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Human malaria caused by Plasmodium falciparum is a major global burden killing between 700,000 and 2.7 million people every year. Africa bears the greatest portion of this burden, with over three quarters of deaths occurring in African children, accounting for 18% of all child deaths in sub-Saharan Africa. Synthesis of tetrahydrofolate through the folate biosynthetic pathway is vital for the survival of P. falciparum parasites and is lacking in the human host. As such, enzymes of this pathway have long presented attractive targets for drug therapy and although increasingly being compromised by resistance, anti-folates such as pyrimethamine and sulfadoxine are still very valuable drugs in many malaria-endemic regions.In this project, further investigation of the enzymes of the folate biosynthetic pathway has been attempted by developing protocols to quantify these proteins and others through proteomic techniques. Two quantification techniques were pursued. The first was quantification using whole, heterologously expressed, stable-isotope labelled forms of P. falciparum proteins for use as heavy standards in mass spectrometry. Great difficulty was experienced in the effort to express and purify P. falciparum enzymes in E. coli expression systems, with only one enzyme successfully expressed and purified in a 13C-labelled form. This one protein was taken forward into quantification experiments. The second quantification technique used a stable-isotope labelled ‘QConcat’ protein, consisting of a number of peptides from 12 P. falciparum proteins of interest, as a heavy standard in mass spectrometry. This was successfully expressed and purified in a 13C-labelled form from an artificial gene using an E. coli expression system. This too was taken forward into quantification experiments.Quantification experiments using the QConcat-based quantification technique were successfully performed on whole P. falciparum extract. Among the proteins quantified were SHMT and DHFR, two proteins of great interest from the folate biosynthetic pathway. Consistent with results from different expression analysis techniques in the literature, the folate enzymes were found to be of lower abundance than housekeeping enzymes and SHMT was found to be more abundant than DHFR.For deep quantitative analysis of the P. falciparum proteome, it was found that fractionation was necessary. Fractionation in this project was performed using a ZOOM™ IEF fractionator (Invitrogen), an OFFGEL™ IEF fractionator (Agilent) and 1D SDS-PAGE. It was found that by using these fractionation techniques, more proteins could be identified within the P. falciparum proteome, with all but one of the enzymes of the folate biosynthetic pathway being identified. Significant advances in the sensitivity of mass spectrometers during this project have also greatly facilitated the investigation of the proteome. In some cases, this meant that proteins which were only previously accessible by prefractionation of the proteome could be seen in whole P. falciparum extract. Unfortunately, QConcat-based quantification using both fractionation and sensitive mass spectrometry could not be successfully achieved in the time available. However, the promising results obtained suggest that, after careful optimisation, such an approach will be valuable.

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

Orientador: Carmen Silvia Bertuzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T17:41:14Z (GMT). No. of bitstreams: 1 Ribeiro_CintiaMarques_M.pdf: 2054933 bytes, checksum: 39195c141e4cd2e600f9454a1cfc8781 (MD5) Previous issue date: 2009 / Resumo: A síndrome de Down (SD) é uma aberração cromossômica atribuída à presença de três cópias dos genes localizados no cromossomo 21. Ocorre com uma freqüência estimada de um indivíduo em 600 nascidos vivos e de uma em 150 concepções. Os mecanismos relacionados com a não-disjunção do cromossomo 21 não foram ainda elucidados e embora a idade materna avançada seja um fator de risco, a maioria das crianças com SD nasce de mães com menos de 30 anos. Um mecanismo proposto para explicar a não-disjunção cromossômica consiste na hipometilação do centrômero levando à formação anormal do cinetócoro e ligação anormal dos microtúbulos. Tal mecanismo teria uma etiologia multifatorial e entre os fatores genéticos estariam as variantes polimórficas de enzimas envolvidas no metabolismo do folato. A via bioquímica do folato pode influenciar a estabilidade do DNA de dois modos: o primeiro está relacionado com a função que o folato desempenha na transferência de unidades de 1-carbono para a biosíntese de novo de nucleotídeos. Baixa concentração intracelular de 5, 10-metilenoTHF leva a uma diminuição da síntese de timidilato e a incorporação errônea de dUTP durante a replicação do DNA; o segundo modo envolve a produção de S-adenosilmetionina (SAM), o principal doador de grupos metil para a maioria das reações de metilação, incluindo a metilação CpG. Baixa concentração intracelular de 5,10-metilenoTHF é associado com baixa produção de SAM e conseqüentemente com a hipometilação do DNA. Diante disso, o objetivo do trabalho foi determinar se os polimorfismos MTHFD-1 1958G>A, TCII 776C>G, MTR 2756A>G, MTRR 66 A>G, RFC-1 80G>A e SHMT-1 1420C>T representam fatores de risco para gestações com SD. Para isso foi realizada a técnica de PCR utilizando 200 amostras de DNAs de mães de portadores de SD (MSD) e 340 amostras de DNAs controles seguida por digestão enzimática dos produtos obtidos e análise estatística dos resultados. Comparando a distribuição genotípica no grupo MSD e controle foi observado que o polimorfismo TCII 776C>G apresentou uma diferença estatisticamente significativa: ?2 (2) = 13,10 e p=0,0014. Nossos resultados indicam que mulheres com genótipo heterozigoto para o polimorfismo TCII 776C>G possuem um risco duas vezes maior de gerarem crianças com SD / Abstract: Down syndrome is a chromosome abnormality caused by the presence of three copies of genes located in the chromosome 21. It occurs in an estimated frequency of one out of 600 liveborns and one out of 150 conceptions. The mechanism related to the non-disjunction of the chromosome 21 has not been totally understood and even though the mother's age is a risk factor, most DS children are born to mothers aged less than 30. One mechanism proposed to explain the chromosome non-disjunction is the centromeic hypomethylation that causes abnormal formation of kinetochore and abnormal links of the microtubules. Such a mechanism is thought to have a multifactorial etiology and among the genetic factors are polymorphic variants of enzymes involved in folate metabolism. The biochemical pathway of the folate influnces the DNA stability in two ways: the first is related to the role of folate in one carbon unit transfer during "de novo" synthesis of nucleotides. Low levels of 5, 10-methylenetetrahydrofolate (5, 10-methyleneTHF) lead to a decrease in thymidylate synthesis and the misincorporation of the dUTP during replication of DNA; the second way involves the production of S-adenosyl methionine (SAM), the main donor of methyl to most methylation processes, incluing CpG methylation. Low intracellular 5,10-methyleneTHF is associated with low SAM production and consequently with DNA hypomethylation. Therefore, the purpose of the study was determining if the polymorfisms MTHFD-1 1958G>A, TCII 776C>G, MTR 2756A>G, MTRR 66A>G, RFC-1 80G>A e SHMT-1 1420C>T represent risk factors for DS pregnancies. So we used techniques of PCR followed by restriction enzyme analysis of 200 DNAs of mothers of DS children (MSD) and 340 DNAs of control mothers with statistic analyses of the results. Comparing the genotypic distribution in the groups MSD and control it has been observed that polymorphism TCII 776C>G showed a statistically remarkable difference: ?2 (2) = 13,10 e p=0,0014. Our results show that women that have heterozigote genotype for polymorphism TCII 776C>G have two times higher risk of generating DS children / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientadores: Helena Teixeira Godoy, Carlos Eugenio Daudt / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T11:15:55Z (GMT). No. of bitstreams: 1 Facco_ElizeteMariaPesamosca_D.pdf: 27687746 bytes, checksum: d51d71b95043f23c20948f904cbac415 (MD5) Previous issue date: 2006 / Doutorado / Doutor em Ciência de Alimentos

Certaines cellules cancéreuses sont méthionine dépendantes cependant les mécanismes de cette méthionine dépendance sont inconnus. Les cellules initiatrices de tumeur, qui représentent un faible pourcentage des cellules d’une tumeur, sont impliquées dans la récidive du cancer, phénocopient les cellules souches cancéreuses et forment des sphéroïdes 3D ou «tumor spheres (TS)» dans des conditions de culture non adhérentes. Nous montrons que, contrairement aux cellules monocouches adhérentes U251, les TS dérivées de cellules de glioblastome U251 ont besoin de méthionine exogène pour se développer. Cette méthionine-dépendance est caractérisée par une courbe en forme de cloche dans laquelle la croissance des TS est ralentie par des concentrations élevées de méthionine (> 0,01mM). Pendant la restriction en méthionine, le 5-méthyle-tétrahydrofolate restaure la formation des TS. Si les TS sont privées de méthionine pendant 24h, puis supplémentées en acide folique, elles présentent des concentrations d'isoformes des folates significativement inférieures à celles retrouvées dans les cellules adhérentes maintenues dans les mêmes conditions. Ceci suggère que le cycle des folates est réprimé dans les TS comparativement aux cellules adhérentes. L'annotation fonctionnelle des données ARN-Seq montre des changements nets dans plusieurs fonctions moléculaires et dévoile dans les TS un cycle cellulaire réduit, une augmentation du caractère « stemness » et une diminution du métabolisme des folates affectant particulièrement DHFR, SHMT et MTFHD. L'analyse du méthylome révèle des changements de méthylation dans le cycle cellulaire, la signature « stemness » et le cycle des folates, malgré des profils globaux de méthylation de l'ADN qui restent stables. Cependant, contrairement à la méthylation importante des promoteurs observée pour le cycle cellulaire et les gènes « stemness » (+ 25%), seuls 10 gènes du cycle des folates sur 139 gènes impliqués dans le métabolisme des mono-carbones sont significativement modifiés. En conclusion, un cycle des folates avec activité réduite fait partie de la reprogrammation métabolique qui déclenche la dédifférenciation en cellules souches cancéreuses et cette répression ne s'explique qu’en partie par la modification de méthylation des promoteurs / Some cancer cells are methionine dependent however little is known about the mechanisms of this dependency. Tumor initiating cells are a rare population of cancer cells, implicated in disease recurrence, that phenocopy cancer stem cells and form 3D spheroids or ‘tumor spheres (TS)» under non adherent conditions. We show that, unlike U251 adherent monolayer cells, TS derived from U251 glioblastoma cells need exogenous methionine to grow. This methionine dependency is characterized by a bell shape curve in which high methionine concentrations (>0.01mM) slow down TS growth. During methionine restriction, 5- methyltetrahydrofolate restores TS formation. When TS are deprived from methionine for 24h, then supplemented with folic acid, they exhibit lower levels of folate isoforms than adherent cells maintained in the same conditions, suggesting that folate cycle is repressed in TS relative to adherent cells. Functional annotation of the RNA-seq data shows clear changes in several molecular functions and reveals in TS a reduced cell cycle, an increased stemness and a diminished folate metabolism affecting particularly DHFR, SHMT and MTFHD. Methylome analysis shows methylation changes in cell cycle, stemness and folate cycle, despite global DNA methylation patterns remaining stable. However, unlike the important promoter methylation observed for cell cycle and stemness genes (+25%), only 10 folate cycle genes out of 139 genes involved in one-carbon metabolism are significantly altered. In conclusion, reduced folate cycle is part of the metabolic reprogramming triggering dedifferenciation into cancer stem cells and this repression is only partly explained by the alteration of promoter methylation

Les folates sont des vitamines importantes dans le développement neurologique d’un enfant puisqu’elles sont impliquées dans deux pathologies : l’autisme et les anomalies de fermeture du tube neural (AFTN). Une carence en vitamine B9 et la présence de certains polymorphismes des gènes du métabolisme des monocarbones sont associées à un risque augmenté d’anomalies de fermeture du tube neural. A l’inverse, une supplémentation périconceptionnelle en vitamine B9 a permis de réduire l’incidence de ces malformations. Dans le cadre de l’autisme, la présence d’anticorps dirigés contre le récépteur aux folates FR aplha au niveau cérébral entraînant une carence en folate cérébral a été décrite avec une fréquence importante chez les enfants autistes. Un traitement par acide folinique permettrait une amélioration des symptômes en corrigeant la carence en folates grâce à un passage médié par le RFC (récépteur non bloqué par les anticorps). La première partie est une étude clinique randomisée versus placebo réalisée au CHU de Nancy dont le but est d’évaluer l’éfficacité d’un traitement par acide folinique pendant 12 semaines sur la réduction des troubles autistiques. 19 enfants ont été inclus dans l’étude.Une amélioration significative des symptômes autistiques est observée pour le score ADOS dans le groupe traité (p= 0,02), plus particulièrement pour les interactions sociales réciproques (p=0,019). La fréquence des Anticorps anti FR alpha au sein du groupe est de 58 %. Il n’y a pas de corrélation observée entre le taux d’anticorps et l’importance de la réponse au traitement. Aucun effet secondaire grave n’a été observé au cours de l’étude. La seconde partie est une étude par séquençage haut débit d’un large panel de gènes chez des patients présentant des anomalies de fermeture du tube neural (SureSelect Focused Exome Plus (Agilent®)). Le séquençage a été complété par une analyse de méthylation pan-génomique (Infinium HumanMethylation Beadchip (Illumina®)). 23 patients ont été inclus dans l’étude. Plusieurs variants rares ont été identifiés comme associés au risque de AFTN dont des variants de gènes du métabolisme des monocarbones : LRP2, rs137983840, p=0,005; MMAA, rs148142853, p= 0,005 ;TCN2, rs35838082, p=0,044, FPGS, rs41306702, p=0,0012, BHMT, rs763726268, p= 0,011 et de la voie Sonic Hedgehog (SHH) (GLI3, rs35364414, p=0,012). Une différence de méthylation significative a été mise en évidence au niveau du gène CFAP46 (hémiméthylation versus absence de méthylation chez les contrôles) chez un patient porteur des 4 variants à risque identifiés (LRP2, MMAA, BHMT et GLI3). Ces résultats renforcent l’implication des folates dans ces deux pathologies du neurodéveloppement que sont l’autisme et les anomalies de fermeture du tube neural. Une recherche des anticorps anti-FRalpha plus systématique chez les enfants autistes pourrait permettre de proposer un traitement par acide folinique ciblé. Dans le cadre des AFTN, notre étude a mis en évidence l’influence de gènes impliqués dans le métabolisme de la vitamine B12 et monocarbone sur le risque de AFTN. Un nouveau gène candidat (GLI3) est identifié ainsi qu’une signature de méthylation mettant en évidence l’influence de la voie SHH / Folates are essentials vitamins in children neurodevelopment with an implication in two pathologies : autism and neural tube defects (NTD). Folates deficiency and some polymorphisms of genes involved in one carbon metabolism (OCM) are associated with NTD. Contrary, periconceptional folate supplementation is associated with decreased NTD frequency.In autism, higher frequency of antibodies against Folate Receptor Alpha (FR alpha) is rapported and associated with folates cerebral deficiency. Folinic acid treatment could improve autistic symptoms by correcting cerebral folate deficiency (cerebral transport mediated by RFC, an other receptor which not blocked by antibodies anti-FR alpha). First part is a randomized controlled trial versus placebo realized in CHU of Nancy in order to evaluate efficiency of folinic acid treatment during 12 weeks on autistic symptoms. 19 children have been included in the study. A significative improvement of autistic symptoms is observed by ADOS score in folinic acid group (p= 0.02) and particularly for mutual social interactions (p=0.012). FRalpha antibodies are present in 58 % of the group. We didn’t observed correlation between antibodies titers and folinic acid response. No serious adverse effects have been observed during the study. Second part is hight throughput next generation sequencing of DNA from patients with NTD using SureSelect Focused Exome Plus (Agilent®). Sequencing has been completed with DNA methylation analysis (Infinium HumanMethylation Beadchip (Illumina®)). 23 patients were included in the study. Six variants have been associated with NTD: from genes of B12 metabolism LRP2, rs137983840, p=0.005; MMAA, rs148142853, p= 0.005 and TCN2, rs35838082, p=0.044), folate cellular metabolism (FPGS, rs41306702, p=0.0012; choline metabolism, BHMT, rs763726268, p= 0.011) and Sonic Hedgehog pathway(SHH) (GLI3, rs35364414, p=0.012). A significative difference of methylation is identified in the vicinity of CFAP46 gene (hemimethylation versus no methylation in pseudo-controls) in one patient exhibited variants of BHMT, LRP2etMMAA. These results highlight implication of folates in these two pathologies of neurodevelopment, wich are autism and NTD. Anti-FRalpha should be routinely evaluated in case of autism in order to propose folinic acid treatment if they are positives. In the NTD study, we identified new variants from gene from one carbon metabolism probably implicated. A new candidate gene is identified (GLI3) and a methylation signature in association with B12 metabolism and OCM gene variants

Since its discovery, it was appreciated that the antifolate pemetrexed had multiple targets within folate metabolism. This laboratory was instrumental in showing that pemetrexed elicited its primary action as a thymidylate synthase inhibitor. Unusual for an antifolate, pemetrexed showed significant clinical activity against malignant pleural mesothelioma and non-small cell lung cancer. Accordingly, the FDA recently issued first-line approvals for pemetrexed in these diseases, leading us to question whether the effects of pemetrexed on other folate-dependent targets could explain this atypical clinical activity of the drug. Studies in this dissertation showed that in addition to thymidylate synthase inhibition, pemetrexed was also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate- dependent enzyme of de novo purine synthesis. Consequent of AICART inhibition, pemetrexed caused robust activation of a key energy-sensing regulatory enzyme of the PI3K-AKT signal transduction pathway, AMP-activated protein kinase (AMPK). AMPK activation resulted from xx accumulation of the AMP-mimetic, ZMP, behind the AICART block. Constituents of the PI3K- AKT cascade are frequently deregulated in human carcinomas, uncoupling nutrient supply from proliferative capacity. Therefore, interventions that reinstate control over aberrant signaling along this axis, such as AMPK activation, are of significant cancer therapeutic interest. The cellular consequences of AMPK activation in response to pemetrexed were assessed. In particular, effects on the downstream target of PI3K-AKT signaling, the mammalian target of rapamycin complex 1 (mTORC1), were studied. Unlike targeted mTORC1 inhibitors, such as rapamycin and its analogs, pemetrexed-mediated activation of AMPK also signaled to mTOR- independent controlling elements of protein and lipid synthesis, highlighting additional benefits of AMPK activating agents that extend beyond effects on mTOR signaling. We therefore propose that the unusual activity of pemetrexed in mesothelioma and non-small cell lung cancer is due in part to effects on signaling processes downstream of AMPK activation. These findings present a novel approach to AMPK activation secondary to an AICART block, define pemetrexed as a molecularly targeted agent, and ultimately extend the utility of antifolates beyond their traditional function.

Les folates (vitamine B9) et la vitamine B12 interviennent comme cofacteurs dans le métabolisme des monocarbones qui régule les réactions de transméthylation impliquées dans les mécanismes épigénétiques. Un déficit en folates et/ou B12 réduit la production de méthionine à partir de l'homocystéine, un acide aminé toxique dont l'accumulation a été associée à la survenue de pathologies du système nerveux central aux différents stades de la vie (spina bifida, maladie d'Alzheimer…). Afin d'explorer les mécanismes cellulaires et moléculaires impliqués dans la réponse à la carence en ces micronutriments, nous avons développé deux nouveaux modèles cellulaires. Ainsi, nous avons étudié les effets d'une déficience en folate sur la prolifération, la différenciation et la plasticité neuronale de progéniteurs neuronaux issus de l'hippocampe d'embryons de rat, la lignée H19-7. Le second modèle correspond à un projet innovant visant à obtenir une déplétion cellulaire en B12 par séquestration membranaire. Il a été obtenu par transfection stable de la lignée de neuroblastome murin NIE-115 dans le but d'induire l'expression d'une protéine chimère contenant le transporteur plasmatique de la vitamine B12, la transcobalamine II, et une protéine d'ancrage membranaire. L'ensemble de ces travaux montre que les altérations du métabolisme des monocarbones associées aux carences répriment la neurogenèse et induisent des troubles de la différentiation neuronale. Ceci suggère l'existence de mécanismes précis par lesquels le déficit en folates, en vitamine B12 et/ou l'homocystéine peuvent affecter le fonctionnement du cerveau et sa plasticité. / Folate (vitamin B9) and vitamin B12 act as cofactors in the one-carbon metabolism that regulates transmethylation reactions involved in epigenetic mechanisms. A deficiency in folate and/or B12 decreases the generation of methionine from homocysteine, a toxic amino acid that has been associated with pathologies of the central nervous system at all ages (spina bifida, Alzheimer's disease…). In order to depict the cellular and molecular mechanisms implicated in the response to the deficiency in these micronutrients, two new cell models have been developed. Thus, we have analyzed the effects of folate deficiency on proliferation, differentiation and neuroplasticity of neuronal progenitors obtained from the hippocampus of rat embryos, i.e. the H19-7 cell line. Regarding the second model, we designed an original approach by stable transfection of NIE-115 murine neuroblastoma cells to impose the anchorage of a chimeric B12 binding protein, transcobalaminoleosin (TO) to intracellular membrane in order to produce intracellular sequestration of B12. Taken together, our results have shown that deficiency-associated alterations of the one-carbon metabolism lead to reduced neurogenesis and to dramatic impairment of neuron differentiation. This suggests the existence of specific mechanisms through which vitamin B9/B12 deficiency and/or homocysteine may affect brain functioning and plasticity.

La carence en donneurs de méthyle est assez fréquente dans la période périnatale et au cours du vieillissement. Les donneurs de méthyle alimentaires, l'acide folique et la vitamine B12, influencent la teneur cellulaire en S-adénosylméthionine et S-adénosylhomocystéine et en homocystéine. Le lien entre les donneurs de méthyle et le métabolisme énergétique n'est pas connu, en dépit de leur rôle dans les voies liées à l'épigénétique et la synthèse de molécules méthylées. Nous avons évalué les conséquences d'un régime alimentaire déficient donneur de méthyle, dans le myocarde des ratons au moment du sevrage, soumis à une carence durant la gestation et la lactation. Le régime carencé augmente l'homocystéine plasmatique et S-adénosylhomocystéine myocardique. Les résultats mettent en évidence une cardiomyopathie hypertrophique et un déficit global de l'oxydation des acides gras avec acylcarnitines plasmatiques. Les liens entre le métabolisme des mono-carbones, l'oxydation mitochondriale des acides gras et de cardiomyopathie ont été constatées par des corrélations entre l'homocystéine et les acylcarnitines et le peptide natriurétique de type B. La carence en donneurs de méthyl peut être une condition aggravante de la cardiomyopathie en altérant l'oxydation des acides gras à travers une expression modifiée de PPAR[alpha] et ERR[alpha] et un déséquilibre de l'acétylation / méthylation de PGC1[alpha]. Nous avons montré que l'Hcy et Apo A-I ont été deux facteurs métaboliques déterminants de l'ABI dans une population ambulatoire de volontaires âgés d'une région rurale de la Sicile. L'influence négative de l'Hcy sur Apo A-I et sur le métabolisme des HDL ouvre des nouvelles perspectives sur l'implication de l'Hcy dans la physiopathologie de l'athérothrombose / The deficiency in methyl donors is prevalent in the perinatal period of life and in aging. Dietary methyl donors, folate and vitamin B12, influence the cellular content in Sadenosylmethionine and S-adenosylhomocysteine and increases homocysteine. The link between methyl donors and energy metabolism is not known, despite their role in pathways related to epigenetics and synthesis of methylated molecules. We evaluated the consequences of a diet lacking methyl donors, in myocardium of weaning rats from dams subjected to deficiency during gestation and lactation. The deficient diet increased plasma homocysteine and myocardium Sadenosylhomocysteine. The results evidenced a hypertrophic cardiomyopathy with a global deficit in fatty acid oxidation with increased plasma acylcarnitines. The links between one-carbone metabolism, mitochondrial fatty acid oxidation and cardiomyopathy were ascertained by correlations between hyperhomocysteinemia, short-, medium- and long-chain acylcarnitines, and type-B natriuretic peptide. Methyl donor deficiency may be an aggravating condition of cardiomyopathy by impairing fatty acid oxidation through altered expression of PPAR[alpha] and ERR[alpha] and imbalanced acetylation/methylation of PGC1[alpha]. Finally, in a clinical study we sshowed that the Hcy and Apo A-I were two metabolic factors that influence the « anckle brachial index » in an ambulatory aged Sicilian population. The influence of homocysteine on Apo A-I and HDL metabolism provides new insights on its role on vascular diseases, at a cross-point between atherosclerosis and atherothrombosis