B cell development is a carefully orchestrated process involving many transcription factors acting in concert with cytokine signals, particularly IL-7. The transcription factor c-Myb has long been implicated in B cell development, however surprisingly little is known about the function of c-Myb in B cell progenitors. I have used several mouse models of c-Myb deficiency to investigate the role of c-Myb in the B cell lineage. Conditional deletion of c-Myb in early B cell progenitors using mb-1Cre (c MybΔmb1/Δmb1) leads to a striking lack of B cells from the pre-pro-B cell stage onwards, demonstrating that c-Myb is absolutely required for B cell development. Mice homozygous for a hypomorphic allele of c-Myb (c MybPlt4/Plt4) also display a severe reduction in B cells; in these mice, defects in lymphoid development can be detected within the multipotent progenitor compartment of bone marrow. c-Myb activates transcription via coactivator proteins, particularly CBP and p300. Mice bearing a point mutation in p300 (p300Plt6/Plt6) that inhibits the interaction of p300 with c Myb display a partial block in B cell development, highlighting the importance of the c Myb-p300 complex for B cell development. Together, these mice demonstrate that c-Myb regulates B cell development by functioning both in multipotent progenitor cells and directly in B cell progenitors. In addition, I show that the B-lymphopenia in c-Myb deficient mice is related to a profound defect in IL-7 signalling. IL-7 normally stimulates the proliferation, survival and differentiation of B cell progenitors, however pro-B cells from c-MybPlt4/Plt4 and c MybΔmb1/Δmb1 mice fail to respond to IL 7. Expression of the IL-7Rα chain is reduced on pro-B cells from c MybPlt4/Plt4 and c-MybΔmb1/Δmb1 mice, suggesting that Il7r may be a c-Myb target gene in B cells. Reporter gene assays show that c-Myb can activate the Il7r promoter in synergy with the transcription factor Pu.1. Overall, this work demonstrates that c-Myb is essential for early B cell development and plays a critical role in linking cytokine signals to the transcription factor networks in B cell progenitors.

Thesis (Ph.D.)--University of Adelaide, Dept. of Orthopaedics Surgery and Trauma, 1998. / Bibliography: leaves 152-223.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1999? / Amendments to thesis in pocket on back cover. Copy of author's previously published article in pocket on back cover. Bibliography: leaves 124-172.

Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2003. / "June 2003" Errata slip inserted in back. Includes bibliographical references (leaves 268-300). Also available online.

Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2003. / Title from screen page; viewed 8 Feb 2005. "June 2003" Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 156-169) Also available in print.

Bibliography: leaves 152-223. Experiments were designed to identify and purify human bone marrow stromal precursor cells by positive immunoselection, based on the cell surface expression of the VCAM-1 and STRO-1 antigens. The data presented demonstrates a hierarchy of bone cell development in vitro.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T12:54:32Z (GMT). No. of bitstreams: 1 Monte-Mor_BarbaradaCostaReis_D.pdf: 7171978 bytes, checksum: 5c73cb6c7edd6138186b17d972fce769 (MD5) Previous issue date: 2007 / Resumo: Policitemia vera (PV) é uma síndrome mieloproliferativa (SMP) crônica que ocorre por proliferação clonal de progenitores hematopoéticos multipotenciais. Pacientes com PV apresentam expansão das três principais linhagens mielóides na medula óssea, levando à produção aumentada de hemácias, granulócitos e plaquetas. Características importantes de PV incluem elevação de hematócrito em presença de níveis normais ou diminuídos de eritropoetina (EPO) e a formação de colônias eritróides endógenas (CEE). A mutação JAK2 V617F, presente na maioria dos pacientes com PV, causa ativação constitutiva de JAK2 e parece ser responsável pelo fenótipo observado. Apesar disso, as mudanças transcricionais desencadeadas pela mutação ainda não foram completamente caracterizadas. Neste trabalho, utilizou-se Serial Analysis of Gene Expression (SAGE) para realizar um estudo de expressão gênica global em células da medula óssea de um paciente com PV portador da mutação, ao diagnóstico e em células normais de doadores saudáveis. Genes com expressão aumentada em PV estão envolvidos em processos biológicos importantes, como transdução de sinal, diferenciação e proliferação celular, ciclo celular, apoptose, resposta imune e regulação da transcrição. JUNB foi um dos genes identificados e, usando reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR), detectou-se expressão aumentada de JUNB em dois outros pacientes com PV JAK2 V617F-positivos. Por meio de linhagens murinas Ba/F3-REPO e culturas primárias de eritroblastos humanos, observou-se que JUNB é expresso após adição de EPO e que a proteína JunB é constitutivamente induzida por JAK2 V617F. Além disso, interferência na expressão de JUNB diminuiu o potencial clonogênico e proliferativo de progenitores eritróides humanos Ainda, em PV, a eritropoese causada por JAK2 V617F mostrou-se mais sensível à diminuição de expressão de JunB do que a eritropoese normal. Assim, esses resultados sugerem que JunB tenha um papel fundamental no desenvolvimento de SMPs causadas por JAK2 V617F / Abstract: Polycythemia vera (PV) is a chronic myeloproliferative disorder that arises through clonal proliferation of multipotent hematopoietic progenitors. PV patients present bone marrow trilineage expansion, leading to increased production of mature red cells, granulocytes and platelets. Important PV features are elevated red cell mass, despite normal or subnormal erythropoietin (EPO) levels, and endogenous erythroid colony (EEC) formation. The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes a JAK2 constitutive activation and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. Serial analysis of gene expression (SAGE) was used to perform a large scale gene expression study in bone marrow cells of a newly-diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells from healthy donors. Genes overexpressed in PV are involved in important biological processes, such as signal transduction, cellular differentiation, cellular proliferation, cell cycle, apoptosis, immune response and transcriptional regulation. JUNB was one of the genes up-regulated in PV and overexpression of JUNB was also detected by quantitative real time polymerase chain reaction (qRT-PCR) in hematopoietic cells of another two JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, JUNB was found to be expressed after Epo addition and JunB protein was shown to be constitutively induced by JAK2 V617F. In addition, JUNB knock down reduced the clonogenic and proliferative potential of human erythroid progenitors. Furthermore, in PV, the JAK2 V617F driven erythropoisis was more sensitive to JunB downregulation than normal erythropoiesis. Hence, these results demonstrate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

O aumento da adiposidade corpórea pode gerar diversos mediadores inflamatórios com capacidade de influenciar a proliferação e a diferenciação hematopoética e, consequentemente, a complexa regulação da hematopoese. Por isso, propusemo-nos, neste trabalho, avaliar a influência do aumento da adiposidade corpórea sobre a proliferação e a diferenciação de células hematopoéticas, bem como sua capacidade em sintetizar citocinas. Ratos Wistar, machos foram alimentados com uma dieta rica em lipídios durante 14 semanas. Após esse período foram avaliados hemograma, mielograma, perfil lipídico, concentrações séricas de leptina, insulina e adiponectina. Citômetria de fluxo foi utilizada para avaliação da porcentagem de células CD34+/CD133+, bem como o ciclo celular de células medulares. Células medulares foram utilizadas para avaliar a atividade proliferativa in vitro e a capacidade de diferenciação, in vitro, na presença de IL-3, EPO, GM-CSF e G-CSF. Animais, alimentados com dieta hiperlipídica, apresentaram maiores concentrações de leptina circulante, com aumento de gordura corporal, aumento da concetração de proteína C reativa, colesterol total, LDL, VLDL e triacilglicerol. O hemograma apresentou neutrofilia absoluta e a medula óssea apresentou-se hipercelular com aumento do número de granulócitos maduros e da população celular CD133-/CD34+. Os resultados dos testes in vitro demonstraram aumento da capacidade de síntese de IL-3 e aumento de G-CSF, com aumento do potencial proliferativo, também evidenciado pelo maior número de células medulares na fase S/G2/M, bem como o aumento da diferenciação granulocítica. Esses resultados sugerem que a leucocitose e neutrofilia observadas em situações de aumento da adiposidade corpórea são decorrentes de uma complexa modulação do sistema hematopoético. / The body fat increase can generate various inflammatory mediators, that are capable to influence the proliferation and differentiation of hematopoietic cells and consequently modulate the complex regulation of the hematopoiesis. In this study we have proposed to evaluate the effect of increase body fat on the proliferation and differentiation of hematopoietic cells, as well as its ability to synthesize cytokines. Male Wistar rats were subjected to a high fat diet during a period of 14 weeks. After that period were evaluated hemogram, mielogram, lipid profile and the serum concentrations of leptin, insulin and adiponectin. Flow cytometry was used to evaluate the percentage of CD34+/CD133+, as well as the cell cycle of bone marrow cells. Bone marrow cells were used to perform the proliferation and differentiation capacity in vitro in the presence of IL-3, EPO, GM-CSF and G-CSF. Animals fed high-fat diet had higher concentrations of circulating leptin with increase body fat, and increase of C-reactive protein, total cholesterol, LDL, VLDL and triacylglycerol concentrations. The hemogram showed absolute neutrophilia and a hypercellular bone marrow with increase of granulocytic mature population and CD133-/CD34+ cells. The results in vitro, showed an increase of IL-3 and G-CSF production, and higher proliferative potential with an increase in S/G2/M bone marrow cell cycle phases, as well as an increase of the granulocytic differentiation. The results suggest that leukocytosis and neutrophilia observed in this model of body fat increase are in fact a result of a complex modulation of the hematopoietic system.

A leishmaniose visceral (LV) à um sÃrio problema de saÃde pÃblica na AmÃrica Latina e vem apresentando uma letalidade crescente no Brasil. à uma doenÃa infecciosa sistÃmica que envolve a medula Ãssea, causando vÃrias manifestaÃÃes hematolÃgicas. Objetivos: Descrever as caracterÃsticas da medula Ãssea na LV e correlacionar os achados citomorfolÃgicos com parÃmetros do sangue perifÃrico e com os sinais de gravidade da doenÃa. Metodologia: Trata-se de um estudo retrospectivo, com levantamento dos dados clÃnicos e laboratoriais de pacientes atendidos em trÃs hospitais de referÃncia no Estado do CearÃ, entre junho de 2001 a agosto 2010. Foram incluÃdos 126 pacientes com diagnÃstico de LV, confirmado pela detecÃÃo do parasito na medula Ãssea e/ou pela sorologia positiva, atravÃs do rK39. As lÃminas dos mielogramas de todos os pacientes foram avaliadas e a carga parasitÃria e os parÃmetros hematolÃgicos foram determinados. A gravidade foi determinada pelo risco de Ãbito, utilizando sistema de escores. Resultados: A idade variou de 5 meses a 79 anos e 74% eram do gÃnero masculino. Os pacientes residentes em regiÃo urbana representaram 68,2% da casuÃstica. Comorbidades estavam associadas em 31,7% dos pacientes. Os achados mais frequentes da medula Ãssea foram carga parasitÃria moderada/alta (57,2%), displasia com predomÃnio de diseritropoese (80,9%), hemofagocitose (30,1%) e granuloma (22,2%). Houve uma associaÃÃo positiva entre a carga parasitÃria com neutropenia grave, neutrÃfilos <500/mm3 (p=0,04) e hemofagocitose (p=0,05). No presente trabalho, utilizando o modelo de prognÃstico baseado em escores, foi observado que 23,8% foram classificados como pacientes com alto risco de Ãbito (&#8805; 3). A maioria dos pacientes com escore considerado de alto risco de Ãbito apresentou medula Ãssea normocelular, carga parasitÃria moderada/alta, diseritropoese e disgranulopoese. Em relaÃÃo à presenÃa de granuloma, houve uma associaÃÃo negativa, estatisticamente significante, nos pacientes com alto risco de Ãbito (p=0,02). O estudo aponta para possÃveis indicadores diagnÃsticos e de prognÃsticos identificÃveis na medula Ãssea de pacientes com LV / The visceral leishmaniasis (VL) is a serious problem of public health in the Latin America and has shown an increase mortality in the Brazil. This is a systemic infectious disease involving the bone marrow, causing many hematologic manifestations. Objetives: to describe LV bone marrow features and correlate cytomorphological findings with peripheral blood parameters and the disease severity: Methodology: It is a retrospective study with gathering clinical and laboratory data of patients attended at three references hospital of Cearà State between june 2001 and August 2010. Were included 126 patients with a LV diagnosis confirmed by detection of the parasite in bone marrow and/or by positive sorology, by rK39. The slides of bone marrow aspiration of all patients were evaluated and the parasite load and the hematologic parameters were determined.The gravity was determined by the risk of death using the scoring system. Results: The age ranged from 5 months to 79 years and 74% were male. The patients from urban area accounted 68,2%. Comorbidities were associated in 31,7% patients. The more frequently finding of bone marrow were moderate/high parasite load (57,2%), dysplasia with predominance of dyserythropoiesis (80,9%), hemophagocytosis (30,1%) and granuloma (22,2%).There was one positive association between parasite load and serious neutropenia, neutrophil <500/mm3 (p=0,04), and hemophagocytosis (p=0,05). In this present study, using the model predictions based on scores was observed that 23,8% were classified as high risk of death. Most patients with a score considered with high risk of death had normocelular bone marrow, parasite load moderate/high, dyserythropoiese and dysgranulopoiese. In relation to the presence of granuloma there was a negative association statistically significant in the patients with high risk of death (p=0,02). The study points to possible diagnostic and prognostic indicators identified in the bone marrow of patients with VL