Multiple myeloma (MM) is a hematological malignancy of antibody producing plasmablasts/plasma cells. MM is characterized by extensive genetic and clonal heterogeneity, which have hampered the attempts to identify a common underlying mechanism for disease establishment and development of appropriate treatment regimes. This thesis is focused on understanding the role of epigenetic regulation of gene expression mediated by the polycomb repressive complexes 1 and 2 (PRC1 and 2) in MM and their impact on disease biology and therapy. In paper I the genome-wide distribution of two histone methylation marks; H3K27me3 and H3K4me3 were studied in plasma cells isolated from newly diagnosed MM patients or age-matched normal donors. We were able to define targets of H3K27me3, H3K4me3 and bivalent (carry both marks) which are, when compared to normal individuals, unique to MM patients. The presence of H3K27me3 correlated with silencing of MM unique H3K27me3 targets in MM patients at advanced stages of the disease. Notably, the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also showed that inhibition of the PRC2 enzymatic subunit EZH2 using highly selective inhibitors (GSK343 and UNC1999) demonstrated anti-myeloma activity using relevant in vitro models of MM. These data suggest an important role for gene repression mediated by PRC2 in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM. In paper II we further explored the therapeutic potential of UNC1999, a highly selective inhibitor of EZH2 in MM. We showed that EZH2 inhibition by UNC1999 downregulated important MM oncogenes; IRF-4, XBP-1, BLIMP-1and c-MYC. These oncogenes have been previously shown to be crucial for disease establishment, growth and progression. We found that EZH2 inhibition reactivated the expression of microRNAs genes previously found to be underexpressed in MM and which possess potential tumor suppressor functions. Among the reactivated microRNAs we identified miR-125a-3p and miR-320c as predicted negative regulators of the MM-associated oncogenes. Notably, we defined miR-125a-3p and miR-320c as targets of EZH2 and H3K27me3 in MM cell lines and patients samples.  These findings described for the first time PRC2/EZH2/H3K27me3 as regulators of microRNA with tumor suppressor functions in MM. This further strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM. In paper III we evaluated the therapeutic potential of targeting PRC1 in MM using the recently developed chemical PTC-209; an inhibitor targeting the BMI-1 subunit of PRC1. Using MM cell lines and primary cells isolated from newly diagnosed or relapsed MM patients, we found that PTC-209 has a potent anti-MM activity. We showed, for the first time in MM, that PTC-209 anti-MM effects were mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, but that other subunits of the PRC1 complex were not affected. We showed that PTC-209 reduced MM cell viability via significant induction of apoptosis. More importantly, we demonstrated that PTC-209 shows synergistic anti-MM activity with other epigenetic inhibitors targeting EZH2 (UNC1999) and BET-bromodomains (JQ1). This work highlights the potential use of BMI-1 and PRC1 as potential therapeutic targets in MM alone or in combination with other anti-MM agents including epigenetic inhibitors.

O objetivo desta Tese foi o de demonstrar que a Teoria do biólogo e epistemólogo suíço Jean Piaget foi vítima de sua própria Representação Social, (na acepção de Serge Moscovici, como explicitaremos já em nossa Introdução). Vítima no sentido de que suas descobertas na área da Biologia e sua criação no campo da construção de modelos formais na mesma ciência, feito inédito até então, caíram no ostracismo na História da ontogênese epigenética do ser humano, tanto nos aspectos biológicos, quanto na área da aquisição do conhecimento científico e lógico matemático. Esses fatos nos mostram Zelia Ramozzi-Chiarottino, (cuja análise e interpretação da Teoria de Jean Piaget constituir-se-á no referencial teórico desta Tese) ao lado de seus colaboradores, no artigo: Jean Piagets unrecognized epigenetic ontogenesis of the logical mathematical thought, (2017). Neste trabalho, restringimo-nos ao Brasil e à produção de Dissertações e Teses de Doutorado sobre a Teoria de Piaget e sua representação social, aqui realizadas nos últimos dez anos. Fizemos um levantamento das Dissertações e Teses a partir do Catálogo de Teses e Dissertações da CAPES. O método estatístico que utilizamos foi o do para analisar os dados coletados. Esta análise confirmou nossa conjectura / Our aim with this Ph.D Thesis was to demonstrate that the Theory of the swiss biologist and epistemologist Jean Piaget was victim of its own Social Representation (within the meaning of Serge Moscovici, as we will still explain in our Introduction). Victim in sense of that his findings in the field of Biology and his creation in the field of the construction of formal models in this same Science, unprecedent feat till then, have fallen in obscurity in the History of epigenetic ontogenesis of the human being, in both biologic and scientific aspects, in terms of the field of acquiring of scientific and logical mathematical knowledgement. These facts Zelia Ramozzi-Chiarottino (whose analysis and interpretation of Piagets Theory will be the theoretical framework of this Thesis) show us next to her co-workers, in the article: Jean Piagets unrecognized epigenetic ontogenesis of the logical mathematical thought, (2017). In the present work, we limited ourselves to Brazil, and to the production of Dissertations and Doctoral Theses on Piagets Theory and its Social Representation, produced here on the last ten years. We made a data survey on the Dissertations and Theses from the Catalogue of Theses and Dissertations from CAPES. Statistic method used was the to analyze the collected data. This analysis validated our conjecture

Le gliome infiltrant du tronc cérébral (en anglais “diffuse intrinsic pontine glioma”, DIPG) est une tumeur pédiatrique rare et très agressive. La durée moyenne de survie après diagnostic est inférieure à un an. Une caractéristique génétique majeure des DIPG est la mutation de l’histone H3 (H3K27M). L’évolution des connaissances en épigénétique a permis de concevoir des inhibiteurs de régulateurs épigénétiques capables de modifier, voire de contrebalancer, l’effet de cette mutation. Ainsi, le panobinostat (PS), un inhibiteur des histone-désacétylases, diminue la croissance cellulaire et conduit à la mort des cellules de DIPG in vitro et in vivo. Son efficacité est en cours d'évaluation dans des essais cliniques. Mon projet de thèse avait pour objectif de déterminer le rôle d’EBP50 et d’IRSp53, deux protéines spécifiquement dérégulées dans les lignées de DIPG après traitement des cellules par le PS. EBP50 est connue pour intervenir dans la progression tumorale mais sa dualité de fonction, à la fois oncogène et suppresseur de tumeur, nous a conduits à étudier plus précisément son rôle dans les cellules de DIPG. IRSp53 a été peu étudiée dans les cancers solides, bien qu'elle semble jouer un rôle important dans la motilité cellulaire et l’invasion. La diminution de l’expression d’IRSp53 et d’EBP50 par ARN interférence dans des lignées DIPG induit la mort des cellules par apoptose et bloque leur croissance ainsi que leur motilité cellulaire, ce qui suggérerait que ces deux protéines sont oncogéniques dans ce modèle. De plus, la localisation cytoplasmique et nucléaire d’EBP50 semble en accord avec son rôle pro-oncogénique dans les cellules de DIPG. En étudiant in vitro l’effet d’un traitement combinatoire du PS avec des inhibiteurs de l’expression d’EBP50 ou d’IRSp53, j’ai mis en évidence une augmentation de la sensibilité des cellules de DIPG au traitement par le PS. Enfin, j’ai validé le traitement ciblant EBP50 in vivo dans un modèle préclinique d’embryon de poulet. En conclusion, ces deux protéines constituent de nouvelles cibles thérapeutiques dans les DIPG et un moyen d’augmenter l’efficacité du PS. / Diffuse Intrinsic Pontine Glioma (DIPG), is a rare and highly aggressive pediatric tumor. The average survival time after diagnosis is less than one year. A major genetic characteristic of this disease is the mutation of histone H3 (H3K27M). The evolution of knowledge in epigenetics has made it possible to design epigenetic regulatory inhibitors able to modify, or even offset, the effect of this mutation. For example, panobinostat (PS), a histone deacetylase inhibitor, reduces cell growth and induces DIPG cell death, both in vitro and in vivo. Its effectiveness is currently being evaluated in clinical trials. My thesis project aimed at determining the role of two proteins, EBP50 and IRSp53, deregulated in different DIPG cell lines after treatment with PS. EBP50 has already been described as involved in tumor progression but its dual function, both oncogenic and tumor suppressor, has led us to further investigate its role in the DIPG cells. IRSp53 has been poorly studied in solid cancers, though it plays an important role in cell motility and invasion. Down-regulation by RNA silencing of these two proteins in DIPG cell lines induces apoptosis, decreases cell growth and motility, leading us to the hypothesis that these two proteins are oncogenic proteins. In addition, the cytoplasmic and nuclear localization of the EBP50 protein is consistent with its oncogenic role in DIPG cells. Then, I investigated the effect of combinatorial therapy that associates PS with EBP50 or IRSp53 expression inhibitors. My results show an increase in the antitumor effect in vitro for both proteins but also in vivo for EBP50, in a preclinical model, the chicken embryo. In conclusion, these two proteins could be the targets of new treatments for DIPG tumors in combination with PS to enhance its efficacy.

Le transfert intergénérationnel de l'immunité est un phénomène dont l'étude remonte à une dizaine d'année. Chez les vertébrés, il prend la forme d'un transfert d'anticorps de la mère aux juvéniles. Pourtant, les processus écologiques et évolutifs du transfert sur plusieurs générations et la persistance des anticorps maternels chez les juvéniles n'ont pas bien étudiés. Ce travail de thèse porte sur l'étude des mécanismes du transfert d'anticorps maternel sur plusieurs générations consécutives et leurs conséquences sur la réponse immunitaire et sur la croissance des juvéniles. En utilisant des approches expérimentales, j'ai pu dans un premier temps montré que le transfert d'anticorps maternel était affecté par la disponibilité en nourriture dans l'environnement. De plus, nos résultats suggèrent que la persistance d'anticorps maternel dépend du niveau initial d'anticorps maternels présents chez le juvénile dans les premiers jours de vie. Dans un second temps, nous avons étudié la réponse immunitaire et la croissance des poussins en tentant de mettre en évidence comment les anticorps maternels influençaient le compromis entre ces deux paramètres physiologiques. Enfin, nos résultats suggèrent que les anticorps maternels pourraient être le messager intergénérationel d'un effet épigénétique sur la réponse immunitaire. En effet, une réponse immunitairre des grand-mères induit par l'exposition à un parasite a été trouvé stimuler la réponse immunitaire des petits-enfants contre ce même parasite. Mes travaux de thèses soulignent une fois de plus, l'importance du transfert de l'immunité dans l'écologie et l'évolution des interactions hôte-parasite. / Intergenerational transfer of immunity is a phenomenon which started to be studied 10 years ago. In vertebrates, it takes the form of a transfer of antibodies from mothers to juveniles. However, the ecological and evolutionary processes of transfer across several generations and the persistence of maternals antibodies in chicks remain poorly understood. This thesis focuses on the study of the mechanisms of maternal antibodies transfer over several generations and on how they impact the immune response and growth in juveniles. By using experimental approaches, I first show that the transfer of maternal antibody was affected by food availability in the environment. In addition, our results suggest that the persistence of maternal antibodies depends on the initial level of maternal antibodies present in the nestlings in the first days of life. Secondly, we studied the immune response and growth of juvenile by investigating how maternal antibodies may affect the resolution of the trade-off between these two physiological parameters. Finally, our results suggest that maternal antibodies would be a messenger of an epigenetic effect on immunty. Indeed, induced immune response in grand-mothers towards a pathogen has been found to stimulate the immune response of grand-children. My thesis emphasizes the importance of maternal transfer of immunity in the ecology and evolution of host-parasite.

A tuberculose representa um sério problema de Saúde Pública para o Brasil e o mundo. Estima-se que cerca de um terço da população mundial seja infectado pelo bacilo, sendo que 95% destes indivíduos desenvolvem a forma latente da tuberculose. Dessa forma, encontrar um marco entre o que faz o bacilo Mycobacterium tuberculosis (Mtb) induzir no hospedeiro uma doença ativa ou sua forma latente tem sido o objeto de estudo de diferentes grupos de pesquisa. Neste contexto, o objetivo deste estudo foi correlacionar mudanças na resposta imune desenvolvida por monócitos de pacientes em diferentes estágios da tuberculose pulmonar com alterações epigenéticas, e então com aspectos clínicos de cada estágio. Nosso estudo incluiu 17 pacientes com a doença ativa, 14 indivíduos com tuberculose latente e 16 controles não infectados. A partir de sangue periférico, foi coletado plasma para quantificação de citocinas, sCD163 e sCD14. Complementarmente, monócitos foram purificados para avaliação da ativação imune através da quantificação de citocinas e espécies reativas de oxigênio mediante estimulação in vitro com Mtb inativado por calor, bem como PCR array para detectar expressão de enzimas de repressão ou ativação epigenética entre os grupos e avaliação do padrão de metilação global. Pacientes com tuberculose ativa apresentaram níveis plasmáticos elevados de citocinas como IL-6, IP-10, TNF-, IL-12, bem como IL-5. Dentre as citocinas, TNF-, IL-5, IL-6 e IP-10 permitiram diferenciar indivíduos latentes dos pacientes com tuberculose ativa. Também demostramos que maior número de correlações entre as citocinas foi observado nos pacientes com a doença ativa, indicando um estado geral mais ativado do sistema imune. Níveis plasmáticos de sCD163 e sCD14 estavam elevados nos pacientes quando comparados com indivíduos latentes e controles, e poderiam também diferenciar a tuberculose ativa. Tais níveis aumentados correlacionam-se com o estado ativado de monócitos dos pacientes, que produzem maior quantidade de espécies reativas de oxigênio e tendem a produzir mais citocinas inflamatórias, como IL-6 e TNF-. Este perfil parece ser modulado por modificações epigenéticas, uma vez que monócitos de pacientes com tuberculose pareceram mostrar menor expressão de enzimas responsáveis por mudanças relacionadas à repressão e maior expressão de enzimas relacionadas à ativação da expressão gênica, bem como redução no padrão global de metilação do DNA genômico, sugerindo maior atividade destas células. Finalmente, uma vez que pacientes com tuberculose apresentaram níveis elevados de alguns mediadores, correlacionamos estes com o grau de lesão pulmonar, e consequentemente, com a gravidade da doença. Nós mostramos que dentre os marcadores, TNF- e sCD163 correlacionaram-se positivamente com a progressão da tuberculose. Assim, concluímos que em relação aos indivíduos controle e com TB latente, os pacientes com tuberculose ativa apresentam o sistema imune em maior estado de ativação, e ainda, que alterações epigenéticas podem ser as responsáveis pela modulação observada. Dentre os fatores aumentados nos pacientes, TNF-, IL-6, IP-10, IL-5, sCD163 e sCD14 podem diferenciar os pacientes com doença ativa dos demais indivíduos analisados, e ainda sCD163 e TNF- podem atuar como indicativos da gravidade da tuberculose. / Tuberculosis is a major public health problem for Brazil and worldwide. It is estimated that about one third of the world population is infected with the bacillus, and 95% of those individuals have the condition in the latent form. Thus, finding a hallmark between what makes the Mycobacterium tuberculosis (Mtb) induces in the host an active disease or its latent form has been the subject of different research groups. In this context, the objective of this study was to correlate changes in the immune response developed by monocytes from patients at different stages of pulmonary tuberculosis with epigenetic alterations and then, with clinical aspects of each stage. Our study included 17 patients with active disease, 14 individuals with latent tuberculosis and 16 uninfected controls. From the peripheral blood, plasma was collected for cytokine and CD163s, sCD14 quantification. In addition, monocytes were purified to evaluation of the immune activation through cytokine and reactive oxygen species quantification upon in vitro stimulation with heat-killed Mtb, as well as PCR array for epigenetic repression or activation enzymes were performed to detect epigenetic changes between the groups and evaluation of the global methylation profile. We have shown that patients with active tuberculosis have increased plasma levels of cytokines such as IL-6, IP-10, TNF-, IL-12, as well as IL-5. Among these cytokines, TNF-, IL-5, IL-6 and IP-10 could differentiate latent-infected from TB patients. We also showed that patients with active disease presented higher number of correlations between plasma cytokines, indicating an activated state of the immune system. The plasma levels of sCD163 and sCD14 were more elevated in patients than latent and control individuals, and might differentiate TB active from these other groups. These increased levels of biomarkers correlate with the activated state of monocytes from patients, which produced raised quantities of reactive oxygen species and tended to produced more pro-inflammatory cytokines, such as IL-6 and TNF-. This profile seems to be modulated by epigenetic modifications, since monocytes from patients with tuberculosis seemed to show reduced expression of enzymes responsible for changes related to repression and enhanced expression of enzymes responsible for activation of gene expression, and in addition, these patients presented reduction in the percentage of global methylation of genomic DNA, suggesting increased activity of these cells when compared to the other groups. Finally, once tuberculosis patients presented increased levels of some mediators, we correlated these with the degree of lung injury, and consequently tuberculosis severity. We have showed that TNF- and sCD163 were correlated with disease progression in tuberculosis. In conclusion, we demonstrated that patients with active tuberculosis are more activated than the other groups, and epigenetic alterations might be responsible for these modulations, indicated by the alteration of the enzymes and methylation pattern analyzed. Among the cytokines/chemokines differentially expressed TNF-, IL-6, IP-10, IL-5 and monocyte activation markers, sCD163 and sCD14 could discriminate between active disease from others, and also sCD163 and TNF- could indicate tuberculosis severity.

Em fêmeas de mamíferos, um dos cromossomos X é inativado proporcionando compensação de dose entre os produtos gênicos de machos e fêmeas. A inativação do cromossomo X (ICX) ocorre no embrião em desenvolvimento, e se caracteriza pela aquisição de marcas heterocromáticas no cromossomo X inativado (Xi), que são mantidas nas células somáticas ao longo das divisões celulares. O melhor modelo para estudo do início da ICX são as células-tronco embrionárias femininas. Provenientes da massa celular interna de blastocistos, elas representam um embrião em desenvolvimento e possuem os dois X ativos; a diferenciação das células promove a ICX in vitro, o que permite a identificação dos fatores e mecanismos moleculares envolvidos. A derivação de linhagens de célulastronco embrionárias humanas (human embryonic stem cells - hESCs) em 1998 permitiu novas possibilidades de estudo da ICX, pois a maioria dos trabalhos procurou esclarecer o mecanismo da ICX no modelo murino. Tradicionalmente, a manutenção da ICX em humanos tem sido investigada em células somáticas híbridas ou transformadas; porém, sabe-se que estas não representam um contexto celular natural. Assim, o presente trabalho teve como objetivos principais explorar a potencialidade de hESCs no estudo do início da ICX, e ainda investigar a função de três fatores na manutenção da ICX em células humanas imortalizadas: DNMT1 (enzima responsável pela manutenção da metilação do DNA), SMCHD1 (proteína da família de coesinas/condensinas), e XIST (um RNA não-codificador que inicia o processo de heterocromatinização do futuro Xi) foram selecionados para este estudo, uma vez que todos participam da manutenção da ICX em camundongos. Até o momento foram derivadas em nosso laboratório quatro linhagens de hESCs, as primeiras da América Latina. A caracterização das linhagens mostrou que, apesar de se manterem indiferenciadas, as hESCs femininas encontram-se em estágio pós-ICX, pois mesmo indiferenciadas já apresentam um dos X inativado. Nossos dados indicam que, submetidas às atuais condições de cultivo, as hESCs não são bons modelos para o estudo do início da ICX, e é possível que a inativação de um cromossomo X durante o cultivo confira alguma vantagem seletiva às células. A estratégia utilizada no estudo da manutenção da ICX foi o silenciamento dos três genes por interferência de RNA (RNAi). Não foi possível diminuir significativamente a expressão dos genes XIST e SMCHD1. Porém, o silenciamento de DNMT1 foi expressivo, e em resposta foi observada reativação do gene MAOA, localizado no cromossomo X e submetido à inativação. Apesar de nossas análises mostrarem que os efeitos da diminuição de DNMT1 foram restritos ao gene MAOA, estes resultados sugerem a existência de diferentes hierarquias de controle epigenético dos genes submetidos à ICX em células humanas / In female mammals, one of the X chromosomes is inactivated to achieve dosage compensation between males and females. The X chromosome inactivation (XCI) occurs early during embryogenesis and is characterized by the acquisition of heterochromatic features on the inactive X (Xi), which are maintained during all the subsequent cell divisions. Embryonic stem cells are the most suitable cells to study the establishment of XCI. They are obtained from the inner cell mass (ICM) of blastocysts, and can represent a developing female embryo, possessing two active X-chromosomes; when differentiated, these cells recapitulate XCI in vitro, and thus one can identify XCI regulators and factors involved. The derivation of human embryonic stem cells (hESCs) in 1998 offered new possibilities to study XCI, since most of the mechanistic studies of XCI have so far been investigated in the mouse model system. Traditionally, maintenance of XCI in humans has been addressed in somatic cell hybrids or transformed cells; however, they do not represent a natural cellular context. The main goals of the present work were to verify the potential of hESCs as models of XCI, and also to study the function of three important factors in XCI maintenance in immortalized human cells. DNMT1 (DNA-methyltransferase 1), SMCHD1 (a cohesin/condensin protein family member) and the XIST gene (a non-coding RNA which triggers XCI and promotes X heterochromatin formation on the future Xi) were selected, as they are key factors in XCI maintenance in the mouse. Until now four hESCs lines were derived in our lab. Their characterization showed that, in spite of been undifferentiated, the female hESCs have already undergone XCI. Our data suggest that, under the actual culture conditions, hESCs are not good models to study XCI, and it is also possible that X inactivation confers selective advantage to hESCs. Knockdown by RNA interference was used to study the roles of three genes in XCI maintenance. We could not efficiently knockdown XIST or SMCHD1. However, the DNMT1 silencing was substantial, and led to the reactivation of MAOA, an X-linked gene subjected to XCI. Although the effect of DNMT1 silencing was restricted to MAOA, our data suggest that there are different epigenetic hierarchies to control the expression of the genes subjected to XCI in human cells.

O linfoma não-Hodgkin (LNH) é bastante prevalente em cães e atualmente é aceito como modelo comparativo da doença em humanos. Padrões aberrantes de metilação de DNA parecem exercer um papel chave no desenvolvimento de tumores hematopoiéticos nos seres humanos, constituem um mecanismo especial de controle transcricional e podem ser influenciados por alterações genéticas e ambientais. Os efeitos da metilação global do DNA têm sido raramente investigados em cães, principalmente em processos neoplásicos. O objetivo do presente estudo foi quantificar a metilação global do DNA em leucócitos sanguíneos de cães portadores de LNH, comparando com a metilação global do DNA de leucócitos sanguíneos de cães sadios e identificar genes diferentemente metilados nas mesmas amostras. Para isto, utilizou-se o DNA obtido da capa leucocitária de amostras de sangue venoso periférico de 10 cães sadios e 9 cães com LNH multicêntrico. Para imunofenotipagem dos linfomas, aplicou-se o painel imunocitoquímico de anticorpos anti-CD79a, anti-PAX5 e anti-CD3. O índice de proliferação celular foi obtido por meio da contagem de núcleos positivos para Ki-67. A metilação global do DNA dos leucócitos foi quantificada pelo método High Performance Liquid Chromatography (HPLC) e visualmente (por escores) em amostras submetidas a imunocitoquímica (ICQ) com o anticorpo anti-5-metil citosina (5MetCyt). Para a identificação de genes diferentemente metilados entre ambos os grupos avaliados, utilizou-se a técnica de beadchip com o ensaio Infinium Methylation EPIC BeadChip humano (850K). Como resultados, em ambos os métodos (HPLC e ICQ), os leucócitos sanguíneos de cães portadores de LNH apresentaram metilação global do DNA significantemente inferior à dos cães sadios (HPLC: p= 0,027 / ICQ: p= 0,015). Das 853.307 ilhas CpGs investigadas no microarranjo, houve hibridização de 34.574 sondas nas amostras caninas. Desse total, observou-se diferença significante em nível de metilação de 606 sondas, e por meio da análise das similaridades homólogas e ortólogas, identificou-se 550 genes diferentemente metilados entre os dois grupos. Nosso estudo foi pioneiro em sugerir que cães com LNH apresentam hipometilação global do DNA de leucócitos circulantes quando comparados a cães sadios. Apesar de termos usado amostras caninas em um ensaio desenvolvido especificamente para o DNA humano (Infinium Methylation EPIC BeadChip) foi possível a identificação de genes diferentemente metilados e possíveis novos alvos com potencial preventivo ou terapêutico. Futuros estudos epidemiológicos são necessários para correlacionar o padrão de metilação de leucócitos com o risco de desenvolver linfomas e utilizá-lo como biomarcador / Non-Hodgkin\'s lymphoma is quite prevalent in dogs and it is currently accepted as a comparative model for the disease in humans. Aberrant patterns of DNA methylation appear to play a key role in the development of hematopoietic tumors in humans, constitute a special mechanism of transcriptional control and may be influenced by genetic and environmental variations. The effects of methylation have been rarely investigated in dogs, especially in neoplastic processes. The aim of the current study is to quantify the global DNA methylation of blood from dogs bearing non-Hodgkin\'s lymphoma, comparing them with the methylation content and pattern of healthy dogs and identify differently methylated genes in the same samples. For this, the DNA obtained from the buffy coat of peripheral venous blood samples from 10 healthy dogs and 9 dogs with multicentric non-Hodgkin\'s lymphoma were used. For immunophenotyping of lymphomas, the immunocytochemical panel of anti-CD79a, anti-PAX5 and anti-CD3 antibodies were applied. The cell proliferation index was performed by counting positive nuclei with anti-Ki-67. The global methylation of leukocyte DNA was quantified by the High Performance Liquid Chromatography (HPLC) method and visually (by scoring) in samples subjected to immunocytochemistry (ICQ) with the anti-5-methyl cytosine antibody (5MetCyt). For the identification of differently methylated genes between both groups, the bead chip technique was used with the Infinium Methylation EPIC BeadChip human assay (850K). As a result, in both methods (HPLC and ICQ), dogs with non-Hodgkin\'s lymphoma had a lower amount of methylation than healthy dogs (HPLC: p = 0.027 / ICQ: p = 0.015). Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 606 probes and through the homologous and orthologous similarities, 550 differently methylated genes were identified between the two groups. Our study was a pioneer in suggesting that dogs bearing non-Hodgkin\'s lymphoma presented DNA global hypomethylation of circulating leukocytes when compared to healthy dogs. Although we used canine samples in an assay developed specifically for human DNA (Infinium Methylation EPIC BeadChip) it was possible to identify differently methylated genes and possible new targets with preventive or therapeutic potential. Future epidemiological studies are needed to correlate the methylation pattern of leukocytes with the risk of developing lymphomas and to use it as a biomarker

A Doença de Parkinson é a segunda doença neurodegenerativa mais comum na atualidade. Cerca de 10% dos casos da doença estão relacionados com fatores genéticos e os outros 90% são devido a fatores ambientais e epigenéticos. Evidências indicam alterações na metilação de genes relacionados ao desenvolvimento da doença de Parkinson. No entanto, não se sabe o efeito de inibidores da metilação de DNA sobre a neurotoxicidade induzida por MPP+, uma neurotoxina que mimetiza processos neurodegenerativos associados ao Parkinson in vitro. Portanto, este trabalho teve como objetivo avaliar o efeito dos inibidores da metilação de DNA (RG108, n-ftaloil-l-triptofano e 5azadC, 5-aza-2´-deoxycytidina) e do doador universal de grupamentos metil (SAM, S-adenosilmetionina) sobre neurotoxicidade induzida por MPP+ em cultura de células imortalizadas (PC12), por meio da análise da viabilidade celular avaliada no teste do MTT (3 - [4,5 dimetiltiazol-2-il] -2,5-difenil-tetrazólio); e da análise de neuritogênese, na presença e na ausência de MPP+. Os resultados demonstraram que: 1. o tratamento com DNMTi (inibidor da DNA metiltransferase) ou com SAM induziram efeito per se sobre a viabilidade celular, apenas quando incubados em altas concentrações e em perídos prolongados (24h); 2. não modificaram a morte celular induzida pelo MPP+, em baixas concentrações, mas agravaram a neurotoxicidade quando incubados em altas concentrações ou por períodos prolongados (24h); 3. essas drogas induziram neuritogênese per se e potencializaram a neuritogênese induzida pelo NGF (fator de crescimento neural); 4. protegeram parcialmente contra a diminuição da neuritogênse induzida pelo MPP+. O conjunto de dados sugere que tanto os DNMTi quanto o SAM podem ser citotóxicos, dependen de suas concentrações e do tempo de exposição à droga. No entanto, essas drogas são capazes de aumentar a neuritogênese (diferenciação celular) e proteger contra a neurotoxicidade celular induzida pelo NGF, em células diferenciadas. / Parkinson\'s disease is the second most common neurodegenerative disease nowadays. About 10% of the disease cases are related to genetic factors and the other 90% are due to environmental and epigenetic factors. Evidence indicates changes in DNA methylation in genes related to the development of Parkinson\'s disease. However, the effect of DNA methylation inhibitors on MPP+-induced neurotoxicity, a drug that mimics neurodegenerative processes associated with Parkinson\'s in vitro, is not known. The aim of this study was to evaluate the effect of DNA methylation inhibitors (RG108, N-phthalyl-L-tryptophan and 5azadC, 5-aza-2?-deoxycytidine) and of the universal donor of methyl group (SAM, S-adenosyl methionine) on: 1. MPP+ -induced neurotoxicity in culture of immortalized cells (PC12), by analysis of cell viability in the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) test; 2. neuritogenesis, in the presence and absence of MPP +. Results indicated that: 1. treatment with DNMTi or SAM induced effects per se on cell viability only at higher concentrations and after prolonged periods of incubation (24h); 2. DNMTi (DNA methyltransferase inhibitors) and SAM increased cell differentiation and neuritogenesis per se, especially when incubated for 30 minutes, as well as they potentiated NGF-induced neurogenesis; 3. the drugs attenuated MPP+-induced effects on neuritogenesis. Altogether, these data suggests short treatment with both DNMTi and SAM do not cause cellular cytotoxicity (cell death), but are able to increase neuritogenesis (cell differentiation) and protect against MPP+-induced neurotoxicity in differentiated cells.

La dystrophie Facio-Scapulo-Humérale (FSHD) est caractérisée par un affaiblissement asymétrique des muscles de la face, de la ceinture scapulaire et des bras. Dans 95% des cas (FSHD1), elle est liée à la contraction d’un élément macrosatellite en 4q35, D4Z4. Dans les 5% des cas qui ne présentent pas de contraction de D4Z4 (FSHD2), des mutations dans le gène SMCHD1 sont retrouvées. Des mutations dans ce gène sont aussi associées à un syndrome développemental très rare, le syndrome de Bosma (BAMS). Les individus présentent des anomalies cranio-faciales mais aucun signe musculaire.Nous avons développé une méthode pour l’analyse de la méthylation de D4Z4 associant modification de l’ADN au bisulfite de sodium et séquençage à haut débit. Nous observons une hypométhylation marquée chez les patients FSHD2 et BAMS au niveau de la partie proximale de cet élément. L’étude de la méthylation au cours de la reprogrammation de fibroblastes primaires en cellules souches pluripotentes induites montre que D4Z4 est reméthylé de façon spécifique dans les cellules contrôles et FSHD1 mais est peu modulée dans les cellules FSHD2 et BAMS. Cette méthylation élevée est une caractéristique de la pluripotence et implique SMCHD1. La reméthylation ne dépend pas non plus de la mémoire épigénétique héritée des fibroblastes ni du nombre de répétitions D4Z4. Ces résultats suggèrent un mécanisme de régulation dynamique. L’hypométhylation de D4Z4 n’est pas observée lors de l’invalidation somatique du gène indiquant que SMCHD1 est impliqué dans la mise en place de la méthylation et non sa maintenance.SMCHD1 est donc impliqué dans la mise en place de la méthylation de D4Z4 dans les cellules pluripotentes. / Facio-Scapulo-Humeral Dystrophy is characterized by the involvement of specific facial, scapulo-humeral and anterior foreleg muscles. In 95% of cases (FSHD1) the disease is associated with a reduction of a macrosatellite element, D4Z4, at the 4q35 locus. In the 5% remaining cases (FSHD2), there is no D4Z4 contraction and patients carry mutations in the SMCHD1 gene. Mutations in SMCHD1 are also involved in a very rare developmental syndrome called Bosma Arhinia Microphtalmia syndrome (BAMS) characterized by cranio-facial abnormalities but no muscular dystrophy.To analyze D4Z4 methylation we developed an approach based on the Sodium Bisulfite treatment method followed by high-throughput sequencing. We showed a significant hypomethylation in the proximal region of D4Z4 in FSHD and BAMS patients compared to controls. Our methylation analysis in primary fibroblasts and corresponding human induced pluripotent stem cells showed that D4Z4 is specifically remethylated upon reprogramming in FSHD1 cells but not in SMCHD1-mutated cells. The high methylation level is a feature of pluripotency likely dependent on SMCHD1. Strikingly, hypomethylation is not observed in somatic cells invalidated for SMCHD1 suggesting a key role for this factor in de novo the methylation. D4Z4 methylation pattern does not depend on the epigenetic memory inherited form donor cells and on the number of the D4Z4 copies and it is a tightly regulated process.In conclusion, SMCHD1 plays a role in the D4Z4 methyl mark deposition at the pluripotency state.

L’hématopoïèse est un processus complexe et extrêmement régulé qui permet la production de l’ensemble des cellules sanguines à partir de cellules souches. Différents acteurs interviennent dans cette régulation et une altération de l’un ou plusieurs de ces régulateurs est souvent à l’origine de leucémies. L’un des acteurs majeurs de cette régulation est le complexe Core Binding Factor (CBF), particulièrement touché dans ces hémopathies. Ce facteur de transcription se compose de la sous-unité CBFβ et d’une sous unité variable RUNX, (habituellement RUNX1 dans l’hématopoïèse). Dans la leucémie aiguë myéloïde 4 à composante éosinophile (LAM4 Eo), le gène CBFβ est retrouvé fusionné au gène MYH11, entraînant la formation d’un gène chimérique codant pour l’oncoprotéine de fusion CBFβ–SMMHC. Cette version altérée du complexe CBF a pour caractéristique de séquestrer RUNX1 dans le cytoplasme et de déréguler l’expression des gènes cible du complexe via diverses mécanismes. Elle est en effet capable d’inhiber l’expression génique par le recrutement d’inhibiteurs transcriptionnels mais a également récemment été décrite comme liée au promoteur de gènes actifs. Ces dérégulations entraînent une altération de la différenciation et/ou une apoptose chez différents progéniteurs hématopoïétiques via divers mécanismes particulièrement étudiés chez la souris. Chez l’homme, les processus oncogéniques par lesquels CBFβ–SMMHC altère la différenciation et induit la leucémogénèse restent cependant peu décrits. Au moyen de deux modèles humains : une lignée ME-1 inductible pour l’inhibition de l’expression de l’oncoprotéine et des blastes leucémiques de patients atteints de LAM4 Eo dérivés de xénogreffes murines, nous avons découvert un nouveau composant cellulaire dérégulé par CBFβ–SMMHC ainsi qu’un nouveau partenaire d’interaction. En effet, dans un premier temps, ce travail révèle que l’oncoprotéine a des effets complexes sur la biogenèse des ribosomes aux niveaux génomique et post-transcriptionnel. Nous avons montré que CBFβ–SMMHC fixe le promoteur des gènes ribosomiques et active leur transcription. Nous avons également observé un niveau d’expression de ces gènes, supérieur dans les LAM dites de type CBFβ–SMMHC comparées aux autres sous-groupes de LAM. Dans la lignée ME-1 cette activation de la transcription ne se traduit cependant pas par une augmentation du contenu cellulaire en ribosomes, expliqué en partie par une maturation du précurseur des ARN ribosomiques moins efficiente en présence de l’oncoprotéine. Dans un second temps nous avons observé que CBFβ–SMMHC interagit directement avec la protéine Polycomb RING1B et BMI1 sous-unité du complexe de répression des gènes PRC1. L’inhibition de CBFβ–SMMHC entraînant une augmentation du niveau de fixation globale de RING1B sur l’ensemble du génome. Nous pensons que de cette altération du niveau de fixation de RING1B induite par CBFβ–SMMHC, découle la dérégulation de nombreux gènes impliqués dans diverses voies ou mécanismes critiques de l’hématopoïèse. Nous avons ainsi mis en lumière deux nouveaux mécanismes oncogéniques médiés par l’oncoprotéine CBFβ–SMMHC ouvrant de nouveaux horizons pour de potentielles cibles thérapeutiques. / Haematopoiesis is a complex process allowing the production of all mature blood cells from stem cells. This process is highly regulated at the transcriptional level, and perturbation of normal transcriptional regulation may cause leukaemia. One of the major actors of this regulation is the Core Binding factor (CBF) complex, which is frequently subject to genetic alteration in leukaemia. This transcription factor consists of a constant CBFβ subunit and a variable RUNX subunit, usually RUNX1 in haematopoiesis. In acute myeloid leukemia 4 with eosinophilic component (AMLM4 Eo), the CBFβ gene is fused to the MYH11 gene, leading to the formation of a chimeric gene encoding the CBFβ–SMMHC oncoprotein. This altered version of the CBF complex sequesters RUNX1 into the cytoplasm, and deregulates wild type CBF target gene expression though diverse mechanisms. While CBFβ–SMMHC can inhibit gene expression by recruiting transcriptional inhibitors, it has also recently been described to bind and activate certain gene promoters. The mechanisms by which these deregulations lead to an alteration of the differentiation and/or an apoptosis of diverse hematopoietic progenitors is best characterised in murine models. In humans, the oncogenic processes by which CBFβ–SMMHC alters differentiation and induces leukaemogenesis remain unclear. Using two human cellular models, namely (i) an ME-1 cell line containing an inducible shRNA directed against the CBFβ- MYH11 fusion transcript and (ii) Patient-derived AML M4Eo murine xenografts, we describe two novel activities of CBFβ–SMMHC. Firstly, we discovered that the oncoprotein has complex effects on ribosome biogenesis at both the genomic and post-transcriptomic levels. We found that CBFβ–SMMHC binds ribosomal gene promoters and activates their transcription, which was corroborated by the observation of higher ribosomal gene expression in human AML M4Eo, compared with other AML subgroups. In the ME-1 cell line this transcriptional activation did not lead to the higher cellular ribosome content, which was explained in part by decreased efficiency of ribosomal RNA maturation in the presence of the oncoprotein. Secondly, we found that CBFβ–SMMHC interacts directly with RING1B and BMI1 protein subunit of the Polycomb gene repression complex PRC1. Depletion of CBFβ–SMMHC lead to increased global binding of RING1B to the genome, resulting in deregulation of numerous genes that are critical for normal haematopoietic differentiation. We have therefore highlighted two new oncogenic mechanisms mediated by the CBFβ–SMMHC oncoprotein, therefore opening new avenues to investigate potential therapeutic targets.