures, erythroblastic islands. Their functioning is determined by complex intracellular and extracellular regulations which include various cell types, hormones and growth factors. The results of modeling are compared with biological and medical data for humans and mice. The purpose of the second part of the thesis is to model some blood diseases, T cell Lymphoblastic lymphoma (T-LBL) and multiple myeloma (MM) and their treatment. TLBL develops in the thymus and it affects the immune system. In MM malignant cells invade the bone marrow and destroy erythroblastic islands preventing normal functioning of erythropoiesis. We developed multi-scale models of these diseases in order to take into account intracellular molecular regulation, cellular level and extracellular regulation. The response to treatment depends on the individual characteristics of the patients. Various scenarios are considered including successful treatment, relapse and development of the resistance to treatment. The last part of the thesis is devoted to a reaction-diffusion model which can be used to describe Darwinian evolution of cancer cells. Existence of pulse solutions, which can describe localized cell populations and their evolution, is proved