Orientador: José Fernando Garcia / Banca: Marcos Vinicius Gualberto Barbosa da Silva / Banca: Gisele Zoccal Mingoti / Banca: Guilherme de Paula Nogueira / Banca: Fabiano Antonio Cadioli / Resumo: RESUMO - Uma das questões mais interessantes que poderiam ser elucidadas empregando a análise de metilação em todo o genoma é como ela afeta a regulação da temperatura corporal em animais domésticos. A resposta a esta questão torna-se imperativa, particularmente no caso de bovinos de leite e corte, uma vez que as raças modernas mais populares descendem de ancestral comum, que derivou em duas subespécies (Bos taurus e Bos indicus) as quais respondem aos estímulos ambientais de formas opostas (raças de zonas temperadas ou adaptadas a regiões tropicais, respectivamente). Mapas de metilação do DNA genômico foram originados usando a técnica de RRBS à partir de células nucleadas do sangue de um grupo de bovinos Angus e Nelore puros, expostos ao estresse ambiental condicionado pelo calor. Os dados de metilação distribuídos ao longo dos cromossomos bovinos puderam ser representados graficamente, enfatizando padrão altamente homogêneo entre as amostras analisadas e a robustez do método. A cobertura de sequenciamento empregada foi suficientemente profunda (em todos os casos superior a 20 vezes o tamanho do genoma), permitindo concluir sobre a ocorrência de eventos de metilação do DNA possivelmente associados a alterações fisiológicas causadas pelo estresse térmico. A análise da metilação do DNA revelou 4.662 janelas metiladas diferencialmente (cada uma com 1.000 pares de bases), sendo a maioria (2.695) relacionada a diferenças entre as raças e não diretamente à resposta ao estresse tér... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: ABSTRACT - One of the most interesting questions that could be solved using genome-wide methylation analysis is how it affects the regulation of body temperature in domestic animals. The answer to this question becomes imperative, particularly in the case of dairy and beef cattle, since modern breeds more frequently descended from a common ancestor, derived in two subspecies (Bos taurus and Bos indicus), which respond to environmental stimuli of different forms (breeds of temperate zones and adapted to tropical regions, respectively). Genomic maps were originated using RRBS DNA methylation data from nucleated blood cells of a group of pure Angus and Nelore cattle exposed to environmental heat strees. Methylation data distributed along the bovine chromosomes could be represented graphically, emphasizing a highly homogeneous pattern between the analyzed samples and the robustness of the method. The sequencing coverage used was sufficiently deep (in all cases higher than 20 times the genome size) leading to the identification of DNA methylation events possibly associated with physiological changes caused by heat stress. The DNA methylation analysis showed a total of 4,662 differentially methylated windows (each with 1,000 base pairs), most of them (2,695) related to differences between breeds and not to the response to heat stress. Analysis of the 214 common windows (comprising 103 genes) revealed epigenetic signals related to the heat stress response and recovery, which were ma... (Complete abstract click electronic access below) / Doutor

La santé et la fertilité des vaches laitières sont au cœur des préoccupations de la filière professionnelle dans un objectif d’efficience, de quantité et de qualité de la production de lait. La mise en place d’une lactation performante se superpose aux profonds changements hormonaux et métaboliques de la période postpartum, se traduisant par une balance énergétique négative. Les conséquences en sont souvent une altération de la fertilité et une immunodépression qui accroit la susceptibilité aux pathologies. Dans les élevages, il existe encore une grande variabilité d’état général et de performances chez les vaches laitières malgré la sélection génomique. Il est proposé que des modifications de la méthylation de l’ADN puissent contribuer à cette variabilité phénotypique individuelle. En effet, la méthylation de l’ADN, en tant que processus épigénétique, est impliquée dans la régulation transcriptionnelle des gènes, et présente une certaine plasticité face aux contraintes environnementales. Nous avons fait l’hypothèse que des signatures épigénétiques portées par les cellules du sang, pourraient refléter l’état de santé des vaches et pourraient être modifiées en réponse à différents facteurs intrinsèques (parité, stades physiologiques…) et aux contraintes environnementales. Ces signatures ont été recherchées dans une population de cellules du sang particulière : les monocytes. Ces cellules, accessibles par prélèvements sanguins et purification en présence d’un anticorps spécifique, constituent la première ligne de réponse d’immunité innée face aux infections aigües participant à la dégradation de l’état de santé des vaches en postpartum. Pour tester l’hypothèse de signatures épigénétiques monocytaires, une analyse du méthylome par « Reduced Representation Bisulfite Sequencing », (RRBS) dans diverses situations d’élevage a été réalisée. En utilisant des ADN génomiques de vaches incluses dans plusieurs protocoles, 22 banques ont été construites et séquencées. Leur analyse a été réalisée en utilisant un pipeline d’analyses bioinformatique et biostatistique développé au laboratoire.En moyenne 1 250 000 CpG sont pris en considération et permettent l’identification et la localisation de cytosines différentiellement méthylées (DMC) : i) 27143 DMC en comparant les méthylomes de différents types cellulaires (monocytes versus fibroblastes et PBMC) ii) 4788 DMC en réponse à un challenge nutritionnel basé sur la distribution du complément alimentaire GENIAL®, fabriqué et distribué en élevage par nos partenaires PILARDIERE et XR-Repro. iii) 2615 et 4616 DMC en réponse au challenge infectieux pour le groupe de vaches témoins et le groupe en restriction alimentaire respectivement (protocole coordonné par Christine Leroux et José Pires (RUMINFLAME, INRA, Theix) combinant une restriction alimentaire et un challenge immunitaire, par injection de LipoPolySaccharide). iv) 4420 DMC issues de la comparaison entre méthylomes de vaches à génome constant (issues du transfert nucléaire, clones) et de vaches à génome variable mais de même âge et élevées dans les mêmes conditions que les clones. Pour certaines régions différentiellement méthylées (DMR) ciblant le promoteur de gènes, le statut de méthylation a été confirmé par conversion bisulfite et pyroséquençage. L’expression des gènes associés a été étudiée. Une anti corrélation significative est observée entre méthylation et expression signant la fonctionnalité de ces régions.En comparant les 11 méthylomes monocytaires, il est montré que 21% des CpG sont extrêmement stables et ne présentent qu’une faible variation de méthylation entre échantillons ( 20%). L’ensemble de ces informations peut être pris en considération pour la conception d’un outil d’épigénotypage. A l’avenir, il serait aussi possible d’utiliser cet outil en routine afin d’appréhender les variations du méthylome monocytaire dans différentes conditions d’élevage. / In dairy breeding, the health and fertility of cows are the main concern with the aims to maintain milk quantity and quality and to reduce the interval between calving in a high competitive economical context. Postpartum period is marked by major hormonal and metabolic changes that affect productivity, immune responses and fertility. The consequences of immune response deterioration are an increasing susceptibility to diseases (mastitis, metritis, endometritis…). Genomic selection in livestock improves the performance of the population but does not exclude phenotypic variability at the level of livestock and the individual. It is proposed that DNA methylation could contribute to this individual phenotype variability. Indeed, DNA methylation is an epigenetic process involved in transcriptional regulation of genes displaying certain plasticity in front of environmental constraints.We assumed the epigenetic signatures carried by blood cells, could reflect overall health and could be modified in response to intrinsic factors (parity, stages …) and to environmental changes. These signatures were researched in a particular blood cells subpopulation: the monocytes. These cells, obtained by blood sampling and purification with a specific antibody, are the first line of defense against acute infections participating in health status deterioration of postpartum cows.To test the monocyte epigenetic signatures hypothesis, monocyte methylome were analyzed by « Reduced Representation Bisulfite Sequencing » (RRBS), in various breeding conditions. Using genomic DNA form cows included in several protocols, 22 libraries were constructed and sequenced. Their analyses were accomplished using a « homemade » pipeline which integrates bioinformatics and biostatistics analyses. On average, 1 250 000 CpGs were analyzed in order to identify differentially methylated cytosines (DMCs): i) 27143 DMC by comparison between different cells types (monocytes versus fibroblasts and PBMC) ii) 4788 DMCs in response to nutritional challenge based on the dietary supplement, GENIAL®, produced and distributed in breeding by our partner PILARDIERE and XR-Repro (in collaboration with Marion Boutinaud, INRA, Rennes). iii) 2615 and 4616 DMCs in response to infectious challenge with LipoPolySaccharide injection for control cows group fed normal diet and for dietary restriction cows group, respectively (collaboration with Christine Leroux and José Pires, RUMINFLAME, INRA, Theix; and Gilles Foucras (ENVT, Toulouse)). iv) 4420 DMCs from the comparison between constant genomic cow (Somatic cell nuclear transfer, clones) and variable genomic cows but with the same age and raised in the same conditions than clones.From DMCs, we identified differentially methylated regions (DMRs) defined as region with at least 3 DMCs inside 100 bp. For some DMRs targeting gene promoter, the methylation status was validated by bisulfite conversion and pyrosequencing. Gene associated expression were also investigated. A significant negative correlation has been observed between methylation and expression, highlighting the functional relevance of these DMRs in gene transcription control.By comparing the 11 monocyte methylomes, 21% of CpGs present a remarkable constant methylation level with weak variability between samples (20%).Taking together, these data can provide a list of relevant DMCs for an epigenetic tool conception. In the future, it would be possible to use this tool for a routine analysis in order to grasp monocyte methylome variations in different breeding management.

Deregulation of the epigenome is associated with the initiation and progression of various types of human cancers. Here we investigated the level of 5-hydroxymethylcytosine (5hmC), expression and function of TET1 and TET2, and DNA methylation in parathyroid tumors and small intestinal neuroendocrine tumors (SI-NETs). In Paper I, an undetectable/very low level of 5hmC in parathyroid carcinomas (PCs) compared to parathyroid adenomas with positive staining, suggested that 5hmC may represent a novel biomarker for parathyroid malignancy. Immunohistochemistry revealed that increased tumor weight in adenomas was associated with a more aberrant staining pattern of 5hmC and TET1. A growth regulatory role of TET1 was demonstrated in parathyroid tumor cells. Paper II revealed that the expression of TET2 was also deregulated in PCs, and promoter hypermethylation was detected in PCs when compared to normal parathyroid tissues. 5-aza-2′-deoxycytidine treatment of a primary PC cell culture induced TET2 expression and further supported involvement of promoter hypermethylation in TET2 gene repression. TET2 knockout demonstrated a role for TET2 in cell growth and migration, and as a candidate tumor suppressor gene. In Paper III, variable levels of 5hmC, and aberrant expression of TET1 and TET2 were observed in SI-NETs. We demonstrated a growth regulatory role for TET1, and cytoplasmic expression with absent nuclear localization for TET2 in SI-NETs. In vitro experiments supported the involvement of exportin-1 in TET2 mislocalization, and suggested that KPT-330/selinexor, an orally bioavailable selective inhibitor of exportin-1 and nuclear export, with anti-cancer effects, could be further investigated as a therapeutic option in patients with SI-NETs. In Paper IV, DNA methylation was compared between SI-NET primary tumors and metastases by reduced representation bisulfite sequencing. Three differentially methylated regions (DMR) on chromosome 18 were detected and chosen for further analyses. The PTPRM gene, at 18p11, displayed low expression in SI-NETs with high levels of methylation in the presumed CpG island shores, and in the DMR rather than the promoter region or exon 1/intron 1 boundary. PTPRM overexpression resulted in inhibition of cell growth, proliferation, and induction of apoptosis in SI-NET cells, suggesting a role for PTPRM as an epigenetically deregulated candidate tumor suppressor gene in SI-NETs.

Repetitive verbal patterns of speech are a trait associated with high-functioning autism (HFA). For some, this higher-order restricted, repetitive behavior impedes learning, social opportunities, and access to work environments. Despite emerging motivation for establishing social relationships, some individuals with HFA lack the behavioral prerequisites to establish meaningful relationships. The purpose of this study is to demonstrate the effectiveness of teaching interaction to decrease higher-order verbal restricted and repetitive behaviors (RRBs) for four adults with HFA. Through a multiple baseline design across participants, individuals were exposed to a function-based intervention (i.e., teaching interaction) that systematically taught and reinforced alternative communicative behaviors while interrupting repetitive speech with specific feedback. Teaching interaction effectively reduced RRBs and increased alternative conversation for all four participants. Three of the four participants elected to participate in post-intervention maintenance sessions that occurred in individualized naturalistic settings. Their conversation behaviors maintained with one participant receiving one booster session.