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.

Chan Sin-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 154-169). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Contents --- p.viii / Abbreviations --- p.xiii / List of Figures and Tables --- p.xvi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Ontogeny of hematopoiesis --- p.1 / Chapter 1.1.2 --- Hiercharay of hematopoiesis --- p.2 / Chapter 1.2 --- Regulation of hematopoiesis --- p.5 / Chapter 1.2.1 --- Bone marrow stromal cell --- p.5 / Chapter 1.2.2 --- Hematopoietic growth factor --- p.6 / Chapter 1.2.3 --- Hematopoietic growth factor receptors and signal transduction --- p.10 / Chapter 1.2.4 --- Transcriptional regulation of myeloid cell development --- p.11 / Chapter 1.3 --- Deregulated hematopoiesis - Leukemia --- p.20 / Chapter 1.3.1 --- Classification of leukemia --- p.20 / Chapter 1.3.2 --- Molecular basis of leukemia --- p.20 / Chapter 1.4 --- Prostaglandin E2 induced WEHI-3B JCS and JCS-14 cell differentiation --- p.22 / Chapter 1.4.1 --- Induced leukemia cell differentiation --- p.22 / Chapter 1.4.2 --- Inducer of cell differentiation - Prostaglandin E2 --- p.22 / Chapter 1.4.3 --- WEHI-3B JCS and subline JCS-14 cells --- p.24 / Chapter 1.5 --- The aims of study --- p.26 / Chapter Chapter Two --- Identification of differentially expressed genes during PGE2-induced WEHI-3B JCS-14 cell differentiation / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Strategy for studying PGE2-induced JCS-14 cell differentiation --- p.28 / Chapter 2.1.2 --- Method for studying differential gene expression: Microarry Technology --- p.29 / Chapter 2.2 --- Materials --- p.32 / Chapter 2.2.1 --- Cell line --- p.32 / Chapter 2.2.2 --- AtlasT M Mouse cDNA Expression Array --- p.32 / Chapter 2.2.3 --- Chemicals --- p.32 / Chapter 2.2.4 --- Solutions and buffers --- p.33 / Chapter 2.2.5 --- Reagents --- p.34 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Morphological study of PGE2-induced JCS-14 cell differentiation --- p.35 / Chapter 2.3.2 --- Preparation of total RNA from PGE2-induced JCS-14 cells --- p.35 / Chapter 2.3.2.1 --- Preparation of cell lysates --- p.35 / Chapter 2.3.2.2 --- Isolation of total RNA --- p.35 / Chapter 2.3.3 --- Preparation of cDNA probes --- p.36 / Chapter 2.3.3.1 --- Probe synthesis from total RNA --- p.36 / Chapter 2.3.3.2 --- Purification of the labeled cDNA probes --- p.37 / Chapter 2.3.4 --- Hybridization cDNA probes to the Atlas Array and stringency wash --- p.37 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Morphological changes in PGE2-treated JCS-14 cells --- p.39 / Chapter 2.4.2 --- Analysis of total RNA from PGE2-induced JCS-14 cells --- p.43 / Chapter 2.4.3 --- Hybridization of cDNA probes to AtlasT M cDNA Expression Array --- p.45 / Chapter 2.5 --- Discussion --- p.73 / Chapter 2.5.1 --- Morphological study of JCS-14 cell differentiation --- p.73 / Chapter 2.5.2 --- Differentiation commitment of JCS-14 cell under PGE2 induction --- p.73 / Chapter 2.5.3 --- Gene expression profile by microarray --- p.74 / Chapter 2.5.4 --- Gene expression profile of 5 hours PGE2-induced JCS-14 cells --- p.74 / Chapter 2.5.5 --- Further analysis of regulatory genes in PGE2-induced JCS-14 cell differentiation --- p.77 / Chapter Chapter Three --- Expression profile of identified genes in WEHI-3B JCS-14 and JCS cell differentiation / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.1.1 --- Quantitation of mRNA by Real time RT-PCR --- p.80 / Chapter 3.1.2 --- Relative quantitation of gene expression --- p.83 / Chapter 3.2 --- Materials --- p.85 / Chapter 3.2.1 --- Cell lines --- p.85 / Chapter 3.2.2 --- SYBR® Green PCR core kit --- p.85 / Chapter 3.2.3 --- Chemicals --- p.85 / Chapter 3.2.4 --- Solutions and buffers --- p.86 / Chapter 3.2.5 --- Enzymes and nucleic acids --- p.86 / Chapter 3.3 --- Methods --- p.88 / Chapter 3.3.1 --- Preparation of total RNA from PGE2-induced JCS-14 and JCS cells --- p.88 / Chapter 3.3.1.1 --- Preparation of cell lysates --- p.88 / Chapter 3.3.1.2 --- Isolation of total RNA --- p.88 / Chapter 3.3.2 --- Reverse transcription (RT) --- p.88 / Chapter 3.3.3 --- Design of real-time PCR primers --- p.88 / Chapter 3.3.4 --- Determination of relative efficiency of target and reference amplification by validation experiment --- p.89 / Chapter 3.3.5 --- Confirmation of expression profile of identified genes in JCS-14 and JCS cells by comparative CT method in real-time PCR --- p.90 / Chapter 3.4 --- Results --- p.91 / Chapter 3.4.1 --- Analysis of total RNA from PGE2-induced JCS-14 and JCS cells --- p.91 / Chapter 3.4.2 --- Validation experiment of real-time PCR primers --- p.93 / Chapter 3.4.3 --- Expression profile of specific genes in JCS-14 and JCS cells by comparative CT method --- p.101 / Chapter 3.5 --- Discussion --- p.114 / Chapter 3.5.1 --- Study of gene expression profiles in JCS-14 and JCS cell differentiation --- p.114 / Chapter 3.5.2 --- Transcription analysis by real-time PCR --- p.114 / Chapter 3.5.3 --- Gene expression profiles during PGE2-induced JCS-14 and JCS cell differentiation --- p.115 / Chapter Chapter Four --- Inhibition of specific gene expression in WEHI-3B JCS-14 and JCS cells using antisense blocking technique / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.1.1 --- Antisense technique --- p.122 / Chapter 4.1.2 --- Design of antisense oligonucleotides --- p.125 / Chapter 4.1.3 --- Transfer of oligonucleotides to cells --- p.128 / Chapter 4.2 --- Materials --- p.129 / Chapter 4.2.1 --- Cell lines --- p.129 / Chapter 4.2.2 --- Chemicals --- p.129 / Chapter 4.2.3 --- Reagents --- p.129 / Chapter 4.2.4 --- Solutions --- p.129 / Chapter 4.3 --- Methods --- p.131 / Chapter 4.3.1 --- Design of antisense oligonucleotides --- p.131 / Chapter 4.3.2 --- Transfection of oligonucleotides into cells --- p.134 / Chapter 4.3.3 --- Morphological study of PGE2-induced JCS-14 and JCS cells --- p.134 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- Effect of antisense oligonucleotides on JCS-14 cell differentiation --- p.135 / Chapter 4.4.2 --- Effect of antisense oligonucleotides on JCS cell differentiation --- p.136 / Chapter 4.5 --- Discussion --- p.146 / Chapter 4.5.1 --- Effects of antisense B-myb on JCS-14 and JCS cell differentiation --- p.146 / Chapter 4.5.2 --- Effects of antisense thyroid hormone receptor (c-erbA) and transcription terminator factor (TTF-1) on JCS-14 and JCS cell differentiation --- p.147 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Introduction --- p.148 / Chapter 5.2 --- Differentiation program triggered by Prostaglandin E2 --- p.148 / Chapter 5.2.1 --- Lineage preference during differentiation --- p.148 / Chapter 5.2.2 --- Differentially expressed genes during PGE2-induced JCS-14 cell differentiation --- p.149 / Chapter 5.2.3 --- Expression patterns of the three differentially expressed genes in PGE2-induced JCS-14 and JCS cells --- p.149 / Chapter 5.2.4 --- Antisense blocking during differentiation --- p.151 / Chapter 5.3 --- Further studies --- p.152 / References --- p.154

Styelid ascidians are diverse in developmental modes, varying from strictly sexual solitary species to highly integrated colonies. Circulatory stem cells (CSCs) accomplish fundamental roles in developmental processes of styelid ascidians. In the colonial styelids, CSCs enable budding and are capable of giving origin to the germline in certain species. The function of these cells have been tested experimentally in models within Styelidae. However, the understanding of coloniality as an evolutionary novelty requires reconstructing the possible ancestral CSCs characteristics in Styelidae. To address this issue, this work analyzes the possible developmental origin and the identity of putative CSCs among blood cell populations. The first chapter of this dissertation aimed to characterize and compare the hemocyte populations in two solitary styelids: Styela plicata and Styela canopus. In addition, the early development, the metamorphosis and the early maturation were compared in both species. After metamorphosis, S. canopus briefly develops a network of extracorporeal vessels with numerous terminal ampullae. These characters are usually associated to colonial ascidians, and were not found in S. plicata. With respect to the hemocyte populations, similar morphotypes were present in both species. However, S. canopus shows a lower frequency of vacuolated cells, which may be due to a reduced level of cytotoxicity in the tunic relative to S. plicata. These differences observed between S. canopus and S. plicata may be related to differences in the degrees of gregariousness or body size among the two species. In order to investigate possible approaches to distinguish and isolate CSC populations in a solitary styelid model, I used imaging flow cytometry. Putative CSCs were identified through measurement of morphological parameters and aldehyde dehydrogenase (ALDH) activity. The correlation between these parameters allowed to determine 2 gates enriched with particular cell types. A significant difference was found on the ALDH+ population within a gate of cells with low granularity, suggesting the presence of cells among circulatory hemocytes. To scrutinize the biogenesis of CSCs in S. plicata, I present a description of a candidate hematopoietic niche in this species. An exhaustive histological survey for hemoblast-like cells was performed, and complemented with immunohistochemistry with stem cell (piwi) and proliferation (pHH3) markers. The morphological and expression profiles of the intestine support the intestinal submucosa (IS) as a hematopoietic niche. At this region there are aggregations of cells with and undifferentiated morphological profile, corroborated by ultrastructural analysis. Furthermore, the IS holds high cellular proliferation and frequency of piwi+ cells. Ascidians are considered interesting models to investigate asexual reproduction and modular development. This study represents an advancement towards understanding the processes, cell populations and structures that may be related to facilitating the appearance of this evolutionary novelty / As ascídias da família Styelidae são diversas em modos de desenvolvimento, variando de espécies estritamente sexuais solitárias até colônias altamente integradas. As células-tronco circulatórias (CTCs) desempenham papéis fundamentais nos processos do desenvolvimento de ascídias styelídeas. Nas especies coloniais deste grupo, as CTCs permitem a brotação e são capazes de originar a linha germinativa em certas espécies. A função dessas células tem sido testada experimentalmente em modelos dentro de Styelidae. No entanto, a compreensão da colonialidade como uma novidade evolutiva requer reconstruir as características das possíveis CTCs ancestrais para Styelidae. Com o fim de abordar essa questão, este trabalho analisa a possível origem do desenvolvimento e a identidade de CSCs putativas entre populações de células sanguíneas de styelídeas solitárias. O primeiro capítulo desta dissertação teve como objetivo caracterizar e comparar as populações de hemócitos em dois espécies solitárias: Styela plicata e Styela canopos. Além disso, o desenvolvimento inicial, a metamorfose e a maturação do juvenil foram comparados em ambas as espécies. Após a metamorfose, S. canopus desenvolve brevemente uma rede de vasos extracorpóreos com numerosas ampolas terminais. Esses caracteres são geralmente associados a ascídias coloniais e não foram encontrados em S. plicata. Com relação às populações de hemócitos, morfotipos semelhantes estavam presentes em ambas as espécies. No entanto, o S. canopos apresenta menor frequência de células vacuoladas, o que pode ser devido a um nível reduzido de citotoxicidade na túnica em relação a S. plicata. Essas diferenças observadas entre S. canopos e S. plicata podem estar relacionadas a diferenças nos graus de gregariedade ou tamanho corporal entre as duas espécies. A fim de investigar possíveis abordagens para distinguir e isolar populações de CTCs em um modelo de styelídeo solitário, usei citometria de fluxo com adquisição de imagem. As CTCs putativas foram identificadas através da medição de parâmetros morfológicos e da atividade da aldeído desidrogenase (ALDH). A correlação entre estes parâmetros permitiu determinar 2 gates enriquecidos com tipos celuláres particulares. Uma diferença significativa foi encontrada na população ALDH+ dentro de um gate de células com baixa granularidade, sugerindo a presença de células-tronco circulatórias. Para examinar a biogênese das CTCs em S. plicata, foi realizada uma descrição de um nicho hematopoiético candidato nesta espécie. Um exame histológico exaustivo para células semelhantes a hemoblastos foi realizado e complementado com imunohistoquímica com marcadores de células-tronco (piwi) e proliferação (pHH3). Os perfis morfológicos e de expressão do intestino sustentam a submucosa intestinal (SI) como nicho hematopoiético. Nesta região há agregações de células com morfolia indiferenciada, corroborada pela análise ultraestrutural. Além disso, a SI mantém alta proliferação celular e freqüência de células piwi+. As ascídias são consideradas modelos interessantes para investigar a reprodução assexuada e o desenvolvimento modular. Este estudo representa um avanço na compreensão dos processos, populações celulares e estruturas que podem estar relacionadas a facilitar o surgimento desta novidade evolutiva

Dans tous les organismes animaux la réponse immunitaire est divisée en deux composantes : la réponse humorale, qui consiste en la production d'un grand nombre de molécules toxiques pour le pathogène, et la réponse cellulaire, qui met en jeu des cellules immunitaires produites lors de l'hématopoïèse. Chez les mammifères adultes, l'hématopoïèse se déroule dans la moelle osseuse, où un microenvironnement particulier appelé " niche hématopoïétique " contrôle l'auto-renouvèlement, la prolifération et la différenciation des Cellules Souches Hématopoïétiques (CSH) à l'origine de l'ensemble des cellules sanguines/immunitaires. Suite à une infection par un pathogène, l'homéostasie du système hématopoïétique est modifiée, afin de permettre la mise en place d'une réponse immunitaire cellulaire adaptée. Le rôle de la niche hématopoïétique dans le contrôle de l'hématopoïèse suite à une infection reste à ce jour mal connu. La drosophile est utilisée comme système modèle pour étudier in vivo l'hématopoïèse et la réponse immunitaire. L'hématopoïèse a lieu chez la drosophile au stade larvaire dans un organe spécialisé appelé Glande Lymphatique (GL). Au sein de cet organe, un petit groupe de cellules, le Centre de Signalisation Postérieur (PSC), contrôle l'équilibre entre progéniteurs hématopoïétiques et cellules immunitaires différenciées, et a donc un rôle équivalent à celui de la niche hématopoïétique des mammifères. Suite à un stress immun, tel que le parasitisme par des guêpes, une différenciation massive de cellules immunitaires spécifiques, les lamellocytes, a lieu dans la GL; puis la dispersion de la GL permet la libération des lamellocytes dans la circulation lymphatique. Lors du parasitisme, la guêpe pond un œuf dans le corps de la larve de drosophile. En absence de réponse immunitaire cellulaire, l'œuf de guêpe se développe au dépend de son hôte, entraînant sa mort. En formant une capsule autour de l'œuf de guêpe, les lamellocytes neutralisent son développement et permettent la survie de l'hôte. Au cours de ma thèse, je me suis intéressée à la réponse immunitaire cellulaire de la larve de drosophile au parasitisme par des guêpes. Je me suis plus particulièrement intéressée au rôle de la " niche hématopoïétique " dans cette réponse. Pour cela, j'ai initié une approche transcriptomique ayant pour but d'identifier les gènes spécifiquement exprimés dans le PSC en réponse au parasitisme. En parallèle, j'ai caractérisé le rôle de la voie de signalisation Toll/NF?B dans la GL lors de la réponse au parasitisme. La voie Toll/NF?B joue un rôle essentiel dans la réponse immunitaire humorale et son rôle dans la réponse immunitaire cellulaire reste à définir. Mes travaux indiquent que la voie Toll/NF?B est activée dans le PSC suite au parasitisme. Son activation est médiée par le facteur de transcription NF?B "Dorsal-related Immunity Factor" (Dif), qui est requis dans le PSC pour permettre la différenciation rapide et massive de lamellocytes et la dispersion des cellules de la GL. De plus, j'ai établi un réseau génique, impliquant les deux voies de signalisation Toll/NF?B et EGFR ainsi que les espèces réactives de l'oxygène (ROS) dans le contrôle de la réponse au parasitisme. Une augmentation du niveau de ROS dans le PSC et l'activation de la voie EGFR dans les cellules immunitaires ont été décrits comme nécessaires à l'encapsulation des œufs de guêpe après parasitisme. Mes données établissent qu'ils sont en plus requis respectivement dans les cellules du PSC et dans les progéniteurs hématopoïétiques pour permettre la dispersion de la GL après parasitisme. Basé sur la forte conservation des voies de signalisation et processus moléculaires contrôlant l'hématopoïèse entre les mammifères et la drosophile, mes résultats posent la question de la conservation du réseau génique établi chez la drosophile et du rôle de la voie NF?B dans la niche hématopoïétique des mammifères lors d'une réponse à une infection. / In all organisms, the immune response is divided into two parts: the humoral response, which consists of producing a large number of molecules to combat the pathogen, and the cellular response, which relies on immune cells produced during hematopoiesis. In adult mammals, hematopoiesis occurs in the bone marrow, where a particular microenvironment called the "hematopoietic niche" controls self-renewal, proliferation and differentiation of Hematopoietic Stem Cells (HSCs), which give rise to all blood cell types. Following a pathogenic infection, the hematopoietic system's homeostasis is modified in order to obtain an adapted cellular immune response. The role that the hematopoietic niche plays during an immune response remains unclear. Drosophila is used as a model system to study in vivo hematopoiesis and the immune response. In drosophila, hematopoiesis occurs at the larval stage in a specialized organ called the Lymph Gland (LG). Within this organ, a small group of cells termed the Posterior Signalling Center (PSC), controls the balance between hematopoietic progenitors and differentiated immune/blood cells, a role similar to the mammalian hematopoietic niche. Following an immune challenge, especially in response to wasp parasitism, a massive differentiation of specific immune cells called lamellocytes occurs in the LG. The LG subsequently disperses to release lamellocytes into the hemolymph. During parasitism, the wasp lays an egg in the drosophila larva. In the absence of a cellular immune response, the wasp egg will develop and kill its host. By forming a capsule around the wasp egg, lamellocytes impede the pathogen's development and permit the host's survival. During my PhD, I studied the drosophila larva cellular immune response to wasp parasitism. I focused my research on the role of the "hematopoietic niche". I therefore initiated a transcriptomic study, in order to identify genes expressed by the PSC in response to parasitism. In parallel, I characterized the role of the Toll/NF?B signalling pathway in the LG during parasitism. The Toll/NF?B pathway plays a key role in the humoral response both in drosophila and mammals; however its role in the cellular immune response remains unknown. My results indicate that the Toll/NF?B pathway is activated in the PSC following parasitism. Its activation is mediated by the NF?B transcription factor " Dorsal-related Immunity Factor " (Dif), which is required in the PSC for rapid lamellocyte production and LG dispersion. Furthermore, I established the existence of a genetic network comprising the Toll/NFkB and EGFR signalling pathways and Reactive Oxygen Species (ROS), in order to control the immune response to parasitism. An increase in ROS levels in the PSC and EGFR pathway activation in the immune cells, have been described as required for wasp egg encapsulation. My data suggest that the ROS and the EGFR pathway are also required for LG dispersion following wasp parasitism, in PSC cells and in hematopoietic progenitors, respectively. Based on the high conservation of signalling pathways and molecular processes controlling hematopoiesis, my results raise the question of whether such a network is conserved in the mammalian hematopoietic niche in response to pathogenic infections.

Understanding the factors that regulate the growth and differentiation of haemopoietic stem cells (HSC) remains a major challenge. In this study, the proliferation and differentiation of CD34+ cells from normal donors and chronic myeloid leukaemia (CML) patients was compared. The proliferation and entry of CML cells into the cell cycle was decreased relative to cells from normal donors, and greater heterogeneity in the phenotype of CML cells at the initiation of culture was observed. Analysis of phenotype concomitant with cell division also demonstrated that the differentiation of normal CD34+ cells was consistent between donors, while marked variability was observed in the differentiation of CD34+ cells from CML patients. This included expression of CD13, CD33, CD38 and HLA-DR, which were linked to cell division in normal but not CML cells. The tyrosine kinase inhibitor, imatinib, is a novel drug displaying promising results in the treatment of CML by specifically inhibiting the growth of leukaemic cells. To examine whether myelosuppression observed in patients treated with imatinib may arise from inhibition of normal haemopoiesis, imatinib was added to colony assays established using cells from normal bone marrow. Suppression of monocyte/macrophage growth, but not that of eosinophils or neutrophils, was observed at therapeutic concentrations of imatinib. Inhibition of monocytic differentiation to macrophages was also observed and was associated with decreased functional capacity such as altered antigen uptake, production of proinflammatory cytokines and stimulation of responder cells. The specific suppression of monocyte/macrophage differentiation and function was not due to blockade of tyrosine kinases known to be inhibited by imatinib and was consistent with an inhibition of the M-CSF/c-fms signalling pathway. This hypothesis was tested using a cell line that was dependent on M-CSF for growth and survival. Cell proliferation and phosphorylation of c-fms were inhibited at an IC50 of 1.9μM and 1.4μM imatinib respectively and this was not attributable to decreased c-fms expression. These important findings therefore identify c-fms as a further target of imatinib, and suggest that imatinib should be considered for treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. Potential side effects resulting from imatinib treatment must also be considered. / Thesis (Ph.D.)--School of Medicine, 2004.

CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Early T-cell development is regulated by a complex interplay between transcription factors and developmental cues which ensure that functional T-cells are produced within the thymus. Early thymocytes integrate these signals in a step-wise fashion that progressively restricts their lineage potential as they transition through the early stages of T-cell development. Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. Furthermore, many additional key regulators such as Notch1 have been identified, but the connections among them and their specific roles in early T-cell development have not been well established. In this thesis, I set out to determine the specific roles of HEB at the beta-selection checkpoint and to establish connections between HEB and the key regulators within the gene regulatory network that orchestrates early T-cell development. To facilitate these studies, I generated a series of new mouse models including HEBAlt transgenic mice that express a short form of HEB called HEBAlt, which enabled me to answer specific questions and examine rare populations. First, my studies of HEB-/- mice allowed me to identify an early block in T-cell development, which was alleviated upon the addition of an HEBAlt transgene. Furthermore, I identified pTa and CD3e signalling as specific targets of HEBAlt during -selection. Second, my studies on HEB-/- mice revealed that they have a defect in T-cell commitment, with compromised Notch1 function and a tendency to become DN1-like cells. Moreover, the DN1-like cells could be induced to differentiate into thymic NK cells, revealing a role for HEB in the T/NK cell lineage decision. This study has revealed a new set of interactions among HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. Unexpectedly, my studies have also provided evidence for a role of HEBAlt in lymphomagenesis, highlighting the strict regulation of E-protein function that is necessary to ensure normal T-cell development.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Early T-cell development is regulated by a complex interplay between transcription factors and developmental cues which ensure that functional T-cells are produced within the thymus. Early thymocytes integrate these signals in a step-wise fashion that progressively restricts their lineage potential as they transition through the early stages of T-cell development. Gene knockout studies have shown that the E-protein transcription factor HEB is required for normal thymocyte development. Furthermore, many additional key regulators such as Notch1 have been identified, but the connections among them and their specific roles in early T-cell development have not been well established. In this thesis, I set out to determine the specific roles of HEB at the beta-selection checkpoint and to establish connections between HEB and the key regulators within the gene regulatory network that orchestrates early T-cell development. To facilitate these studies, I generated a series of new mouse models including HEBAlt transgenic mice that express a short form of HEB called HEBAlt, which enabled me to answer specific questions and examine rare populations. First, my studies of HEB-/- mice allowed me to identify an early block in T-cell development, which was alleviated upon the addition of an HEBAlt transgene. Furthermore, I identified pTa and CD3e signalling as specific targets of HEBAlt during -selection. Second, my studies on HEB-/- mice revealed that they have a defect in T-cell commitment, with compromised Notch1 function and a tendency to become DN1-like cells. Moreover, the DN1-like cells could be induced to differentiate into thymic NK cells, revealing a role for HEB in the T/NK cell lineage decision. This study has revealed a new set of interactions among HEB, Notch1, and GATA3 that regulate the T-cell fate choice in developing thymocytes. Unexpectedly, my studies have also provided evidence for a role of HEBAlt in lymphomagenesis, highlighting the strict regulation of E-protein function that is necessary to ensure normal T-cell development.