Thesis (Ph.D.)--Ohio University, November, 2006. / Title from PDF t.p. Includes bibliographical references.

Negative regulatory factors inhibit the proliferation of haematopoietic stem cells thus protecting them from differentiation pressures. One of the negative regulators of stem cell proliferation is the tetrapeptide Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP). This peptide is endogenously produced in vivo and long term bone marrow cultures and is degraded by angiotensin 1-converting enzyme (ACE) both in vivo and in vitro. The aim of these investigations was to study the role of ACE on haematopoietic stem and progenitor cell proliferation. Since the N-domain ACE active has been implicated in AcSDKP degradation, an analysis of two ACE inhibitors (captopril and lisinopril) shown to have differential effects on the N-domain ACE active site was conducted. Both captopril and lisinopril equally reduced ACE activity in plasma in vitro. However, captopril had a lesser effect on reducing serum ACE activity in vitro than lisinopril. Captopril and AcSDKP together reduced the proportion of GM-CFC in S-phase after 7 hours of in vitro incubation. In addition, ACE resistant AcSDKP analogue (AcSDPψKP) when incubated with bone marrow cells in the absence of captopril also reduced the proportion of GM-CFC in S-phase. This finding suggest that the effect of captopril and AcSDKP on GM-CFC proliferation was due to AcSDKP alone. Haematopoietic stem cells were induced into cell cycle by in vivo administration of either 2 Gy-γ-irradiation or the two cytotoxic drugs, cytosine arabinoside (Ara-C) (100 mg/kg i.p.) or 5 flourouracil (5 FU) (150 mg/kg i.v). Bone marrow cells were sampled and incubated in vitro for up to 24 hours. Captopril together with AcSDKP reduced the proportion of high proliferative colony forming cells-1 (HPP-CFC-1) in S-phase following 2 Gy-γ-irradiation. Lisinopril together with AcSDKP had no such effect. In addition, captopril alone in vitro reduced the proportion of HPP-CFC-1 in S-phase induced into cell cycle by cytotoxic drugs. Lisinopril had no such effect. Incubation alone reduced the proportion of HPP-CFC-1 in S-phase in cytotoxic drug treated bone marrow cells. When cultures, which were incubated with captopril, were assayed for AcSDKP levels, captopril induced an increase in AcSDKP levels in both control normal bone marrow cells and cells derived from Ara-C treated mice. However, it did not affect AcSDKP levels in cultures derived from 5 FU and 2 Gy treated mice, AcSDKP together with captopril were shown to inhibit S-phase cell entry of HPP-CFC-1 when they were incubated with bone marrow cells derived from mice treated with either 2 Gy-γ-irradiation or cytotoxic drug insults. Interestingly, captopril was unable to reduce the proportion of SA2 leukaemic cells in S-phase Captopril on its own at therapeutic doses reduced the proportion of HPP-CFC- 1 in S-phase in vivo regardless of the insult used to induce HPP-CFC-1 into cell cycle. Lisinopril slightly reduced the proportion of HPP-CFC-1 in S-phase following Ai-a-C treatment only. Captopril induced an in vivo increase in AcSDKP levels in all the models tested. Captopril also reduced the proportion of HPP-CFC-1 and GM-CFC in S-phase following fractionated doses of Ara-C. Captopril's inhibitory effect on GM- CFC proliferation following fractionated dose of Ara-C was diminished after 7 days while it was sustained with HPP-CFC-1. Long-term bone marrow cultures revealed that captopril and AcSDxj/KP had the same effect on cellularities of both layers and on the proliferation of HPP-CFC and GM-CFC in both layers. From the present investigations, it can be concluded that captopril is a potent inhibitor of HPP-CFC-1 proliferation. This effect may in part be mediated by AcSDKP mechanism.

The description of the molecular events responsible for the control of cell division and differentiation is, currently, one of the major goals of molecular and cellular biologists. Cell and tissue culture techniques have proved to be promising laboratory tools for the study of the regulators of cellular growth and differentiation. Most cells in culture require specific polypeptide growth factors which are supplied by the addition of a complex biological fluid such as serum or, in some instances, by the cells themselves. These growth factors usually act on their target cell via a membrane receptor to which they bind. The events which occur after the growth factor binds to the membrane receptor have not been fully described, but the phosphorylation of tyrosine residues in certain proteins has been observed. A study was made of the polypeptide growth factors responsible for the growth and differentiation of haematopoietic cells in vitro. These growth factors, called colony - stimulating factors (C.S.F.'s) were prepared from human placental conditioned medium, giant cell tumour conditioned medium and pokeweed mitogen stimulated spleen conditioned medium. A C.S.F. from human placental conditioned medium was radioiodinated and the binding of the labelled growth factor to an anti-C.S.F. antiserum was studied. The binding studies indicated that a purer C.S.F. preparation and/or a more specific antiserum was necessary in order to establish a radioimmunoassay for C.S.F. The C.S.F.'s from giant cell tumour conditioned medium were purified by ultrafiltration, hydrophobic - interaction chromatography, gel filtration and thiolpropyl - sepharose 6B chromatography. Two peaks of biological activity were observed on gel filtration. One of these peaks gave an apparent MW of 63,000 and the other peak gave an apparent MW of 30,200. The C.S.F. from pokeweed mitogen stimulated spleen conditioned medium was labelled with peroxidase and the binding of the labelled-C.S.F. to bone marrow cell membranes studied. The labelled-C.S.F. bound to the membranes and the binding exhibited a linear relationship with membrane protein content. Also a defined growth medium for chick embryonic heart cells was developed. These cells were observed to differentiate from primitive foetal cells into mature "adult-type" cells. The cells grew as a monolayer, had spontaneous activity and were seen to beat.

An attempt has been made to purify an acid proteinase from human plasma, which is capable of dissolving fibrin clot in 1% (w/v) monochloroacetic acid. On SDS gel electrophoresis the enzyme isolated from plasma contains two protein bands with molecular weights of approximately 90,000 and 54,000. Different substrates were used to evaluate the proteolytic activity of the plasma enzyme, including fibrin clot, azo-casein, acid denatured haemoglobin (Hb), haemoglobin polyacrylamide gel (insoluble Hb), haemoglobin electrophoresis (soluble Hb) and the synthetic substrate N-acetyl-L-phenylalanyl-L-diiodotyrosine. The plasma enzyme seems to belong to the group of aspartyl proteinases as its activity against the above substrates was maximal at low pH, and was inhibited by pepstatin which is a powerful inhibitor of aspartyl proteinases.

Current therapeutic regimes for the treatment of acute myeloid leukaemia employ aggressive cytotoxic strategies which have limited success and are not suitable for all patients. Manipulating myeloid leukaemic cells according to their responses to pharmacologically tolerable agents rather than employing aggressive non specific cell kill would be desirable. Differentiation induction of myeloid leukaemic cells as an alternative therapeutic regime was evaluated. To elucidate the growth and differentiation abnormalities of myeloid leukaemic cells the differentiation induction and growth factor responses of bone marrow from three myeloid leukaemic models (SA2, SA7, SA8) and a leukaemic cell line (SA2 CL) were investigated in microtitre suspension cultures and compared with normal. The regulators of normal haemopoiesis occupy a key position in attempts to understand the nature of the abnormal state existing in myeloid leukaemia and could potentially play an important role therapeutically by suppressing myeloid leukaemic populations. To evaluate differentiation induction as an alternative therapeutic regime for the treatment of myeloid leukaemia both the physiological regulators and the differentiation inducer β-all trans retinoic acid (βatRA) alone and in combination with ara-C were investigated. The effects of these agents directly on the leukaemic clonogenic cell populations in vitro were measured following in vivo transplantation. The proliferative effects of WEHI-3B CM, as a source of IL-3, L929 CM as a source of M-CSF, and recombinant murine GM-CSF alone and in combination were investigated. Bone marrow cells from the SA7 and SA8 transplanted leukaemias were growth factor dependent for proliferation in vitro. The SA2 transplanted leukaemia proliferated autonomously at low passage numbers, the dominant leukaemic clone changed characteristics with progressive transplantation and became growth factor dependent at high passage numbers. A cell line (SA2 CL) was derived from leukaemic bone marrow cells of a low passage number of the SA2 leukaemia and proliferated at a high rate autonomously with minimal spontaneous differentiation in culture. The growth factor dependent leukaemias were induced to proliferate with each of the growth factors. No differences were observed in the dose response relationships between normal and leukaemic cells. Differences were observed in the proliferative rates, the day to which exponential cell growth was sustained in culture and the growth factor which induced maximal proliferation. WEHI-3B CM induced the maximal proliferative response of normal bone marrow cells and two of the leukaemias whereas rGM-CSF induced a maximal response of the third leukaemia. Leukaemic cells proliferated at a higher rate than normal cells in the first two to three days in culture but were not sustained in culture for as long as normal bone marrow cells. It appeared therefore, that in the case of some leukaemias the haemopoietic growth factors were effective at inducing a high initial but short term proliferative response of leukaemic cells and were more effective in sustaining normal bone marrow cells in vitro.

Activities associated with, or interacting with, leukaemic cell populations were assayed for the ability to influence in vitro haematopoiesis. The first of these, the glycoprotein leukaemia inhibitory factor (LIF), has a role in aspects of murine, non human primate and human haematopoiesis. It is thought to be particularly important in the development of megakaryocytes and is also known to induce the terminal differentiation of certain leukaemic cell lines. LIF was assayed both for direct and indirect effects on the proliferation of haematopoietic precursor cell populations in vitro. As a direct acting agent in semi-solid agar culture of haematopoietic cell populations derived from normal bone marrow or 15 day foetal liver, LIF was unable to support colony formation. In cultures of cells derived from normal bone marrow stimulated with single, or combinations of, growth factors, the addition of LIF had no statistically significant effect on the level of colony formation. In cultures of cells derived from foetal liver, stimulated with particular growth factor combinations (medium conditioned by the Wehi3B leukaemic cell line + medium conditioned by the lung fibroblast cell line, L929); GM-CSF + M-CSF; IL-la + IL-3 + M-CSF), LIF, was shown to decrease the level of colony formation. LIF did not directly alter the proportion of the population in DNA synthesis in cell populations derived from normal femoral marrow, 15 day foetal liver or y- irradiated femoral marrow. As an indirect acting agent LIF failed to block the synthesis of a stem cell stimulator, or it's action, on a population of high proliferative potential colony forming cells derived from normal femoral marrow, cloned in the presence of Wehicm+L929cm. (HPP-CFC (Wehicm + L929cm)) LIF's actions on clones of a murine myeloid leukaemia (SA2JMB1) were also assessed. LIF had no statistically significant effect on colony formation or the level of DNA synthesis in populations of SA2JMB1 leukaemic cells. A second group of associated activities was produced by the X- irradiation induced murine myeloid leukaemia (SA2JMB1). Medium conditioned by the leukaemic cells was assayed in vitro both for direct and indirect effects on the proliferation of haematopoietic cells derived from femoral marrow. As a direct acting agent in 7 and 14-day semi-solid agar culture of femoral marrow, leukaemic conditioned medium alone stimulated limited colony formation. In 7 and 14 day cultures stimulated with single and combinations of specific colony stimulating factors: (rmGM-CSF, rhM-CSF, rhIL-1a) a significant increase in colony number was noted in all cases when cultures were supplemented with leukaemic conditioned medium. SA2JMBlcm was shown to support the proliferation of an IL-3 dependent cell line (FDCP-A4 cells). The colony enhancing ability of SA2JMBlcm was shown to be blocked by pretreatment with antibodies to IL-3. This suggested that SA2JMB1 conditioned medium contained IL-3 or an IL-3 like activity, as one of its components. The conditioned medium failed to directly alter the level of DNA synthesis in a population of HPP-CFC (Wehicm+L929cm) derived from normal bone marrow or y- irradiated bone marrow. As an indirect acting agent the conditioned medium did block the action of a stem cell proliferation stimulator on normal bone marrow derived HPP-CFC (Wehicm+L929cm). This leukaemia associated activity was shown to be larger than 50KD, sensitive to heat treatment and able to act in a different manner to the stem cell inhibitor MIP-1-a. Thus this novel activity may be important in blocking stimulator action in haematopoietic stem cells and thus contribute to the haematopoietic insufficiency seen in leukaemia.

Haematopoietic activity in the murine fetal liver was observed to increase from the 12th day of gestation to reach a peak on the 15th day of gestation. Hepatic haematopoiesis is predominantly erythroid. As maturation proceeds the numbers of myelopoietic cells increase. Haematopoietic activity declines after the 15th day of gestation, though haematopoietic cells were still obvious in the liver of the 10 day neonate. Cellular interactions were studied in vivo with the electron microscope and in vitro using long term cultures. Haematopoietic cell clusters were abundant in fetal liver both in vivo and in vitro. Macrophages and hepatocytes act as the central supporting cells of these clusters. There was evidence of communication between the central supporting cells and the blood cells precursors, which may be essential for the maintenance of haematopoiesis both in vivo and in vitro. A high proportion of early HPP-CFC derived from fetal liver are synthesising DNA, while HPP-CFC derived from adult bone marrow are relatively quiescent. The kinetic properties of the HPP-CFC population are very similar to those of the CFU-S population both in fetal liver and in adult bone marrow. A high proportion of GM-CFC which are a more mature class of progenitor cells were cycling in both fetal liver and adult bone marrow. Fetal liver extract was shown to have an overall inhibitory effect on haematopoiesis in normal bone marrow and irradiated bone marrow and on colony formation by leukemic cells. Detrimental effects on normal bone marrow cells were observed only with high concentrations of fetal liver extract. Fetal liver blood cell precursors were found to adhere preferentially to bone marrow stromal layers, which could not be attributed to the kinetic properties of these cells. This preferential adhesion may promote the in vivo accumulation of haematopoietic stem cells derived from fetal liver in the medullary cavities.

The murine lympho-myeloid complex is depleted by a large dose of nitrogen mustard and the pattern of recovery of the haemopoietic tissues and cells followed to establish the relationships ox the precursor and mature cells. Culture of bone marrow in agar and stathmokinetic techniques are used to examine the controlled proliferation for the granulocyte elements in the bone marrow recovering from nitrogen mustard and the influence of the spleen is also considered. The control of the proliferation of the granulocyte cells is lost in myeloid leukaemia. Stathmokinetic methods and spleen colon assays are used to asses cell proliferation in irradiation-induced myeloid leukaemias in mice and the relevance of these parameters so measured to those demonstrated by normal bone marrow is discussed.

The cellular integrity of the blood is maintained by the cellular output of the haematopoietic stem cell population which produces the specialized precursors and differentiated cells which constitute the blood. The investigation of haematopoietic stem cell behaviour and regulation has been hampered by both the difficulty in their identification and the development of relevant assay systems. The purpose of this investigation was to study the behaviour and regulation of the haematopoietic stem cell population in normal and leukaemic haematopoiesis using an in vitro assay of a primitive haematopoietic precursor. The use of a combination of haematopoietic colony-stimulating factors [interleukin 3 (IL3)/multi-CSF and macrophage colony-stimulating factor (M-CSF/CSF-1)] in semi-solid agar culture of murine haematopoietic tissue, stimulated the proliferation of a haematopoietic colony-forming cell, defined as the "HPP-CFCIL3+CSF-1" population, which was characterized by a high proliferative potential, a multipotency and behavioural and regulatory properties consistent with its being a primitive haematopoietic precursor and possibly a component of the haematopoietic stem cell population. The proportion of the in vitro HPP-CFCIL3+csf-1 population in S-phase in normal murine marrow, was determined to be relatively low at approximately 10%, increasing to approximately 40% in sublethally X-irradiated, regenerating murine marrow and the respective presence of the haematopoietic stem cell proliferation inhibitor and stimulator was demonstrable by the induction of appropriate kinetic changes in the in vitro HPP-CFCIL3+CSF-1 population. In leukaemic haematopoiesis, leukaemic proliferation often occurs at the expense of apparently suppressed normal haematopoiesis. In vitro HPP-CFCIL3+CSF-1 assay of the haematopoietic stem cell proliferation regulators in a number of murine, myeloid leukaemic cell lines, failed to demonstrate either increased levels of the haematopoietic stem cell proliferation inhibitor, or evidence of a direct-acting, leukaemia- associated proliferation inhibitor, however, evidence of a leukaemia- associated impairment of inhibitor and stimulator production was observed and this may be a possible mechanism by which the leukaemic population develops a proliferative advantage over normal haematopoietic tissue. The identification of a possible mechanism of leukaemic progression and suppression of normal haematopoiesis may subsequently allow the development of potentially more effective disease treatment and management regimes. The endogenous haemoregulatory tetrapeptide: Acetyl-N-Ser- Asp-Lys-Pro [AcSDKP, Mr=487 amu] is reported to prevent the G0-G1 transition of haematopoietic stem cells into S-phase. The mechanism of action of AcSDKP and a number of related peptides, was investigated in relation to the stem cell proliferation stimulator and inhibitor. AcSDKP demonstrated no direct haemoregulatory role against the in vitro HPP-CFCIL3+CSF-1 population, which is consistent with reports that AcSDKP is not active against cells already in late G1, or S-phase, rather it appeared to act indirectly by impairing the capacity of the haematopoietic stem cell proliferation stimulator to increase the proportion of the in vitro HPP-CFCIL3+CSF-1 population in S-phase. An apparent impairment of stimulator action may explain the reported AcSDKP-associated 'block' of haematopoietic stem cell recruitment. A putative endogenous AcSDKP precursor and synthetic and degradative enzyme systems have been reported and the possible physiopathological role of AcSDKP in a number of myeloproliferative disorders has been implicated. The potential application of AcSDKP as a 'haemoprotective' agent administered prior to the use of S-phase- specific chemotherapy may be of clinical significance. The in vitro HPP-CFCIL3+CSF-1 assay of a primitive haematopoietic precursor cell population, which may be a component of the haematopoietic stem cell population, should play a significant role in the investigation of haematopoietic stem cell behaviour and regulation in both normal and aberrant haematopoiesis. With the characterization of the mechanism(s) of action of the haematopoietic stem cell proliferation inhibitor and stimulator and the haemoregulatory tetrapeptide AcSDKP, the manipulation of the haematopoietic system to clinical advantage can be envisaged, while the identification of the aberrant regulatory mechanism(s) in haematopoietic dysfunction may allow, the development of more effective disease treatment and management regimes.

Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T23:55:35Z (GMT). No. of bitstreams: 1 Marques_VanessaAlvesArruda_D.pdf: 1717936 bytes, checksum: 37ac1f747e37b8fb133778d014bd61d5 (MD5) Previous issue date: 2006 / Resumo: Com os recentes progressos na compreensão dos complexos mecanismos moleculares relacionados a diferentes aspectos da modulação imunológica, cada vez mais vem se tornando possível ampliar os conhecimentos acerca das potenciais interferências do ponto de vista imunopatogênico induzidas pela presença e evolução tanto de processos infecciosos quanto tumorais. Modelos experimentais vêm sendo desenvolvidos e aprimorados com a finalidade de criar condições controladas para avaliações de diferentes aspectos relativos a potenciais modificações no comportamento imunológico de linhagens celulares frente à ação de novos agentes. O modelo do Tumor Ascítico de Erlich apresentou-se como uma opção de interesse, uma vez que os conhecimentos experimentais sobre diferentes aspectos relativos a sua manipulação já fazem parte de consagrada linha de pesquisa nacional. Um dos comportamentos mais recentemente estudados a seu respeito se refere a condições onde ocorre a migração do fenótipo Th1 para Th2, relacionadas ao agravamento da doença. A busca de fontes naturais para conhecimento e aplicação de novos princípios ativos vem se caracterizando como uma das mais promissoras soluções do ponto de vista farmacêutico, e pode corresponder a uma importante área de exploração da biodiversidade nacional. Dessa forma, a proposta do presente estudo foi, unindo linhas de pesquisa bastante consolidadas e conhecimentos relativos à nossa biodiversidade, avaliar o potencial terapêutico, com foco em abordagens imunopatológicas, do extrato da planta Tabebuia avellanedae (ETA) através do modelo experimental Tumor ascítico de Ehrlich. Neste trabalho investigamos os efeitos do ETA e seu constituinte isolado, a ß-lapachona (ß-lap), sobre o crescimento e diferenciação de progenitores hematopoéticos [células formadoras de colônias de granulócitos e macrófagos (CFU-GM)], a capacidade funcional das células ¿Natural Killer¿(NK) e a produção das citocinas do perfil Th1 [fator de necrose tumoral- a (TNF-a ) e interferon- ? (IFN-?)] e do perfil Th2 [interleucina (IL)-10] de animais normais e portadores do TAE. Como esperado, o TAE produziu, concomitante à mielosupressão, um aumento no número de CFU-GM esplênico. O tratamento destes animais com a dose de 120mg/kg de ETA, administrado por 7 dias após a inoculação do tumor, demonstrou uma maior eficácia, produzindo aumento na mielopoiese, redução no numero de colônias esplênicas e reversão dos fatores estimuladores de colônias (CSF) induzidos pelo TAE. Além disso, esta dose de ETA produziu a melhor eficácia na sobrevida de animais portadores do TAE, aumentando em 42% a duração da sobrevida. No entanto, doses de 30 e 500mg/kg não afetaram a mielosupressão e a hematopoese extramedular induzidas pelo tumor. O tratamento dos animais portadores do TAE com 1, 2 ou 5mg/kg de ß-lap por 3 dias após a inoculação do tumor resultou em um padrão semelhante de dose resposta, sendo a mielosupressão prevenida apenas com a dose de 1mg/kg. Interessante observar que esta dose prolongou a duração na sobrevida de animais portadores do TAE de maneira similar à dose mais eficaz do ETA, ambos em 42%. Nosso estudo também demonstrou importantes diferenças entre os tratamentos com ETA e ß-lap. Neste sentido, animais normais tratados com 2 e 5mg/kg de ß-lap apresentaram hematopoese extramedular, respectivamente 2 e 3 vezes maior que o CFU-GM de animais normais. Além disso, uma drástica mielosupressão foi observada após tratamento com ß-lap 5mg/kg, atingindo valores similares aos observados nos animais portadores do TAE e não tratados. Quanto à produção de citocinas, o tratamento com 120mg/kg de ETA impediu a polarização Th1-Th2 encontrada nos animais portadores do TAE. Neste sentido, o tratamento destes animais aumentou os níveis de secreção de TNF-a e IFN-? e reduziu os níveis de IL-10, aumentados durante a evolução temporal do TAE. O estudo da atividade funcional das células NK em animais portadores do tumor e tratados com EAT demonstrou um aumento na capacidade citotóxica das células NK, a qual se encontrava reduzida. O tratamento destes animais com ß-lap 1mg/kg impediu parcialmente a polarização Th1-Th2 em animais portadores, assim como demonstrou aumento parcial na capacidade citotóxica das células NK. Estes resultados sugerem que a eficácia antitumoral demonstrada pelo ETA pode estar relacionada com seu constituinte, a ß-lap. Por outro lado, a ausência de constituintes associados presentes no extrato bruto pode ser responsável pelos efeitos inibitórios dose-dependentes deste composto na hematopoese de animais normais. A comparação entre efeitos de um extrato vegetal e um de seus constituintes ativos isolado é outro aspecto a ser considerado, sendo importante etapa no desenvolvimento de novos fármacos de origem vegetal / Abstract: In accordance with the recent progress in the understanding of immunological and molecular mechanisms related to the different aspects of immunological modulation, the improvement of knowledge about the potential interferences, at the immunopathogenic point of view, induced by the presence and evolution of infectious diseases or neoplastic process has increased. Experimental models have been developed and refined in order to create appropriate conditions to evaluate different aspects related to potential changes in the immunological behaviors of determinate cell lines with the presence of new pharmacological agents. The Ehrlich ascites tumor (EAT) model is an option of interest, since the accumulated experimental knowledge about its manipulation is part of a consolidated research line. One of its more recently studied behaviors is concerned with situations where the migration from Th1 to Th2 phenotype occurs, at the same time with the disease impairment. The search for new natural sources of pharmaceutical active principles and the careful and detailed studies about them, has been characterized as one of most promising therapeutic solutions at the moment, and would be seen as a strategic way to explore the national biodiversity. Therefore, the aim of the present study was to combine the well known techniques related to the Ehrlich ascites tumor changing behavior with a potential active principle from our botanical biodiversity, the Tabebuia avellanedae extract (TACE), in order to evaluate its immunological therapeutic potential. The effects of TACE and its isolated naphtoquinone, ß-lapachone (ß-lap), on the growth and differentiation of granulocyte-macrophage progenitor cells in normal and EAT-bearing mice were studied. Myelosupression concomitant with an increased number of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with the dose of 120mg/Kg TACE given for seven days after tumour inoculation elicited optimal modulatory effects on bone marrow formation and also produced a favourable response for the reduction of splenic haematopoiesis. Increased serum colony-stimulating activity induced by the tumour was also reversed with this dose-regimen. In addition, this dose-schedule showed the better efficacy in prolonging life span of EAT-bearing mice, with an extension of 42% in the duration of survival. Conversely, the doses of 30 and 500mg/kg hardly affected the myelosuppression and extramedular hematopoiesis induced by the tumor. Treatment of tumour-bearing mice with 1, 2 and 5mg/kg ß-lap given for 3 days after tumour inoculation resulted in a similar pattern of dose response, with tumour-induced myelosupression being prevented only with the 1mg/kg dose. Surprisingly, this dose-schedule prolonged the duration of survival by the same rate of 42% observed with the optimal dose of TACE. However, an important divergent response of ß-lap was its stimulating effect on the extramedular hematopoiesis of normal mice treated with the doses of 2 and 5mg/kg, which produced, respectively, 2 and 3-fold increases of CFU-GM in the spleen. In addition, in the group of normal mice exposed to the dose of 5mg/kg, a drastic reduction in the number of bone marrow CFU-GM was observed, reaching levels as low as those found in non-treated tumour bearers. We also investigated the effects of TACE and ß-lap on the natural killer cell activity (NK) and on Th1 [tumor necrosis factor- a (TNF-a ) and interferon- ? (IFN-?)] and Th2 [interleukin (IL)-10] responses in normal and EAT-bearing mice. Regarding the production of cytokines, the treatment with TACE blocked the Th1-Th2 polarization found during the EAT progression. In this respect, the treatment of these animals with TACE 120mg/kg increased the TNF-a and IFN-? levels and reduced the levels of IL-10, increased during the temporal evolution of EAT. In relation to the functional activity of NK cells, the treatment with this same dose of TACE enhanced the NK cell function, reduced during the EAT growth. The treatment of these animals with ß-lap 1mg/kg produced a significant, but partial response on NK cell activity on EAT-bearing mice. In this respect, this compound only partially increased the TNF-a and IFN-? levels and reduced the levels of IL-10, increased during the temporal evolution of EAT. These findings suggest that the antitumoral efficacy demonstrated by the crude extract of T. avellanedae might be related to its constituent, the ß-lapachone. On the other hand, the absence of the associated constituents present in the crude extract might be responsible for the dose-dependent inhibitory effect of this biosynthetic compound on normal murine hematopoiesis. The comparison between a total vegetal extract and some of its isolated compounds remain another significant aspect in phytochemical research, specially focused in new medicines discovery / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica