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A Leucose Enzoótica Bovina é uma doença crônica de ocorrência mundial que acomete bovinos. Seu agente etiológico é um retrovírus do mesmo grupo dos que causam a leucemia linfocítica crônica e a AIDS, importantes doenças que acometem humanos. Os retrovírus são vírus que se integram ao DNA do hospedeiro e desta forma são potencialmente mutagênicos. Os bovinos infectados podem desenvolver uma linfocitose persistente e/ou linfoma. A gênese das neoplasias está nas mutações de classes de genes como os proto-oncogenes, genes supressores de tumor, genes de reparo do DNA e genes que controlam a apoptose. O gene p53 acumula algumas funções como de parada do ciclo celular, estimulo ao reparo do DNA ou indução da célula a apoptose. O gene bcl-2 é conhecidamente um gene que regula a apoptose. A interferência no processo de apoptose através de mutações provocadas pelos retrovírus nos genes p53 e bcl-2 é um dos mecanismos carcinogênicos associados a LEB. As mutações no gene p53 acabam promovendo o aumento de sua meia vida e em bcl-2 o aumento em sua expressão. Devido a essas alterações o aumento na expressão do gene bcl-2 e o aumento da meia vida da p53 podem ser avaliadas pela técnica de imunoistoquímica. O presente trabalho avaliou as proteínas p53 e bcl-2 em amostras de bovinos infectados pelo VLB através da técnica de imunoistoquímica. Foram pré-definidos escores de imunomarcação em amostras de animais não infectados. Os escores apresentados pelos animais infectados pelo VLB foram significativamente maiores que os animais do grupo controle. Os valores percentuais de imunomarcação apresentados para bcl-2 foram de 50,26 ± 12,25 do grupo afetado contra 10,86 ± 3,30 do grupo controle. Para p53 somente o grupo afetado apresentou imunomarcação, ficando em zero a imunomarcação do grupo controle. O valor percentual médio do grupo afetado foi de 55,95 ± 7,07. A diferença significativa entre os percentuais dos grupos controle e afetado confirma que os genes p53 e bcl-2 podem estar envolvidos no processo de linfomagênese da LEB. / The Enzootic Bovine Leukosis (EBL) is a chronic disease with mundial occurrence in bovines. Its etiologic agent is a retrovirus of the same group that promotes the chronic lymphocytic leukemia and HIV, that wich are important diseases in humans. The retrovirus integrates into the host DNA and thus they are potential mutagenic. The infected bovines can develop a persistent lymphocytosis and/or lymphoma. The neoplasm genesis is associated with genes mutation like the protooncogenes, tumor suppressor, DNA repair and apoptosis control. The p53 gene has some functions how cell cycle arrest, DNA repair stimulus or apoptosis induced. The interference in the apoptosis process through mutations stimulates about the retrovirus in the p53 and bcl-2 genes are the carcinogenic mechanisms associated to EBL. The mutations in the p53 gene promote the increase in its half life and increase of bcl-2 expression. Due this alterations the increase of bcl-2 gene expression and p53 half-life can be evaluate by immunohistochemistry. This work evaluated the bcl-2 and p53 proteins in EBL infected bovines tissue samples by immunohistochemistry. We determined scores of immunomarked in non-infected animal\'s samples. The scores showed by the EBL infected animals were significantly higher than in control group. The infected bovines showed 50.26 ± 12.25% of positive cells to bcl-2 while the control group showed 10.86 ± 3.3%. Only the infected animals presented reactivity against p53, whereas the control group no showed reaction. These results confirm that the p53 and bcl-2 genes can be involved in a process of EBL leukemogenesis.

A Leucose Enzoótica Bovina é uma doença crônica de ocorrência mundial que acomete bovinos. Seu agente etiológico é um retrovírus do mesmo grupo dos que causam a leucemia linfocítica crônica e a AIDS, importantes doenças que acometem humanos. Os retrovírus são vírus que se integram ao DNA do hospedeiro e desta forma são potencialmente mutagênicos. Os bovinos infectados podem desenvolver uma linfocitose persistente e/ou linfoma. A gênese das neoplasias está nas mutações de classes de genes como os proto-oncogenes, genes supressores de tumor, genes de reparo do DNA e genes que controlam a apoptose. O gene p53 acumula algumas funções como de parada do ciclo celular, estimulo ao reparo do DNA ou indução da célula a apoptose. O gene bcl-2 é conhecidamente um gene que regula a apoptose. A interferência no processo de apoptose através de mutações provocadas pelos retrovírus nos genes p53 e bcl-2 é um dos mecanismos carcinogênicos associados a LEB. As mutações no gene p53 acabam promovendo o aumento de sua meia vida e em bcl-2 o aumento em sua expressão. Devido a essas alterações o aumento na expressão do gene bcl-2 e o aumento da meia vida da p53 podem ser avaliadas pela técnica de imunoistoquímica. O presente trabalho avaliou as proteínas p53 e bcl-2 em amostras de bovinos infectados pelo VLB através da técnica de imunoistoquímica. Foram pré-definidos escores de imunomarcação em amostras de animais não infectados. Os escores apresentados pelos animais infectados pelo VLB foram significativamente maiores que os animais do grupo controle. Os valores percentuais de imunomarcação apresentados para bcl-2 foram de 50,26 ± 12,25 do grupo afetado contra 10,86 ± 3,30 do grupo controle. Para p53 somente o grupo afetado apresentou imunomarcação, ficando em zero a imunomarcação do grupo controle. O valor percentual médio do grupo afetado foi de 55,95 ± 7,07. A diferença significativa entre os percentuais dos grupos controle e afetado confirma que os genes p53 e bcl-2 podem estar envolvidos no processo de linfomagênese da LEB. / The Enzootic Bovine Leukosis (EBL) is a chronic disease with mundial occurrence in bovines. Its etiologic agent is a retrovirus of the same group that promotes the chronic lymphocytic leukemia and HIV, that wich are important diseases in humans. The retrovirus integrates into the host DNA and thus they are potential mutagenic. The infected bovines can develop a persistent lymphocytosis and/or lymphoma. The neoplasm genesis is associated with genes mutation like the protooncogenes, tumor suppressor, DNA repair and apoptosis control. The p53 gene has some functions how cell cycle arrest, DNA repair stimulus or apoptosis induced. The interference in the apoptosis process through mutations stimulates about the retrovirus in the p53 and bcl-2 genes are the carcinogenic mechanisms associated to EBL. The mutations in the p53 gene promote the increase in its half life and increase of bcl-2 expression. Due this alterations the increase of bcl-2 gene expression and p53 half-life can be evaluate by immunohistochemistry. This work evaluated the bcl-2 and p53 proteins in EBL infected bovines tissue samples by immunohistochemistry. We determined scores of immunomarked in non-infected animal\'s samples. The scores showed by the EBL infected animals were significantly higher than in control group. The infected bovines showed 50.26 ± 12.25% of positive cells to bcl-2 while the control group showed 10.86 ± 3.3%. Only the infected animals presented reactivity against p53, whereas the control group no showed reaction. These results confirm that the p53 and bcl-2 genes can be involved in a process of EBL leukemogenesis.

Mon travail a été d'étudier le rôle de l'expression de FZD9, un récepteur Wnt, dans l'hématopoïèse normale et maligne. Frizzled 9 (FZD9) est un composant clé de la voie de signalisation Wnt, une voie connue pour jouer un rôle important dans l'hématopoïèse normale et maligne. La méthylation d'un site CpG proximal de site d'initiation de la transcription du gène FZD9 est un facteur de mauvais pronostic dans les LAMs et sa déméthylation est un facteur prédictif de réponse aux thérapies épigénétiques. Sa fonction dans l'hématopoïèse n'est cependant pas connue. Au cours de ma thèse, j'ai démontré que le promoteur du gène FZD9 est dans un état non-permissif dans les cellules leucémiques. L'induction de la différenciation des cellules leucémiques restaure l'état permissif du promoteur, le recrutement du facteur E2F et de l'histone H3 acétyle permettant l'expression de FZD9. L'expression de FZD9 expression est également retrouvée au cours de la différenciation myéloïde d'une lignée IPS Dans les cellules de la lignée THP1 différenciées en monocytes, FZD9 est retrouvé dans un complexe comprenant LRP5/6 et Wnt5a. L'incubation des cellules différenciées par Wnt5a, un ligand de FZD9, déclenche la diminution de l'expression de -caténine et sa localisation nucléaire ainsi que l'expression des gènes cibles de la voie Wnt canonique (c-myc, Cyclin Dl and CD44). Nous n'avons détecté aucune augmentation des taux intracellulaires de calcium. Ces travaux suggèrent que la méthylation du promoteur de FZD9 dans les LAMs pourrait participer à la leucémogénèse en maintenant la voie b-caténine active / My goal was to study the importance of the expression of the FZD9, a Wnt receptor, in both normal and malignant hematopoiesis. Frizzled 9 (FZD9) is a key component of the Wnt signaling pathway, a pathway which has been shown to play a role in both normal and malignant hematopoiesis. Methylation of the CpG proximal to the transcription start site of the FZD9 gene is recognized as a prognostic factor in AML and its demethylation is a predictive factor for response to epigenetic therapy. Its function in hematopoiesis is however not known. The results show that the FZD9 promoter is in a non-permissive state in leukemic cells. Induction of myeloid differentiation in human myeloid leukemic cell fines restores FZD9 promoter permissiveness with recruitment of E2F and acetylated histone H3 and upregulation of FZD9 mRNA expression. FZD9 expression was progressively increased through the stages of myeloid differentiation in an IPS cell line. In differentiated monocytic THP1 cells, FZD9 was found in the LRPS/6 Wnt receptor complex. Incubation of differentiated cells with Wnt5a, a ligand of FZD9, triggered the decreased expression of - catenin and its nuclear localisation and the canonical Wnt-target genes (c-myc, Cyclin D1 and CD44). We detected no increase in calcium intracellular levels and thus activation of classical non-canonical pathway was not noted upon WntSa incubation. The results of my PhD suggest that the reported methylation of FZD9 promoter in AML and HR-MDS patients may participate in leukemogenesis by the maintenance of an activated Wnt canonical pathway.

Hematopoietic stem and progenitor cells proliferate and differentiate to reconstitute all lineages of functional blood cells. They are regulated by intricate cellular and molecular signals, on both genetic and epigenetic levels. Alterations in these regulatory signaling networks can lead to hematopoietic dysfunction, as well as transformation of hematopoietic cells and induction of leukemogenesis. This thesis focuses on uncovering molecular modulators that are crucial for the proper regulation of hematopoietic stem/progenitor cells. In Chapter II, I describe studies investigating functional roles of the histone demethylase UTX in normal and malignant hematopoiesis, using a short hairpin RNA-mediated knockdown approach. My data revealed that UTX is required for proliferation, self-renewal and differentiation of hematopoietic progenitor cells ex vivo through transcriptional regulation of hematopoiesis- specific transcriptional factors. I also discovered that UTX is critical for the proliferation of leukemia cells, implicating UTX as a possible target for clinical therapy. In Chapter III, I focus on understanding the process of leukemogenesis by generating and characterizing a novel model of myeloid sarcoma and acute myeloid leukemia in mice. This model induces these hematopoietic malignancies by introduction of multiple oncogenetic lesions (specifically, p16/p19-/-;Kras(G12V)) into bone marrow cells, and subsequent transplantation of these gene-modified cells into immunodeficient NOD.SCID mice. This model is very rapid and reproducible, and represents the first transplantable myeloid sarcoma model reported. Moreover, the disease induced in mice recapitulates the pathological progression of myeloid sarcoma in patients, providing a powerful model for dissection of critical leukemogenic events and discovery of new candidate therapeutic targets. Together, these studies help to reveal novel molecular modulators required for normal hematopoiesis, and offer potential animal model and drug target for therapeutic applications.

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No. of bitstreams: 2 Tese - Lysa Bernardes Minasi - 2013.pdf: 3405021 bytes, checksum: 8697a199b1d732fc69f73a28ed8848a2 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-01-13 / The ALL is a malignant disorder that originates from a precursor cell lympho-hematopoietic system compromised for the development of B or T lymphocyte lineage.The precursor cell acquisition by a series of genetic abnormalities alters the normal process of maturation leading to cellular differentiation arrest and leukemic clone proliferative advantage on cells of hematopoietic tissue. More than 50 recurrent genetic alterations have been identified in the ALL. These often involve genes known or putative role in the development of lymphocytes and in the case of leukemogenesis.In this study, the variation in the expression of molecular markers was analyzed using PCR methodology array on 16 children with ALL before treatment and at the end of induction chemotherapy (D +28). These patients were diagnosed in HAJ and SCMG, from May 2012 to January 2013. Samples of bone marrow or peripheral blood were sent to NPReplicon-PUC-GO. In the present study we observed a negative correlation between gender and immunophenotype (p = 0.016), females have a greater association with immunophenotype B and less associated with immunophenotype T. We also observed a positive correlation between immunophenotype and age (p = 0.04), immunophenotype and marker CD10 + (p = 0.03), immunophenotype and risk group (p = 0.015) and marker CD10 + and risk group (p = 0.043). Before treatment the gene RUNX1 met with increased expression by 5.0 times compared to the control group and after induction chemotherapy was observed a reduction in its expression. The expression pattern of the gene TAL1 showed significant decrease, with the exception of post-treatment analysis showed that an increase in its expression. A positive correlation was observed between the expression of BAX and BCL2 (r = 0.94 and p <0.0001. We demonstrated a significant difference in gene expression pattern of ALL at diagnosis and after induction therapy. We conclude our observation regarding the gene expression profile in patients with ALL enrolled in this study, but to define a panel of molecular markers is necessary to evaluate several other genes involved in the process of leukemogenesis in ALL with the help of other methodologies. / A LLA é uma desordem maligna que origina de uma célula precursora do sistema linfo-hematopoético comprometida para o desenvolvimento da linhagem de linfócitos B ou T. A aquisição pela célula precursora de uma série de anormalidades genéticas, altera seu processo normal de maturação, conduzindo a parada na diferenciação celular e vantagem proliferativa do clone leucêmico sobre as células do tecido hematopoético. Mais de 50 alterações genéticas recorrentes tem sido identificadas na LLA. Estas frequentemente envolvem genes com papel conhecido ou putativo no desenvolvimento dos linfócitos e no processo da leucemogênese.Neste estudo, a variação da expressão dos marcadores moleculares, foi analisada utilizando a metodologia de PCR array, em 16 crianças com LLA ao diagnóstico e no final da quimioterapia de indução. Tais pacientes foram diagnosticados no HAJ e na SCMG, no período de maio de 2012 a janeiro de 2013. As amostras de medula óssea ou sangue periférico foram enviadas ao NPReplicon-PUC-GO. No presente estudo foi possível observar uma correlação negativa entre as variáveis sexo e o imunofenótipo (p = 0.016), ou seja, pacientes do sexo feminino tem uma maior associação com o imunofenótipo B e menor associação com o imunofenótipo T. Foi observado também uma correlação positiva entre imunofenótipo e idade (p = 0.04), imunofenótipo e marcador CD10+ (p = 0.03), imunofenótipo e grupo de risco (p = 0.015) e marcador CD10+ e grupo de risco (p = 0.043). Antes do tratamento o geneRUNX1 encontrou-se com um aumento da expressão em 5,0 vezes em relação ao grupo controle e após a quimioterapia de indução foi observado uma redução na sua expressão. O padrão de expressão do gene TAL1apresentou significativa diminuição, com exceção da análise pós tratamento que demonstrou um aumento da sua expressão. Uma correlação positiva foi observada entre a expressão de BAX e BCL2 (r = 0,94 e p < 0,0001). A partir do perfil de expressão de cada gene analisado, demonstramos no presente estudo uma diferença significativa no padrão de expressão gênico da LLA ao diagnóstico e após a terapia de indução. Concluímos nossa observação com relação ao perfil de expressão gênica nos pacientes com LLA incluídos neste estudo, mas para definirmos um painel de marcadores moleculares é necessário a avaliação de diversos outros genes que participam do processo da leucemogênese na LLA com auxílio de outras metodologias.

Despite recent advances in the understanding of the molecular bases of hematological malignancies, the specific mechanisms on how they originate and why some subtypes are more prevalent than others still remain to be elucidated. These two important aspects have been even more difficult to analyze when dealing with individuals under immune suppression because other factors must be considered. Questions still remain as to why individuals with AIDS tend to develop lymphoproliferative disorders differently from those observed in individuals under iatrogenic immunosuppressive therapy. Most of lymphomas occurring in transplant recipients are B-cell neoplasias typically associated with Epstein-Barr virus (EBV) infection. In contrast, only about 50% of lymphomas of patients with AIDS are associated with lymphotrophic herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). No known infectious agent has been detected in the remaining 50% of AIDS-associated lymphomas, suggesting the involvement of novel viruses or unique molecular mechanisms. Since most oncogenic viruses persist as episomal circular viral genomes in the nuclei of tumor cells, we developed a method to visualize and identify covalently closed circular DNA (cccDNA) in lymphoma samples. Although this study revealed no novel viruses, we identified concatemers of the mitochondrial genome in all lymphoma samples tested. We further studied in detail one AIDS-associated lymphoma (denominated EL) whose mitochondrial DNA primarily consisted of tandem head-to-tail genome duplications. Insertion of cytosine residues was noted in the EL mitochondrial genome sequence near the origin of replication. EL cells responded weakly to Fas-apoptotic stimulus, displayed reduced mitochondrial activity and mass, and produced higher levels of reactive oxygen species (ROS) than control cells. Screening of several other AIDS-associated lymphomas and established lymphoma cell lines revealed a different kind of mitochondrial genome concatemers consisting of interlinks of DNA monomer molecules. Concatemers were not detected in normal T-lymphocytes suggesting an association with neoplastic transformation. This dissertation describes the two distinct types of mitochondrial genome concatemers identified in transformed lymphoid cells and presents a detailed analysis of their structure and implications in cellular homeostasis.

Markéta Sypecká CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years. However, every stage of this disease has significantly higher level of miR-155, which is known as oncomiR. Micro RNAs represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets. We hypothesized that by gene editing of CLL B - cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Method we use for gene editing is CRISPR/Cas9, which enables to delete sequence of mature miR-155 in genome of leukemic B-cells. Methods: CRISPR/Cas9, nucleofection, qRT-PCR, FACS Results:We achieved to isolate clone that bears one allelic deletion (miR-155-/+) in sequence for mature...