Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.

Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus. In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro. Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity. In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP. Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 737

Thesis (Ph.D.)--University of Melbourne, The Walter and Eliza Hall Institute of Medical Research, The Division of Immunology and the Division of Molecular Medicine, Dept. of Medical Biology, Faculty of Medicine and Dentistry, 2009. / Typescript. Includes bibliographical references (p. 133-165)

Le développement placentaire est un processus finement contrôlé dans le temps et dans l'espace. Il est caractérisé par une invasion précoce et profonde, de l'endomètre et du premier tiers du myomètre, par les cytotrophoblastes extravilleux, cellules responsables du remodelage des artères spiralées utérines et de l'établissement de la circulation fœto-maternelle. Tout déficit dans ces processus physiologiques conduit à des complications de la grossesse, telles que la Pré-Eclampsie (PE), ou le Retard de Croissance Intra-Utérin (RCIU). Les travaux récents de l'équipe suggèrent l'implication d'un nouveau facteur angiogène, nommé EG-VEGF (Endocrine Gland Derived Vascular Endothelial Growth Factor), dans le développement de la PE. L'objectif de ma thèse fut de mieux caractériser le rôle de l'EG-VEGF dans le développement placentaire normal au cours du premier trimestre de la grossesse. Pour cela trois axes ont été explorés : i) l'étude de la régulation de l'EG-VEGF par le récepteur nucléaire PPARγ (Peroxisome proliferator-activated receptor gamma), ii) la détermination de son rôle dans les différenciations hématopoïétique et angiogénique placentaires et iii) la contribution au développement d'un modèle murin de la PE. A l'issue de cette thèse, mes travaux ont montré que non seulement l'expression de l'EG-VEGF et de son récepteur PROKR2 étaient régulées positivement par PPARγ, mais aussi, que ce récepteur nucléaire est directement impliqué dans la mise en place de la vascularisation intra-placentaire, avec la participation des deux récepteurs PROKR1 et PROKR2, et que l'inhibition de l'invasion trophoblastique par PPARγ, seraient en partie contrôlée par l'EG-VEGF, via PROKR2. Mon travail a également mis en évidence que l'EG-VEGF serait impliqué dans le contrôle de la différenciation hématopoïétique et endothéliale placentaire. Il aurait un effet inhibiteur sur la différenciation des cellules hématopoïétiques et endothéliales, mais plus particulièrement sur les cellules endothéliales hémogéniques. Enfin, ma contribution au développement d'un modèle in vivo de la PE a permis de montrer qu'un maintien de la libération de l'EG-VEGF, au-delà de sa période normale de production, serait responsable du développement de la PE, suite à un défaut de l'invasion trophoblastique, entraînant la libération par le placenta, de sFlt-1 et de sEndogline. Ces derniers vont induire un dysfonctionnement rénal et une hypertension artérielle. L'ensemble de ces trois projets a contribué à l'avancée de nos connaissances actuelles sur les mécanismes physiologiques du développement placentaire, ainsi que sur un facteur clé de la placentation, l'EG-VEGF, et a également permis de mieux appréhender les causes de l'établissement des pathologies de la grossesse, comme la PE et le RCIU. / Placental development is a process that is finely controlled. It is characterized by early and deep invasion of the endometrium and the first third of the myometrium by extravillous cytotrophoblasts that participate to the remodeling of the spiral arteries and to the establishment of the feto-maternal circulation. Poor remodeling of spiral arteries by trophoblastic cells, leads to the development pregnancy pathologies such as, Preeclampsia (PE) and Intra-Uterine Growth Restriction (IUGR). During the last decade, our team has gathered interesting data that propose the new factor, EG-VEGF (Endocrine Gland Derived Vascular Endothelial Growth Factor) as a potential marker for PE. My thesis project aimed at further characterizing the role of EG-VEGF during pregnancy. Three main axis were addressed, i) The study of the regulation of EG-VEGF by PPARγ (Peroxisome proliferator-activated receptor gamma), ii) The study of the role in hematopoietic and angiogenic placental cells differentiations and iii) The development of an in vivo model of PE. My thesis showed that 1) EG-VEGF and PROKR2 expression are upregulated by PPARγ, 2) that the regulation of intra-placental vascularization and trophoblastic invasion by PPARγ is mediated by EG-VEGF through PROKR1 and PROKR2 and through PROKR2 receptors, respectively, 3) that EG-VEGF controls hematopoietic and endothelial cell differentiation and 4) that maintenance of EG-VEGF production beyond its normal period of secretion during pregnancy leads to the development of PE in a gravid mouse model. Altogether, these projects contributed to have a better knowledge about physiological mechanisms of placental development and about a key factor of placentation EG-VEGF. Moreover they improved our understanding of the origins of pregnancy diseases establishment such as PE and RCIU.

Hematopoietic stem cells (HSCs) have the ability of both self-renewal and differentiation. After bone marrow damage, surviving host HSCs or transplanted donor HSCs are able to restore hematopoiesis and maintain it for a long time due to the self-renewal potential. HSCs reside in a specific microenvironment in the bone marrow, in stem cell niche, which supports their survival and controls their functioning. In this study, we investigated the impact of bone marrow damage induced by increasing doses of irradiation on engraftment efficiency of transplanted donor repopulating cells. Using the CD45.1/CD45.2 congenic mouse model, we developed a new approach enabling estimation of surviving HSCs in damaged hematopoietic tissue. Its principle is in measuring of the donor chimerism resulting from transplantation of a defined dose of normal congenic bone marrow cells. The transplanted donor cells contain repopulating cells, progenitors (STRCs) and HSCs (LTRCs) that give rise to blood cell production which proceeds in parallel with that present in the host hematopoietic tissue. We applied this approach to monitor spontaneous regeneration of repopulating cells, including HSCs, in mice irradiated with a sublethal dose of 6 Gy or by a lethal dose of 9 Gy and rescued by syngenic bone marrow cells. This was...

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T08:29:05Z (GMT). No. of bitstreams: 1 Torello_CristianeOkuda.pdf: 8596128 bytes, checksum: e07457446c814c5f191e0c629491876d (MD5) Previous issue date: 2009 / Resumo: Neste trabalho, investigamos os efeitos do extrato padronizado de Rhodiola rosea L. (ERR) sobre a resposta imunohematopoética de camundongos infectados com Listeria monocytogenes. Os resultados demonstram que o ERR aumenta a resistência dos animais frente uma dose letal de Listeria quando administrado profilaticamente nas doses de 100 e 250 mg/kg por sete dias consecutivos anteriores à inoculação. Paralelamente, reversão da mielossupressão induzida pela infecção, concomitante ao aumento na atividade de células natural killers (NK) e na produção das citocinas TNF-a, IFN-? e fatores estimuladores de colônias foram observados. Os resultados obtidos demonstram que o ERR compartilha da habilidade de regular positivamente os desequilíbrios hematopoiéticos e imunológicos envolvidos nos estágios iniciais da infecção com L. monocytogenes. Estes efeitos podem ser atribuídos à recuperação no equilíbrio da resposta hematopoiética através da produção de IL-1a e IL-6 pelas células estromais no microambiente medular e também da produção de fatores estimuladores de colônias a partir das 24 horas de infecção, promovendo aumento no número de progenitores de macrófagos e granulócitos na medula óssea. Além disso, a eficácia do ERR também depende do aumento na produção das citocinas TNF-a e IFN-?, do aumento na atividade funcional das células NK e polarização da resposta celular para Th1. Juntos, estes efeitos contribuem para o aumento na resistência a L. monocytogenes. / Abstract: In this work, we have investigated the effects of Rhodiola rosea L. extract (ERR) in Listeria monocytogenes infected mice. Our results demonstrated that ERR protects mice from a lethal dose of L. monocytogenes, when administered prophylactically at 100 and 250 mg/kg, for seven consecutive days prior to the infection. In addition, prevention of myelosuppression induced by infection, concomitant to increasing natural killers (NK) cells activity and production of TNF-a, IFN-? and colony-stimulating factors were observed. The results showed that ERR share the ability of regulating positively the hematopoietic and immunological unbalance involved in the initial stages of infection with this pathogen. These effects could be attributed to recovering of the hematopoietic response through the production of IL-1a and IL-6 by stromal cells in the bone marrow microenvironment and the production of colony-stimulating factors at 24 hours of infection, promoting an increase in the number of granulocytes and macrophages progenitors in the bone marrow microenvironment. Moreover, the efficacy of ERR depends on high levels of TNF-a and IFN- ?, increased NK cells activity and polarization to Th1 response. Together, these effects contribute to increasing resistance to L. monocytogenes. / Universidade Estadual de Campi / Farmacologia / Doutor em Farmacologia

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T00:13:54Z (GMT). No. of bitstreams: 1 Calgarotto_AndranaKarla_D.pdf: 1646916 bytes, checksum: ca2ab5d61b64cf7aa060ac3c410f1029 (MD5) Previous issue date: 2012 / Resumo: Neste trabalho avaliamos os efeitos de compostos derivados do nitroestireno, NTS1 e NTS2, sobre a resposta imunohematopoética de camundongos normais e portadores do tumor ascítico de Ehrlich (TAE). O estudo dos mecanismos envolvidos no tratamento de camundongos com NTS1 e NTS2 frente às alterações induzidas pelo TAE mostra efeitos imunológicos distintos dos dois compostos. O composto NTS1 induziu aumentos significativos na atividade de células NK, na proliferação de células mononucleares esplênicas e na produção de citocinas com padrão Th1 (IL-2 e IFN-?) liberadas por células mononucleares do baço. Em contrapartida, a atividade de NTS2 esteve relacionada com ativação macrofágica. Nossos resultados demonstram que o tratamento com NTS2 promove aumento significativo nos níveis de TNF-a e IL-1 no sobrenadante da cultura de macrófagos peritoneais. Outro efeito importante de NTS2 sobre macrófagos peritoneais foi o estímulo na produção de NO2-. Além dos parâmetros imunológicos citados, avaliamos, os efeitos de NTS1 e NTS2 sobre o crescimento e diferenciação de precursores hematopoéticos. Tanto NTS1 como NTS2 foram capazes de reverter a mielossupressão provocada pela evolução do tumor, além de aumentar a atividade estimuladora de colônias (CSA) no soro de camundongos Balb/c. Os resultados obtidos demonstram que os compostos compartilham da habilidade de regular positivamente os desequilíbrios hematopoéticos e imunológicos envolvidos na evolução temporal do TAE. A partir disso, testamos a capacidade dos compostos no processo de diferenciação mielocítica utilizando um sistema de cultura ex- vivo no qual células humanas CD34+ foram tratadas com NTS1 e NTS2. Nossos resultados apresentaram atividade dose-dependente de NTS1 e NTS2 na proliferação e viabilidade de células CD34+. Além de aumentar significativamente o número de progenitores mielóide comum e para granulócitos de macrófagos. Os compostos apresentaram diferentes efeitos durante o processo de diferenciação. Inibiram a formação de neutrófilos maduros, porém, NTS1 aumentou significativamente a produção de metamielócitos e NTS2 de monócitos. Estes efeitos fenotípicos na proliferação e na diferenciação observados após o tratamento com NTS1 e NTS2 podem estar relacionados com a via p38MAPK e o fator transcricional CEBP-a / Abstract: In this work, we have investigated the effects of two nitrostirene derivatives compounds, NTS1 and NTS2, in the immune-hematopoeitic system in normal and Ehrlich ascites tumor (EAT)-bearing mice. The study of the mechanisms involved in the treatment produced by the NTS1 and NTS2 against induced alterations by EAT were different, showing significant improvements in the NK cells activity, proliferation and Th1 (IL-2 and INF-?) by mononuclear spleen cells. On the other hand, NTS2 activity was related to macrophage activation. Our results show that treatment with NTS2 promotes significant increase in the TNF-a and IL-1 levels in supernatants of the cultures of peritoneal macrophages. Another important effect of NTS2 on peritoneal macrophage was the stimulation in the production of NO2-. Beyond immunological parameters, we investigated the NTS1 and NTS2 effects on hematopoietic progenitors. Treatment with NTS1 and NTS2 protected the host of myelosuppression caused by tumor development and increase the colony-stimulating activity (CSA) in the serum of Balb/c mice. The results showed that NTS1 and NTS2 share the ability of regulating positively the hematopoietic and immunological unbalance involved in the TAE development. Concerning their effects on myelopoiesis, we investigated the compounds utilizing an ex-vivo differentiation system in which umbilical cord blood derived CD34+ cells were treated with NTS1 and NTS2. Our results show that NTS1 and NTS2 have concentration dependent effects on proliferation and viability of CD34+ cells. Moreover, NTS1 and NTS2 significantly increase common myeloid and granulocyte/macrophage progenitors. The compounds have differential effects on terminal differentiation, inhibiting mature neutrophil, however, NTS1 significantly increased metamyelocytes and NTS2 monocytes. The phenotypic effects on proliferation and differentiation observed after NTS1 and NTS2 treatment can be explained by changes in p38MAPK cell signaling and with CEBP-a transcription factor / Doutorado / Farmacologia / Doutor em Farmacologia

Les molécules d’adhésion jonctionnelles JAM-B et JAM-C forment une paire récepteur/ligand impliquée dans la régulation de nombreux mécanismes biologiques dont l’inflammation, l’hématopoïèse et la spermatogénèse. Dans la moelle osseuse, l’interaction entre JAM-C et JAM-B, respectivement exprimée par les cellules souches hématopoïétiques (CSH) et les cellules stromales, joue un rôle dans la rétention et la quiescence des CSH. Dans le testicule, JAM-C participe à la polarisation des spermatides en différenciation en interagissant avec JAM-B exprimée par les cellules de Sertoli. GRASP55 (Golgi ReAssembly and Stacking Protein of 55 kDa), identifiée au laboratoire comme un interacteur intracellulaire des protéines JAMs, est une protéine de l’appareil de Golgi participant à l’architecture et la dynamique de celui-ci ainsi qu’au transport protéique non-conventionnel.Le but de mon travail de thèse a été d’étudier le rôle de GRASP55 in vivo par des approches génétiques et pharmacologiques. Nous avons ainsi pu mettre en évidence que l’expression de GRASP55 par la spermatide ronde permet la localisation polarisée de JAM-C et le déroulement correct de la spermatogénèse. A contrario, GRASP55 n’est pas essentiel à l’hématopoïèse en conditions basales ou de stress. Toutefois, la délétion de GRASP-55 dans les cellules leucémiques diminue le progression de la pathologie in vivo. Ces résultats montrent un rôle non redondant de GRASP55 dans la spermatogenèse et la prolifération de cellules leucémiques et ouvrent des pistes possibles pour un ciblage thérapeutique de GRASP55 en hématologie. / The junctional adhesion molecules JAM-B and JAM-C form a receptor / ligand pair involved in regulation of many biological mechanisms including inflammation, hematopoiesis and spermatogenesis. In the bone marrow, the interaction between JAM-C and JAM-B, expressed by hematopoietic stem cells (HSC) and stromal cells respectively, is involved in HSC retention and quiescence. Similarly, in the testis, JAM-C participates in the polarization of differentiated spermatids by interacting with JAM-B expressed by Sertoli cells. GRASP55 (Golgi ReAssembly and Stacking Protein of 55 kDa), identified in our laboratory as a new intracellular interactor of JAM, is a Golgi apparatus protein involved in Golgi architecture and dynamics as well as unconventional secretion.The aim of my thesis was to study the role of GRASP55 in vivo by genetic and pharmacological approaches. We demonstrate that GRASP55 expression by round spermatid allows polarized localization of JAM-C and the correct course of the spermatogenesis. In contrast, GRASP55 is not essential for hematopoiesis in basal or stress conditions. However, deletion of GRASP-55 in leukemic cells decreases the progression of the pathology in vivo. These results show a non-redundant role of GRASP55 in the spermatogenesis and proliferation of leukemic cells and allow us to consider GRASP55 as a potential target in hematology.

Orientadores: Fabíola Traina, Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T21:37:05Z (GMT). No. of bitstreams: 1 Campos_PauladeMelo_D.pdf: 6042513 bytes, checksum: b12134db0721f177eb0c2116e7fdf5e2 (MD5) Previous issue date: 2015 / Resumo: As neoplasias mieloproliferativas crônicas BCR-ABL1 negativas (NMP) apresentam como característica comum a ocorrência de proliferação celular exacerbada, mantendo a capacidade de diferenciação mieloide terminal. Em parcela significativa dos casos, a ativação da proliferação celular ocorre pelo aumento da atividade tirosinoquinase de proteínas específicas. Entretanto, a heterogeneidade molecular observada nos pacientes e as respostas clínicas insatisfatórias observadas em parte dos casos com os tratamentos vigentes sugerem que mecanismos adicionais, como interações proteicas não descritas e novas mutações, possam estar envolvidos na fisiopatologia destas neoplasias. Neste trabalho, objetivamos estudar as vias de ativação tirosinoquinase em policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) (subprojeto 1), e em mastocitose sistêmica (subprojeto 2). O foco principal do subprojeto 1 consistiu em avaliar, em NMP, a associação JAK2/IRS2, previamente descrita em células não hematológicas após estímulo, bem como o envolvimento de IRS2 em vias de proliferação celular e apoptose. Utilizamos modelos de linhagens celulares leucêmicas humanas com JAK2 mutado (JAK2V617F) e JAK2 selvagem (JAK2WT), submetendo-as a inibição gênica por shRNA entregue por lentivírus e ao tratamento com o inibidor seletivo de JAK1/2 ruxolitinib. As células foram então submetidas à avaliação da viabilidade celular por MTT, e da apoptose por citometria de fluxo (anexina V/PI e caspase-3) e por imunobloting (caspase-3 clivada). A expressão do mRNA de IRS2 foi avaliada por PCR em tempo real em amostras de células CD34+ de sangue periférico de 99 pacientes com diagnóstico de PV, TE e MFP, e em 28 doadores normais. Através de imunoprecipitação e microscopia confocal, observamos a associação constitutiva entre JAK2 e IRS2 nas células JAK2V617F, mas não nas células JAK2WT. Em células JAK2V617F, a inibição de IRS2 por lentivírus diminuiu significativamente a fosforilação de STAT5, reduziu a viabilidade celular, aumentou as taxas de apoptose, e potencializou os efeitos de ruxolitinib. Não houve mudanças nas taxas de viabilidade celular e apoptose nas células JAK2WT inibidas para IRS2. A expressão do mRNA de IRS2 foi significativamente maior nos pacientes com TE em relação aos doadores normais, e em pacientes com NMP portadores da mutação JAK2V617F em relação aos pacientes com JAK2WT. Estas evidências sugerem que IRS2 possa participar das vias de sinalização celular nas NMP através de interação direta com JAK2. A inibição farmacológica de IRS2, isoladamente ou em conjunto com ruxolitinib, é ferramenta potencial no tratamento de pacientes com PV, TE e MFP. No subprojeto 2, nosso objetivo consistiu em investigar mutações no gene KIT em um caso de mastocitose sistêmica familiar seguido em nosso serviço, em que mãe (caso 1) e filha (caso 2) apresentavam extensa infiltração cutânea e da medula óssea por mastócitos, bem como avaliar a sensibilidade dos mastócitos neoplásicos ao tratamento com os inibidores de tirosinoquinase (ITK) imatinibe, dasatinibe e PKC412. Através de sequenciamento por Sanger, identificamos a mutação KITK509I em células de medula óssea total, CD3+ de sangue periférico e mucosa oral de ambas pacientes. Os pais do caso 1 apresentaram KIT selvagem. O tratamento in vitro por 4, 8 ou 12 dias de células totais de medula óssea dos casos 1 e 2 com os ITK avaliados resultou em menor viabilidade celular, avaliada por MTT, e em redução da fosforilação de P70S6K com todas as drogas testadas. Entretanto, apenas o imatinibe evidenciou resposta consistente na indução de apoptose. Foi iniciado tratamento dos casos 1 e 2 com imatinibe 400mg/dia via oral. Três meses após o início, houve normalização da pele e da medula óssea; após dois anos de seguimento, as pacientes mantêm-se em remissão. Embora rara, a mutação KITK509I deve ser pesquisada em todos os casos de mastocitose sistêmica familiar. O imatinibe pode ser considerado como droga de primeira escolha nestes casos / Abstract: The BCR-ABL1 negative chronic myeloproliferative neoplasms (MPN) are characterized by increased cellular proliferation with preserved terminal myeloid differentiation. In most cases, the activation of cell proliferation is caused by an increased tyrosine kinase activity of specific proteins. However, patients' molecular heterogeneity and the incomplete clinical responses observed in part of the cases using the current treatments suggest that additional mechanisms, such as unknown protein interactions and new mutations, can be involved in the pathophysiology of MPN. In this study, our main goal was to investigate tyrosine kinase activation pathways in polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) (subproject 1), and in systemic mastocytosis (subproject 2). The focus of subproject 1 consisted in the investigation of JAK2/IRS2 association in MPN, already described in non-hematological cells following extrinsic stimulus, and in evaluating IRS2 function in MPN cell proliferation and apoptosis. JAK2 wild-type (JAK2WT) and JAK mutated (JAK2V617F) human leukemia cell lines were transduced with lentivirus-mediated shRNA targeting IRS2, and treated with vehicle (DMSO) or with the selective JAK1/2 inhibitor ruxolitinib. Cells were then submitted to evaluation of cell viability (MTT) and apoptosis (anexin V/PI and caspase-3 by flow cytometry, and cleaved caspase-3 by immunoblotting). IRS2 mRNA expression was evaluated by real time quantitative PCR in CD34+ peripheral blood cells of 99 patients with PV, ET and PMF, and in 28 healthy donors. Through immunoprecipitation/immunobloting and confocal microscopy, we observed the constitutive JAK2/IRS2 association in JAK2V617F cells, but not in JAK2WT cell lines. In JAK2V617F, IRS2 silencing significantly decreased phospho-STAT5, reduced cell viability, induced apoptosis, and potentiated the effects of ruxolitinib treatment. No differences in cell viability and apoptosis ratios were observed in IRS2 silenced JAK2WT cells. IRS2 mRNA expression was significantly higher in ET patients when compared to healthy donors, and in patients harboring JAK2V617F mutation in relation to JAK2WT. These evidence suggest that IRS2 participate in MPN cell signaling pathways through its interaction with JAK2. IRS2 pharmacological inhibition, alone or in combination with ruxolitinib, may be a potential tool in the treatment of PV, ET and PMF patients. In subproject 2, our main goal was to seek for KIT mutations in a case of systemic familial mastocytosis, followed in our outpatient clinics, in which the mother (case 1) and the daughter (case 2) had extensive skin and bone marrow infiltration by mast cells. Also, we aimed to evaluate in vitro sensitivity of neoplastic mast cells to the treatment with the tyrosine kinase inhibitors (TKI) imatinibe, dasatinibe and PKC412. Using Sanger sequencing analysis, we identified the KITK509I mutation in total bone marrow, peripheral blood CD3+ and oral mucosa cells in both patients. The parents of case 1 had wild type KIT. The in vitro treatment of total bone marrow cells of cases 1 and 2 with TKI for 4, 8 and 12 days resulted in reduced cell viability, as evaluated by MTT, and in reduced phosphorylation of P70S6K for all tested drugs. However, only imatinibe consistently induced apoptosis in both cases. Patients were started on imatinibe 400mg orally per day. Three months following imatinibe treatment, there was a complete reversion of skin and bone marrow mast cells infiltration; after two years of follow-up, cases 1 and 2 remain in complete remission of the systemic mastocytosis. Although rare, KITK509I mutation should be investigated in all cases of familial systemic mastocytosis. Imatinibe is a good first choice for the treatment of these cases / Doutorado / Fisiopatologia Médica / Doutora em Ciências

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.