Abstract 2-oxoglutarate-dependent dioxygenases (2-OGDDs) are an enzyme family that contains many enzymes that modify chromatin in extensive ways. These enzymes include several histone lysine demethylases (KDMs) and TET enzymes that convert methylated cytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) ultimately leading to DNA demethylation. Disturbed DNA and histone methylation are found in many cancers. However, the role of KDMs and TETs behind these oncogenic changes has so far not been fully investigated. This study focused on the role of these chromatin-modifying enzymes in cancers with special emphasis on enzyme kinetic studies. Cancers with inactivating fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) mutations accumulate fumarate, succinate and R-2-hydroxyglutarate, respectively. In this study we showed how these cancer-associated 2-oxoglutarate (2-OG) analogues can inhibit the TET enzymes and many of the KDMs leading to lower 5-hmC levels and increased H3K27 and H3K9 methylation on chromatin, respectively. We also characterized kinetic properties of acute myeloid leukaemia (AML)-associated TET2 mutants and found that their ability to bind 2-OG or iron was impaired leading to diminished catalytic activity. Tumours are often hypoxic due to inadequate vasculature and blood supply. The TET enzymes and KDMs require oxygen for the reactions they catalyse. We determined the oxygen affinity of TETs and many KDMs and found that a H3K27 demethylase KDM6A has a remarkably low affinity for oxygen indicating that it is inactivated in hypoxic tumours and tissues. H3K27 methylation was found to be increased in hypoxic cells and this blocked cell differentiation. Altogether, these studies shed light on the mechanisms behind the altered DNA and histone methylation found in several cancers with hypoxic conditions or FH, SDH and IDH mutations. Altered DNA and histone methylation has previously been associated with progression of cancer, such as epithelial-to-mesenchymal transition (EMT). We now linked catalytic inhibition of 2-OGDDs to disturbed DNA and histone methylation that can account for altered cell differentiation, EMT and increased aggressiveness and invasiveness of cancers. / Tiivistelmä 2-oksoglutaraatista riippuvaiset dioksygenaasit ovat entsyymiperhe, johon kuuluu useita entsyymejä, jotka muokkaavat kromatiinin epigeneettisiä merkkejä monin tavoin. Näitä entsyymejä ovat mm. DNA:n demetylaatioon vaikuttavat TET-entsyymit sekä useat histonidemetylaasit. Vaikka muutoksia DNA:n ja histonien metylaatiotasoissa on havaittu useissa syövissä, ei näiden entsyymien roolia muutosten taustalla ole vielä tutkittu. Tämä tutkimus kohdistui näiden epigenetiikkaan vaikuttavien entsyymien roolin ymmärtämiseen syövissä keskittyen erityisesti kyseisten entsyymien kinetiikkaan. Useissa syövissä on havaittu fumaraattihydrataasin, sukkinaattidehydrogenaasin ja isositraattidehydrogenaasien aktiivisuuteen vaikuttavia mutaatioita, jotka johtavat fumaraatin, sukkinaatin ja R-2-hydroksiglutaraatin kertymiseen syöpäsoluihin. Tässä tutkimuksessa osoitimme, kuinka nämä karsinogeeniset 2-oksoglutaraattianalogit voivat inhiboida TET-entsyymejä ja histonidemetylaaseja, mikä alentaa 5-hydroksimetyylisytosiinitasoja ja lisää histonien metylaatiota. Näytämme myös, kuinka tietyillä akuutissa myelooisessa leukemiassa esiintyvillä TET2-mutanteilla on heikentynyt kyky sitoa 2-oksoglutaraattia tai rautaa, mikä johtaa entsyymien aktiivisuuden laskuun. Kasvainkudoksissa happipitoisuudet ovat usein matalia nopean kasvun ja puutteellisen verisuonituksen vuoksi. TET-entsyymit ja histonidemetylaasit vaativat happea katalysoimissaan reaktioissa. Määritimme TET-entsyymien ja monien histonidemetylaasien riippuvuutta hapesta ja osoitimme, että H3K27-histonidemetylaasi KDM6A on erittäin riippuvainen hapesta, mikä osoittaa, ettei se pysty toimimaan kasvaimissa ja kudoksissa, joissa happipitoisuudet ovat matalia. Huomasimme, että vähähappisissa olosuhteissa solujen H3K27 metylaatio on lisääntynyt, mikä johti erilaistumisen estymiseen soluissa. Tämä tutkimus paljasti uusia mekanismeja useista syövistä löytyneiden muuntuneiden DNA:n ja histonien metylaatiotasojen taustalla. Häiriintynyt DNA:n ja histonien metylaatio on aiemmin yhdistetty syöpien etenemiseen, erityisesti solujen erilaistumisen häiriintymisen kannalta. Tässä tutkimuksessa yhdistimme 2-oksoglutaraatista riippuvaisten entsyymien inhibition häiriintyneeseen DNA:n ja histonien metylaatioon, joka voi johtaa muuntuneeseen solujen erilaistumiseen ja lopulta lisääntyneeseen syöpien aggressiivisuuteen ja invasiivisuuteen.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, the 5-year survival rate being less than 5%. At the time of diagnosis, 90% of PDACs extend beyond the pancreas and distant metastases are often present. Due to aggressive growth, local expansion and early appearance of metastasis, primary PDAC tumours are local enough for curative surgical resection in only 10–20% of the cases. Adjuvant chemotherapy is indicated in these curative-treated cases, with slight improvement in survival. PDAC is considered to represent a heterogeneous group of biologically and prognostically different malignancies. Characterization of these subgroups is essential and there is an urgent need for more accurate biomarkers and targeted treatments in PDAC. In the current work, we immunohistochemically investigated the expression levels and prognostic values of oxidative stress markers (8-OHdG, Keap1, Prx I, II, III, V and VI), epigenetic histone modifiers (KDM4A, KDM4B, KDM4D and SIRT1–4), and cell-cycle regulators (p16, Rb, CDK4) and DNA-repair enzymes (FEN1 and MGMT) in the cohort of surgically treated PDAC patients. We found that Keap1 expression was associated with better pancreatic cancer-specific survival. Expression of antioxidative peroxiredoxins I, III, V and VI was also connected with a more favourable tumour characteristics and Prx I and VI showed prognostic value. When considering the biology of PDAC, we noticed that pivotal epigenetic regulation also occurred in exocrine pancreatic tissue adjacent to resection margins. Overexpression of the cell-cycle regulator CDK4 and the DNA-repair enzyme FEN1 in the whole population, and elevated expression level of MGMT in the most high-risk patients were connected with worse prognosis. The results of the study can be utilized in the future when individualized therapies are being designed for PDAC patients. Due to occurrence of the epigenetic regulation also in exocrine pancreatic tissue adjacent to resection margins, it could be evaluated in future for routine diagnostics and treatment optimization. The potential role of MGMT in the development of PDAC chemoresistance should be studied in the future. / Tiivistelmä Haiman duktaalinen adenokarsinooma (PDAC) on yksi aggressiivisimmista syöpäsairauksista. Viiden vuoden elossaoloennuste on vain lähellä 5 prosenttia. Diagnoosihetkellä 90% haiman adenokarsinoomista yltää haiman ulkopuolelle ja usein kasvain on jo lähettänyt etäpesäkkeitä. Kasvutaipumuksen sekä metastasoinnin takia kuratiivinen kirurginen hoito on mahdollista vain 10–20% tapauksista. Liitännäissolunsalpaajahoito on aiheellista näissä kuratiivistavoitteisesti hoidetuissa tapauksissa. Kuitenkin vaikutus kokonaiselossaoloaikaan on melko vähäinen. Uusimman tutkimustiedon valossa PDAC:aa pidetäänkin heterogeenisenä ryhmänä biologisesti ja ennusteellisesti erilaisia tautiryhmiä. Näiden tautiryhmien tunteminen ja tunnistaminen riittävän tarkkojen merkkiaineiden avulla olisi ensiarvoisen tärkeää, jotta hoitoja voitaisiin kohdentaa niistä hyötyville potilaille. Väitöskirjatutkimuksessa selvitimme immunohistokemiallisin menetelmin oksidatiivisen stressin merkkiaineiden (8-OHdG, Keap1, Prx I, II, III, V ja VI), epigeneettisten histonimodifikaattorien (KDM4A, KDM4B, KDM4D ja SIRT1–4) sekä solusyklin säätelijöiden (p16, Rb, CDK4) ja DNA-korjausentsyymien (FEN1 ja MGMT) ilmentymistä ja ennusteellista arvoa kirurgisesti hoidetuilla PDAC-potilailla. Tutkimuksessamme totesimme, että kasvainkudoksen Keap1-ilmentymä yhdistyi parempiennusteiseen taudinkuvaan. Antioksidatiivisten peroksiredoksiinien I, III, V ja VI ilmentyminen yhdistyi niin ikään suotuisampaan kasvaimen fenotyyppiin ja Prx I ja VI osoittivat ennusteellista arvoa. Havaitsimme lisäksi, että PDAC:n biologiaan keskeistesti vaikuttavaa epigeneettistä säätelyä tapahtuu myös malignin haimakudoksen viereisessä eksokriinisessä haimakudoksessa. Solusyklin säätelijä CDK4:n ja DNA-korjausentsyymi FEN1:n voimakas ilmentyminen koko tutkimuspopulaatiossa sekä kohonnut MGMT:n ilmentyminen korkeimman riskin potilailla yhdistyivät huonompaan taudin ennusteeseen. Väitöskirjatyön tutkimustuloksia voidaan tulevaisuudessa hyödyntää, kun tutkitaan yksilöllisiä hoitomuotoja PDAC-potilailla. Koska epigeneettistä säätelyä tapahtuu myös syövän viereisessä eksokriinisessa haimakudoksessa, voidaan tulevaisuudessa tämän kudoksen arviointia mahdollisesti käyttää rutiinisti diagnostiikassa sekä hoidon optimoinnissa. MGMT:n mahdollinen rooli PDAC:n kemoresistenssin kehittymisessä tulisi tulevaisuudessa selvittää.

O aneurisma de aorta abdominal (AAA) é uma doença assintomática na maioria dos casos, podendo acometer 5% das pessoas do gênero masculino com idade superior a 65 anos, predispondo ao risco de ruptura com mortalidade em torno de 80%. O presente estudo teve como objetivo avaliar os perfis de expressão gênica e de metilação do DNA no tecido, como também os microRNAs no plasma e no tecido de indivíduos com e sem AAA na tentativa de identificar marcadores biológicos e alvos terapêuticos para o diagnóstico, monitoramento e tratamento precoce do AAA. Os perfis de expressão gênica, miRNA e metilação de DNA dos tecidos da aorta abdominal (n=6) obtidos durante a cirurgia aberta para correção de AAA foram comparados com tecidos da aorta abdominal de doadores de órgãos sem AAA (n=6). Também foram comparados os perfis de miRNAs circulantes no plasma do grupo AAA (n=6) com o grupo-controle de voluntários com as características semelhantes, porém sem AAA (n=6). Para a análise da expressão gênica, utilizou-se a qPCR Array, analisando-se genes relacionados ao endotélio vascular humano (PAHS-015Z, QIAGEN®). A análise do perfil de miRNA foi realizada utilizando-se Human miFinder 384HC miScript miRNA PCR Array (MIHS-3001Z, QIAGEN®) e, para análise de metilação do DNA, utilizou-se a qPCR array com 22 genes das vias de estresse e toxicidade EpiTect Methyl II (EAHS-581Z, QIAGEN®). O software Ingenuity Pathway analysis (IPA®) foi utilizado para identificação das prováveis relações entre os microRNAs e a expressão gênica realizada nesta pesquisa. No estudo da expressão gênica, quatro genes (SPHK-1, TYMP, ALOX5 e HIF1A) foram identificados como mais expressos e outros 6 genes (PTGIS, CX3CL1, ITGB1, COL18A-1, FN1 e AGTR1) apresentaram expressão reduzida nos tecidos de AAA. Na análise do perfil de miRNAs, 24 miRNAs foram significantemente mais expressos e 35 miRNAs menos expressos no tecido. No plasma de indivíduos com AAA, 8 miRNAs apresentaram-se mais expressos e 9 miRNAs menos expressos. Dois miRNAs, miR-328-3p e let-7c-5p demonstraram expressões comuns entre tecido e plasma. Quanto ao padrão de metilação de DNA, somente o gene GDF15 teve grau de metilação maior nos tecidos de AAA quando comparado ao grupo-controle. A análise funcional revelou que o gene PTGIS (prostaciclina sintetase), um potente vasodilatador e inibidor da atividade plaquetária, foi reprimido pelo miR-150-5p, que se mostrou 7,5 vezes mais expresso no tecido de AAA, e teve uma possível interação com o miR-328-3p, cuja expressão foi 3,7 vezes mais baixa no tecido. Os genes com expressão reduzida nos tecidos do AAA foram alvos de miRNAs com expressão aumentada, evidenciando a importância e influência dos fatores epigenéticos tanto para o desenvolvimento quanto para a gravidade do AAA. / Abdominal aortic aneurism (AAA) is an asymptomatic disease in the majority of cases that may occur in 5% of males over age 65, predisposing to the risk of rupture leading to a mortality rate of 80%. The aim of this study was to evaluate the DNA metilation and gene expression profile in tissue, and microRNA expression pattern in both plasma and tissue samples from individuals with and without AAA to identify biological markers and therapeutic targets for an early diagnosis and treatment of AAA, respectively. Genes and miRNA expression and DNA metilation profiles in AAA tissues (n= 6) were compared to abdominal aortic tissues obtained from organ donators without AAA (n = 6). We also compared circulating miRNAs profiles in plasma samples, between AAA (n = 6) and the control group without AAA (n = 6). For the gene expression analysis we used a qPCR Array (PAHS-015Z, QIAGEN®) to analyze genes related to human vascular endothelium. For the miRNA expression pattern and for DNA methylation analysis we used the Human miFinder 384HC miScript miRNA PCR Array (MIHS-3001Z, QIAGEN®) and EpiTect Methyl II (EAHS-581Z, QIAGEN®), respectively. The Ingenuity software was used to identify the interactions between the miRNAs and genes evaluated in this study. Four genes (SPHK-1, TYMP, ALOX5 and HIF1A) were upregulated and six other genes (PTGIS, CX3CL1, ITGB1, COL18A-1, FN1 e AGTR1) were downregulated in AAA tissues. In addition, the miRNAs analysis showed 24 miRNAs more expressed and 35 miRNAs less expressed in AAA tissue than controls. Although in plasma samples, AAA group presented 8 miRNAs more expressed and 8 miRNAs less expressed than controls. Only, miR-328-3p and let-7c-5p were differently expressed between AAA and controls in both tissue and plasma samples. DNA methylation analysis showed that the gene GDF15 was hypermethylated in AAA tissues when compared to the control group. Functional analysis revealed that PTGIS, a potent vasodilator and platelet activity inhibitor was supressed by miR-150-5p, which had a seven-fold increase in AAA tissues. Moreover, a possible interaction between PTGIS and miR-328-3p, about 4-fold decreased in AAA tissues, was showed. Thus, the downregulated genes in AAA tissues are targets of miRNAs with increased expression in the same biological sample. These results highlight the importance and influence of epigenetic factors for both development and severity of AAA.

L’augmentation de température que subit la planète ces dernières décennies a de nombreuses conséquences dont la recrudescence de maladies infectieuses aussi bien chez l’homme que chez les animaux. Certaines populations de l’ormeau européen Haliotis tuberculata, vivant dans les zones les plus chaudes de Bretagne et de Normandie ont ainsi subi de très importantes mortalités depuis 1997, dues à la bactérie Vibrio harveyi. Cependant, certaines des populations les plus sévèrement touchées se sont aujourd’hui reconstruites et les mortalités semblent s’être arrêtées dans certaines de ces zones. La question se pose donc de l’apparition d’une résistance de l’ormeau face à cette maladie émergente. Pour répondre à cette question, les réponses à l’infection de plusieurs populations naturelles par cette bactérie ont été analysées. Une population présentant une forte résistance à la maladie a été identifiée.La voie d’entrée de la bactérie (ie. les branchies) a été identifiée comme jouant un rôle dans la résistance à l’infection. Par ailleurs, des infections successives ont permis de démontrer un effet d’amorçage immunitaire. Suite à une première exposition, une protection durant jusqu’à deux mois intervient contre l’effet d’inhibition de la phagocytose, provoquée normalement par une infection à V. harveyi. La différence d’expression de gènes des hémocytes d’ormeaux sensibles et résistants a été quantifiée par RNAseq pendant une infection expérimentale. Cette comparaison a montré une reconnaissance plus efficace du pathogène chez les résistants, par des récepteurs tels que les TLR ou les PGRP. La forte surexpression chez la population résistante, d’un gène impliqué dans la synthèse de mucine qui est l’un des composants principaux du mucus renforce l’hypothèse d’une forte implication des branchies dans la résistance. Enfin, une analyse in silico des séquences obtenues en RNAseq a permis d’apporter des preuves de l’existence d’un système de méthylation de l’ADN chez H. tuberculata ainsi qu’une possible implication de ce système dans l’adaptation de l’ormeau à son milieu. / Increasing global temperatures have numerous consequences for marine ecosystems, including the rise of infectious diseases. Certain populations of the European abalone Haliotis tucerculata have suffered from severe and recurrent mortality since 1997 due to infection caused by the bacterium Vibrio harveyi, particularly in areas with higher average summer temperatures. Given the spatial heterogeneity in mortalities, and the observation that the historically most severely impacted populations have recovered in recent years, the question of the emergence of resistance to the disease was addressed. The mortality rate in response to infection by V. harveyi was quantified experimentally in abalone originating from three natural populations, and one population exhibiting resistance to the disease was identified. In a subsequent experiment, the immune response of abalone was compared between infected individuals from a resistant and from a susceptible population. The portal of entry of the bacterium (ie. gills) was identified as playing a role in resistance. Furthermore, successive exposures of abalone to the bacterium demonstrated an immune priming effect, such that following a first exposure, phagocytosis was no longer inhibited by infection with V. harveyi, and that this improved protection against the disease lasted for at least two months. Differences in gene expression was quantified by RNAseq in the hemocytes of resistant and susceptible abalone following exposure to the pathogen. This comparison showed that resistant abalone had more effective recognition of the bacterium by receptors as the TLR or PGRP. The substantial over-expression of a gene involved in the synthesis of mucin, the main component of mucus, (UDP-GalNAC) in the resistant population, supports the interpretation of a strong involvement of gills in the resistance. Finally, an in-silico analysis of the sequences obtained from RNAseq indicate the existence of a DNA methylation system in H. tuberculata and suggested an involvement of epigenetic mechanisms in the adaptation of abalone to its environment.

The genus Mycobacterium represents more than 120 species of bacteria including the pathogenic M. tuberculosis, the etiological agent of tuberculosis. The host mounts a robust inflammatory and cell-mediated response to contain the spread of pathogenic mycobacteria. While macrophages, dendritic cells (DCs) and neutrophils are known to facilitate early responses, the effector functions of CD4+ and CD8+ T cells are critical for containment of the mycobacteria. The type I T helper (Th1) subset of CD4+ T cell population orchestrates the protective immunity through cytokines like interferon (IFN)-γ, interleukin (IL)-12, IL-23 and tumor necrosis factor (TNF)-α However, it is known that despite such responses, host can only contain but not eradicate the infection. Additionally, infection of over one-third of the world’s population with pathogenic mycobacteria is a testimony of its success as a pathogen. Much of its success is attributed to the multiple evasion strategies employed such as inhibition of phagosome-lysosome fusion, secretion of reactive oxygen intermediates antagonistic proteins like superoxide dismutase and catalase, downregulation of antigen presentation to T cells, downregulation of the pro-inflammatory cytokines, skewing the immune balance toward the less effective Th2 responses, inhibition of autophagy, induction of regulatory T cells (Tregs) and immunosuppressive cytokines etc. Thus, an effective check on the infection would be possible if we understand the mechanisms underlying such evasion and survival strategies. In this perspective, evaluation of the host-pathogen interactions in terms of integration of key signaling centers, particularly that during mycobacteria-macrophage or mycobacteria-DC interactions, would underscore as a critical requisite to detail the immune responses and its regulation. This study addresses three such immune evasion and survival strategies employed by the mycobacteria; downregulation of IFN-γ-induced autophagy in macrophages, expansion of Tregs by modulating DC phenotype and finally epigenetic regulation of genes involved in foamy macrophage generation. Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. It contributes to both innate and adaptive immune responses to infections and plays an essential role in restricting intracellular pathogens and delivering pathogen-derived antigens for major histocompatibility complex class II presentation. Nonetheless, several pathogens, especially viruses such as herpes simplex virus, human immunodeficiency virus, influenza; and bacteria like Mycobacteria, Shigella and Listeria exhibit multiple mechanisms to evade autophagy. However, the identities and contributions of host signaling molecules and mechanisms by which pathogens modulate autophagy have not been explored in depth. Here, we demonstrate that M. bovis BCG, Shigella flexneri and Listeria monocytogenes but not Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli inhibit IFN-γ-induced autophagy in macrophages by evoking selective and robust activation of WNT and sonic hedgehog (SHH) pathways via mechanistic target of rapamycin (mTOR). Utilization of macrophages derived from mir155-null mice or by conventional siRNA or miRNA mimics emphasized the role for mTOR-responsive epigenetic modifications in the induction of microRNAs, miR-155 and miR-31 to fine-tune autophagy. Importantly, cellular levels of PP2A, a phosphatase, were regulated by miR-155 and miR-31. Diminished expression of PP2A led to inhibition of glycogen synthase kinase (GSK)-3β, a negative regulator and a nodal link that regulate WNT and SHH pathways. This facilitated the prolonged activation of WNT and SHH signaling pathways. Further, sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases (ALOX5 and ALOX15), which in tandem inhibited IFN-γ-induced janus kinase (JAK)- signal transducer of activated (STAT) signaling and contributed to evasion of autophagy. Together, we have identified novel molecular mechanisms and host factors that are crucial to control autophagy and help the bacterial pathogens like mycobacteria to evade the host immune responses. Much of the protective immunity against mycobacterial infection is mediated by Th1 CD4+ T cells. However, suppressive T cell populations such as CD4+CD25+FoxP3+ Tregs or a less effective Th2 cells are exploited by mycobacteria to subvert the protective host immune response. In this perspective, the molecular mechanisms underlying mycobacteria-induced Treg expansion are unclear. Utilizing cues from the previous reports from others’ and our laboratory, we explored the role for host signaling pathways such as SHH, WNT and NOTCH1 signaling during mycobacteria-mediated DC maturation and Treg generation/expansion. We demonstrate that while inhibition of SHH signaling markedly reduced the ability of the infected DCs to expand Tregs, NOTCH1 signaling functioned to suppress M. bovis BCG-induced Treg expansion. Though SHH and NOTCH1 signaling did not regulate the DC maturation during infection in terms of the maturation markers CD1a, HLA-DR, CD40, CD83, CD80 and CD86, pro-inflammatory cytokines such as TNF-α, IL-2, IL-1β and IL-6 were moderately NOTCH1-responsive and suppressed by SHH signaling. Further, M. bovis BCG-induced SHH signaling and Treg expansion was mediated by the classical phosphoinositide 3-kinase (PI3K)-mTOR-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) cascade. Recent studies have attributed the role for programmed death ligand (PD-L)1 and cyclooxygenase (COX)-2-catalyzed prostaglandin (PG)E2 during expansion of Tregs. Experiments utilizing pharmacological inhibitors and conventional siRNAs indicated that both PD-L1 and COX-2/PGE2 were induced upon M. bovis BCG and M. tuberculosis infection in DCs and were regulated by SHH signaling. While SHH-responsive transcription factor, GLI1 arbitrated COX-2 expression, mycobacteria-stimulated SHH signaling was found to suppress miR-324 and miR-338, bonafide miRNAs that target PD-L1, to aid increased expression of PD-L1 and Treg expansion. This highlights the bi-functional role of SHH signaling during mycobacterial infection of DCs. Further, we found interesting cross-regulation of NOTCH and SHH pathway functions during M. bovis BCG infection of DCs. Inhibition of NOTCH1 signaling resulted in elevated expression of infection-induced PD-L1 whereas inhibition of SHH signaling showed increased transcripts of JAGGED2 (JAG2), a NOTCH1 ligand, and NOTCH intracellular domain (NICD), a marker for NOTCH activation. Thus, our results demonstrate that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in DCs to determine the functional outcome of the immune responses including Tregs expansion that favours its survival. Foamy macrophages (FMs) are integral components of granulomas during mycobacterial pathogenesis. FMs are one of the morphotypes differentiated from macrophages characterized by the presence of lipid bodies (LBs)/droplets. The lipids provide nutrients to mycobacteria, leading to an enhanced ability to survive and replicate in host FMs. LBs are also known to regulate lipid metabolism, membrane trafficking, intracellular signaling and inflammatory mediator production. Interestingly, LBs are stores for various immune mediators including arachidonic acid, COX-2, ALOX5, ALOX15 and leukotrienes, underscoring the significance of FMs in the current study. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs in the course of mycobacterial infection are not clear. Here, we analyzed the role for one of the histone modifications widely implicated in shaping the immune responses, Histone H3 lysine 27 trimethylation (H3K27me3), a known marker for gene silencing. While the trimethylation of H3K27 is catalyzed by EZH2, a component of Polycomb-repressive complex (PRC)2, Jumonji C (JmjC) domain protein (JMJD3) is a well-established H3K27me3 demethylase. Unlike M. smegmatis, infection of macrophages with M. tuberculosis or M. bovis BCG displayed JMJD3-dependent LB formation. Supporting this observation, the genes involved in lipid biosynthesis (Ascl1, Adrp, Psap) and uptake (Fat (CD36) and Msr1) were significantly upregulated with M. tuberculosis or M. bovis BCG infection of macrophages in a JMJD3- and TLR2-dependent manner. Abca1 and Abcg1, genes assisting in lipid export were downregulated or remained unchanged with M. tuberculosis or M. bovis BCG infection. Chromatin immunoprecipitation analysis revealed a reduced H3K27me3 mark on the promoters of the selected genes that were upregulated on mycobacterial infections. Corresponding, elevated recruitment of JMJD3 to these promoters was observed. Interestingly, NOTCH1 signaling-responsive MUSASHI (MSI), an evolutionarily conserved RNA-binding protein that inhibits translation of the mRNA, was found to positively regulate infection-induced JMJD3 expression. MSI targeted a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein (MINT/ SPEN), in the infected macrophages to aid in FM formation. Immunohistochemistry and immunofluorescence experiments utilizing in vivo murine granuloma model using M. bovis BCG substantiated these observations. Thus, our study has unveiled novel roles for JMJD3 and its regulators in epigenetic regulation of LB generation in FMs. Altogether, we have established significant roles for several new host factors and inhibitory, survival mechanisms employed by pathogenic mycobacteria. Emphasis on functions of miRNAs and epigenetic regulation in the study has underscored the importance of fine-tuning immune responses during mycobacterial pathogenesis to determine the cell-fate and shape the course of infection. Further understanding and evaluation of these molecular regulators bears potential importance in disease control by aiding the search for effective drugs and therapeutics.

La maladie d’Alzheimer (MA) est caractérisée par une perte progressive des capacités mnésiques et des fonctions cognitives accompagnée du développement de dégénérescences neurofibrillaires (DNFs) et de plaque séniles dans le parenchyme cérébral. La découverte de perturbations épigénétiques dans des cerveaux de patients Alzheimer ont mené la communauté scientifique à se tourner vers de nouvelles voies thérapeutiques. De telles altérations, notamment au niveau de l’acétylation des histones de la chromatine, pourraient rendre compte de dysfonctionnements dans l’expression des programmes génétiques. En utilisant les souris THY-Tau22 (développement progressif de DNFs), nous avons évalué l’efficacité de deux stratégies visant à augmenter l’acétylation des histones : activer les HATs et inhiber les HDACs. Ces deux stratégies permettent une récupération des performances en mémoire spatiale chez les souris THY-Tau22, ouvrant ainsi de nouveaux axes thérapeutiques et de recherches dans la MA. / Alzheimer’s disease (AD) is characterized by a progressive loss of memory and cognitive functions associated with the development of neurofibrillary tangles (NFTs) and amyloid plaques in the brain. Nowadays, there is no satisfactory cure for AD. The discovery of epigenetic alterations in AD brain led the scientist community to think about new therapeutic options. Such modifications, and notably those on histone acetylation of the chromatin, could be associated with the genetic dysfunctions observed in AD. The reestablishment of proper epigenetic regulations, and thus gene expression, appears as an original therapeutic option. Using THY-Tau22 mice, we assessed the effect of two strategies aimed at increasing histone acetylation with a HAT activator (CSP-TTK21) or an HDAC inhibitor (phenylbutyrate). We show that both therapeutic strategies allow the rescue of spatial memory performances in the THY-Tau22 mouse model. These results open new leads for AD therapeutics and research.

La procréation médicalement assistée (PMA) permet à beaucoup d’individus infertiles de concevoir un enfant qui leur est génétiquement lié. Cependant, des données scientifiques émergentes suggèrent que la PMA pourrait entraîner des risques épigénétiques pour les futurs enfants. Conformément à l'hypothèse des origines développementales de la santé et des maladies, la PMA pourrait augmenter le risque de développer des maladies à apparition tardive par des mécanismes épigénétiques, car l’hyperovulation, les méthodes de fécondation et la culture embryonnaire pourraient nuire à la reprogrammation épigénétique de l'embryon. De tels risques épigénétiques soulèvent des enjeux éthiques pour toutes les parties prenantes: les futurs parents et enfants, les professionnels de la santé, et la société. Ce mémoire se concentre sur les questions éthiques soulevées par la prise en compte de ces risques lors de l'utilisation de la PMA. Pour mettre en lumière ces enjeux, nous utilisons l’approche principiste. Nous argüons qu'une tension éthique peut émerger entre le respect de l'autonomie procréative des parents d’intention et le devoir de minimiser les risques pour les enfants potentiels. Une seconde tension éthique peut émerger entre le droit des parents d’intention de faire un choix éclairé, et la réticence que peuvent avoir les professionnels de la santé de communiquer l’information sur les risques épigénétiques de la PMA, étant donné la validité incertaine de ces informations. Nous explorons aussi le risque de conflits d’intérêts pour les cliniciens des cliniques de PMA. Nous soutenons que les parents d’intention et les professionnels de la santé ont la responsabilité partagée de promouvoir les meilleurs intérêts du futur enfant. Nous plaidons pour que plus de recherche soit faite sur les effets de la PMA sur la santé des futurs enfants, pour que soient énoncées des lignes directrices priorisant le recours à des techniques moins risquées au niveau épigénétique, et pour que d’autres lignes directrices guident les professionnels de la santé dans la communication des risques épigénétiques associés à la PMA. Enfin, nous suggérons que cette communication se fasse dans le cadre d’une approche centrée sur le patient. Nous explorons aussi l’apport d’une approche narrative pour aborder les tensions éthiques soulevées par l’approche principiste. / The use of assisted reproductive technologies (ART) allows many coping with infertility to conceive. However, an emerging body of evidence suggests that ART could carry epigenetic risks for those conceived through the use of these technologies. In accordance with the Developmental Origins of Health and Disease (DOHaD) hypothesis, ART could increase the risk of developing late-onset diseases through epigenetic mechanisms, since superovulation, fertilization methods and embryo culture could impair the embryo’s epigenetic reprogramming. Such epigenetic risks raise ethical issues for all stakeholders: prospective parents and children, health professionals, and society. This thesis focuses on ethical issues raised by the consideration of these risks when using ART. To highlight these issues, we use the principlist approach. We argue that an ethical tension can emerge between respect for the reproductive autonomy of prospective parents and the duty to minimize the risks for potential children. A second ethical tension can emerge between the parents' right to make an informed choice about the use of ART, and the reluctance of health professionals to communicate epigenetic risk given its uncertain validity. We also explore the risks of conflicts of interests for health professionals in ART clinics. We argue that prospective parents and health professionals have a shared responsibility to promote the best interests of the future child. We also argue in favor of further research on the effects of ART on the health of future children, and in favor of clinical guidelines that prioritize the use of techniques that carry less epigenetic risk and that assist health professionals in communicating the epigenetic risks associated with ART. Finally, we suggest that this communication be done within the patient-centered approach. We also explore the contribution of a narrative approach to address the ethical tensions raised by the principlist approach.

Abstract Colorectal cancer is the third most common cancer in the developed countries. Originally, development of CRC was thought to proceed by a sequence of steps known as an adenoma-carcinoma sequence. At present CRC is recognized as a disease developing through diverse pathways. Serrated adenocarcinoma represents an endpoint of tumors developing from serrated pathway. This thesis focuses on studying the molecular alterations in serrated adenocarcinoma. Microsatellite instability, hypermethylation of promoter region in DNA repair genes hMLH1 and MGMT, frequency of KRAS and BRAF mutations and mutation spectrum of PTCH1 was determined in serrated adenocarcinomas (n=42) and compared to non-serrated adenocarcinomas (n=75). MSI, particularly low level of MSI (p=0.02) and methylation of both hMLH1 and MGMT promoters (p=0.004, p=0.026) were found to be more prevalent for serrated CRC. BRAF mutation was frequent and specific to serrated adenocarcinomas (p&lt;0.001) and KRAS mutations were more frequent in serrated adenocarcinomas than in non-serrated cancers (p=0.002). A significant association between BRAF mutation, hMLH1 and MGMT methylation and MSI-H phenotype was found in serrated carcinomas. KRAS mutation was seen in association with MSS/MSI-L phenotype; in fact, if serrated adenocarcinoma presents with MSI-L there always seems to be a KRAS mutation as well. Negative immunohistochemical staining of the hMLH1 enzyme was in association with methylation of the gene and proved reliable in the detection of MSI-H phenotype (p&lt;0.0001). Sequencing analysis of the whole coding regions of the PTCH1 gene did not reveal any truncating mutation to explain the previously detected downregulation of the gene in serrated CRCs. In conclusion, serrated adenocarcinomas proved to be an independent, but heterogeneous subtype of CRCs. High combined mutation rate (79–82%) of KRAS and <I>BRAF</I> in serrated adenomas and adenocarcinomas indicates that MAPK activation is a crucial part of the serrated pathway. BRAF mutations are specific for serrated adenocarcinoma, and identify a subset of serrated adenocarcinomas with gene methylation and a tendency for MSI-H. High frequency of KRAS mutations in serrated adenocarcinomas suggests that a significant proportion of KRAS-mutated CRCs originate from serrated precursors. / Tiivistelmä Paksu- ja peräsuolisyöpä eli kolorektaalisyöpä on Suomessa kolmanneksi yleisin syöpätyyppi. Syöpää edeltävien muutosten tunnistaminen on tärkeää, jotta sen ehkäisy ja seuranta olisi tehokasta. Tavallisia adenoomapolyyppeja on pidetty tärkeimpinä kolorektaalisyövän esiastemuutoksina. 2000-luvulla on havaittu, että nk. sahalaitapolyypit edustavat tärkeää osaa esiastemuutoksista, ja näistä kehittyvää syöpää kutsutaan sahalaitaiseksi syöväksi. Sahalaitaisen syövän kehittymismekanismit eroavat huomattavasti tavallisesta kolorektaalisyövästä. Tässä väitöskirjassa keskityttiin tutkimaan sahalaitaiselle syövälle tyypillisiä morfologisia piirteitä sekä geneettisiä muutoksia. Työssä selvitettiin DNA mikrosatelliitti-instabiliteetin sekä DNA korjausgeenien hMLH1 ja MGMT promoottorialueiden hypermetylaation esiintyminen, nk. MAPK –signaalinsiirtoreitin komponenttien, KRAS ja BRAF -geenien, mutaatioiden yleisyys sekä PTCH1 geenin mutaatiokirjo sahalaitaisissa (n=42) ja tavallisissa kolorektaalisyövissä (n=75). DNA:n mikrosatelliitti-instabiliteetti, erityisesti matala-asteisena (MSI-L) (p=0.02) sekä <I>MLH1</I> ja hMGMT -geenien metylaatio (p=0.004, p=0.026) olivat yleisempiä sahalaitaisissa syövissä. <I>BRAF</I> mutaatio oli yleinen sekä spesifinen sahalaitasyöville (p&lt;0.001). Myös KRAS -mutaatiot olivat yleisempiä sahalaitaisissa syövissä (p=0.002). BRAF mutaatio, hMLH1 sekä MGMT metylaatio ja korkea-asteinen mikrosatelliitti-instabiliteetti (MSI-H) esiintyivät hyvin usein yhdessä sahalaitaisissa syövissä. Sahalaitaisissa syövissä KRAS –mutaatiot liittyivät MSI-L fenotyyppiin. hMLH1 geenin ilmentyminen tutkittiin myös immunohistokemiallisesti. Sahalaitaisissa syövissä MLH1 –proteiinin häviäiminen oli yhteydessä metylaatioon ja liittyi spesifisesti MSI-H:n esiintymiseen (p &lt; 0.0001). PTCH1 geenin sekvensointi ei paljastanut proteiinin toimintaa vahingoittavia muutoksia, eikä tuloksen perusteella pystytä selittämään aikaisemmin havaittua geenin ilmentymisen häviämistä sahalaitaisessa syövässä. Tulosten perusteella sahalaitainen syöpä on oma, mutta heterogeeninen kolorektaalisyövän alatyyppi. KRAS ja BRAF –geenien aktivoivien mutaatioiden yleisyys (79–82%) osoittaa, että MAPK -reitin aktivaatio on tärkeää sahalaitaisen syövän kehityksessä. BRAF -mutaatiot ovat spesifisiä sahalaitaisille syöville, ja yhdessä metylaation sekä MSI-H:n kanssa identifioi osan sahalaitasyövistä omaksi ryhmäkseen. <I>KRAS</I> –mutaatioiden yleisyys sahalaitaisissa syövissä antaa aiheen epäillä, että merkittävä osa KRAS –mutaation sisältävistä kolorektaalisyövistä kehittyy sahalaitapolyypeista.

L'obésité maternelle peut prédisposer aux pathologies métaboliques à l'âge adulte. Une perte de poids préconceptionnelle est recommandée aux femmes obèses, mais ses effets sur la croissance fœto-placentaire et la santé de la descendance adulte sont encore peu connus. Les objectifs de cette thèse étaient d’étudier les effets des trajectoires pondérales maternelles sur le phénotype de la descendance à terme et à l’âge adulte, ainsi que sur l’expression génique. Les descendants de mères obèses présentent une restriction de croissance fœtale, associée à des altérations d’expression des gènes de la machinerie épigénétique dans le foie fœtal et le labyrinthe placentaire. Notre étude souligne la sensibilité particulière de la machinerie d’acétylation des histones au métabolisme maternel. À l'âge adulte, les mâles nés des mères obèses développent une obésité aggravée lorsqu'ils sont exposés à un environnement obésogène. La perte de poids maternelle préconceptionnelle améliore la croissance fœtale et normalise le poids à l’âge adulte. Elle est donc bénéfique pour la descendance. Cependant, certains effets de l’obésité, corrigée par l’intervention nutritionnelle, sont conservés car le poids fœtal et l’expression d’une partie de gènes restent altérés. Ce travail apporte des premiers éléments sur les mécanismes du conditionnement développemental par les trajectoires pondérales maternelles. / Maternal obesity (OB) impacts fetal growth and adult offspring phenotype. It is still unknown whether the currently recommended preconceptional weight loss (WL) for obese women is beneficial for feto-placental growth and adult offspring health. The objectives of this thesis were to assess the effects of maternal weight trajectories on offspring phenotype at term and in adulthood, as well as gene expression in placenta and fetal liver. At E18.5, fetuses from obese females presented a fetal growth restriction (FGR); this FGR was almost completely abolished by maternal WL. Placental and hepatic expression of epigenetic machinery genes was affected by maternal OB, especially the histone acetylation pathway. Maternal WL normalized the expression of only a subset of these genes. Males born to OB mothers gained weight faster under high-fat diet than males born to control mothers; maternal WL rescued this phenotype. These results show that expression of epigenetic machinery genes and in particular histone acetylation regulators, is highly sensitive to maternal obesity. Preconceptional WL alleviates the effects of OB on fetal and adult weight but some effects of obesity cured by nutritional intervention were retained in offspring phenotype at term. This study is an important step toward understanding the mechanisms linking maternal nutrition to fetal growth and adult health.

Pesquisas recentes em medicina genômica sugerem que fatores de risco que incidem desde a concepção de uma criança até o final de sua adolescência podem influenciar no desenvolvimento de doenças crônicas da idade adulta. Artigos científicos com descobertas e estudos inovadores sobre o tema indicam que a epigenética deve ser explorada para prevenir doenças de alta prevalência como doenças cardiovasculares, diabetes e obesidade. A grande quantidade de artigos disponibilizados diariamente dificulta a atualização de profissionais, uma vez que buscas por informação exata se tornam complexas e dispendiosas em relação ao tempo gasto na procura e análise dos resultados. Algumas tecnologias e técnicas computacionais podem apoiar a manipulação dos grandes repositórios de informações biomédicas, assim como a geração de conhecimento. O presente trabalho pesquisa a descoberta automática de artigos científicos que relacionem doenças crônicas e fatores de risco para as mesmas em registros clínicos de pacientes. Este trabalho também apresenta o desenvolvimento de um arcabouço de software para sistemas de vigilância que alertem profissionais de saúde sobre problemas no desenvolvimento humano. A efetiva transformação dos resultados de pesquisas biomédicas em conhecimento possível de ser utilizado para beneficiar a saúde pública tem sido considerada um domínio importante da informática. Este domínio é denominado Bioinformática Translacional (BUTTE,2008). Considerando-se que doenças crônicas são, mundialmente, um problema sério de saúde e lideram as causas de mortalidade com 60% de todas as mortes, o presente trabalho poderá possibilitar o uso direto dos resultados dessas pesquisas na saúde pública e pode ser considerado um trabalho de Bioinformática Translacional. / Genomic medicine has suggested that the exposure to risk factors since conception may influence gene expression and consequently induce the development of chronic diseases in adulthood. Scientific papers bringing up these discoveries indicate that epigenetics must be exploited to prevent diseases of high prevalence, such as cardiovascular diseases, diabetes and obesity. A large amount of scientific information burdens health care professionals interested in being updated, once searches for accurate information become complex and expensive. Some computational techniques might support management of large biomedical information repositories and discovery of knowledge. This study presents a framework to support surveillance systems to alert health professionals about human development problems, retrieving scientific papers that relate chronic diseases to risk factors detected on a patient\'s clinical record. As a contribution, healthcare professionals will be able to create a routine with the family, setting up the best growing conditions. According to Butte, the effective transformation of results from biomedical research into knowledge that actually improves public health has been considered an important domain of informatics and has been called Translational Bioinformatics. Since chronic diseases are a serious health problem worldwide and leads the causes of mortality with 60% of all deaths, this scientific investigation will probably enable results from bioinformatics researches to directly benefit public health.