Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

No description available.

La contribution des Cellules Souches/Stromales Mésenchymateuses (CSM) dans le développement des Leucémies Aiguës Myéloblastiques (LAM) n’est pas encore clairement établie. L'objectif de ce travail a été de rechercher de potentielles modifications phénotypiques et fonctionnelles au sein des CSM médullaires de patients atteints de LAM de novo au diagnostic. Nous montrons que ces cellules présentent un défaut prolifératif accompagné d’une augmentation de l’apoptose et d’un déficit d’expression de certains facteurs de la niche (Ang-1, SCF, TPO et VCAM-1). De façon intéressante, ce défaut prolifératif est indépendamment associé à une évolution péjorative de la maladie. Néanmoins, ces anomalies des CSM de LAM ne semblent pas affecter leur capacité de soutien de l’hématopoïèse physiologique ou leucémique in vitro. En effet, comme les CSM normales, elles protègent les cellules leucémiques de l’apoptose, induisent leur quiescence (principalement par contact direct) et ainsi diminuent la proportion des cassures double-brin d’ADN. Ces données suggèrent que les modifications des CSM de LAM, probablement une des conséquences délétères de la prolifération tumorale, n'auraient pas un rôle spécifique dans le développement du processus leucémique. / The contribution of Mesenchymal Stem/Stromal Cells (MSCs) to the development of Acute Myeloid Leukemias (AMLs) remains poorly understood. In the present study, we investigated potential functional and phenotypic modifications of Bone Marrow (BM)-derived MSCs from patients with AML de novo at diagnosis. We showed that BM-derived MSCs from most of AML patients display proliferative defect, had increased apoptosis levels and demonstrated defective expression of several niche-related factors (Ang-1, SCF, TPO and VCAM-1). Interestingly, this proliferative defect was independently associated with disease progression. Nevertheless, these abnormalities in AML MSCs did not affect their in vitro capacity to support physiological but also leukemic hematopoiesis. Indeed, as normal MSCs do, they protect blast cells from apoptosis, induce their quiescence (mainly by direct contact), and decreased yields of DNA double-strand breaks. Consequently, in AML de novo these stromal cell alterations, probably a consequence of the deleterious effect of the tumor cell growth on BM MSCs, do not appear to have a specific role in the development of the leukemic process.

Submitted by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2014-10-03T18:02:20Z No. of bitstreams: 1 TESE YEIMI ALZATE LOPEZ. 2014.pdf: 1273705 bytes, checksum: ff739cf53b4a176758fd9e9cf081c526 (MD5) / Approved for entry into archive by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2014-10-07T13:54:41Z (GMT) No. of bitstreams: 1 TESE YEIMI ALZATE LOPEZ. 2014.pdf: 1273705 bytes, checksum: ff739cf53b4a176758fd9e9cf081c526 (MD5) / Made available in DSpace on 2014-10-07T13:54:41Z (GMT). No. of bitstreams: 1 TESE YEIMI ALZATE LOPEZ. 2014.pdf: 1273705 bytes, checksum: ff739cf53b4a176758fd9e9cf081c526 (MD5) / Estudos sócio-antropológicos que abordam experiências e repercussões das doenças crônicas têm apontado o caráter heterogêneo das contingências da vida cotidiana no enfrentamento da doença por parte dos sujeitos e os complexos processos de busca de cuidado para resolver seus problemas. No caso do câncer, a sua construção sociocultural envolve o status de doença maligna, assim como uma doença que ameaça a vida, o que se coloca nas experiências dos pacientes como fonte de incerteza, medo e estigmas. No caso específico da Leucemia Mieloide Crônica (LMC), o conhecimento da sua patogênese (mas não da sua etiologia) e os desenvolvimentos tecnológicos recentes em tratamentos têm levado a uma reconstrução do discurso científico biomédico, mudando a forma de conotá-la, denotá-la, explicá-la e tratá-la como uma forma diferenciada de outros tipos de leucemia e de câncer. A fase crônica da LMC, há pouco mais de uma década, tinha uma duração estimada de três a seis anos, seguida pela transformação para as fases acelerada e blástica de curta duração, estimadas entre 3 a 6 meses (Kantarjian, et.al. 1993, 2002), colocando um panorama de mortalidade alcançada em pouco tempo. Devido ao desenvolvimento de medicamentos conhecidos como Inibidores de Tirosino Quinase (ITK), substância importante na progressão da célula leucêmica, a LMC é reconhecida na atualidade como uma “mudança de paradigma” no tratamento da Leucemia e do câncer (Goldman & Melo, 2003; Stephen et.al., 2003, Souza e Pagnano, 2004, Rüdiger, et.al, 2007). Estes medicamentos têm alcançado o controle e remissões duradouras da doença na fase crônica, que só tinham sido alcançados através do Transplante de Medula Óssea (TMO) (Schiffer et.al. 2007, Jabbour, et.al. 2007, Lopes et.al. 2009). Este panorama tem redefinido os protocolos de tratamento, colocando o TMO (e observando suas taxas de mortalidade) como tratamento de segunda ou terceira linha em caso de resistência aos ITK, mas continua sendo considerada a única terapia de cura da LMC (Rüdiger, et.al, 2007, Aranha, 2008). Alguns estudos recentes apontam para a possibilidade de “cura” a partir do tratamento continuado com os ITK, porém a suspensão do tratamento só é recomendado sob certa condições e com um monitoramento e controle citogenético e molecular constante (Branford, 2012; Jabbour, et.al, 2013). Esse contexto permite evidenciar uma reconstrução constante do discurso (e práticas) biomédico da doença, a partir de novas descobertas científicas e tecnológicas, gerando velhas e novas incertezas, ambiguidades e significados da “cronicidade da LMC”, assim como coloca as diferentes práticas de cuidado, nas quais se ressaltam, especialmente, as “trilhas” e barreiras de acesso dos pacientes a atenção e medicamentos. O objetivo geral dessa pesquisa foi o de analisar e interpretar as experiências de enfermidade e os itinerários terapêuticos de dez (10) portadores de Leucemia Mieloide Crônica (LMC), nas cidades de Medellín, Colômbia e Salvador – BA, Brasil. Para compreender o fenômeno estudado, foi realizado um metaestudo da literatura qualitativa nacional e internacional sobre a categoria Itinerários Terapêuticos (IT) e sua articulação com a categoria experiência de enfermidade (Artigo 1). Ao realizar essa meta-análise e síntese das teorias, métodos e dados produzidos pelas pesquisas, foi possível discutir sobre as correntes teórico-metodológicas, limites, alcances e dimensões que esta categoria permite para a abordagem e compreensão dos diferentes e complexos processos de escolha, avaliação e aderência a certos tipos de cuidados e tratamentos por parte dos sujeitos. Na sequência, foi realizada a interpretação das experiências de enfermidade de sujeitos da Colômbia e do Brasil (Artigo 2) assim como a análise da construção dos itinerários terapêuticos dos sujeitos na busca de cuidados (Artigo 3). Em ambos os artigos empíricos utilizou-se a estratégia metodológica do estudo de casos coletivos. O interesse num grupo (limitado) de casos, mais do que um caso individual, coloca a ênfase menos em “generalizações” a partir dos casos (Stake 2007) e mais na compreensão da unicidade de cada caso, sem perder a riqueza das suas particularidades e diferenças, mas também das semelhanças que com certeza aparecem para os leitores durante a análise. No total, foram incluídos dez (10) casos (5 de Medellín e 5 de Salvador), seguindo os seguintes critérios: mulheres e homens de idades entre 30 e 80 anos, com um tempo de diagnóstico da doença de no mínimo um ano, em fase crônica; ser usuários dos sistemas de saúde público ou privado e receber atendimento em diferentes instituições. Os critérios escolhidos não pretendiam incluir casos típicos ou atípicos (Yin, 2013), mas sim, casos “representativos” que, de alguma forma, informavam dimensões trazidas por outros pacientes. Como resultados, no artigo 2 se apresentam as diferentes dimensões das experiências de enfermidade dos casos analisados, apresentando fenômenos como a ruptura biográfica causada pela doença, os complexos processos de “incorporação” do social nas suas experiências. A análise de cada caso permitiu evidenciar um processo no qual as mudanças nas experiências podem avançar de uma fase, na qual prevalece os sentidos de morte, sofrimento, desconstrução e rupturas (das relações sociais, do cotidiano etc.), para outra, quando vai se apresentando um processo de normalização (Bury, 1982), onde se coloca, em alguns casos, sentidos positivos, ambiguidades e incertezas sobre as mudanças e o aprendizado que a LMC traz para suas vidas. As narrativas apontaram para um desconhecimento geral” sobre a LMC e a leucemia como tal, colocando a questão levantada por Comaroff e Maguire (1981) sobre as implicações sociais dos avanços tecnológicos para o tratamento da leucemia que ficam no terreno do conhecimento científico e médico. Isto leva os pacientes e seus familiares a se confrontarem com as ambiguidades e incertezas apontados nos sentidos do “controle” e de “risco”. Ao seguir a construção do IT dos sujeitos, no artigo 3, se apresenta a análise dos casos em cada contexto evidenciando sua influência no percurso que se inicia desde a “descoberta” de uma alteração até depois de conseguir o “controle” da doença. Assim, o processo de diagnóstico permite discutir como uma condição “grave” às vezes não é percebida pelos pacientes, mas sim pela sua rede social, que é quem os leva à procura da consulta especializada. O acesso à atenção no nível terciário (alta complexidade) permite, em ambos os contextos, discutir os modelos de atenção, as falhas nos serviços de saúde, as diversas fragmentações dos sistemas que levam os pacientes e suas famílias por diversas peregrinações, barreiras, burocracias, colocando processos que foram concebidos como “causas estruturais do sofrimento”. / Socio-anthropological studies that discuss experiences and the impact of chronic diseases have shown the heterogeneous character of the everyday life’s contingencies in the coping with the illness by the subjects and the complex processes of searching for care to solve their problems. In the case of cancer, its sociocultural construction involves the status of malignant disease, as well as a life-threatening disease, which is placed on the experiences of patients as a source of uncertainty, fear and stigma. In the specific case of Chronic Myeloid Leukemia (CML), the knowledge of its pathogenesis (but not its etiology) and recent technological developments in treatment have led to a reconstruction of the biomedical scientific discourse, changing the way it connotes, denote it, explain it and treat it as a different way from other types of leukemia and cancer. The chronic phase of CML, a little over a decade, had an estimated duration of three to six years, followed by transformation to the phases accelerated and blastic of short duration, estimated between 3 to 6 months (Kantarjian, et.al. 1993 , 2002), placing a panorama of mortality achieved in a short time. Due to the drugs’ development known as Tyrosine Kinase Inhibitors (ITK), an important substance in the progression of leukemic cells, CML is recognized today as a “paradigm shift” in the treatment of leukemia and cancer (Goldman & Melo, 2003; Stephen et.al., 2003 and Souza Pagnano, 2004 Rüdiger, et.al, 2007). These medications have gained control and durable remissions in the chronic phase of the disease, which had only been achieved through Bone Marrow Transplantation (BMT) (Schiffer et.al. 2007 Jabbour, et.al., 2007, Lopes et.al. 2009). This overview has redefined the treatment protocols, putting BMT (and watching their mortality rates) as treatment for second or third line in case of resistance to ITK, but still considered the only healing therapy of CML (Rüdiger et. Al, 2007 Spider, 2008). Some recent studies point to the possibility of “cure” from continuing with ITK treatment, but treatment discontinuation is recommended only under certain conditions and with a monitoring and constant control cytogenetic and molecular (Branford, 2012; Jabbour, et. Al, 2013). This context will show a constant discourse’s reconstruction (and practice) of biomedical disease, from new scientific and technological findings, generating old and new uncertainties, ambiguities and meanings of “chronic CML”, and puts the different care practices, in which they point out, especially the “trails” and barriers to patient access to care and medications. The overall objective of this research was to analyze and interpret the experiences of illness and therapeutic itineraries of ten (10) patients with Chronic Myeloid Leukemia (CML) in the cities of Medellín, Colombia, and Salvador – BA, Brazil. To understand the phenomenon studied, a national and international qualitative literature’s meta-analisys on Therapeutic Itineraries (IT) category and its articulation with the experience of illness category (Article 1) was performed. In performing this meta-analysis and synthesis of theories, methods and data produced by researches, it was possible to discuss the theoretical and methodological perspectives, limits, scope and dimensions that this category allows for this approach and understanding of different and complex processes of choice, evaluation and adherence to certain types of care and treatment by subjects. Following on, the interpretation of the subjects’s illness experiences from Colombia and Brazil (Article 2) as well as the analysis of the construction of subjects’s therapeutic itineraries in seeking care (Article 3) was performed. In both empirical articles, it was used the methodological approach of the collective cases’s study. The interest in a group (limited) cases, more than one individual case, the emphasis is less on “generalizations” from the cases (Stake 2007) and more on understanding the uniqueness of each case, without losing the richness of its particularities and differences, but also the similarities that certainly appear to readers during analysis. Were included in the total ten (10) cases (5 of 5 Medellin and Salvador), using the following criteria: women and men aged 30 to 80, with disease time of diagnosis, at least, one year in chronic phase; be users of public or private health systems and receive care in different institutions. The criteria chosen did not intend to include typical or atypical cases (Yin, 2013), but “representative” cases that, somehow, informed dimensions brought by other patients. As a result, Article 2 present the different dimensions of the illness experience of the cases analyzed, presenting the phenomena such as biographical disruption caused by the disease, the complex processes of “incorporation” of the social in their experiences. The analysis of each case has highlighted a process where changes in the experiences can move from one phase, in which prevails senses of death, suffering, and deconstruction and ruptures (social relations, everyday etc..), to another, when it is being presented a standardization process (Bury, 1982), which arises, in some cases, positive way, ambiguities and uncertainties about changes and learning that the LMC brings to their lives. The narratives indicated a “general unknown” of CML leukemia, putting the issue raised by Comaroff and Maguire (1981) on the social implications of technological advances for the treatment of leukemia that are in the field of scientific and medical knowledge. This faces to patients and their families to the ambiguities and uncertainties indicated in the directions of the “control” and “risk”. By following the construction of IT’s subjects, in Article 3, presents the analysis of cases in each context highlighting its influence on the path that begins from the “discovery” of a change until after getting the “control” of the disease. Thus, the diagnosis’s process allows discuss how a “serious” condition is sometimes not perceived by patients, but by their social network, which is who leads them to search for specialized consultation. The access to care at the tertiary level (high complexity) enables, in both contexts, discuss the models of care, the failures in the health services, the various systems’s fragmentations that lead patients and their families for several pilgrimages, barriers, bureaucracies, putting processes that were designed as “structural causes of suffering”.

O presente estudo compreende 96 transplantes de medula óssea (TMO) de doadores HLA-idênticos em pacientes portadores de Leucemia Mielóide Crônica, no período de Junho de 1986 a Junho de 1998. A autora selecionou diversas covariáveis para serem estudadas como fatores prognósticos de cinco desfechos principais: ocorrência de doença enxerto contra hospedeiro (DECH) aguda e crônica, incidência de recaída, sobrevida livre de doença (SLD) e sobrevida global (SG). As covariáveis estudadas foram: idade, sexo, escolaridade, tempo entre o diagnóstico e o transplante, fase da doença ao transplante, regime de condicionamente, profilaxia de DECH, compatibilidade de sexo entre doador e receptor, sexo do doador, tamanho do baço e do fígado, percentagem de blastos e número de plaquetas no sangue periférico na primeira consulta ao CEMO, ocorrência e grau de DECH aguda, ocorrência de DECH crônica e tempo para recuperação de plaquetas após o TMO. Não foi encontrada associação estatisticamente significativa num nível de 95% de confiança entre qualquer das covariáveis e a ocorrência de DECH crônica ou de recaída. A ocorrência de DECH aguda mostrou-se associada apenas com a fase da doença ao transplante. As covariáveis que se mostraram associadas com a sobrevida global e a sobrevida livre de doença foram: a percentagem de blastos no sangue periférico e tamanho do baço na primeira consulta ao CEMO, a fase da doença ao transplante, o tipo de profilaxia de DECH, a ocorrência e o grau de DECH aguda e o tempo para recuperação de plaquetas num nível acima de 20 x 103/mm3. / This study comprises 96 bone marrow transplantations (BMT) from HLA-identical siblings in patients with Chronic Myeloid Leukaemia (CML), from June 1986 to June 1998. The author selected several covariates to be studied as prognostic factors for five main endpoints: acute and chronic graft versus host disease (GVHD) occurrence, relapse incidence (RI), disease free survival (DFS) and overall survival (OS). The covariates studied were: age, sex, years of schooling, time from diagnosis to transplantation, disease phase at BMT, conditioning regimen, GVHD prophylaxis, sexmatch between donor-receptor, donor sex, spleen and liver size, blasts percentage and platelet counts in peripheral blood at first consultation at CEMO, acute GVHD occurrence and grade, chronic GVHD occurrence and time to platelet recovery after BMT. No statistically significant association was found between the covariates studied and the occurrence of acute or chronic GVHD or of relapse. The covariates found to have an association with overall survival (OS) and disease free survival (DFS) were the percentage of blasts in peripheral blood and the spleen size at first consultation, the disease phase at transplant, type of GVHD prophylaxis, the occurrence and grade of acute GVHD and the time to platelet recovery above 20 x 103/mm3.

Orientadora: Profa. Dra. Ana Carolina Santos de Souza Galvão / Nanoparticulas derivadas de oxidos metalicos, em suas formas livres ou associadas a moleculas organicas ou drogas antineoplasicas, tem sido empregadas no diagnostico e tratamento do cancer. Neste cenario, destaca-se o uso de oxidos nanoestruturados derivados de zinco. Recentes estudos demonstraram a capacidade de nanoparticulas de ZnO, individualmente ou em associacao a agentes quimioterapicos, de sensibilizacao de celulas tumorais resistentes a multiplas drogas (fenotipo MDR), que possuem em sua membrana proteinas de efluxo que impedem o acumulo destas drogas no interior das celulas. A falta de seletividade e dificuldade de internalizacao pelas celulas de alguns compostos utilizados no tratamento de cancer, sao considerados os maiores empecilhos para o tratamento quimioterapico, tornando a modificacao da superficie de materiais nanoestruturados com atividade antitumoral, atraves da funcionalizacao, forma de aumentar a incorporacao e consequente atividade citotoxica destes compostos em celulas tumorais. Neste trabalho, as nanoparticulas foram funcionalizadas com L-glutamina, devido a grande importancia deste aminoacido no metabolismo de celulas cancerigenas e ao fato destas apresentarem maior captacao desse aminoacido que celulas nao tumorais. A citotoxicidade de nanoparticulas funcionalizadas com L-glutamina ou nao funcionalizadas foi avaliada em celulas de leucemia mieloide cronica K562 e sua versao com fenotipo MDR Lucena-1. A atividade citotoxica de ZnO foi comprovada pelos ensaios de exclusao de Trypan Blue e reducao de MTT, com IC50 de 20 evalencia de dupla marcacao nas celulas K562 tratadas com as nanoparticulas ZnO e ZnO_Gln, enquanto que para Lucena-1 observou-se porcentagens semelhantes de celulas duplamente marcadas e marcadas apenas com Anexina V. Para avaliar se ha influencia de especies reativas de oxigenio (EROs), geradas peLucena-1. Atraves das analises das possiveis vias de morte celular via analise de Western Blotting, estudando principalmente as proteinas e os eventos envolvidos no processo de morte celular programada (Bcl2, Bad, Bid, Caspase-3 e PARP) e autofagia (Beclin-1 e P62), observou-se que mais de uma via de morte foi ativada nas celulas tratadas com as nanoparticulas, o que corrobora com o descrito em literatura, considerando autofagia e apoptose sao as principais vias de morte ativadas pela acao citotoxica das nanoparticulas de ZnO. Outro evento observado foi a alteracao da atividade da proteina PgPieloide cronica K562 e a linhagem com fenotipo MDR Lucena-1 e a utilizacao deste aminoacido na funcionalizacao de nanomateriais pode ser uma importante ferramenta a ser explorada. / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2015. / Nanoparticles derived from metal oxides, in their free forms or associated with organic molecules or antineoplastic drugs, have been employed in the diagnosis and treatment of cancer. In this scenario, we highlight the use of zinc-based nanostructured oxides. Recent studies demonstrated the ability of ZnO nanoparticles, alone or in combination with chemotherapeutic agents, sensitization of tumor cells multidrug resistant (MDR phenotype), which have on their membrane efflux proteins that prevent the accumulation of these drugs within cells. The lack of selectivity, and difficulty internalization by cells of some compounds used in the treatment of cancer, are considered the major obstacles to chemotherapy, making the nanostructured surface modification of materials having anti-tumor activity through the functionalization way to increase the cytotoxic activity and subsequent incorporation of these compounds in tumor cells. In this work, the nanoparticles were functionalized with L-glutamine, due to the great importance of this amino acid in cancer cell metabolism and the fact that these have higher uptake of this amino acid to non-tumor cells. The cytotoxicity of nanoparticles functionalized with L-glutamine functionalized or not was evaluated in chronic myelogenous leukemia cells K562 and its version with MDR phenotype-Lucena 1. The cytotoxic activity of ZnO was confirmed by Trypan Blue exclusion and MTT reduction assays, with IC50 of 20 d K562 with ZnO nanoparticles and ZnO_Gln, while for Lucena-1 was observed similar percentages of doubly labeled cells and marked only with Annexin V. To assess whether there is influence of reactive oxygen species (ROS) generated by nanoparticles of ZnO and ZnO_Gln in their cytotoxic activity in ceC promoted protection against the cytotoxicity of nanoparticles even in higher concentrations of nanoparticles (30 ug / ml) in both the lines cell. In the potentiation of cell death caused by ZnO nanoparticles in cell lines studied, functionalization with L-glutamine promoted greater incorporation o562 cells treated with pure functionalized nanoparticles and in relation to the control compared to cells of Lucena-1 strain. Through analysis of the possible ways of cell death via Western blot analysis, the proteins and particularly studying the events involved in programmed cell death (Bcl2, Bad, Bid, caspase-3 and PARP) and autophagy (beclin-1 and P62), there was more than one death pathway was activated in cells treated with nanoparticles, which corroborates with what is described in literature, considering autophagy and apoptosis are the main routes of death activated cytotoxic action ofytotoxic effect of ZnO in the chronic myelogenous leukemia line K562 and the line Lucena MDR-1 phenotype and use this amino acid in the functionalization of nanomaterials can be an important tool to be exploited.

L'utilisation des ressources en fer et les variations du potentiel redox sont des processus impliqués dans la prolifération et la différenciation cellulaire. Ils participent à l'hématopoïèse normale et leur dérégulation peut être associée à des conditions pathologiques. Les hémopathies, telles que la leucémie aiguë myéloïde (LAM), témoignent du lien entre disponibilité en fer, signalisation redox et leucémogenèse. La déplétion en fer induit un arrêt de la prolifération suivi de la mort cellulaire, et pour des cellules primaires leucémiques (blastes) de patients LAM, elle peut conduire à un réengagement de la différenciation vers la lignée monocytaire. Cependant, les besoins en fer des clones leucémiques restent mal définis. Dans les cellules animales, le cœur du réseau de régulation du fer est organisé à travers le système régulateur IRE-IRP. Les Iron Regulatory Proteins (IRP), agissent sur la traduction de nombreuses protéines impliquées dans la gestion du fer par interaction avec les Iron Responsive Elements (IRE) localisés sur les régions non codantes des ARN messager (ARNm) régulés. A partir de lignées cellulaires leucémiques (KG1, K562), de blastes de patients LAM et de progéniteurs CD34+ contrôles issus de sang de cordon et de moelle osseuse de donneurs sains, le statut du système de gestion cellulaire du fer a été caractérisé pour les premières étapes de l'hématopoïèse normale et pathologique. A travers la manipulation des apports cellulaires en fer, notamment par l'utilisation de chélateurs à usage thérapeutique, la réponse du système homéostatique a été suivie. Nos données soulignent les faibles besoins en fer des progéniteurs hématopoïétiques, et d'autres cellules, pour proliférer. Dans les lignées cellulaires le régulateur IRP est en excès par rapport à ses cibles IRE, ce qui pourrait être une caractéristique générale du contrôle de la traduction pour des ARNm spécifiques par fixation de régulateurs translationnels. La régulation semble exclusivement le fait d' IRP1, puisqu' IRP2 n'a pas été détecté dans les progéniteurs hématopoïétiques, qu'ils soit pathologiques ou non. De subtiles différences ont été identifiées dans les quantités des composants du réseau gérant le fer dans les cellules leucémiques en comparaison des cellules saines témoins, ainsi que des capacités différentes à croître dans un milieu minimal comportant des concentrations en fer précisément définies. Les informations obtenues à travers ce travail pourraient bénéficier à l'élaboration de protocoles thérapeutiques, incluant notamment la manipulation du fer, dans les LAM ou d'autres pathologies. / Use of iron resources and variations of the redox balance are processes involved in cell proliferation and differentiation. They participate to normal hematopoiesis and their disturbance may be associated with pathological conditions. Hematological neoplasms, such as acute myeloid leukemia (AML), provide clinical evidence of the link between iron availability, redox signaling, and malignancy. Stringent iron depletion induces arrest of proliferation followed by cell death, and deprived primary leukemic cells of AML patients (blasts) have been previously shown to engage into the monocytic lineage. Yet, the iron needs of leukemic clones are unknown. The core network of cellular iron regulation in mammals is organized around the IRE-IRP system. The Iron Regulatory Proteins (IRP) act on the translation of many proteins involved in iron management by interacting with Iron Responsive Elements (IRE) located on the untranslated regions of messenger RNA (mRNA) coding these proteins. Using leukemic cell lines (KG1, K562), blasts of AML patients and CD34+ progenitors isolated from cord blood or the bone marrow of healthy donors, the status of the iron management system was established in the first stages of normal and pathological hematopoiesis. The response of the homeostatic system upon manipulation of iron provision, including with clinically implemented chelators, has been monitored. Our data emphasize the weak iron requirements of hematopoietic progenitors, and other cells, to proliferate. In cell lines the IRP regulator is in excess of its IRE targets, which may be a general feature of translational control for specific mRNA. The regulation seems exclusively mediated by IRP1, as the IRP2 regulator has not been detected in normal or malignant hematopoietic progenitors. Subtle differences have been found in the iron handling system of leukemic cells as compared to normal cells, together with different abilities to grow on a minimal medium containing precisely defined iron concentrations. The design of improved therapeutic regimens including iron manipulation, in AML and other pathologies, may benefit from considering the information obtained in this work.

Les protéines de choc thermiques (HSP) sont des chaperons moléculaires qui stabilisent le pliage et la conformation de protéines normales et oncogéniques, prévenant la formation d'agrégats protéiques. Elles sont impliquées dans la régulation de l'apoptose, de la survie cellulaire et dans la cancérogénèse. HSP90 est la protéine chaperone majeure de stabilisation d'oncogènes impliqués dans les hémopathies malignes. L'objectif de notre travail était de déterminer l'implication de HSP90 dans différents types d'hémopathies malignes, les Leucémies Aiguës Myéloïdes (LAM), les Syndromes Myélodysplasiques (SMD) et les Leucémies Aiguës Lymphoblastiques (LAL), et de tester son inhibition par un inhibiteur spécifique, la tanespimycine (17- AAG). Dans les LAM, nous avons évalué l'implication des différentes isoformes de la protéine dans la résistance aux chimiothérapies et aux inhibiteurs de HSP90. Ce travail met en évidence la valeur péjorative de l'expression de HSP90 dans les différents sous types d'hémopathies, corrélant avec un risque de rechute élevé ou d'évolution vers des formes plus agressives. L'utilisation de la tanespimycine a permis de déclencher l'apoptose dans les cellules immatures impliquées dans ces pathologies. HSP90 constitue donc une protéine majeure de la cellule leucémique, et son ciblage offre des perspectives intéressantes dans le traitement des hémopathies malignes / Heat shock proteins (HSP) are molecular chaperones that stabilize the folding and conformation of normal and oncogenic proteins, preventing the formation of protein aggregates. They are involved in the regulation of apoptosis, cell survival and carcinogenesis. HSP90 is the major chaperone implicated in stabilization of oncogenes involved in hematologic malignancies. The aim of our study was to determine the involvement of HSP90 in various types of malignancies, Acute Myeloid Leukemia (AML), Myelodysplastic Syndromes (MDS) and Acute Lymphoblastic Leukemia (ALL) and to test its inhibition by a specific inhibitor, the tanespimycine (17-AAG). In acute myeloid leukemia, we evaluated the involvement of different isoforms of the protein in resistance to chemotherapy and inhibitors of HSP90. This work highlights the pejorative value of HSP90 expression in different subtypes of malignancies, correlated with a high risk of relapse or progression to more aggressive forms. Use of tanespimycine has triggered apoptosis in immature cells involved in these diseases. HSP90 is therefore a major protein of the leukemic cell and its targeting offers interesting perspectives in the treatment of hematologic malignancies

O presente estudo compreende 96 transplantes de medula óssea (TMO) de doadores HLA-idênticos em pacientes portadores de Leucemia Mielóide Crônica, no período de Junho de 1986 a Junho de 1998. A autora selecionou diversas covariáveis para serem estudadas como fatores prognósticos de cinco desfechos principais: ocorrência de doença enxerto contra hospedeiro (DECH) aguda e crônica, incidência de recaída, sobrevida livre de doença (SLD) e sobrevida global (SG). As covariáveis estudadas foram: idade, sexo, escolaridade, tempo entre o diagnóstico e o transplante, fase da doença ao transplante, regime de condicionamente, profilaxia de DECH, compatibilidade de sexo entre doador e receptor, sexo do doador, tamanho do baço e do fígado, percentagem de blastos e número de plaquetas no sangue periférico na primeira consulta ao CEMO, ocorrência e grau de DECH aguda, ocorrência de DECH crônica e tempo para recuperação de plaquetas após o TMO. Não foi encontrada associação estatisticamente significativa num nível de 95% de confiança entre qualquer das covariáveis e a ocorrência de DECH crônica ou de recaída. A ocorrência de DECH aguda mostrou-se associada apenas com a fase da doença ao transplante. As covariáveis que se mostraram associadas com a sobrevida global e a sobrevida livre de doença foram: a percentagem de blastos no sangue periférico e tamanho do baço na primeira consulta ao CEMO, a fase da doença ao transplante, o tipo de profilaxia de DECH, a ocorrência e o grau de DECH aguda e o tempo para recuperação de plaquetas num nível acima de 20 x 103/mm3. / This study comprises 96 bone marrow transplantations (BMT) from HLA-identical siblings in patients with Chronic Myeloid Leukaemia (CML), from June 1986 to June 1998. The author selected several covariates to be studied as prognostic factors for five main endpoints: acute and chronic graft versus host disease (GVHD) occurrence, relapse incidence (RI), disease free survival (DFS) and overall survival (OS). The covariates studied were: age, sex, years of schooling, time from diagnosis to transplantation, disease phase at BMT, conditioning regimen, GVHD prophylaxis, sexmatch between donor-receptor, donor sex, spleen and liver size, blasts percentage and platelet counts in peripheral blood at first consultation at CEMO, acute GVHD occurrence and grade, chronic GVHD occurrence and time to platelet recovery after BMT. No statistically significant association was found between the covariates studied and the occurrence of acute or chronic GVHD or of relapse. The covariates found to have an association with overall survival (OS) and disease free survival (DFS) were the percentage of blasts in peripheral blood and the spleen size at first consultation, the disease phase at transplant, type of GVHD prophylaxis, the occurrence and grade of acute GVHD and the time to platelet recovery above 20 x 103/mm3.

A leucemia mielóide aguda ainda apresenta altos índices de mortalidade em adultos, exceção feita à leucemia promielocítica. A otimização dos protocolos de tratamento tem sido muito discutida há 3 décadas, com resultados ainda insatisfatórios. Fatores prognósticos como idade, cariótipo e tolerância à consolidação com altas doses de citarabina guardam relação com a melhor sobrevida. Com o objetivo de avaliar diferentes protocolos de tratamento e validar estes e outros fatores prognósticos, conduzimos um estudo retrospectivo no Hospital das Clínicas da Universidade de São Paulo, analisando prontuários médicos e os eventos relacionados à leucemia mielóide aguda, de 1978 a 2007. Analisamos 400 pacientes tratados curativamente e achamos que idade abaixo de 60 anos (27% vs 7%), cariótipo favorável (53% vs 28% vs 5%) e administração de doses totais de citarabina, principalmente se acima da mediana de 45,45 gramas (68% vs 44% vs 21%) tem impacto positivo na sobrevida global em 5 anos, sendo o uso de altas doses de citarabina um fator independente. A positividade para mieloproxidase, classificação FAB e protocolo de tratamento não mostraram associação estatisticamente significante para melhores índices de sobrevida. Pudemos concluir que, se os protocolos de indução não apresentam diferenças estatísticas, a consolidação intensiva com altas doses de citarabina em pacientes abaixo de 60 anos tem impacto independente na sobrevida global, com resultados ainda melhores quando a dose total é maior ou igual a 45,45 gramas. O cariótipo também foi validado em nossa população / Acute myeloid leukemia in adults is still a highly fatal disease, except for acute promyelocitic leukemia. The optimization of treatment protocols has been debated for three decades, without satisfactory results. Prognostic factors like age, kariotype and consolidation with cytarabine in high dosis seem to correlact with a better overall survival. We conducted a retrospective study in the General Hospital of University of Sao Paulo analyzing medical records and acute myeloid leukemia outcomes to compare different treatment protocols used through 1978 to 2007. We also intended to validate international prognostic factors as the ones cited in our population. We analyzed 400 patients treated with curative intention and found better overall survival in 5 years regarding age less than 60 years (27% vs 7%), favorable karyotipe (53% vs 28% vs 5%) and high dosis cytarabine in consolidation, meanly if total dose was at least the median of 45,45 g (68% vs 44% vs 21%). Consolidation with high dosis cytarabine was an independent predictor of better overall survival. No estatistical differences were seen regarding myeloperoxidase positivity, induction protocol and FAB classification. We concluded that, if the induction protocols seem to be no different in results, consolidation with high dosis cytarabine for patients under 60 years has impact in overall survival, being even better when the total dosis is at least 45,45 g. Karyotipe has also been validated in our study population