Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Orientadores: Fabiola Traina, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T21:51:25Z (GMT). No. of bitstreams: 1 MachadoNeto_JoaoAgostinho_M.pdf: 23849071 bytes, checksum: df78354ffdd25c98c274422937e03f93 (MD5) Previous issue date: 2011 / Resumo: A ocorrência da leucemia aguda resulta de uma combinação de mutações e alterações em funções protéicas que conferem a capacidade de proliferação, defeito na diferenciação e apoptose celular. Síndromes mielodisplásicas (SMD) são desordens hematopoéticas resultantes de alterações na célula pluripotente, caracterizadas por hematopoese ineficaz e alta taxa de evolução para leucemia mieloide aguda (LMA). Células leucêmicas expressam uma variedade de receptores de fatores de crescimento e citocinas, como o receptor do Insulin-like growth factor 1 (IGF-1R). A via de sinalização do IGF-1 inicia-se através da ativação de seu receptor e subsequente ativação de seus substratos, como os substratos do receptor de insulina (IRS). Algumas evidências indicam a participação das proteínas IRS em doenças hematológicas: (1) IRS1 foi descrito como constitutivamente fosforilado e associado ao BCR-ABL em células K562; (2) a expressão de IRS1 foi relacionada com pior prognóstico em leucemia linfóide aguda (LLA) BCR-ABL positiva; (3) IRS2 associa-se ao receptor de eritropoetina; (4) a expressão de IRS2 foi modulada durante estímulos com eritropoetina e IGF-1 e em processos de diferenciação em células hematopoéticas normais e leucêmicas. Neste estudo, foi observada a presença da expressão gênica e protéica de IRS1 e IRS2 em células hematopoéticas normais, mielodisplásicas e leucêmicas, entretanto, o padrão de expressão das duas proteínas foi diferente. Em linhagens celulares de leucemia aguda, IRS1 foi expresso em linhagens de leucemia aguda mieloide (P39, K562, NB4, KG-1, e HL60) e linfoide (MOLT4, Jurkat, Raji e Daudi), enquanto que IRS2 foi expresso preferencialmente em linhagens mieloides. Em células hematopoéticas primárias, não houve diferença na expressão de IRS1 entre células hematopoéticas de pacientes com SMD e LMA e controles normais, e a expressão gênica de IRS1 apresentou-se aumentada em amostras de medula óssea de pacientes com LLA em relação aos controles normais. A expressão de IRS2 foi menor nas amostras de medula óssea de pacientes com SMD, LMA e LLA em relação aos controles normais, e a expressão de IRS2 foi menor em pacientes com SMD de alto risco se comparados com SMD baixo risco, de acordo com a classificação FAB, WHO e com número de citopenias. A participação de IRS2 na diferenciação eritróide de células hematopoéticas normais e mielodisplásicas foi evidenciada através da avaliação da expressão de IRS2 durante a diferenciação eritroide de células progenitoras de medula óssea de doadores normais e de pacientes com SMD. O estudo evidenciou o aumento da expressão de IRS2 durante a diferenciação eritroide, sendo que nas células mielodisplásicas, IRS2 apresentou um menor aumento se comparado às células hematopoéticas normais. Em células BCR-ABL positivas, a inibição da expressão de IRS1 (realizada através do uso de shRNA mediado por lentivírus específico para IRS1) resultou em inibição da proliferação celular e crescimento clonal, acúmulo de células na fase G0/G1 e redução de células na fase S do ciclo celular. A inibição de IRS1 resultou na inibição da fosforilação das proteínas Akt, P70S6K e ERK. Entretanto, a inibição de IRS1 não modulou a apoptose e as proteínas BCL2, BAX e BAD, assim como não modulou a fosforilação de BCR-ABL e CRKL e não apresentou sinergismo quando associado ao inibidor de tirosina quinase do BCR-ABL (imatinib). Estes dados indicam que IRS1 participa dos processos celulares de proliferação celular e clonogenicidade em células BCR-ABL positivas, através da modulação de Akt, P70S6K e ERK. Os achados aqui descritos sugerem que IRS1 é expresso em células hematopoéticas normais, mielodisplásicas e leucêmicas, destacando-se sua elevada expressão em células de LLA e sua participação na via de sinalização BCR-ABL. Estes dados indicam que IRS1 pode ser um alvo terapêutico em LLA e leucemia mieloide crônica (LMC), especialmente nas leucemias BCR-ABL positivas e resistentes a inibidores da atividade tirosina quinase do BCR-ABL. Adicionalmente, IRS2 é expresso em células hematopoéticas, destacando-se a sua expressão reduzida em células mielodisplásicas e leucêmicas quando comparadas às células hematopoéticas normais. A reduzida expressão de IRS2 em células hematopoéticas de pacientes com SMD de alto risco quando comparados aos de baixo risco e o reduzido aumento da expressão de IRS2 na diferenciação eritroide de progenitores de pacientes com SMD sugerem que a expressão de IRS2 participa da fisiopatologia das SMD e pode ser um marcador prognóstico nesta doença / Abstract: Acute leukemia results from a combination of mutations and changes in protein functions that confer the ability of proliferation, and defect in differentiation and apoptosis. Myelodysplastic syndromes (MDS) are hematopoietic disorders caused by alterations in pluripotent cells, characterized by ineffective hematopoiesis and a high rate of progression towards acute myeloid leukemia (AML). Leukemia cells express a variety of receptors for growth factors and cytokines, such as Insulin-like growth factor 1 (IGF-1R). The signaling pathway is initiated by activating its receptor and subsequent activation of its substrates such as insulin receptor substrate (IRS). There is evidence that suggests an involvement of IRS proteins in hematopoeitic disease: (1) IRS1 was described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, (2) the IRS1 expression was associated with a poorer prognosis in BCR-ABL positive acute lymphoblastic leukemia (ALL), (3) IRS2 bind to erythropoietin receptors, (4) IRS2 expression was modulated during stimulation with erythropoietin and IGF-1 during cell differentiation in normal and leukemia hematopoietic cells. In this study, we observed the presence of the gene and protein of IRS1 and IRS2 in normal hematopoietic, leukemia, and myelodysplastic cells, however, the expression pattern of these proteins was different. In acute leukemia cell lines, IRS1 was expressed in myeloid leukemia cells (P39, K562, NB4, KG-1 e HL60) and lymphoid leukemia cells (MOLT4, Junkat, Raji e Daudi), whereas IRS2 expression was more evident in myeloid cell lines. In primary hematopoietic cells, no difference was observed in IRS1 expression between normal, MDS and AML cells, and the IRS1 expression was increased in bone marrow samples from ALL patients compared to normal controls. IRS2 expression was lower in bone marrow samples from patients with MDS, AML and ALL compared to normal controls, and the IRS2 expression was lower in high risk compared with low risk MDS patients, according to FAB and WHO classification, and number of cytopenias. The participation of IRS2 in erythroid differentiation of normal and myelodysplastic hematopoietic cells was evidenced by evaluating IRS2 expression during erythroid differentiation of progenitor cells from the bone marrow of normal donors and patients with MDS. The study demonstrated an increase in IRS2 expression during erythroid differentiation, whereas in myelodysplastic cells, IRS2 showed a smaller increase compared to normal hematopoietic cells. In BCR-ABL positive cells, IRS1 inhibition (by lentivirus-mediated shrunk specific for IRS1) resulted in inhibition of cell proliferation and clonal growth, accumulation of the cells in G0/G1 phase and reduction of cells in S phase of cell cycle. The IRS1 silencing resulted in inhibition of Akt, P70S6K and ERK phosphorylation. However, IRS1 inhibition did not modulate apoptosis; and the proteins BCL2, BAX and BAD, did not modulate the phosphorylation of BCR-ABL and CRKL, nor did they show synergism when combined with tyrosine kinase inhibitor of BCR-ABL (Imatinib). These data indicate that IRS1 participates in the proliferation and clonogenic of BCRABL positive cells by modulation of Akt, P70S6K and ERK. The findings reported herein suggest that IRS1 is expressed in normal hematopoietic, leukemia and myelodysplastic cells, highlighting its high expression in ALL cells and involvement in the BCR-ABL pathway. These data indicate that IRS1 may be a therapeutic target in ALL and chronic myeloid leukemia (CML), especially in BCR-ABL positive leukemias resistant to inhibitors of tyrosine kinase activity of BCRABL. In addition, IRS2 is expressed in hematopoietic cells, highlighting its reduced expression in myelodysplastic and leukemia cells compared to normal hematopoietic cells. The reduced IRS2 expression in high risk when compared to low risk MDS and the lower increase in IRS2 expression during the erythroid differentiation of progenitor cells from MDS patients suggest that the expression of IRS2 participates in the pathophysiology of MDS and may be a prognostic marker in this disease / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T16:35:29Z (GMT). No. of bitstreams: 1 Fattori_Andre_D.pdf: 5773591 bytes, checksum: 1c28ebb312b8a424e862317faa2d4a1b (MD5) Previous issue date: 2006 / Resumo: No presente estudo analisou-se a expressão gênica por microarray em 17 casos de Leucemia Mielóide Aguda (LMA), 5 casos de Leucemia Linfóide Aguda (LLA), tendo como controle 4 amostras de células progenitoras de sangue periférico coletadas de doadores de transplante de medula óssea após estimulação com G-CSF. Para isto foram utilizadas membranas de microarray contendo cerca de 4700 fragmentos de cDNA correspondendo a genes conhecidos ou a ESTs, construídas no Instituto Ludwig de Pesquisa do Câncer/FAPESP a partir dos clones de cDNA gerados pelo Projeto Genoma Humano do Câncer. Suas principais vantagens são (1) a utilização da metodologia ORESTES na construção da biblioteca de ESTs, o que a torna mais representativa da parte central dos genes; (2) ser desenvolvida com clones cDNA provenientes do Projeto Genoma Humano do Câncer e, portanto, com potencial de conter genes não disponíveis nas membranas comerciais e, (3) sendo produzida a partir de tecidos neoplásicos variados, teoricamente pode apresentar seqüências relacionadas à tumorigênese. A metodologia foi eficiente na caracterização de um conjunto de genes diferencialmente expressos nas amostras de LMA, LLA, LMA subtipo M3 e dos casos-controle, pela análise estatística pelo método de Wilcoxon e clusterização hierárquica supervisionada e não-supervisionada desenvolvidas por Eisen. Na comparação entre LMA e LLA, sob o aspecto funcional, foi possível observar genes particularmente afetados relacionados a processos celulares específicos, como as vias de regulação do metabolismo de nucleotídeos, adesão e sinalização celulares (SS2XIP, MUC4, THBS1 e RGL2), transdução de sinal especialmente pela via Wnt e via Ras e Rho das ?small GTPases? (RGL2, ARGHAP1 e CDKN1B e beta-catenina) entre as LMA, e um conjunto de genes reguladores apoptóticos como os genes BIRC6, SH3GLB1, PSEN1 e NFKB1 entre as LLA. Os microarrays evidenciaram resultados importantes na separação de genes relacionados às LMA-M3 e às LMA-não M3, mostrando expressão diferencial de genes antes não associados ao subtipo específico M3, tais como TLE1, PSCDBP, SMG1, TALDO1, CUL1, TBX1 e UBXD8), alguns relacionados à regulação do ciclo celular e morte programada, e componentes do complexo Groucho/TLE que funciona como repressor da atividade transcricional induzida pela via Wnt.Os resultados foram validados através da utilização de PCR em tempo real em 5 genes selecionados e testados em uma população maior de casos (n=50 amostras de leucemias). Em 4 destes genes, os resultados foram completamente confirmados, demonstrando que a tecnologia de microarrays foi eficiente no rastreamento de genes potencialmente relacionados aos de subtipos de Leucemias Agudas, em especial as LLA e as LMA-M3, nesta membrana 3.7K do Instituto Ludwig/FAPESP. Assim, a utilização da metodologia de microarray com seqüências derivadas do Projeto genoma do Câncer (Instituto Ludwig/FAPESP) permitiu a discriminação de vias metabólicas importantes no processo de tumorigênese, alguns até mesmo associados a tipos leucêmicos específicos, e o reconhecimento de genes diferencialmente expressos antes não descritos na literatura / Abstract: The development of gene expression assays, such as cDNA microarrays, has permitted the simultaneous study of several genes; one of this methods is the cDNA microarrays. These investigations contribute to the classification of diseases, comprehension of tumoral biology and, finally, the determination of genetically related prognostic factors. In this present study, the gene expression profiles were analysed for 17 cases of Acute Myeloid Leukemia (AML), 5 cases of Acute Lymphocytic Leukemia (ALL) and 4 samples of peripheral blood apheresis, after stimulation with G-CSF for alogenic transplantation donors. For the microarray assays, membranes containing approximately 4700 spots were developed by the Ludwig Institute for Cancer Research, with contribution and financial support of FAPESP, using cDNA cloned from the Human Cancer Genome Project. The main advantages of these membranes include (1) availability of a cDNA library constructed by the ORESTES methodology, which makes the cDNA clones more representative of the whole transcriptome, (2) the non-comercial characterization of human transcripts, which may be vantageous for finding new genes and/or ESTs not diffusely disponible in large-scale production and, (3) since the library is contructed from different tumoral tissues, it may constitute the best representation of the tumorigenesis process. The methodology efficiently distinguished a group of genes, supporting the identification of the ALL, AML and control samples by the Wilcoxon Statistical Analysis and the supervised and non-supervised Hierarchical Clusterization developed by Eisen. Among the genes that were differentially expressed when comparing AML and ALL, it was possible to observe genes related to specific cellular processes such as: in the AML group nucleotides metabolism regulation pathways, adhesion and cell signaling (SS2XIP, MUC4, THBS1 e RGL2), signal transduction, especially in the Wnt pathway and Ras and Rho from the ?Small GTPases? pathway (RGL2, ARGHAP1, CDKN1B and beta-catenin), and in the ALL a group of apoptotic regulators genes such as BIRC6, SH3GLB1, PSEN1 e NFKB1 among. The microarray efficiently to distinguished genes related to M3-AML and non-M3-AML, showing differentially expressed genes not previosly described in the literature to be related to the specific sbtype M3-AML, such as TLE1, PSCDBP, SMG1, TALDO1, CUL1, TBX1, UBXD8, genes associated with programmed cell death and cell cycle, and components of Groucho/TLE complex, which functions like a repressor of transcriptional activity induced by the Wnt pathway. The results were validated through the utilization of Real Time PCR in five selected genes, tested in a greater number of cases (n=50 acute leukemia samples). In 4 of these genes the results were completely confirmed, demonstrating that the microarray technology with the 3.7K Ludwig Institut/FAPESP membranes was efficient for screening genes possibly related to specific leukemias subtypes, particularly the ALL and M3-AML groups. As such, the utilization of microarray methodology with sequences derived from the Cancer Genome Project (Ludwig Institut/FAPESP) permitted the distinction of important metabolic pathways for the tumorigenesis process, some of them associated to specific leukemic subtypes, and the recognition of differentially expressed genes not before described in the literature / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Clínica Médica

Orientador: Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T16:46:11Z (GMT). No. of bitstreams: 1 Palodetto_Bruna_M.pdf: 1523635 bytes, checksum: 0dc5f0194b2dd973ee8eaeac9a384983 (MD5) Previous issue date: 2012 / Resumo: As síndromes mielodisplásicas (SMD) são um grupo heterogêneo de doenças hematopoiéticas caracterizadas pela hematopoiese ineficaz resultando em citopenia no sangue periférico; cerca de 30% das SMDs evolui para leucemia mielóide aguda secundária. Leucemias agudas (LA) são doenças malignas do sangue caracterizadas por acúmulo de blastos podendo ser classificadas em mielóide agudas (LMA), quando há envolvimento de mieloblastos, ou linfóides agudas (LLA), quando há envolvimento de linfoblastos. A sobrevida média dos pacientes com leucemia aguda ainda é muito baixa e muitos deles são resistentes ao tratamento ou apresentam recaída. O melhor entendimento sobre os mecanismos de progressão da mielodisplasia e da resposta ao tratamento em leucemias agudas poderia melhorar a taxa de resposta ao tratamento e aumentar a sobrevida dos pacientes. O metabolismo e o efluxo de drogas são mecanismos de defesa responsáveis pela proteção contra agentes tóxicos e estão envolvidos na biotransformação de diversos xenobióticos. O metabolismo de drogas pode ser divido em duas fases (Fase I: Oxidação; Fase II: Conjugação), sendo ambas mediadas por enzimas metabolizadoras de drogas. O efluxo de drogas é outro mecanismo de proteção contra tóxicos, similar ao metabolismo de drogas, porém mediado por proteínas de membrana. Essas proteínas são polimórficas e esses polimorfismos alteram a atividade enzimática, podendo modificar a resposta ao tratamento e a sua resistência. O gene CYP2B6 codifica uma enzima da fase I do metabolismo responsável pela ativação dos fármacos. Esse gene possui o polimorfismo G15631T onde há troca do aminoácido (Gln172His) resultando em perda da atividade enzimática. Os genes GSTM1, GSTT1 e NQO1 codificam enzimas da fase II do metabolismo, responsáveis pela conjugação com outras substâncias para facilitar a excreção. Os genes GSTM1 e GSTT1 possuem um polimorfismo que causa deleção homozigota do gene; e o gene NQO1 possui o polimorfismo C609T que resulta em troca do aminoácido codificado (Pro187Ser). Esses polimorfismos levam a perda da atividade enzimática. O gene MDR-1 codifica a P-glicoproteína que é uma proteína de membrana responsável pelo efluxo de drogas. Esse gene possui o polimorfismo C3435T que apesar de ser silencioso (Ile1142Ile) diminui a expressão de P-glicoproteína. Assim, o objetivo deste estudo foi identificar a influência dos polimorfismos CYP2B6 G15631T, GSTT1, GSTM1, NQO1 C609T e MDR-1 C3435T no risco de leucemias agudas e SMD, na progressão de SMD e resposta ao tratamento de leucemia aguda. Foram analisados 90 pacientes com leucemia aguda (66 LMA e 24 LLA), 68 pacientes com SMD e 100 controles normais utilizando os métodos de PCR-RFLP e Multiplex. Não houve diferença estatística na freqüência dos polimorfismos entre pacientes e grupo controle. Em SMD encontramos maior frequência de deleções de GST em pacientes que progrediram comparados aos pacientes que não progrediram: 50% e 21% (P=0,019). Também encontramos menor frequência do alelo polimórfico T do polimorfismo MDR-1 C3435T em pacientes que progrediram comparada a dos pacientes que não progrediram: 50% e 81% (P=0,012). Na resposta ao tratamento de leucemias agudas, encontramos uma tendência à maior frequência do polimorfismo NQO1 C609T em pacientes com falha de indução quando comparados a pacientes com remissão em leucemias agudas, em geral, (P=0,093) e pacientes somente com LMA (P=0,125); e quando comparamos falha de indução com o grupo controle em leucemias agudas, em geral, (P=0,101) e somente em LMA (P=0,08). Observamos a mesma tendência quando comparamos a frequência do polimorfismo NQO1 C609T em pacientes com óbito precoce versus a população normal (P=0,058). Em conclusão, estes resultados sugerem que os polimorfismos não estão relacionados ao risco de leucemia aguda e SMD, embora a amostra aqui analisada possa ter sido insuficiente; as deleções GST e o polimorfismo MDR-1 C3435T estão envolvidos na progressão de SMD e o polimorfismo NQO1 C609T tem uma tendência a estar relacionado à falha de indução e ao óbito precoce em pacientes com leucemias agudas, em geral, e somente LMA / Abstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by ineffective hematopoiesis resulting in peripheral blood cytopenia, about 30% of MDS patients progresses to acute myeloid leukemia. Acute leukemia (AL) are malignant blood diseases characterized by accumulation of blasts and they can be classified into acute myeloid (AML), when there is myeloblasts involvement or acute lymphoid (ALL), when there is lymphoblasts involvement. The median survival of acute leukemia patients is very low and many of them are resistant to treatment or relapsed. The better understanding of the myelodysplasia progression mechanisms and the acute leukemia response to treatment could improve the treatment response rate and patients survival. The metabolism and drug efflux are defense mechanisms responsible for protection against toxic agents and are involved in the biotransformation of various xenobiotics. The drug metabolism can be divided into two phases (Phase I: Oxidation; Phase II: Conjugation), both being mediated by drug metabolizing enzymes. The drug efflux is a similar mechanism of protection but is mediated by membrane proteins. These enzymes are polymorphic and these polymorphisms alter the enzyme activity and may modify treatment response and resistance. The CYP2B6 gene encodes a phase I enzyme responsible for drug activation. This gene has the G15631T polymorphism where there is exchange of the amino acid (Gln172His) resulting in loss of enzyme activity. The GSTM1, GSTT1 and NQO1 genes encoding phase II metabolizing enzymes that are responsible for combining with other substances to facilitate drug excretion. GSTM1 and GSTT1 genes have a polymorphism that causes homozygous deletion of the gene; and the NQO1 gene has the C609T polymorphism that results in amino acid changes (Pro187Ser). These polymorphisms lead to loss of enzyme activity. The MDR-1 gene encodes P-glycoprotein (P-gp) which is a membrane protein responsible for drug efflux. This gene has the C3435T polymorphism that despite being silent (Ile1142Ile) leads to lower P-gp expression. The aim of this study was to identify the influence of CYP2B6 G15631T, GSTT1, GSTM1, NQO1 C609T and MDR-1 C3435T polymorphisms in acute leukemia and MDS risk, MDS progression and acute leukemia response to treatment. We analyzed 90 patients with acute leukemia (66 AML and 24 ALL), 68 MDS patients and 100 normal controls using the PCRRFLP and Multiplex methods. There was no statistical difference in the frequency of polymorphisms between patients and control group. In MDS we found higher frequency of GST deletions in patients who progressed compared to patients who did not progress: 50% and 21% (P = 0.019). We also found less frequently polymorphic allele T of MDR-1 C3435T polymorphism in patients who progressed compared to patients who did not progress: 50% and 81% (P = 0.012). In acute leukemia response to the treatment, we found a trend toward a higher frequency of NQO1 C609T polymorphism in patients with induction failure compared to patients in remission, with acute leukemia in general, (P = 0.093) and AML patients only (P = 0.125); and induction failure when compared with the control group in acute leukemia in general (P = 0.101) and only in AML patients (P = 0.08). We observed the same trend when comparing the frequency of NQO1 C609T polymorphism in patients with early death versus normal population (P = 0.058). In conclusion, these results suggest that theses polymorphisms are not related to acute leukemia and MDS risk, although the sample analyzed here may have been insufficient; GST deletions and MDR-1 C3435T polymorphism are involved in MDS progression and NQO1 C609T polymorphism has a tendency to be related to induction failure and early death in patients with acute leukemia, in general, and AML only / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Les progrès thérapeutiques réguliers ont transformé le pronostic des enfants atteints de leucémie aiguë, posant avec une grande acuité le problème des effets secondaires tardifs, de l'insertion sociale mais aussi de la qualité de vie des patients et de leur entourage, ainsi que des déterminants mis en jeu. L'ensemble des acteurs du système de soins ont la responsabilité de l'étude de ces effets secondaires tardifs et de leur prise en compte. En 2004, le projet LEA (Leucémie de l'Enfant et de l'Adolescent) a débuté avec pour objectif d'évaluer l'état de santé et la qualité de vie à moyen et long terme de patients traités pour une leucémie aiguë de l'enfance après 1980. La volonté était de mettre en oeuvre un système pouvant produire de la connaissance dans une démarche de recherche traditionnelle, mais aussi de s'inscrire rapidement dans une démarche pragmatique de production d'informations susceptibles de modifier les pratiques de prise en charge et de suivi. L'objectif général de ce travail cherche à démontrer l'utilité de dispositifs lourds comme la mise en oeuvre d'une cohorte, dans la double logique de fournir une information d'une part directement utile à la décision clinique, et d'autre part susceptible d'éclairer la décision publique. Les travaux scientifiques s'articulent autour de : 1. La visibilité de la cohorte LEA au regard des autres dispositifs existants au niveau international ; 2. Les conséquences à distance des traitements lourds reçus par les patients ; 3. La qualité de vie de l'entourage à distance de l'épisode aigu de la maladie ; 4. L'utilité d'un suivi systématisé dans la diminution des inégalités d'accès au système de santé entre les classes sociales. / Regular advances in cancer treatment have dramatically improved the prognosis of children with acute leukemia, raising with a great acuity the problem of the late physical side effects, social integration, quality of life of the patients and their family as well as identification of the determinants of these outcomes. It is the responsibility of all the care system actors to consider these objective and subjective late effects. The LEA project (Leucémie de l'Enfant et de l'Adolescent - childhood and adolescent leukemia) was initiated in 2004 with the aim of studying the long-term health status and quality of life of children treated for leukemia after January 1980. As soon as the project began, the aim was to implement a system that can produce knowledge in a traditional research approach, but also to rapidly become a pragmatic approach of producing information that could affect both care and monitoring practices. The general objective of this manuscript seeks to demonstrate the utility of heavy plan such as the implementation of a cohort, in the double approach of providing information on the one hand directly relevant to clinical decision, and on the other hand likely to enlighten public decision. The present scientific works are based on: 1. The visibility of LEA in relation to other cohorts of childhood cancer survivors existing internationally; 2. The long-term impact of the heavy modalities of treatment, as the hematopoietic stem cell transplantation or irradiation; 3. The quality of life of the family long after the completion of cancer therapy; 4. The usefulness of a systematic follow-up in reducing inequalities in access to health care among social classes.

Orientadores: João Agostinho Machado Neto, Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T20:37:02Z (GMT). No. of bitstreams: 1 MachadoNeto_JoaoAgostinho_D.pdf: 13204649 bytes, checksum: a00ab26c9097cb10817c14fe2bdb2356 (MD5) Previous issue date: 2015 / Resumo: A identificação de genes/proteínas diferencialmente expressos em neoplasias hematológicas e a investigação funcional destas proteínas no fenótipo neoplásico são essenciais para o entendimento da biologia da doença. ANKHD1 é altamente expressa em células de leucemia aguda e tem potencial para regular vários processos celulares através de seus domínios de repetição de anquirina. Nosso grupo de pesquisa havia previamente identificado a interação entre ANKHD1 e SIVA através de ensaio de duplo híbrido. Outras proteínas potencialmente relacionadas à ANKHD1 que foram de interesse deste trabalho foram a Stathmin 1 que interage com SIVA e a YAP1 que interage com ANKHD1 em células não hematológicas. O objetivo principal do presente trabalho foi investigar a participação funcional de ANKHD1 e proteínas relacionadas em neoplasias hematológicas. Utilizamos modelos de linhagens leucêmicas humanas e células hematopoéticas primárias provenientes de indivíduos normais (n=52) e de pacientes com diagnóstico de síndrome mielodisplásica (SMD; n=65), neoplasia mieloproliferativa (NMP; n=82), leucemia mieloide aguda (LMA; n=60) e leucemia linfoide aguda (LLA; n=19). Técnicas para avaliação de expressão gênica (qPCR), expressão e interação proteica (western blotting), ensaios funcionais de migração (ensaio transwell), proliferação (MTT e clonogenicidade in vitro, formação de tumor in vivo) e apoptose (Anexina V/IP, TUNEL), e inibição de proteínas de interesse através do uso de inibidores farmacológicos ou ferramentas de terapia gênica foram utilizados. A interação entre ANKHD1 e SIVA foi confirmada por ensaios de coimunoprecipitação. Em linhagens celulares leucêmicas U937 e Jurkat, o silenciamento de ANKHD1 reduziu a proliferação celular, migração e o crescimento tumoral, enquanto que a inibição de SIVA aumentou a migração e o crescimento tumoral e reduziu a sensibilidade à apoptose induzida por radiação UV de células U937. A inibição de ANKHD1 reduziu a atividade de Stathmin 1 e a interação entre SIVA/Stathmin 1. Grande parte das linhagens leucêmicas e amostras primárias de pacientes com neoplasias hematológicas não apresentou a expressão de YAP1, e o silenciamento de ANKHD1 não modulou a atividade de YAP1 em células U937. A expressão de Stathmin 1 foi significativamente elevada em amostras de células hematopoéticas de pacientes com neoplasisa hematológicas (SMD AREB-1/AREB-2, LMA, LLA e mielofibrose primária) quando comparada às amostras de doadores saudáveis. O silenciamento de Stathmin 1 reduziu a proliferação celular e clonogenicidade em células leucêmicas U937, Namalwa e células HEL JAK2V617F. Especificamente em células HEL JAK2V617F, a inibição de Stathmin 1 ou o tratamento com o agente estabilizador de microtúbulos paclitaxel teve um efeito potencializador ao tratamento com ruxolitinib, inibidor de JAK1/2, na indução de apoptose. Em conclusão, propomos que a ANKHD1 participa do fenótipo neoplásico de células leucêmicas através de sua interação com SIVA, inibição de SIVA e ativação de Stathmin 1. Os nossos resultados indicam que a função de ANKHD1 na leucemogênese independe de sua interação com YAP1. Em células humanas com ativação constitutiva da via JAK2/STAT, identificamos a relevância funcional da participação de Stathmin 1 no fenótipo neoplásico / Abstract: The identification of genes/proteins differentially expressed in hematopoietic neoplasms and the functional investigation of these proteins in the neoplastic phenotype are essential for the understanding of disease biology. ANKHD1 is highly expressed in acute leukemia cells and has a potential role in regulating many cellular processes through its ankyrin repeat domains. Our research group has previously identified the interaction between ANKHD1 and SIVA through two-hybrid assay. Other proteins potentially related to ANKHD1 that were of interest of this research are Stathmin 1 that interacts with SIVA, and YAP1 that interacts with ANKHD1 in non hematologic cells. The aim of this study was to investigate the functional role of ANKHD1 and its related-proteins in hematologic malignancies. We used human leukemia cell lines and primary hematopoietic cells from healthy donors (n=52) and patients with myelodysplastic syndromes (MDS; n=65), myeloproliferative neoplasms (MPN; n=82), acute myeloid leukemia (AML; n=60) and acute lymphoid leukemia (ALL; n=19). Techniques for the evaluation of gene expression (qPCR), protein expression and interaction (western blotting), functional assays of migration (transwell assay), proliferation (in vitro MTT and clonogenicity, in vivo tumor formation) and apoptosis (Annexin V/PI, TUNEL), and inhibition of proteins of interest through pharmacologic agents or gene therapy tools were used. The interaction between ANKHD1 and SIVA was confirmed by co-immunoprecipitation assays. In leukemia cell lines U937 and Jurkat, ANKHD1 silencing reduced cell proliferation, migration and tumor growth, while SIVA inhibition increased migration and tumor growth, and reduced sensitivity to UV-induced apoptosis of U937 cells. Inhibition of ANKHD1 reduced Stathmin 1 activity and the interaction between SIVA/Stathmin 1. A large proportion of leukemia cell lines and primary samples from patients with hematologic malignancies did not present YAP1 expression, and ANKHD1 silencing did not modulate YAP1 activity in U937 cells. Stathmin 1 expression was significantly higher in primary hematopoietic cells from patients with hematological malignancies (MDS RAEB-1/RAEB-2, AML, ALL and primary myelofibrosis) compared to samples from healthy donors. Stathmin 1 silencing reduced cell proliferation and clonogenicity of U937 and Namalwa leukemia cells, and HEL JAK2V617F cells. Specifically in HEL JAK2V617F cells, Stathmin 1 silencing or treatment with the microtubule-stabilizing agent paclitaxel had a potentiating effect to treatment with the JAK1/2 inhibitor ruxolitinib on apoptosis induction. In conclusion, we propose that ANKHD1 participates in the leukemia phenotype through its interaction with SIVA, SIVA inhibition, and Stathmin 1 activation. Our results indicate that ANKHD1 plays a role in leukemogenesis independently of its interaction with YAP1. In human cells with a constitutive activation of the JAK2/STAT pathway, we identified the relevant role of Stathmin 1 in the malignant phenotype / Doutorado / Clinica Medica / Doutor em Clínica Médica

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13326号 / 論医博第2194号 / 新制||医||1043(附属図書館) / (主査)教授 小川 誠司, 教授 滝田 順子, 教授 河本 宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

v anglickém jazyce Leukemia is the most common malignant childhood disease, which occurs in Czech Republic every year in about 100 cases. That's why has been devoted over the last 5 decades much attention and funding to this research and the children with leukemia have a high chance for achieving complete remission. Nevertheless, for about 10% of patients leukemia still remains lethal and these cases are subjects of current studies. Leukemia is a complex disease in its pathology involving lots of aberrations, which pathologically manifest at the DNA, mRNA and protein level. Nevertheless some symptoms of this disease are only at the protein grade (and not DNA or mRNA). Since a broad part of the protein profile in leukemic cell changes, it is necessary to develop newer, more sensitive and especially more efficient methods, which are able to follow up changes in large sets of proteins and detect prognostic and diagnostic markers. Within diploma study a new method - multiplex antibody array - has been tested. Our laboratory tests this method in collaboration with a Norwegian laboratory (Rikshospitalet University Hospital, University of Oslo) under leading by Dr. Lund-Johansen, who is the author of this method. Antibody array presents a set of 1152 latex bead populations labeled with fluorescent dyes in...

Treatment of acute myeloid leukemia, an aggressive hematopoietic malignancy of myeloid progenitors, has remained rather stagnant over the course of several decades. Infusions of cytarabine and anthracycline antibiotics have dominated the landscape of AML therapy, with minor changes to dosing schedule occasionally making slight adjustments to efficacy or tolerability. Improvements in prognosis have been bittersweet, with most progress seen in younger populations less likely to get the disease, and already more likely to achieve remission and to meet survival milestones. Much of this progress is attributed to other factors, such as improved supportive care and availability of hematopoietic stem cell and platelet transfusion. In most patients, occupying the 60-and-above demographic, improvements in survival have not been significant. In turn, the population impact of AML has changed little over time. While accounting for about one-third of total leukemia cases and one percent of total cancer cases, AML accounts for about one half of total leukemia deaths and two percent of total cancer deaths. Most advances straying away from standard treatment have been in important pathways that could be impactful in subsets of the overall AML patient population. Tyrosine kinases are implicated in numerous cancers including AML, with activity-enhancing mutations conferring growth advantages to malignant cells. About one-third of AML patients have mutations in one such kinase, FLT3, and may benefit from inhibitors to tyrosine kinases overall and from FLT3- specific agents. Mutations in isocitrate dehydrogenases highlight another subpopulation, about one-fifth of AML patients, who might benefit from emerging agents that inhibit these pathways from creating a leukemia-favoring environment in the bone marrow. Other pathways similarly implicated in numerous cancers including AML are being targeted with new agents that can benefit some AML patients, such as Hedgehog signaling and apoptotic regulation. Still, breakthroughs are needed that can help most AML patients, particularly in the cases of relapsed leukemia that occurs in most patients within a year or two after remission is achieved. CD33 is among a few molecular targets for AML, though it is just as ubiquitously expressed on healthy myeloid cells. Antibody-drug conjugates like Mylotarg have made progress in this approach, though hematopoietic toxicities have made treatment difficult in older populations. Clever techniques such as ablation of CD33 from healthy myeloid progenitors may be supportive in CD33-based approaches, and immunotherapy involving CD33-targeting is a rapidly growing research focus. This dissertation describes a new type of bispecific antibody that binds CD33 on AML and CD3 on cytotoxic T cells in a proof-of-concept study. Various formats for bifunctional molecules have been created and used clinically, including antibody-drug conjugates and bispecific antibodies that simultaneously engage antigens on two different types of cells. Those like the one described here, bispecific T-cell engagers, have typically taken the form of single-chain fusion proteins containing the variable regions binding to both antigens of interest. Other bispecific antibodies have imitated naturally-occurring immunoglobulin structures, boasting superior pharmacokinetics while facing steep obstacles in large-scale production. The single-chain fusions, easier to produce, can face difficulties in full engagement, with loss of function sometimes seen in fusion partners at the C-terminus. We propose a new format, believed to present two antigen-binding domains in N-terminal positions on a two-chain heterodimeric structure. Capitalizing on an elegantly designed system of hydrophobic cores and hydrogen-bonding networks generating an orthogonal heterodimer, we added an immunoglobulin hinge region to secure a permanently-bound heterodimer, and attached domains binding to CD3 and CD33. We hypothesized that this design, ensured to present its antibody components at N-termini, could bind two antigens at a distance appropriate for facilitating T cell cytotoxicity to AML. After expressing and purifying these proteins in mammalian cells, we demonstrated their ability to persist as a bispecific heterodimer. We showed in vitro that our bispecific heterodimers could bind both CD3+ and CD33+ cells, and that they bolstered T cell cytotoxicity to AML cell lines in a dose-dependent manner. Monomeric components bound only CD3+ or CD33+ cells depending on antibody variable domain present, and had no effect on T cell cytotoxicity. In a mouse model of minimal residual disease, T cells alone did not have a significant effect on the growth of AML, nor did they have an effect on overall survival. T cells with bispecific heterodimer greatly extended survival, and mice of this treatment group were free of leukemia. These findings suggest that this format for bispecific proteins allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. We believe this presents a compelling modular system for bispecific antibodies, where CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens, and can be further developed into a trispecific system engaging other immune cells or extending half-life with anti-albumin or Fc domains.

La télomérase est présente dans un nombre limité de cellules, telles que la plupart des cellules cancéreuses où son activité est indispensable pour l’immortalisation de ces dernières. C’est pourquoi cette enzyme a été proposée comme cible prometteuse pour des thérapies anticancéreuses. Ainsi, il est d’un intérêt certain d’identifier les mécanismes par lesquels elle est régulée, notamment via sa sous-unité catalytique, hTERT. Les rétinoïdes sont des inducteurs bien connus de la maturation granulocytaire associée à une répression de hTERT dans les blastes de leucémie aiguë promyélocytaire (LAP). Dans une lignée cellulaire LAP résistant à la maturation induite par les rétinoïdes (NB4-LR1), a précédemment été identifiée une nouvelle voie de répression transcriptionnelle de hTERT en absence de différenciation. De plus, mon laboratoire d’accueil a isolé un variant de la lignée NB4-LR1 résistant à cette répression transcriptionnelle de hTERT, la lignée NB4-LR1SFD. Ensemble, ces lignées cellulaires, qui se comportent différemment face à un traitement à l’ATRA, fournissent un outil unique et puissant pour obtenir plus d’informations sur plusieurs problèmes de la régulation de la biologie moléculaire de la télomérase. Dans cette étude, en utilisant plusieurs approches complémentaires (immunoprécipitation de la chromatine combinée à une technique d’analyse à haut débit du positionnement des nucléosomes et de la méthylation de l’ADN et une approche de FISH), j’ai pu obtenir une vue intégrée des événements épigénétiques qui promeuvent la transition du promoteur de hTERT d’un état silencieux à un état actif et inversement. Cette information sera cruciale pour le développement de stratégies anticancéreuses ciblant l’expression de hTERT. / Telomerase is present in a limited number of cells, most of them cancerous where its activity is crucial for their immortalisation. Thus, that enzyme has been proposed as a promising target for anticancer therapies. Therefore, it is of great interest to identify the mechanisms by which it is regulated, notably through its catalytic subunit hTERT. Retinoids are well-known inducers of granulocytic maturation associated with hTERT repression in acute promyelocytic leukemia (APL) blasts. However, in NB4-LR1, a maturation-resistant APL cell line, was previously identified a new pathway of retinoid-induced hTERT transcriptional repression independent of differentiation. Furthermore, my host lab reported the isolation of a variant of NB4-LR1 cells resistant to this repression: NB4-LR1SFD. These two cell lines, which behave distinctly in response to ATRA treatment, provide a unique and interesting tool to gain further insights into the molecular biology of telomerase regulation. In this thesis project, using several complementary approaches (chromatin immunoprecipitation combined to a high-resolution, single-molecule nucleosome positioning assay and DNA methylation, and a FISH approach) allowed me to shed more light on the integrated epigenetic events that lead to hTERT promoter transition from its silent to its active state and vice versa. The information obtained could be crucial for the development of anticancer strategies targeting hTERT expression.