Alterations of the epigenome in brain cancer: loss of 5-hydroxymethylcytosine

Abstract : 5-Methylcytosine is an epigenetic mark, which can be oxidized to 5-hydroxymethylcytosine (5hmC) in DNA by ten-eleven translocation (TET) oxygenases. It is an initial step in the demethylation of 5mC. Levels of 5hmC is relatively high in the brain compared to other organs, but these levels are known to be significantly reduced during the development of a brain tumor, especially in glioblastoma multiforme (GBM). However, no known mechanisms may fully explain this abnormality. The objectives of my project were to (1) understand the implications of the demethylation pathway mediated by TET, and (2) gain a deeper insight in the epigenetic make-up of brain tumors. (1) U87 cells were incubated with 5mC, 5hmC, 5-formylcytosine (5fC) or co-incubated of 5hmC with 3,4,5,6-tetrahydro-2’-deoxyuridine (dTHU) over a timeline of 0, 24, 48 and 96 hours. (2) 130 brain tumors (GBM= 79; grade II/III= 51) were obtained directly from surgery and immediately suspended in DNA extraction buffer. Both cell samples and tumor tissues underwent DNA extraction and DNA digestion protocols. The percent per cytosine (%/C) was obtained by quantification of 5mC, 5hmC, 5fC, 5-hydroxymethyluracil (5hmU) and 5formyluracil (5fU) using LC-MS/MS. (1) Cellular incubations showed that it is possible to increase levels of 5hmC in DNA, but also a slight increase in 5mC levels throughout the experiment. 5HmC levels dramatically increased by 1.9-fold after 96h. On the other hand, no increase was observed in 5fC levels. Both 5hmC and 5fC incubations were accompanied by high increases in 5hmU and 5fU levels respectively. The addition of dTHU to the 5hmC incubation decreased 5hmU incorporation by 65%. (2) The average levels of 5mC, 5hmC and 5fC, in brain tumors, were 4.0, 0.15 and 0.021 %/C respectively. 5HmU and 5fU levels were present at comparable levels of 5hmC and 5fC. Levels of 5hmC, 5hmU and 5fU were significantly lower in the DNA of GBM specimens. There was a strong correlation between 5mC with 5hmC and 5fC in GBM, but this was absent in low grade tumors. The presence of 5hmU and 5fU in brain tumor and the increase in their levels during cell incubations indicate a deamination activity in these cancerous cells, which may impinge on the cellular levels of 5hmC, in particular. Furthermore, upon the incubations with 5hmC, downstream levels of 5fC did not increase suggesting a TET malfunction. TET activity is maintained in GBMs, but impaired in low grade tumors due to isocitrate dehydrogenase-1 (IDH1) mutations. Therefore, in brain tumors, a strong deamination activity and TET impairment may lead to epigenetic reduction of 5hmC.

Memoires de Soie en Inde: La Ville de Kanchipuram et son art de Tissage

A cidade de Kanchipuram, no Estado de Tamilnadu, sul da Índia, é sinónimo de templos hindus, mas também do sari em seda. A migração de comunidades de tecelão para esta cidade começou durante o reinado da Dinastia Chola nos Séculos 8-13 d. C. Anteriormente, o pano de seda foi considerado como tecido dos Deuses e os tecelões satisfizeram as necessidades religiosas do templo da cidade. Paulatinamente, um tecido de seda torna-se, tanto um tecido dos "mortais", como dos Deuses, e houve um aumento da procura dos têxteis em seda, especialmente, do sari em seda. A particularidade dos tecelões de Kanchipuram reside na sua técnica complicada de tecelagem e nos ricos motivos que são uma expressão da paixão do tecelão. Nosso trabalho de investigação centra-se nas técnicas de tecelagem e no seu produto final - o sari - para destacar a sua singularidade. Neste contexto, quero propor um projecto de documentação dos motivos do sari, para ilustrar um dos principais meios de valorização desta tradição de tecelagem que remonta a vários séculos. RÉSUMÉ: La ville de Kanchipuram, située dans l'état du Tamilnadu, dans le sud de l'Inde, fait souvent écho aux temples hindous mais également au sari en soie. Les tisserands ont migré à l'époque Chola (850- 1279 ap.J.C.) pour répondre aux besoins religieux de la ville car au départ, la soie était un tissu destiné à I 'usage des dieux. Au fur et à mesure, la soie est devenue aussi bien un tissu destiné aux 'mortels' qu'aux dieux. Ces tisserands ont connu par la suite, une forte demande, plus particulierement, pour les saris en soie. La particularité des tisserands de la ville de Kanchipuram réside dans sa technique laborieuse de tissage et dans les motifs élaborés parle billet desquels s'exprime la passion du tisserand dans sa tâche. Le présent mémoire s'attache à la technique de tissage et à son produit final - le sari - pour mettre en valeur son unicité. Dans ce cadre, je propose un projet de documentation des motifs des saris pour illustrer un des vecteurs clés de valorisation de cette tradition de tissage qui remonte à plusieurs siecles. ABSTRACT: The city of Kanchipuram, located in the State of Tamilnadu in southern India, is synonymous with Hindu temples and silk saris. The migration of weaver communities to the city started during the Chola reign (81 131 centuries A.D). Early on, silk was considered the cloth of the gods and these weavers met the needs of the temple city by producing silk textiles for religious use. Gradually, silk became a cloth as much for the 'mortais' as for the gods and demand increased for silk textiles, especially saris. The importance of Kanchipuram weaving lies in its complex techniques and rich motifs as expressions of the. weaver’s passion. This text examines the weaving techniques popularly known as the korvai technique, as well as the saris produced using this technique. ln addition, it attempts to catalogue certain motifs woven into the saris as a first step in promoting and valorizing the cultural richness of an art dating back several centuries.