Orientador: Mary Luci de Souza Queiroz / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-14T06:41:55Z (GMT). No. of bitstreams: 1 Perhs_SimoneMariaCipas_M.pdf: 3198961 bytes, checksum: 7dcc68b78919337c618a237e6794be07 (MD5) Previous issue date: 2009 / Resumo: O modelo experimental de infecção por Listeria monocytogenes tem provado ser útil na investigação dos efeitos de novos compostos na resposta imunológica primária contra bactérias intracelulares. Em particular, o número de células hematopoéticas na medula óssea e baço é de fundamental importância na resistência a esta infecção. No presente estudo, investigamos o efeito da solução hidro alcoólica de própolis (SHAP), sobre o crescimento e diferenciação de precursores hematopoéticos para granulócitos e macrófagos (CFU-GM) da medula óssea e do baço de animais infectados com Listeria monocytogenes. Além disso, quantificamos os níveis de fatores estimuladores de colônias (CSFs). A eficácia da SHAP frente a uma dose letal da bactéria também foi avaliada. Demonstramos que a SHAP protegeu os animais contra uma dose letal de L. monocytogenes, quando administrada profilaticamente na dose de 4 mg/kg durante 10 dias, com aumento de 20% na sobrevida. Este tratamento também preveniu a mielossupressão e hematopoese extramedular causadas por uma dose subletal de L. monocytogenes, devido a um aumento no número de CFU-GM na medula óssea. Além disso, observamos que os animais somente tratados (não infectados) não apresentaram diferenças significativas em relação ao grupo controle. Adicionalmente, a investigação da produção de fatores estimuladores de colônias no soro dos animais revelou um aumento de CSA nos grupos infectados após 48 e 72h pré-tratados com SHAP, sugerindo que a estimulação da mielopoese pelo produto seja mediada pela produção de fatores de crescimento. Além disso, observamos o aumento da citocinas interferon- gamma IFN-g no grupo infectado/tratado com o produto nas 48 e 72h. / Abstract: Infection with Listeria monocytogenes has proven to be a useful model to investigate early effects of new compounds on the immune response to intracellular bacteria. In particular, the number of hematopoietic cells in the bone marrow and spleen is critically important to resistance to this infection. In this study, we demonstrated the protective effects of hydro alcoholic solution of propolis in mice infected with a lethal dose of Listeria monocytogenes, when administered prophylactically at 4mg/kg for 10 days, with survival rates up to 20%. This dose also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with Listeria monocytogenes, due to the maintenance of granulocytemacrophage progenitors (CFU-GM) numbers in the bone marrow. Non-infected mice treated with hydro alcoholic solution of propolis presented similar numbers of CFU-GM in the bone marrow when compared to the controls. Furthermore, investigation of the production of colony-stimulating factors (CSF) revealed increased colony-stimulating activity (CSA) in the serum of infected mice pre-treated with hydro alcoholic solution of propolis 48 and 72 hours after the infection, suggesting that its effects on the production of growth factors mediate stimulation of myelopoiesis by hydro alcoholic solution of propolis. Moreover, we observed increased level of IFN-g in infected/treated mice. These results sustain our proposal of hydro alcoholic solution of propolis use as an adjuvant agent in the prophylaxis of opportunistic infections and reduction of side effects caused by immunosuppressive therapies. / Mestrado / Farmacologia / Mestre em Farmacologia

O nicho endosteal da medula óssea abriga as células-tronco hemopoéticas (CTH) em quiescência/autorrenovação. As CTH podem ser classificadas em dois grupos: células que reconstituem a hemopoese em longo prazo (LT-CTH) e curto prazo (CT-CTH). Investigamos, neste trabalho, os efeitos da desnutrição proteica (DP) no tecido ósseo e a participação do nicho endosteal na sinalização osteoblasto-CTH. Para tanto, utilizamos camundongos submetidos à DP induzida pelo consumo de ração hipoproteica. Os animais desnutridos apresentaram pancitopenia e diminuição nas concentrações de proteínas séricas e albumina. Quantificamos as CTH por citometria de fluxo e verificamos que os desnutridos apresentaram menor porcentagem de LT-CTH, CT-CTH e de progenitores multipotentes (PMP). Avaliamos a expressão das proteínas CD44, CXCR4, Tie-2 e Notch-1 nas LT-CTH. Observamos diminuição da expressão da proteína CD44 nos desnutridos. Isolamos as células LT-CTH por cell sorting e avaliamos a expressão gênica de CD44, CXCR4 e NOTCH-1. Verificamos que os desnutridos apresentaram menor expressão de CD44. Em relação ao ciclo celular, verificamos maior quantidade de LT-CTH nas fases G0/G1. Caracterizamos as alterações do tecido ósseo femoral, in vivo. Observamos diminuição da densidade mineral óssea e da densidade medular nos desnutridos. A desnutrição acarretou diminuição da área média das seções transversais, do perímetro do periósteo e do endósteo na cortical do fêmur dos animais. E na região trabecular, verificou-se diminuição da razão entre volume ósseo e volume da amostra e do número de trabéculas, aumento da distância entre as trabéculas e prevalência de trabéculas ósseas em formato cilíndrico. Avaliamos a expressão de colágeno, osteonectina (ON) e osteocalcina (OC) por imuno-histoquímica, e de osteopontina (OPN) por imunofluorescência no fêmur e verificamos diminuição da marcação para OPN, colágeno tipo I, OC e ON nos desnutridos. Evidenciamos, pela técnica do Picrosírius, desorganização na distribuição das fibras colágenas e presença de fibras tipo III nos fêmures dos desnutridos, além de maior número de osteoclastos evidenciados pela reação da fosfatase ácida tartarato resistente. Os osteoblastos da região femoral foram isolados por depleção imunomagnética, imunofenotipados por citometria de fluxo e cultivados em meio de indução osteogênica. Observamos menor positividade para fosfatase alcalina e vermelho de alizarina nas culturas dos osteoblastos dos desnutridos. Avaliamos, por Western Blotting, a expressão de colágeno tipo I, OPN, osterix, Runx2, RANKL e osteoprotegerina (OPG), e, por PCR em tempo real, a expressão de COL1A2, SP7, CXCL12, ANGPT1, SPP1, JAG2 e CDH2 nos osteoblastos isolados. Verificamos que a desnutrição acarretou diminuição da expressão proteica de osterix e OPG e menor expressão gênica de ANGPT1. Avaliamos a proliferação das células LSK (Lin-Sca1+c-Kit+) utilizando ensaio de CFSE (carboxifluoresceína succinimidil ester). Foi realizada cocultura de células LSK e osteoblastos (MC3T3-E1) na presença e ausência de anti-CD44. Após uma semana, verificamos menor proliferação das LSK dos desnutridos. O bloqueio de CD44 das LSK do grupo controle diminuiu a proliferação destas em três gerações. Entretanto, nos desnutridos, esse bloqueio não afetou a proliferação. Concluímos que a DP promoveu alterações no tecido ósseo e nas CTH. Entretanto, não podemos afirmar que as alterações observadas no sistema hemopoético foram decorrentes de alterações exclusivas do nicho endosteal. / The bone marrow endosteal niche hosts hematopoietic stem cells (HSC) in quiescence/self-renewal. HSC can be classified into two groups: cells capable of renewing indefinitely (LT-HSC) or repopulating in the short term (ST-HSC). In this work, we investigated the effects of protein malnutrition (PM) on bone tissue and the involvement of the endosteal niche in osteoblast-CTH signaling. Therefore, we used mice subjected to PM induced by the consumption of hypoproteic feed. Malnourished animals presented pancytopenia and decreased concentration of serum protein and albumin. We quantified the HSC by flow cytometry and found that the malnourished ones had lower percentage of LT-HSC, ST-HSC and multipotent progenitors (MPP). We assessed the expression of the CD44, CXCR4, Tie-2 and Notch-1 proteins in LT-HSC. We observed decreased expression of CD44 protein with the malnourished ones. We isolated the LT-HSC cells by means of cell sorting and assessed the gene expression of CD44, CXCR4 and NOTCH-1. We found that malnutrition had lower expression of CD44. Regarding the cell cycle, we see greater amount of LT-HSC in the G0 and G1 phases. We characterized the changes of the femoral bone tissue in vivo. We observed a decrease in the bone mineral density and medullar density in malnourished animals. As for malnourished animals, the femoral cortical region showed a significant decrease in tissue area, periosteal and endosteal perimeter. The femoral trabecular region of malnourished animals showed decreased bone volume/tissue volume ratio, decreased trabecular number, increased trabecular separation and prevalence of rod-like trabeculae. We investigated the expression of collagen, osteonectin (ON) and osteocalcin (OC) by means of immunohistochemistry and the expression of osteopontin (OPN) by immunofluorescence and we found that malnourished animals showed decreased labeling for OPN, type I collagen, OC and ON in the cortical region of the femur. Picrosirius staining was used to analyze disorganization of collagen fibers and presence of type III fibers in the femurs of the malnourished. Cortical and trabecular regions of malnourished animals presented a higher number of osteoclasts as shown by tartrate-resistant acid phosphatase reaction. Moreover, osteoblasts were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry and cultured in osteogenic induction medium. Results proved less positive for alkaline phosphatase and alizarin red in the cultures of osteoblasts of malnourished animals. We assessed, by means of Western blotting, type I collagen expression, OPN, osterix, Runx2, RANKL and osteoprotegerin (OPG) and, by real time PCR, the expression of COL1A2, SP7, CXCL12, ANGPT1, SPP1, JAG2 and CDH2 with the isolated osteoblasts. We found that malnutrition led to osterix and OPG decreased protein expression and lower ANGPT1 gene expression. We evaluated LSK cell (Lin-Sca1+c-Kit+) proliferation by CFSE (carboxyfluorescein succinimidyl ester). LSK cells and osteoblasts (MC3T3-E1) cocultures were performed in the presence and absence of anti-CD44. After a week, we found lower proliferation of LSK in the malnourished. The LSK CD44 blocking in the control group decreased the proliferation of these three generations. However, as for the malnourished, such blockage did not affect proliferation. We concluded that the PM has promoted changes in bone tissue and the CTH. However, we can\'t claim that the alterations observed in hematopoietic system were due to endosteal niche-only changes.

Camundongos AIRmax e AIRmin exibem diferenças significativas no número médio de leucócitos migrantes e no conteúdo protéico do exsudato inflamatório produzido por partículas de poliacrilamida. Um dos fatores preponderantes para a maior capacidade inflamatória da linhagem AIRmax é a maior produção de neutrófilos maduros pela medula óssea. Assim, considerando a diferente capacidade de produção leucocitária, entre as linhagens AIRmax e AIRmin, nos propomos a estudar comparativamente nestas linhagens, o processo de mielopoese in vitro em resposta ao GM-CSF e ATRA. Verificamos que as células da medula óssea dos animais AIRmax apresentaram maior potencial proliferativo e maiores níveis de expressão de genes envolvidos nos estágios iniciais da mielopoese, do que os animais AIRmin. Além disso, o conteúdo protéico das células em cultura revelou diferenças quantitativas e qualitativas de proteínas provavelmente envolvidas no processo de mielopoese. / Mice AIRmax and AIRmin exhibit significant differences in the average number of migrating leukocytes and in the protein content of inflammatory exudate produced by polyacrylamide particles. This higher inflammatory capacity of the AIRmax mice is due to three convergent factors: higher local production of chemotactic factors, increased resistance of locally infiltrated neutrophils to spontaneous apoptosis and larger production of mature neutrophils by the bone marrow. Thus, considering the differential capacity of leukocyte production between AIRmax and AIRmin mice, we are studying comparatively the myelocytic differentiation process in vitro. We verified that the BM cells of AIRmax mice showed higher proliferation levels. In addition by qPCR technique it was verified a larger expression of genes involved in the initial stages of myelopoiesis in the AIRmax BM cultures. The analysis of BM cellular protein content by 2D gel electrophoresis revealed quantitative and qualitative differences between two mouse lines.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T10:07:43Z (GMT). No. of bitstreams: 1 Costa_LauremiliaRiconG.R.da_D.pdf: 3335092 bytes, checksum: 1187b50ec4df532e027b86fabe029bc9 (MD5) Previous issue date: 2014 / Resumo: Os eventos de migração e adesão das células progenitoras hematopoéticas são cruciais para a sua manutenção nos nichos da medula óssea e são regulados por diversos fatores, dentre eles as RhoGTPases. ARHGAP21 é uma proteína RhoGAP (RhoGTPase activating protein, que regulam negativamente as RhoGTPases), e atua na migração e adesão celulares. O papel da Arhgap21 na hematopoese ainda é desconhecido. O objetivo geral do presente estudo foi estudar a função da Arhgap21 na migração e adesão das células progenitoras hematopoéticas. Neste trabalho, mostramos que ainda não foi possível a obtenção do camundongo nocaute para Arhgap21, pois parece que morrem antes do nascimento. O estudo foi realizado com os camundongos Arhgap21+/-, cuja expressão gênica e protéica de Arhgap21 está reduzida. As células progenitoras hematopoéticas Arhgap21+/- apresentaram menor quimiotaxia induzida por CXCL-12 e redução da adesão à fibronectina, o que provavelmente levou a um homing ineficiente para a medula óssea e para o baço. Observou-se também que o microambiente da medula óssea e do baço com redução de Arhgap21 leva à ineficiência do homing das células progenitoras hematopoéticas normais. Não foi observada alteração na expressão gênica e na freqüência de células expressando CXCR-4, bem como não houve diferença na secreção de CXCL-12. As células da medula óssea Arhgap21+/- apresentaram maior expressão de Cdc42 e parecem possuir maior atividade desta RhoGTPase. Pela primeira vez se descreve o papel da Arhgap21 na hematopoese normal e ela parece ser um importante regulador da migração, adesão e homing das células progenitoras hematopoéticas, provavelmente pela regulação negativa de Cdc42, através da sua função GAP / Abstract: Migration and adhesion of hematopoietic progenitor cells are crucial events for their maintenance on bone marrow niches. The role and mechanisms underlying hematopoietic progenitors migration and adhesion has been extensively studied and RhoGTPases have been implicated in these events. Regulators and effectors of RhoGTPases functions also have been studied in biology of hematopoietic progenitor cells. ARHGAP21 is a RhoGAP member, which function as negative regulators of RhoGTPases, and plays role in cell migration and adhesion. The aim of this study was to investigate the role of Arhgap21 in migration and adhesion of hematopoietic progenitor cells. It was not possible to generate a knockout mouse until now, because they die before birth, as results indicate. Heterozygous mice (Arhgap21+/-)v have reduction in Arhgap21 gene and protein expression and we used it as model. Hematopoietic Progenitor cells from Arhgap21+/- mice showed reduction in CXCL-12- induced chemotaxis as well as in fibronectin adhesion, which probably leaded to inefficient homing to wild-type bone marrow and spleen observed. In addition, homing of wild-type hematopoietic progenitor cells to Arhgap21+/- bone marrow and spleen was also reduced. It was not observed differences in CXCR-4 gene expression and frequency of progenitor hematopoietic cells expressing this receptor as well as in CXCL-12 secretion in Arhgap21+/- bone marrow or serum. It is the first time that the role of Arhgap21 in hematopoiesis is described and it seems to be an important regulator of hematopoietic progenitor cell migration, adhesion and homing, probably due to Cdc42 negative regulation by its RhoGAP function / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Fisiopatologia Médica

Orientadores: Mary Luci de Souza Queiroz, Mario Jose Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T12:21:04Z (GMT). No. of bitstreams: 1 Vecina_JulianaFalcato_D.pdf: 5354544 bytes, checksum: 42b3bf9f058841b4f83202596e841387 (MD5) Previous issue date: 2013 / Resumo: A obesidade é um problema de epidemia mundial. Considerada um processo inflamatório crônico, favorece a resistência à insulina e o desenvolvimento do Diabetes Mellitus tipo 2. Além disso, pode gerar diversos mediadores capazes de influenciar a complexa regulação hematopoética. A procura de agentes naturais que minimizam os transtornos da obesidade está recebendo uma atenção da comunidade científica. Nesse sentido, a Chlorella vulgaris (CV) surge como uma alternativa terapêutica e profilática das complicações da obesidade. Estudos realizados por nosso grupo demonstraram o efeito da alga na restauração da mielossupressão em diferentes modelos experimentais. Considerando estes aspectos, o presente estudo teve como objetivo avaliar a ação profilática da CV na modulação imunohematopoética, metabólica e de sinalização da insulina em animais obesos. Para tanto, camundongos Balb/C foram divididos em 4 grupos: controle (C), dieta padrão e CV (C+CV), dieta hiperlipídica (DH) e DH+CV, em diferentes períodos. A administração de CV restaurou a redução da hematopoese medular e o aumento na hematopoese extramedular promovidos pela DH. A CV aumentou os níveis de atividade estimuladora de colônias (CSA) em todos os grupos estudados, sendo que o grupo DH+CV produziu um aumento adicional de 2 vezes em todos os períodos avaliados. O grupo DH apresentou diminuição no número de progenitores de granulócitos-monocítico na medula, a CV reverteu tal alteração. Em relação ao metabolismo e sinalização de insulina, o grupo DH demonstrou glicemia significativamente elevada em relação ao grupo C, CV foi capaz de reduzi-la neste grupo. Além disso, o grupo DH+CV mostrou maior tolerância à glicose e insulina, paralelamente a melhora na fosforilação de IR?, IRS-1 e AKT no fígado, músculo esquelético e tecido adiposo. Como esperado, o grupo DH apresentava níveis elevados de citocinas pró-inflamatórias (IL-1, IL-6, TNF-?, TGF-? e INF?) confirmando a presença de inflamação subclínica nesses animais. A CV reduziu significativamente os níveis séricos dessas citocinas. Além disso, o grupo DH apresentou diminuição dos níveis de IL-10, sendo que a CV reverteu tais valores. A CV manteve os níveis de ácidos graxos livres, corpos cetônicos, triglicérides, colesterol e suas frações dentro dos valores fisiológicos no grupo DH+CV. Portanto, tais resultados mostram que a CV foi capaz de modular os parâmetros imunohematopoéticos e prevenir os efeitos deletérios induzidos pela DH em camundongos obesos. Sendo assim, a alga Chlorella vulgaris surge como uma promissora alternativa terapêutica e/ou complementar no tratamento da obesidade e suas complicações / Abstract: Obesity is a worldwide epidemic problem. Considered a chronic inflammatory process, promotes insulin resistance and development of Diabetes Mellitus type 2 (DM2). In addition, could generate several mediators, which are capable to influence the complex hematopoiesis regulation, including tumor necrosis factor-? (TNF-?), Interleukin-1 (IL-1), Interleukin-6 (IL-6) and leptin. The search for natural agents that minimize obesity-associated disorders is receiving attention from the scientific community. In this sense, Chlorella vulgaris (CV) has emerged as an alternative treatment and prophylaxis agent against obesity-related complications. Studies by our group have demonstrated the alga effect in myelosuppression recovery in different experimental models. Considering these aspects, the present study aimed to evaluate the prophylactic effect of CV on immunohematopoietic modulation, metabolic and insulin signaling in obese animals. To this, Balb/C mice were divided into 4 groups (n=6): control (C), standard diet and CV (C+CV), high-fat diet (HFD), HFD and CV (HFD+CV) in different periods. The administration of CV restored medullar hematopoiesis reduction and increased extramedullar hematopoiesis promoted by HFD. The CV increased the CSA levels in all groups. While, HFD+CV group produced a twice additional increase in the CSA levels in all periods studied. Only HFD group showed decrease in the number of GMP in BM, CV reverted this change. In relation to metabolism and insulin signaling, HFD group showed a significant increase in blood glucose compared to C, CV was able to decrease blood glucose on this group. In addition, HFD+CV group improve tolerance of glucose and insulin, together with the improvement of IR?, IRS-1, and AKT phosphorylation in the liver, skeletal muscle and adipose tissue. As expected, HFD group showed increased levels of pro-Inflammatory cytokines (IL-1, IL-6, TNF-?, TGF-? and IFN?), confirming the presence of subclinical inflammation in these animals. The CV causes a significant reduction in serum levels of these cytokines in this group. Moreover, HFD group showed reduced levels of IL-10, and the CV reverted such values. The CV maintained the levels of free fatty acids, ketone bodies, triglycerides, cholesterol and its fractions within the physiological values in HFD+CV group. Therefore, these results show that CV was able to modulate the immunohematopoietic parameters and prevent the deleterious effects of diet-induced obese mice. Thus, the alga Chlorella vulgaris appears as a promising prophylactic and therapeutic agent against obesity-related complications / Doutorado / Farmacologia / Doutora em Farmacologia

Des mises au point de nouvelles stratégies d’expansion ex vivo des cellules souches hématopoïétiques (CSH) sont développées depuis quelques années afin de pallier le problème du faible nombre de ces cellules pour le traitement des hémopathies ou de certaines tumeurs solides. Notre équipe avait établi un modèle d’expansion des CSH via leur exposition aux homéoprotéines HOXB4 ou HOXC4. L’étude comparative des transcriptomes de ces cellules a permis l’identification de cibles précoces des facteurs HOXB4/C4 parmi lesquels les gènes codant les régulateurs post-transcriptionnels Pumilio (de la famille PUF). Les facteurs PUF sont impliqués en particulier dans le maintien des cellules souches germinales dans différents modèles animaux, chez les vertébrés ou les invertébrés. Cependant, le rôle des facteurs PUF humains (hPum1 et hPum2) dans les cellules hématopoïétiques humaines n’avait jamais été étudié.Mon travail de thèse exposé ici a consisté, d’une part, en l’étude du profil d’expression des facteurs hPum1 et hPum2 dans différentes lignées hématopoïétiques et au cours de l’hématopoïèse humaine, démontrant une expression plus importante de ces gènes dans les cellules les plus immatures ainsi que dans les progéniteurs dont la prolifération est activée. D’autre part, l’étude fonctionnelle des facteurs hPum1 et hPum2 a mis en évidence leur implication dans l’expansion et la survie des cellules CD34+. L’inhibition spécifique de hPum1 ou de hPum2 in vitro par des shARN, induit une diminution significative du nombre absolu des cellules ainsi qu’une augmentation de leur apoptose. Cela corrèle avec une accumulation des CSH en phase G0-G1 du cycle cellulaire. Par ailleurs, la répression de l’expression de hPum1 ou de hPum2 diminue la reconstitution de l’hématopoïèse in vivo dans des souris immunodéficientes NOD-SCID-γC-/-. L’analyse des ARNm cibles des facteurs Pum par une étude comparative des transcriptomes des CSH transduites ou non par des vecteurs lentiviraux contenant des shARN hPum1 ou hPum2, a permis l’identification de nombreux gènes impliqués dans le contrôle de la croissance, de la survie ou du cycle cellulaire. L’ensemble de nos résultats montre l’indispensable implication des facteurs Pumilio dans le maintien de l’état souche, la prolifération et la survie des CSH humaines. Nous avons démarré des études fonctionnelles dans les cellules leucémiques myéloïdes primaires afin d’évaluer le rôle éventuel des facteurs Pumilio dans la leucémogenèse. Ultérieurement, la caractérisation de hPum1 et hPum2 comme de nouvelles molécules impliquées dans l’expansion des CSH permettra d’envisager leur étude dans la perspective de nouvelles stratégies thérapeutiques. / Ex vivo expansion of hematopoietic stem cells (HSCs) could improve new therapeutic strategies for the treatment of hematopoietic malignancies and solid tumors. Our team had developed an original method to expand human HSCs, consisting in the transfer into these cells of active HOXB4 or HOXC4 homeoproteins. The comparative transcriptomic analysis of CD34+ cells exposed or not to HOXB4 or HOXC4 proteins induced over-expression of Pumilio (PUF) genes. PUF proteins are post-transcriptional regulators of gene expression. They are involved in different biological functions among which the maintenance of stem cells. However, the function of human PUF factors (hPum1 and hPum2) in hematopoietic stem cells has never been investigated. The work that I developed during my thesis first consisted in analyzing the expression of PUF factors in different hematopoietic cell lines and during human hematopoiesis. The results highlighted a high expression of the hPum1 en hPum2 genes in the most immature cells and in the proliferating active progenitors. The study of human PUF factors by inducing their inhibition using specific shRNAs revealed their involvement in proliferation and survival of CD34+ cells. In vitro, inhibition of hPum1 or hPum2 decreases the expansion of human HSCs and increases cell apoptosis. The hPum1 or hPum2 repression also increases the number of HSCs in G0-G1 phase of the cell cycle. Moreover, the inhibition of hPum1 or hPum2 reduces the capacity of human HSCs to reconstitute in vivo hematopoiesis of immunodeficient NOD-SCID-γC-/- mice. The identification of PUF target mRNAs by a comparative transcriptomic analysis of human HSCs infected or not with lentiviral vectors containing hPum1/2 shRNAs, revealed a large number of genes involved in the regulation of cell growth, survival or cell cycle. On the whole, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of human HSCs. Functional studies in primary myeloid leukemic cells are in progress to assess the potential role of the Pum factors in the leukemogenic process. Later on, identification of Pumilio factors as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.

AIF fait partie des protéines mitochondriales inductrices de mort mais possède aussi un rôle vital nécessaire à la respiration cellulaire. Les recherches menées lors de cette thèse portent sur ces deux fonctions. D'une part, j'ai approfondi l'étude de la nécrose régulée induite par un agent alkylant de l'ADN. J'ai découvert l'importance de RIP1 dans cette voie de mort cellulaire et ainsi conduit à la définir comme nécroptose. J'ai aussi mis en évidence le rôle de BID, BH3-only de la famille BCL-2, dans la libération d'AIF des mitochondries. J'ai montré que les protéases calpaïnes clivaient BID permettant à sa forme tronquée de relocaliser aux mitochondries et d'y activer le facteur pro-apoptotique BAX. Cette étude contribue à replacer le rôle des BH3-only dans des voies de mort cellulaire au delà de l'apoptose. D'autre part, j'ai étudié le rôle d'AIF dans l'hématopoïèse grâce à un modèle murin invalidé pour AIF dans ce système. J'ai observé un blocage de différenciation thymique et le développement d'une pancytopénie sévère. J'ai démontré que cette dernière est associée à la perte des cellules souches hématopoïétiques dont j'ai testé les capacités ex vivo et in vivo. Pour comprendre les raisons de ce défaut, j'ai caractérisé les conséquences associées à la perte d'AIF : perte du complexe I de la chaine respiratoire, diminution d'activité de phosphorylation oxydative, diminution de la production d'ATP, augmentation des espèces réactives de l'oxygène. Cette deuxième étude démontre l'importance d'une phosphorylation oxydative fonctionnelle et de mitochondries saines pour une hématopoïèse normale et particulièrement pour le maintien des cellules souches hématopoïétiques. / AIF is one of the cell death effectors released from mitochondria but it also possess a vital role by regulating the cellular respiration. Throughout this thesis work, I have focused my studies on these two functions. On one hand, I have performed a deeper characterization of the DNA alkylating agent induced regulated necrosis. I have identified RIP1 as a crucial determinant of this cell death pathway, hence linking it to necroptosis. I have also highlighted the role of BID, a BH3-only member of the BCL-2 family, in the mitochondrial release of AIF. I have shown that calpains proteases cleave BID into tBID which relocalize to mitochondria where it helps activating the pro-apoptotic factor BAX. This study contributes to reconsider the role of BH3-only proteins in cell death pathways beyond apoptosis. On the other hand, I have studied AIF role in hematopoiesis thanks to a mouse model with hematopoietic lineage-specific deletion of AIF. I have observed a block in T-cell development and the rapid development of severe pancytopenia. I have demonstrated that this pancytopenia is associated with the loss of hematopoietic stem cells whom capacities were tested both ex vivo and in vivo. In order to understand the underlying determinants of these defects, I have characterized the cellular consequences related to AIF deletion : loss of the respiratory chain complex I, decrease of the oxidative phosphorylation capacity, decreased levels of ATP, increased levels of reactive oxygen species. This second study reveals the importance of a proper oxidative phosphorylation system combined with healthy mitochondria for a normal hematopoiesis and hematopoietic stem cells maintenance.

The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis. The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.

Le tissu adipeux (TA) contient un grand nombre de leucocytes qui jouent un rôle fondamental dans la régulation de l'activité métabolique du TA. Chez l'individu sain, les leucocytes du TA ont un profil majoritairement anti-inflammatoire (macrophages M2, polynucléaires éosinophiles, lymphocytes T CD4 Th2 et T régulateurs). Chez le sujet obèse, on observe une modification des populations immunitaires vers un phénotype majoritairement pro-inflammatoire (macrophages M1, polynucléaires neutrophiles, lymphocytes T CD8 et T CD4 Th1). Cet état inflammatoire participe au développement du syndrome métabolique. Chez l'adulte, les leucocytes circulants sont principalement produits dans la moelle osseuse (MO) par des cellules souches hématopoïétiques (CSH). Notre équipe a montré qu'une partie des leucocytes du TA sont produits in situ grâce à la présence de CSH tissulaires spécifiques, dont l'activité hématopoïétique diffère selon le dépôt adipeux. Ce résultat suggère que les CSH du TA pourraient être contrôlées par leur niche, comme c'est le cas dans la MO. Considérant le rôle prépondérant des leucocytes dans la physiopathologie du TA et le rôle des CSH dans ce tissu, les objectifs de cette thèse ont été les suivants : 1) Caractériser le rôle de l'hématopoïèse du TA dans le développement des maladies métaboliques. 2) Caractériser d'un point de vue cellulaire et moléculaire la niche des CSH du TA et la régulation de leur activité par cet environnement. 3) Mettre en évidence et caractériser l'activité hématopoïétique du TA chez l'homme. Concernant le premier objectif, nos résultats montrent que dans un modèle de diabète induit par un régime riche en gras, les CSH du TA produisent des macrophages pro- inflammatoires ayant un rôle direct dans le développement des altérations de l'homéostasie glucidique. La greffe de CSH du TA issues d'une souris diabétique dans une souris maintenue sous régime standard induit le transfert de la pathologie. Inversement, la greffe de CSH de TA d'une souris saine dans une souris diabétique améliore le phénotype métabolique. Concernant le second objectif, nous montrons que les CSH du TA se localisent préférentiellement dans le cœur du TA sous-cutané, région principalement composée d'adipocytes beiges, alors que la périphérie, constituée d'adipocytes blancs uniloculaires héberge moins de CSH. L'activation ou l'inhibition des adipocytes beiges diminue la quantité de CSH au cœur du tissu, montrant qu'un déséquilibre du métabolisme des adipocytes beiges a un impact sur les CSH, suggérant que ces adipocytes pourraient alors faire partie d'une niche hématopoïétique. Les approches in vitro ne nous ont pas permis d'aller plus loin dans la caractérisation des acteurs cellulaires et/ou moléculaires de cette niche. Concernant le troisième objectif, nous montrons pour la première fois la présence de CSH dans le TA chez l'homme. La fonctionnalité de ces CSH a été testée in vitro et in vivo. En culture en milieu semi-solide, les CSH de TA humain sont capables de donner des clones myéloïdes, comme chez la souris. In vivo, chez des souris immuno-déficientes reconstituées avec des CSH de TA humain, on retrouve des cellules immunitaires humaines dans le tissu adipeux, ce qui démontre leur capacité à reconstituer une partie du système immunitaire de ce tissu. En conclusion, ce travail de thèse a permis de montrer que l'activité hématopoïétique du TA joue un rôle crucial dans le maintien de la balance énergétique. Les CSH du TA résideraient préférentiellement dans une niche localisée au cœur du tissu, composée d'adipocytes beiges. La caractérisation des signaux moléculaires présents dans les différentes zones du TA permettra de proposer de nouvelles hypothèses sur la régulation de l'activité des CSH du TA. Chez l'homme, notre travail a permis de mettre en évidence une hématopoïèse tissulaire endogène au tissu adipeux, renforçant ainsi l'importance physiopathologique de nos précédents résultats obtenus chez la souris. / The adipose tissue (AT) contains a lot of leukocytes that play a fundamental role in the regulation of AT metabolic activity. In a physiological situation, AT-leukocytes mostly display an anti-inflammatory profile (M2 macrophages, eosinophils, CD4 Th2 T cells and regulatory T cells). Obesity induces a shift in AT immune cells towards a pro-inflammatory phenotype (M1 macrophages, neutrophils, CD8 and CD4 Th1 T cells). This inflammatory state contributes to the development of the metabolic syndrome. In adults, circulating leukocytes are mostly produced in the bone marrow (BM) by hematopoietic stem cells (HSC). A few years ago, we have shown AT harbors a specific resident HSC population that can renew innate immune cells and especially macrophages in the AT, via in situ differentiation. This endogenous hematopoietic activity differs according to the localization of the fat pad, suggesting that like BM-HSC, AT HSC might be controlled by their environment. Considering the important role of leukocytes in the AT physiopathology and the role of resident HSC in this tissue, the objectives of this work were the followings: 1) To characterize the role of the AT hematopoiesis in the onset of metabolic diseases. 2) To characterize the AT HSC niche from a cellular and a molecular point of view, and the regulation of their activity by this environment. 3) To demonstrate the presence of an endogenous AT-hematopoiesis in humans. First, by using transplantation of sorted AT-HSC and gain and loss of function studies we showed that some of the inflammatory AT-macrophages inducing metabolic disease originate from resident AT-HSC. Transplantation of AT-HSC sorted from high fat diet-fed (HFD) mice is sufficient to induce AT-macrophage accumulation, and to transfer metabolic disease in control mice. Conversely, the transplantation of control AT-HSC improves both AT-inflammation and glucose homeostasis in HFD mice. Second, we showed that AT-HSC are preferentially localized in the core of sub-cutaneous AT that contains beige adipocytes, instead of the periphery that mostly harbors unilocular white adipocytes. Activation or inhibition of beige adipocytes induces a loss of this preferential localization, suggesting that modifications of the subcutaneous AT core region metabolism impact HSC behavior. This suggests that beige adipocytes might be a part of a hematopoietic niche in the AT. However, we were unable to characterize the cellular and/or molecular constituants of this niche. Finally, we showed for the first time that as in mice, human AT contains resident HSC. In methylcellulose semi-solid medium, human AT-HSC are able to produce myeloid clones. In vivo, after transplantation of human AT-HSC in immunodeficient mice, human immune cells are observed in the AT. These results show that human AT exhibit a functional endogenous hematopoietic activity. Altogether, we show in this study that the AT hematopoietic activity plays a crucial role in the control of energy balance. Although AT HSC are localized preferentially at the vicinity of beige adipocytes, molecular signals controlling this population remain to be characterized. Finally, we demonstrate for the first time an endogenous hematopoiesis in human AT, highlighting the physiopathological importance of our previous results obtained in mice.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:19:15Z (GMT). No. of bitstreams: 1 XavierFerrucio_JulianaMartins_D.pdf: 4222784 bytes, checksum: e2845997ddb128f2d29a2cabc667da17 (MD5) Previous issue date: 2015 / Resumo: A hematopoiese, processo pelo qual a célula-tronco hematopoiética (CTH) dá origem a todas as células do sangue, é regulada através do seu contato com diversos tipos celulares que compõem o estroma da medula óssea, mantendo o balanço entre autorrenovação e diferenciação das CTHs, processos também regulados por vias de sinalização como a via Hedgehog e proteínas reguladoras de citoesqueleto como RhoGTPases. Sendo assim, os objetivos gerais do trabalho foram investigar a via Hedgehog na medula óssea de pacientes com síndromes mielodisplásica (SMD) e a função da ARHGAP21 na autorrenovação das CTH. Através de imunohistoquímica de bióspias de medula óssea de pacientes com SMD em comparação com anemia megaloblástica (usada como controle) observamos o aumento de células marcadas para os ligantes Sonic e Dessert Hedgehog e células positivas para c-Kit nas amostras de SMD. Em seguida, analisamos a expressão gênica dos membros da via Hedgehog em células totais de medula óssea de pacientes SMD e doadores normais. Não houve diferença na expressão de Patched (PTCH) em SMD em comparação com doadores saudáveis, porém quando as amostras são classificadas de acordo com a WHO 2008, observamos aumento significativo de PTCH nos pacientes com <5% de blastos na medula óssea (AR, ARSA e CRDM). As expressões tanto de GLI1 como de SUFU foram reduzidas nos pacientes SMD e no grupo de pacientes com ARSA, CRDM e CRDU quando comparados aos doadores normais. A expressão de SUFU também foi reduzida em grupo de pacientes SMD com > 5% de blastos na medula óssea (AREB-1 e 2). Observamos aumento de 7 vezes na expressão de SMO no grupo de SMD e de 14 vezes no grupo AREB-1 e 2 em comparação com doadores normais. Análises de sobrevida de Cox demonstraram que o aumento na expressão de SMO e a redução na expressão de PTCH são fatores independentes na sobrevida global dos pacientes com SMD. Além disso, confirmamos o aumento da ativação da via Hedgehog em células CD34+ de pacientes com SMD através do aumento da expressão dos alvos da via: GLI1, BMI1 e Nanog. Nossos dados indicam que a via Hedgehog está desregulada na medula óssea dos pacientes com SMD e o possível envolvimento dessa via na progressão da doença. Através do modelo murino Arhgap21+/- estudamos a importância dessa proteína na hematopoiese normal e observamos que esses animais apresentam redução nas diferenciações eritroide e megacariocítica juntamente com aumento da mobilização mielóide. Observamos ainda o aumento na frequência das CTHs de curta e longa duração na medula óssea dos heterozigotos. Esse aumento foi responsável pela maior recuperação no número de neutrófilos após indução de estresse hematopoiético nos animais Arhgap21+/-. Durante o transplante seriado de células de medula óssea observamos menor reconstituição após 4 semanas, juntamente com menor reconstituição a longo prazo nos animais que receberam células dos heterozigotos, sugerindo menor autorrenovação das CTHs nas células com redução da Arhgap21. O nicho medular dos animais Arhgap21+/- demonstrou redução no suporte inicial da hematopoiese o que foi relacionado à maior produção de ROS após irradição subletal dos heterozigotos. Juntos, esses resultados indicam que a ARHGAP21 tem importante papel na hematopoiese normal, pois participa de processos como a diferenciação, mobilização e autorrenovação das CTHs / Abstract: Hematopoiesis is a process by which hematopoietic stem cell (HSC) gives rise to all blood cells and it is regulated by HSC contact with different cell types that compose the bone marrow stroma and maintain the balance between HSC self-renewal and differentiation, processes that are also regulated by signaling pathways such as the Hedgehog and regulatory proteins of the cytoskeleton as RhoGTPases. The overall aims of this work were to investigate the Hedgehog pathway in myelodysplastic syndrome (MDS) bone marrow and the ARHGAP21 role in hematopoiesis. Immunohistochemistry assays revealed that MDS bone marrow biopsies showed increased Sonic and Dessert Hedgehog together with c-kit positive stained cells compared to megaloblastic anemia (used as control).We also analyzed gene expression of the Hedgehog pathway members in total cells from MDS bone marrow and healthy donors. There was no difference in Patched (PTCH) expression in MDS compared to healthy donors, however, when MDS samples were classified according to WHO 2008, we observed a significant increase in PTCH1 expression in MDS patients <5% bone marrow blast (RCUD, RCMD, ARSA). GLI1 and SUFU expressions were significantly reduced in the MDS patients and in the group of patients with RCUD, RCMD, ARSA, when compared to healthy donors. SUFU expression was also decreased in MDS patients > 5% bone marrow blast (RAEB-1 and RAEB-2) compared to controls. We also observed 7-fold increase of SMO transcripts in the MDS group and 14-fold increase in RAEB-1 and RAEB-2. Cox survival analyses showed that increased SMO and decreased PTCH expression were considered as independent factors influencing the survival of these patients. We also confirmed the enhanced Hedgehog activation in CD34+ cells from MDS patients through increased expression of the pathway targets: GLI1, BMI1 and Nanog. Together, our data indicate that the Hedgehog pathway is deregulated in MDS bone marrow and the possible role of that pathway in disease progression. Arhgap21+/- murine model study showed the importance of this protein in normal hematopoiesis as these animals showed alterations in erythroid and megakariocyte differentiation along with increased myeloid mobilization. We also observed increased short and long term HSC frequency in the heterozygous bone marrow. This increase was responsible for enhanced neutrophil reconstitution during induced hematopoietic stress in Arhgap21+/-. Serial bone marrow transplantation assay showed lower chimerism in peripheral blood 4 weeks after the transplant together with lower long term reconstitution in animals who received Arhgap21+/- bone marrow, indicating decreased HSC self-renew from heterozygous cells. Bone marrow niche of Arhgap21+/- animals showed a reduction in initial support hematopoiesis and this result was correlated with the increased ROS production from Arhgap21+/- bone marrow after sublethally irradiation. Taken together, our data indicate that ARHGAP21 has an important role in hematopoiesis regulating process such as differentiation, mobilization and self-renewal of HSC / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Fisiopatologia Médica