Les folates et la vitamine B12 sont essentiels au bon déroulement du développement cérébral. Ils agissent comme donneurs de méthyles dans le cycle des monocarbones où ils influencent les mécanismes épigénétiques. Les carences en folate et vitamine B12 sont fréquentes chez la femme enceinte et constituent un facteur de risque pour diverses pathologies neurologiques et les défauts développementaux ; ainsi de nombreux pays recommandent une supplémentation périconceptionnelle en acide folique. Une carence gestationnelle chez le rat est associée à un retard de croissance, à un défaut de sutures cérébrales, et à une atrophie de couches cérébrales qui s’accompagnent de troubles à long terme. Let-7a, miR-34a et miR-124a sont des microRNAs régulés par méthylation qui participent au développement cérébral. Leur expression est augmentée en condition de carence. L’expression de leurs cibles, respectivement Trim71, Dll1/Notch et Stat3, est réduite dans le cerveau d’embryons carencés, de même que l’expression et l’activité des récepteurs au glutamate AMPAR1/2 et NMDAR1/2, acteurs de la plasticité synaptique, à 21 jours post-natals. Une supplémentation périnatale en folate (3 mg/kg/j) normalise l’expression des microARN et de leurs cibles, et réduit les défauts structuraux et fonctionnels. Leur répression par siRNA améliore le phénotype de progéniteurs hippocampiques déplétés en B9, et stimule la croissance neuritique. Ces résultats soulignent le rôle potentiel de let-7a, miR-34a, miR-124a et de leurs voies de signalisation dans les anomalies développementales associées à une carence, et montre l’intérêt d’une supplémentation périnatale en folate chez les femmes à risque / Folate and vitamin B12 are essential for proper cerebral development. They act as methyl donors in the one-carbon metabolism and influence epigenetic mechanisms. Low dietary intakes of folate and vitamin B12 are frequent in pregnant women, and deficiency constitutes a risk factor for various neurological and developmental disorders. In several countries, public health policies recommend periconceptional supplementation with folic acid. Gestational deficiency in rats was associated with growth retardation, brain suture defects and atrophy of cerebral layers with long-term brain disabilities. The microRNA let-7a, miR-34a and miR-124a are regulated by methylation and required for brain development. Their expression was augmented in deficiency condition. Protein levels of their targets Trim 71, Dll1/Notch and Stat3, respectively, were decreased in the brains of deprived fetuses, as well as glutamate receptors AMPAR1/2 and NMDAR1/2 in 21d-old rats. Perinatal folate supplementation (3 mg/kg/d) restored the levels of microRNA and their downstream targets, with reduction of structural and functional defects. Silencing by siRNA improved the phenotype of deprived cells, and neurite outgrowth. The data outline the potential role of let-7a, miR-34a, miR-124a and their signaling pathways in developmental defects related to methyl donor deficiency, and support the likely usefulness of perinatal folate supplementation in at risk women

Les dérivés du tétrahydrofolate (THF), plus connus sous le nom de folate(s) ou vitamine B9, sont des cofacteurs indispensables au métabolisme cellulaire puisqu'ils sont à la base des réactions de transfert d'unités monocarbonées, regroupées sous le terme de "métabolisme C1". Chez tous les organismes, ces réactions sont impliquées dans des processus cellulaires clés comme la synthèse des nucléotides (purines, thymidylate), la synthèse de certains acides aminés (sérine, glycine, méthionine) et, indirectement, dans la synthèse de S-adénosylméthionine. Cette dernière constitue le donneur universel de groupements méthyles et intervient donc dans l'ensemble des réactions de méthylation. Les plantes, les champignons et certains micro-organismes possèdent la capacité de réaliser la synthèse de novo de THF alors que les animaux en sont incapables et sont contraints de puiser cette vitamine dans leur alimentation.<br /> Lors du développement de la plante, la capacité de synthèse du THF, le pool global et la nature des folates ainsi que la demande en unités C1 varient de façon importante. Pourtant, la littérature reste succincte en ce qui concerne les mécanismes qui permettent de contrôler l'homéostasie en folates en fonction des besoins fluctuants en unités C1 de la plante et sur la manière dont sont distribuées ces unités entre les différentes voies utilisatrices.<br /> Le travail réalisé dans le cadre de cette thèse a permis de mettre en évidence différents niveaux de régulation du métabolisme C1 permettant à la plante de répondre à une diminution du pool de folates. Ainsi, l'étude transcriptomique de la réponse de cellules d'Arabidopsis à un traitement par un antifolate, le méthotrexate, a révélé une absence de processus de compensation de la baisse de la quantité de folates puisque les gènes impliqués dans la synthèse, le transport et la dégradation du THF ne présentent pas de modification de leur expression. La régulation transcriptionnelle mise en place suite à une limitation en folates concerne un nombre restreint de gènes qui vont influencer la composition en dérivés du THF et non l'abondance de ce cofacteur vitaminique. En effet, alors qu'en situation physiologique le flux d'unités C1 alimente majoritairement les réactions de méthylation, il se trouve que le déficit en folates provoque une réorientation de ce flux vers la synthèse des nucléotides.<br /> La diminution des méthylations cellulaires en situation de déficit en folates a été illustrée lors de l'étude d'une méthyltransférase particulière qui intervient dans la synthèse des chlorophylles, la Mg-protoporphyrine IX méthyltransférase. Des feuilles de pois déficientes en folates présentent une forte diminution de l'index de méthylation qui se traduit par une régulation métabolique de l'activité de la Mg-protoporphyrine IX méthyltransférase, conduisant ainsi à une baisse de la synthèse des chlorophylles. Cette étude démontre que le statut en folates influence, via les réactions de méthylations, des processus physiologiques essentiels comme la biogenèse de l'appareil photosynthétique.<br /> Ce travail de thèse a également mis en évidence un mécanisme de régulation post-traductionnelle de la synthèse de méthionine en situation de carence en folates. Ce mécanisme consiste en un clivage protéolytique de l'extrémité N-terminale de la première enzyme dédiée à la synthèse de méthionine, la cystathionine γ-synthase. L'élimination de ce domaine régulateur de l'enzyme permet, par un mécanisme encore inconnu, d'accroître la biosynthèse de méthionine en situation de déficit en folates.

Orientador: Helena Teixeira Godoy / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T07:36:05Z (GMT). No. of bitstreams: 1 Rybka_AnaCeciliaPoloni_D.pdf: 1201100 bytes, checksum: 1057a41b652a7de5fa122bc5691bea97 (MD5) Previous issue date: 2010 / Resumo: No presente trabalho foram adaptados e aplicados métodos para a determinação do teor de folatos, avaliação de capacidade antioxidante e quantificação do composto trans-2-nonenal em cervejas brasileiras. Foram avaliadas 5 marcas de cervejas tipo Pilsen, sem álcool e Malzbier, nas embalagens lata e garrafa âmbar. Para determinação dos folatos (ácido fólico - AF, 10-metil-ácido fólico - 10Metil, 5-metiltetraidrofolato - 5Metil, 5-formiltetraidrofolato - 5Formil e 10-formil-ácido fólico - 10Formil) vitaminas do complexo B, empregou-se um método utilizando a cromatografia líquida de alta eficiência (CLAE). Na etapa cromatográfica utilizou-se coluna C18 e eluição por gradiente com fase móvel composta por ácido acético 2,0% ajustado a pH 2,8 e acetonitrila, a uma vazão de 0,5 mL/min. A detecção foi realizada com o detector de arranjo de diodos a 290nm para o AF e o 10Metil; e com detector de fluorescência a ?exc. 290nm e ?emis. 360nm na detecção do 5Metil e 5Formil; e a ?exc. 290nm e ?emis. 445nm para o 10Formil. Após sua validação, o método apresentou coeficientes de correlação variando de 0,9970 a 0,9999, limites de detecção de 0,07 a 2,42 mg/100mL, e de quantificação entre 0,22 e 8,07 mg/100mL. Os coeficientes de variação (CV) ficaram entre 0,75 e 5,01% e a precisão intermediária entre 1,20 e 9,99%. Os ensaios de recuperação nos níveis avaliados estiveram entre 92 e 107%. Os folatos encontrados nas cervejas analisadas foram o 10Metil, o 5Metil e o 10Formil e os níveis totais da vitamina variaram de 18,26 a 23,99 µg/100mL. Estas vitaminas foram monitoradas durante a vida de prateleira da bebida (6 meses), sendo avaliadas na cerveja recém fabricada e a cada 2 meses subseqüentes. De modo geral o teor de folatos decresceu ao longo da vida de prateleira, e não foi possível constatar o efeito do tipo de embalagem sobre a vitamina. A avaliação em diferentes lotes resultou em grande variação, com CVs de até 52,42%. Para estabelecer o teor de fenólicos totais foi aplicado o método de Folin-Ciocalteau, pelo qual foram encontrados 47,05, 18,67 e 21,83 mg/100mL em equivalentes de ácido gálico (EAG), na cerveja Malzbier, Pilsen sem álcool e Pilsen, respectivamente. O tipo de embalagem utilizada, lata ou garrafa, apresentou relevância para o teor de fenólicos totais somente para a cerveja Pilsen, e a cerveja sem álcool foi a que mais apresentou variação do teor entre marcas. Para avaliação da capacidade antioxidante foram realizados ensaios utilizando o radical 2,2- difenil -1- picrilhidrazil (DPPH) e o poder antioxidante de redução do ferro (FRAP). Os dois ensaios indicaram a cerveja Malzbier com maior capacidade antioxidante, corroborando com o teor de fenólicos totais. Para identificação e quantificação do trans-2- nonenal, um dos principais responsáveis pelo sabor envelhecido em cervejas, foi utilizado o método de headspace com microextração em fase sólida e cromatografia gasosa acoplada à espectrometria de massas (HS-SPME-GC-MS). Foi utilizada coluna 5%-fenilmetilpolisiloxano (HP-5MS) e a condição cromatográfica empregada teve inicio em temperatura de 60ºC, variando 3 ºC/min até atingir 100ºC. Em seguida, uma rampa de 10 ºC/min até atingir 200ºC e permanência a 200ºC por sete minutos para limpeza da coluna. O gás de arraste (hélio) teve vazão constante de 1,0 mL/min. Os íons monitorados para a identificação (m/z) foram 55, 70 e 83 (fonte de ionização de 70eV), após verificação do espectro de massas do composto. O teor de trans-2-nonenal encontrado variou de 0,11 a 0,46 µg/L nas cervejas em lata, estando acima de seu threshold. Para este composto foi encontrada diferença significativa entre amostras de um mesmo tipo de cerveja, porém não houve diferença significativa entre os três tipos de cerveja analisados. Este trabalho demonstrou que a cerveja brasileira possui substâncias benéficas à saúde humana, com teor similar a de outros alimentos, e que o trans-2-nonenal ainda é presente em quantidades acima do threshold na bebida, podendo comprometer sua qualidade. De modo geral, a embalagem, lata ou garrafa, não influenciou no teor das substâncias analisadas e a cerveja Malzbier se destacou pela maior capacidade antioxidante / Abstract: In the present study methods were adapted and applied to quantify levels of folate, antioxidant capacity assessment and E-2-nonenal quantification in Brazilian beers. 5 brands of Pilsen, non-alcoholic and Malzbier beers were used, in can and amber bottle. For determination of folates (folic acid - FA, 10-methyl-folic acid - 10Methyl, 5-methyltetrahydrofolate - 5Methyl, 5-formyltetrahydrofolate - 5Formyl and 10-formyl-folic acid - 10Formyl) B vitamins, a method using liquid chromatography (HPLC) was employed. In the chromatographic step a C18 column was used and was applied a gradient elution with mobile phase consisting of an acetic acid 2.0% solution adjusted to pH 2.8 and acetonitrile, at a flow rate of 0.5 mL / min. The detection was performed with the diode array detector at 290nm for FA and 10Methyl, and with a fluorescence detector at ?exc. 290nm and ?emis. 360nm in the detection of 5Methyl and 5Formyl, and at ?exc. 290nm and ?emis. 445nm for the 10Formyl. After its validation, the method presented correlation coefficients ranging from 0.9970 to 0.9999 mg/100 mL, detection limits from 0.07 to 2.42 mg/100 mL and quantification limits between 0.22 and 8.07 mg/100 mL. The standard deviation (SD) was between 0.75 and 5.01% and intermediate precision was from 1.20 to 9.99%. Tests of recovery levels were resulted between 92 and 107%. Folates found in beers were 10Methyl, 5Methyl and 10Formyl. The total folate levels found among types of fresh beers ranged from 18.26 to 23.99 µg/100mL. These vitamins were monitored during beer shelf life (6 months), being evaluated in freshly brewed beer and at every subsequent 2 months. Overall levels of folate found decreased over the shelf life, but it was not possible to verify the effect of package to protect beer against degradation of the vitamins. Evaluation in different batches showed a great variation, with SD up to 52.42%. To determine the total phenolic content was applied the Folin-Ciocalteau method, by which they were found 47.05, 18.67 and 21.83 in mg/100mL equivalents gallic acid (EGA) for Malzbier beer, alcohol-free Pilsen and Pilsen, respectively. The type of packaging used, can or bottle, had relevance to the total phenolic content only for Pilsen beer. Nonalcoholic beer showed the highest variation. To evaluate the antioxidant capacity, tests using the radical 2,2 - diphenyl -1 - picrylhydrazyl (DPPH) and the ferric reduction antioxidant power (FRAP) were performed. The two tests indicated the beer Malzbier has the highest antioxidant capacity, which agrees with the content of total phenolics. For identification and quantification of E-2-nonenal, a major cause of aged flavor in beers, it was used a headspace method with micro solid phase extraction and gas chromatography technique coupled with mass spectrometry (HS-SPME-GC-MS). A 5%- phenyl-methylpolisiloxane (HP-5MS) column was used. The chromatographic conditions used beginning in temperature of 60 ºC, ranging from 3 ºC / min up to 100 ºC. Then, ramp of 10 °C / min until 200 ºC and then stay at 200 ºC for seven minutes to clean the column. The carrier gas (helium) had constant flow of 1.0 mL / min. The monitored ions were 55, 70 and 83 (m/z) (source of ionization 70eV), after verification of mass spectra of the compound. Levels of E-2-nonenal ranged from 0.11 to 0.46 µg/L in beer cans, being above its threshold. For this compound significant difference was found between samples of the same type of beer, but there was no significant difference between the three types of analyzed beer. This study showed that the Brazilian beer have beneficial substances to human health, with similar content to other food, and that the E-2-nonenal is still present in quantities above the threshold in the beverage, which can compromise its quality. In general, the packaging, can or bottle, did not influence the content of the analyzed substances, and Malzbier beer stood out for greater antioxidant capacity / Doutorado / Doutor em Ciência de Alimentos

Orientador: Helena Teixeira Godoy / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T11:39:02Z (GMT). No. of bitstreams: 1 Cunha_EleniceCarlaEmidio_M.pdf: 877481 bytes, checksum: 0115f62caab877a3e3e74dfe26ca0466 (MD5) Previous issue date: 2013 / Resumo: No Brasil, o feijão obteve ampla difusão cultural e atualmente o país é considerado o maior produtor e consumidor do grão. Essa leguminosa é citada internacionalmente como uma fonte de folatos, um grupo de vitaminas pertencentes ao complexo B. Entretanto, é quase inexistente o levantamento de dados sobre o teor de folatos em feijões brasileiros. Os folatos participam de dois processos biológicos importantes: o ciclo de metilação, que consiste na transferência de carbonos e a biossíntese do DNA, fundamental para a manutenção das células. Sua deficiência está relacionada a problemas na formação do tubo neural em fetos, doenças cardiovasculares e alguns tipos de câncer. Deste modo, torna-se necessário um método eficiente para detectar e quantificar os folatos presentes nas diferentes variedades de feijão consumidas no país. O objetivo do trabalho foi determinar o teor de folatos por cromatografia líquida de alta eficiência (CLAE), utilizando diferentes métodos de hidrólise enzimática, além de aplicar o método em quatro variedades de feijão comum (carioca, preto, vermelho e branco) e uma variedade de feijão de corda, avaliando o efeito do cozimento sobre os folatos presentes. Em comparação como os outros tratamentos, apenas o tratamento tri-enzimático foi mais eficiente na identificação e quantificação dos folatos. Nos feijões, foram detectatos somente o tetraidrofolato (THF) e o 5-metiltetraidrofolato (5-MTHF). Os folatos foram separados utilizando uma coluna C18 como fase estacionária, solução acidificada com ácido acético/acetonitrila como fase móvel, e detectados por fluorescência (?exc. 290 nm e ?emis 360 nm). Os limites de detecção e quantificação foram, respectivamente, 0,9 ng/mL e 5,4 ng/mL para o THF, e 0,7 ng/mL e 4,2 ng/mL para o 5-MTHF. O método apresentou linearidade na faixa de trabalho, além de repetitividade e reprodutibilidade. Nos feijões crus o teor de THF variou de 20,3 a 1484 µg/100g e o teor de 5-MTHF variou de não determinado a 60 µg/100g. Já nos feijões cozidos, que foram processados de acordo com a recomendação dos fabricantes, o teor de THF variou de 15,3 a 1207 µg/100g, e de 5-MTHF variou de não determinado a 31,3 µg/100g. Observou-se que as perdas por processamento foram significativas e diferentes entre as variedades de feijões, sendo o 5-MTHF o composto mais suceptível. Além disso, as perdas foram ainda maiores quando o feijão foi submetido ao remolho antes do cozimento, como no feijão branco. No feijão de corda foram encontrados os maiores teores de folatos, embora tenha sido encontrado apenas o THF. Houve diferenças significativas entre os três lotes de cada variedade, que pode ser devido à diversidade das condições de cultivo e variação de genótipos do feijão. Os dados mostram que o feijão brasileiro é uma boa fonte de folatos presentes na dieta / Abstract: The beans have been widespread used as a traditional food in Brazil and nowadays the country is the largest producer and consumer of this grain. This legume is referred internationally as a source of folates, which belong to the group of vitamins B complex. However, the data about folates from Brazilian beans are almost nonexistent. Folates take part in two important biological processes: the methylation cycle, that is the transfer of carbon, and the biosynthesis of the DNA, essential for cell maintenance. The folates deficiency is related to problems in neural tube formation in fetuses, cardiovascular disease and some cancers. Thus, it becomes necessary to develop an efficient method for detecting and quantifying folate present in different varieties of beans consumed in Brazil. The aim of this study was to determine the content of folates by high performance liquid chromatography (HPLC), using different enzymatic hydrolysis methods, and apply the method on four common bean (carioca, black, white and red) and a cowpea, as well as evaluating the effect of cooking process on the content of folates. The tri-enzyme treatment compared to other treatments was the most efficient for identification and quantification of the folates. Only the tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF) were detected in the beans. Folates were separated using a C18 column as the stationary phase, acidified solution with acetic acid/acetonitrile as the mobile phase, and were detected by fluorescence (290 nm and ?exc. ?emis 360 nm). The limits of detection and quantification were, respectively, 0.9 ng/mL and 5.4 ng/mL for THF, and 0.7 ng/mL and 4.2 ng/mL for 5-MTHF. The method was linear in the working range including repeatability and reproducibility. In raw beans the THF content ranged from 20.3 to 1484 µg/100g and the amount of 5-MTHF ranged from 60 µg/100g to not determined. Whereas for the cooked beans, which were cooked according to the manufacturer's recommendations, the content of THF ranged from 15.3 to 1207 µg/100g and the amount of 5-MTHF ranged from 31.3 µg/100g to not determined. It was observed that the losses due the process were significant between the different varieties of beans, were the 5-MTHF compound was more susceptible to degradation. Furthermore, the losses were even greater when the beans were subjected to soaking before cooking, as in white beans. In cowpea were found the highest levels of folates, although it has been found only THF. There were significant differences among the three batches of each variety, which may be due to the different growing conditions and variation in the bean genotypes. The data show that the Brazilian beans are a good source of folate present in the diet / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Orientador: Carmen Sílvia Bertuzzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T06:53:07Z (GMT). No. of bitstreams: 1 Rezende_LucianaMontes_M.pdf: 2160530 bytes, checksum: b9f9b53af213aa35b20cf6a49d746a91 (MD5) Previous issue date: 2015 / Resumo: O Câncer de Mama (CM) é a forma de neoplasia mais diagnosticada e a principal causa de morte por câncer em várias regiões do mundo, sendo a maior parte CM esporádico. O estilo de vida reprodutivo representa importante fator de risco relacionado à exposição ao estrógeno durante a vida, visto que o hormônio é responsável por estimular a proliferação celular mamária. Por outro lado, a enzima metilenotetrahidrofolato redutase (MTHFR) regula o balanço celular entre a metilação e a síntese de ácidos nucléicos. Polimorfismos nos genes dos receptores de estrógeno (ESR1 e ESR2) e do metabolismo do folato (MTHFR) têm sido associados ao risco de CM em diferentes populações. Objetivos: Avaliar a associação entre CM esporádico e os polimorfismos c.2014G>A (ESR1), c.1730G>A (ESR2), c.677C>T e c.1298A>C (MTHFR), incluindo as variáveis clínicas. Material e Métodos: Foram selecionadas 253 amostras de DNA de mulheres com CM esporádico do Laboratório de Genética Molecular do Câncer (FCM-Unicamp) e 257 controles femininos com idade superior a 50 anos e sem história familiar para CM ou câncer de ovário. As amostras foram genotipadas por RFLP-PCR. Foram incluídas variáveis clínicas relacionadas ao indivíduo, aos tumores e ao estilo de vida reprodutivo. Para a análise estatística, foi utilizado o software SPSS vs21.0, empregado o teste ?2 para a distribuição dos polimorfismos e, quando observada diferença no percentual destes, foi calculado a Razão de Prevalência. Resultados: Não foi observada diferença significativa entre os grupos em relação à ocorrência de CM para os polimorfismos: c.2014G>A (p=0,281), c.1730G>A (p=0,241), c.677C>T (p=0,443) e c.1298A>C (p=0,805). Ao associar os genótipos às variáveis clínicas, o alelo A (c.2014G>A) foi menos prevalente em tumores com expressão do receptor de estrógeno (RE positivos) (OR=0,469; 95% IC=0,262 ¿ 0,841) e mais prevalente no estadio 0 em relação aos demais (OR=3,911; 95% IC=1,394 ¿ 10,97), enquanto no genótipo GA teve expressão diminuída de RE e no GG, maior expressão de receptor de estrógeno (RP). Para o c.1730G>A, o genótipo GA foi mais prevalente entre as mulheres que amamentaram (OR=2,257; 95% IC=1,254 ¿ 4,061), menos prevalente em hipertensão arterial sistêmica (HAS) (OR=0,578; 95% IC=0,339 ¿ 0,987) e conferiu proteção do estadio 0 em relação aos demais (OR=4,383; 95% IC=1,606 ¿ 11,96). Na análise do haplótipo c.2014G>A/c.1730G>A, GG/GG foi menos prevalente entre as mulheres que amamentaram (OR=0,525; 95% IC=0,298 ¿ 0,924) e teve maior expressão de RP. Para os polimorfismos no MTHFR, o genótipo CC (c.677C>T) representou um fator de risco para metástases a distância (OR=5,311; 95% IC=1,124 ¿ 25,09) e teve menor expressão de RE, enquanto o genótipo AA (c.1298A>C) foi menos prevalente no estadio 0 em relação aos demais (OR=0,034; 95% IC=0,067 ¿ 0,669) e apresentou maior expressão de RE. Na análise do haplótipo c.677C>T/c.1298A>C, o CC/AA foi associado ao uso de contraceptivo hormonal (OR=3,671; 95% IC=1,344 ¿ 10,03) e HAS (OR=1,979; 95% IC=1,036 ¿ 3,782); e o CT/AC foi menos prevalente no carcinoma invasivo de tipo não especial (NST) (OR=0,032; 95% IC=0,243 ¿ 0,918) e conferiu proteção do estadio 0 em relação aos demais (OR=3,476; 95% IC=1,341 ¿ 10,47). Conclusões: Os polimorfismos parecem não alterar o risco para o desenvolvimento de CM esporádico, no entanto, podem estar associados à sua gravidade / Abstract: The Breast Cancer (BC) is the most frequently diagnosed form of cancer and the main cause of cancer death in worldwide. The reproductive lifestyle is an important risk factor related to exposure to estrogen during the life, being the hormone is responsible for stimulating cell proliferation in the breast. On the other hand, the enzyme methylenetetrahydrofolate reductase (MTHFR) regulates cell balance between synthesis and methylation of nucleic acids. Polymorphisms in the genes of the estrogen receptor (ESR1 and ESR2) and folate metabolism (MTHFR) have been associated with risk of breast cancer in different populations. Objectives: To evaluate the association between sporadic BC and c.2014G>A (ESR1), c.1730G>A (ESR2), c.677C>T and c.1298A>C (MTHFR) polymorphisms, including the risk factor and the association with clinical variables. Material and methods: We enrolled 253 DNA samples from women with sporadic BC from Molecular Genetics of Cancer Laboratory (FCM/Unicamp) and 257 female controls over the age of 50 and without family history of BC or ovarian cancer. The samples were genotyped by PCR-RFLP. Clinical variables were included related to the individual, to tumors and reproductive lifestyle. For statistical analysis, SPSS software vs21.0 was used, the ?2 test was applied for the distribution of polymorphisms and when significant differences was observed, we calculated the odds ratio. Results: There was no significant difference between the groups in the occurrence of BC for the polymorphisms: c.2014G>A (p=0.281), c.1730G>A (p=0.241), c.677C>T (p=0.043) and c.1298A>C (p=0.805). By associating genotypes to clinical variables, the A allele (c.2014G>A) was less prevalent in tumors with positive ER (OR=0.469; 95% CI=0.262 to 0.841) and more prevalent in stage 0 compared to the other (OR=3.911; 95% CI=1.394 to 10.97), while the GA genotype had lower expression of ER and GG, most PR expression. For c.1730G>A, the GA genotype was more prevalent among women who breastfed (OR=2.257; 95% CI=1.254 to 4.061), less prevalent in hypertension (OR=0.578; 95% CI=0.339 to 0.987) and conferred protection to stage 0 in relation to others (OR=4.383; 95% CI=1.606 to 11.96). In the haplotype analysis c.2014G>A/c.1730G>A, GG/GG was less prevalent among those who breastfed (OR=0.525; 95% CI=0.298 to 0.924) and had higher PR expression. For polymorphisms in MTHFR, the CC genotype (c.677C>T) was a risk factor for distant metastasis (OR=5.311; 95% CI=1.124 to 25.09) and had lower expression of SR, while the genotype AA (c.1298A>C) was less prevalent in stage 0 in relation to others (OR=0.034; 95% CI=0.067 to 0.669) and showed a higher expression of ER. In the haplotype analysis for c.677C>T and c.1298A>C, the CC/AA combination was associated with hormonal contraceptive use (OR=3.671, 95% CI=1.344 to 10.03) and hypertension (OR=1.979; 95 % CI=1.036 to 3.782). The CT/AC was less prevalent in invasive carcinoma NST (OR=0.032; 95% CI=0.243 to 0.918) and conferred protection to stage 0 in relation to others (OR=3.476; 95% CI=1.341 to 10.47). Conclusions: The polymorphisms analyzed do not appear to alter the risk for developing sporadic BC, however, may be associated with its severity / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

Les folates sont des vitamines hydrosolubles, impliqués dans la limitation d’apparition de pathologiestelles que les anomalies de fermeture du tube neural. L’homme est dépendant de son alimentation pourcouvrir ses besoins en folates. Les légumes sont une des sources importantes de folates. Dans un mêmetemps, les légumes verts sont principalement consommés à partir de produits transformés, cuits,surgelés ou appertisés. Il est donc important de maitriser les paramètres de la transformation afin depréserver au mieux les teneurs finales en folates. L’objectif de ce travail a donc été d’identifier l’impactdu procédé de transformation (industriel et domestique) sur la perte en folates. Deux procédésindustriels ont été étudiés : la surgélation des épinards et l’appertisation des haricots verts, lestraitements domestiques ont consisté en la cuisson de légume dans de l’eau à ébullition ou à la vapeur.Les traitements thermiques (blanchiment et cuisson vapeur) n’induisent pas de diminutionssignificatives de la concentration en folates. En revanche, des pertes sont observées lorsque les légumessont en contact étroit avec l’eau. Cette étude a permis de mettre à jour deux mécanismes de perte, ladiffusion et la dégradation thermique. La diffusion des folates a été étudiée à l’échelle du laboratoire, lesconstantes de diffusion des folates ont été déterminées. La diffusion est largement influencée par lamatrice elle-même, plus que par la température ou le pH. La part de la dégradation thermique, etnotamment l’oxydation, ne peut être exclue au cours de la diffusion. C’est pourquoi des cinétiques dedégradation ont été réalisées à différentes température et différentes teneurs en oxygène, à partir depurées d’épinards et de haricots verts ainsi que sur des solutions modèles. L’oxygène joue un rôleimportant dans la dégradation chimique, notamment dans les solutions modèle. Les matrices végétalesconstituraient donc une barrière à l’oxydation des folates. Les folates sont globalement assez bienconservés au cours du procédé de transformation. Une des pistes de recherche qui prendrait un sens dansla continuité de ce travail serait l’étude de l’impact des procédés de transformation sur labioaccessibilité des folates / Folates are water-soluble vitamins, involved in the limitation of diseases such as neural tube defects.Man is dependent on his food to cover its folates’ needs. Fruits and vegetables, particularly greenvegetables are one of the major dietary sources of folates. In the same time, green vegetables are mainlyconsumed as processed products, cooked frozen or canned. It is therefore important to controlprocessing parameters in order to preserve folates content in final products. The aim of this study was toidentify the impact of processing (industrial and domestic) on folates loss. Two industrial processingchains were studied: spinach freezing chain and green beans canning chain, home treatments consistedin vegetables boiling or steaming. Thermal treatments (blanching and steaming) did not induce asignificant decrease in folates concentrations. In contrast, losses were observed when vegetables were inclose contact with the water. This study identifies two mechanisms of losses, leaching and thermaldegradation. Folates diffusion was studied on a laboratory scale, the diffusion constants for folates weredetermined. Diffusion was more influenced by the matrix, than by temperature or pH. Some of thermaldegradation, including oxidation, can not be excluded during leaching experiments. Degradationkinetics were performed at different temperature, with different oxygen contents, from spinach andgreen beans puree as well as model solutions. Oxygen plays an important role in chemical degradation,especially in model solutions. Plant matrix seemed to have protective effect, maybe as a barrier tooxygen. Folates are generally fairly well preserved during processing. One of the perspectives would bethe study of the impact of processing on folates bioaccessibility

The present PhD thesis, entitled "Use of silica supports for enhancing the stability of folates and developing antimicrobial agents", focuses on the development and evaluation of new smart systems based on the use of silica nano- and microparticles as inorganic supports for the encapsulation or immobilization of two compounds types of interest to the food industry: vitamins and antimicrobials. The first chapter shows the effect of encapsulation of folic acid and 5-formyltetrahydrofolate in mesoporous silica microparticles functionalized with polyamines on the bioaccessibility and stability of both vitamers. The use of this hybrid organic-inorganic support allows the modification of the delivery of the vitamin dependent on the pH of the medium (inhibition of the release at an acidic pH, e.g. stomach, controlled release at a neutral pH, e.g. intestine) in in vitro systems. Also, the mesoporous support can protect the vitamin against degradation after exposure to external agents, such as pH, temperature or light. Furthermore, the incorporation of encapsulated folic acid into fruit juices (apple and orange) allowed us to study the modulating and protective capability of the delivery system in a real food matrix. This study has demonstrated that the amine-functionalized support is able to not only maintain folic acid inside pores after its incorporation into juices and to properly modify the delivery of the vitamin after simulated in vitro digestion, but to also protect the vitamin after the simulating the processing and storage of juices. The second chapter describes development of different antimicrobial agents based on the combination of organic active compounds with diverse silica materials. To this end, two different methodologies were used: encapsulation of the antimicrobial agent in the pores of mesoporous silica particles, and immobilization of the active compound on the surface of silica particles. The effect of encapsulation on the enhancement of the antimicrobial properties of a compound was studied by evaluating the antimicrobial activity of free and MCM-41 nanoparticles encapsulated caprylic acid against diverse pathogen food-borne bacteria. The results of the in vitro bacterial susceptibility assays and the determination of cellular damage by microscopy showed that the nanodevice maintains antimicrobial properties in relation to the free caprylic acid. The effect of the immobilization of an active compound on the surface of a support on the improvement of its antimicrobial properties was studied with two types of different molecules: polyamines (used as a molecular gate in the folates delivery system) and essential oils. In both cases, immobilization of the bioactive compound increased the antimicrobial effect of free molecules between 1- and 100-fold in the in vitro assays and in real food systems (fruit juices and pasteurized milk), in which immobilized compounds had a bacteriostatic effect during storage, while the equivalent free compound concentrations allowed microbial growth. In summary, it is concluded that the present thesis has evaluated the versatility of silicon oxide particles to solve two of the biggest problems in the food industry: alteration of active compounds during food processing and current antimicrobial systems losing of efficacy. Thus the developed devices may be used as alternative methods to traditional encapsulation systems or traditional food safety treatments. / La presente tesis doctoral que lleva por título "Uso de soportes de sílice para la mejora de la estabilidad de los folatos y el desarrollo de agentes antimicrobianos", se centra en el desarrollo y evaluación de nuevos sistemas inteligentes basados en el uso de nano- y micropartículas de sílice como soporte inorgánico para la encapsulación o inmovilización de dos tipos de compuestos de interés para la industria alimentaria: vitaminas y antimicrobianos. El primer capítulo muestra el efecto de la encapsulación de ácido fólico y 5-formiltretahidrofolato en micropartículas mesoporosas de sílice funcionalizadas con poliaminas sobre la bioaccessibilidad y estabilidad de ambos vitámeros. Por una parte, el uso de este soporte híbrido orgánico-inorgánico permite modular la liberación de la vitamina en función del pH del medio (inhibición de la liberación a pH ácido -estómago- y liberación controlada a pH neutro -intestino-) en sistemas in vitro. Así mismo, el soporte mesoporoso es capaz de proteger a la vitamina frente a la degradación tras la exposición a diversos agentes externos, como el pH, la temperatura o la luz. Además, la incorporación de ácido fólico encapsulado a zumos de frutas (manzana y naranja) ha permitido estudiar la capacidad moduladora y protectora del sistema de liberación en una matriz alimentaria real. Este estudio ha demostrado que el soporte funcionalizado con poliaminas no sólo es capaz de mantener el ácido fólico en el interior de los poros tras la incorporación a los zumos y modular correctamente la liberación del mismo tras la simulación de una digestión in vitro, sino que también es capaz de proteger la vitamina tras la simulación del procesado y almacenamiento de los zumos. En el segundo capítulo se describe el desarrollo de diferentes agentes antimicrobianos, basados en la combinación de compuestos activos orgánicos con diversos materiales de sílice. Para ello, se emplearon dos metodologías diferentes: la encapsulación del agente antimicrobiano en los poros de las partículas mesoporosas de sílice, y la inmovilización del compuesto activo sobre la superficie de las partículas de sílice. El efecto de la encapsulación sobre la mejora de las propiedades antimicrobianas de un compuesto se estudió determinando la actividad antimicrobiana de ácido caprílico libre y encapsulado en nanopartículas mesoporosas tipo MCM-41 frente a diversas bacterias patógenas presentes en alimentos. Los resultados de los ensayos in vitro de susceptibilidad bacteriana y la determinación del daño celular mediante microscopia mostraron que el nanodispositivo mantiene las propiedades antimicrobianas respecto al ácido caprílico libre. El efecto de la inmovilización de un compuesto activo sobre la superficie de un soporte en la mejora de sus propiedades antimicrobianas se estudió con dos tipos de moléculas diferentes: poliaminas (usadas como puerta molecular en el sistema de liberación de folatos) y aceites esenciales. En ambos casos la inmovilización del compuesto bioactivo incrementó entre 1-100 veces el poder antimicrobiano de las moléculas libres tanto en ensayos in vitro, como en ensayos en alimentos reales (zumos de frutas y leche pasteurizada) donde se comprobó que los compuestos inmovilizados tienen un efecto bacteriostático a lo largo del período de almacenamiento mientras que concentraciones equivalentes de compuesto libre permiten el crecimiento del microorganismo. En resumen, se puede concluir que en la presente tesis se ha evaluado la versatilidad de los sólidos de óxido de silicio para solventar dos de los grandes problemas de la industria alimentaria: alteración de compuestos bioactivos durante el procesado del alimento y la pérdida de la eficacia de los sistemas antimicrobianos actuales. Así, los dispositivos desarrollados podrían ser usados como métodos alternativos a los sistemas tradicionales de encapsulación o los tratamientos tradiciona / La present tesi doctoral, que porta per títol "Ús de suports de sílice per a la millora de l'estabilitat dels folats i el desenvolupament d'agents antimicrobians", es centra en el desenvolupament i avaluació de nous sistemes intel·ligents basats en l'ús de nano- i micropartícules de sílice com a suport inorgànic per a l'encapsulació o immobilització de dos tipus de compostos d'interès per a la indústria alimentària: vitamines i antimicrobians. El primer capítol mostra l'efecte de l'encapsulació d'àcid fòlic i 5-formiltretahidrofolat en micropartícules mesoporoses de sílice funcionalitzades amb poliamines sobre la bioaccessibilitat i estabilitat d'ambdós vitàmers. D'una banda, l'ús d'aquest suport híbrid orgànic-inorgànic permet modular l'alliberament de la vitamina en funció del pH del medi (inhibició de l'alliberament a pH àcid -estómac- i alliberament controlat a pH neutre -intestí-) en sistemes in vitro. Així mateix, el suport mesoporós és capaç de protegir a la vitamina enfront de la degradació després de l'exposició a diversos agents externs, com el pH, la temperatura o la llum. A més, la incorporació d'àcid fòlic encapsulat a sucs de fruites (poma i taronja) ha permès estudiar la capacitat moduladora i protectora del sistema d'alliberament en una matriu alimentària real. Aquest estudi ha demostrat que el suport funcionalitzat amb poliamines no sols és capaç de mantindre l'àcid fòlic a l'interior dels porus després de la incorporació als sucs i modular correctament l'alliberament del mateix després de la simulació d'una digestió in vitro, sinó que també és capaç de protegir la vitamina després de la simulació del processat i emmagatzemament dels sucs. En el segon capítol es descriu el desenvolupament de diferents agents antimicrobians, basats en la combinació de compostos actius orgànics amb diversos materials de sílice. Per a això, es van emprar dues metodologies diferents: l'encapsulació de l'agent antimicrobià en els porus de les partícules mesoporoses de sílice, i la immobilització del compost actiu sobre la superfície de les partícules de sílice. L'efecte de l'encapsulació sobre la millora de les propietats antimicrobianes d'un compost es va estudiar determinant l'activitat antimicrobiana d'àcid caprílic lliure i encapsulat en nanopartícules mesoporoses tipus MCM-41 enfront de diversos bacteris patògens presents en aliments. Els resultats dels assajos in vitro de susceptibilitat bacteriana i la determinació del dany cel·lular per mitjà de microscòpia van mostrar que el nanodispositiu manté les propietats antimicrobianes respecte a l'àcid caprílic lliure. L'efecte de la immobilització d'un compost actiu sobre la superfície d'un suport en la millora de les seues propietats antimicrobianes es va estudiar amb dues tipus de molècules diferents: poliamines (usades com a porta molecular en el sistema d'alliberament de folats) i olis essencials. En ambdós casos la immobilització del compost bioactiu va incrementar entre 1-100 vegades el poder antimicrobià de les molècules lliures tant en assajos in vitro, como en assajos en aliments reals (sucs de fruites i llet pasteuritzada) on es va comprovar que els compostos immobilitzats tenen un efecte bacteriostàtic al llarg del període d' emmagatzemament mentre que concentracions equivalents de compost lliure permeten el creixement del microorganisme. En resum, es pot concloure que en la present tesi s'ha avaluat la versatilitat dels sòlids d'òxid de silici per a resoldre dos dels grans problemes de la indústria alimentària: alteració de compostos bioactius durant el processat de l'aliment i la pèrdua de la eficàcia dels sistemes antimicrobians actuals. Així, els dispositius desenvolupats podrien ser usats com a mètodes alternatius als sistemes tradicionals d'encapsulació o els tractaments tradicionals per assegurar la innocuïtat dels aliments. / Ruiz Rico, M. (2016). Use of silica supports for enhancing the stability of folates and developing antimicrobial agents [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/76805 / TESIS

Les erreurs innées du métabolisme sont des maladies héréditaires pouvant affecter, entre autres, la synthèse et le transport des neurotransmetteurs. Le diagnostic de ces pathologies repose essentiellement sur l’analyse par chromatographie d’un fluide biologique [plasma, urine, liquide céphalorachidien (LCR)]. L’objectif de cette thèse était de développer des méthodes simples et rapides pour le diagnostique des troubles de la neurotransmission. Dans un premier temps, nous avons développé et validé le dosage simultané, en moins de 10 minutes, des métabolites de la dopamine, de la sérotonine et de la tétrahydrobioptérine (BH4) par chromatographie liquide ultra-haute pression (UHPLC) couplée à une détection séquentielle électrochimique et fluorimétrique. Cette méthode a été appliquée à l’analyse de 1372 échantillons de LCR, permettant ainsi d’établir les valeurs fréquentes de la population française. Afin de transposer la méthode précédente en UHPLC couplée à la spectrométrie de masse (MS), nous avons étudié les mécanismes d’auto- et d’électro- oxydation de la BH4, par mobilité ionique différentielle couplée à la MS haute résolution (résonance cyclonique ionique FTICR) et à la photodissociation Infra-rouge. Ce travail nous a permis d’isoler et de caractériser pour la première fois la qBH2, intermédiaire réactionnel fugace de la BH4, impliqué dans le mécanisme d’action de cette dernière. La méthode UHPLC-MS/MS développée, permet en outre le dosage simultané du 5-Méthyl-tétrahydrofolate. / Inborn errors of metabolism are inherited diseases that can alter the synthesis and transport of neurotransmitters. The diagnosis of these conditions is currently based on the chromatographic analysis of a biological fluid [plasma, urine, cerebrospinal fluid (CSF)]. The aim of this thesis was to develop simple and rapid methods for the diagnosis of neurotransmitter disorders. Firstly, we developed and validated the simultaneous determination of dopamine, serotonin, and tetrahydrobiopterin (BH4) metabolites by ultrahigh pressure liquid chromatography (UHPLC) coupled to sequential electrochemical and fluorimetric detection. This method was applied to the analysis of 1372 CSF samples, thus establishing the frequent ranges of the French population. In order to transpose the previous method into UHPLC coupled to mass spectrometry (MS), we studied the mechanisms of auto- and electro- oxidation of BH4, by Differential Ion Mobility coupled to high resolution MS (FTICR) in conjunction with Infra-Red photo-dissociation. This work allowed us to isolate and characterize qBH2, the transient reaction intermediate of BH4, involved in the mechanism of action of the latter. The proposed UHPLC-MS/MS method also allows the simultaneous determination of 5-methyltetrahydrofolate.

Les donneurs de méthyles (B12 et folates) régulent le cycle des monocarbones qui joue un rôle primordial dans les régulations épigénétiques/épigénomiques par méthylation. Une carence en donneurs de méthyles produit un retard de croissance intra-utérine et favorise les anomalies du développement, principalement du système nerveux central. De plus, des taux élevés d'homocystéine associés à une telle carence constituent un facteur de risque pour diverses pathologies neurodégénératives. Nous avons étudié les conséquences d'une carence péri-conceptionnelle et gestationnelle sur le développement cérébral embryonnaire de rats Wistar. L'étude morphométrique a montré un retard de croissance des embryons carencés qui affectait également le cerveau, avec une atrophie de structures telles que l'hippocampe, le cortex et la zone subventriculaire. En raison de la forte sensibilité de l'hippocampe, les effets de la carence ont par ailleurs été étudiés sur un modèle cellulaire de progéniteurs neuronaux hippocampiques. L'utilisation de ces deux modèles a permis de montrer in vivo et in vitro la régulation négative par la carence de la voie Stat3, qui influence prolifération et survie, via une régulation épigénomique post-transcriptionnelle impliquant miR-124. La dérégulation du programme neurogénique impliquant les histones désacétylases affecte la différenciation cellulaire. Par ailleurs, nous avons démontré que la carence en donneurs de méthyles était associée à une modification post-traductionnelle correspondant à une N-homocystéinylation irreversible de protéines neuronales, en particulier associées au cytosquelette. Cette modification induit l'agrégation des protéines, phénomène impliqué dans de nombreuses maladies neurodégénératives. La combinaison de ces différents mécanismes apporte un éclairage nouveau sur les défauts de développement et les troubles cognitifs associés à une carence précoce en donneurs de méthyles, soulignant l'importance de la « programmation foetale » dans la survenue de certaines pathologies neurologiques / Methyl donors (B12 and folate) regulate the one-carbon cycle that plays an important role in the epigenetic/epigenomic regulations by methylation. Methyl donor deficiency (MDD) leads to intrauterine growth retardation and promotes neurodevelopmental abnormalities. Also, high levels of homocysteine associated with such a deficiency are a risk factor for various neurodegenerative diseases. We have studied the consequences of a periconceptional and gestational deficiency on the development of the embryonic brain of Wistar rats. Morphometric studies showed retardation in the development of deficient embryos which also affected the brain, with an atrophy of some structures including hippocampus, cortex and subventricular zone. Given the high sensitivity of the hippocampus, the effects of MDD have been additionally studied in a cellular model of hippocampal neuronal progenitors. Using these two models, we showed both in vivo and in vitro the downregulation of Stat3 pathway regulating cell proliferation and survival, through an epigenomic post-transcriptional process involving miR-124. Disruption of the neurogenic program implying histone deacetylases was shown to alter cell differentiation. Furthermore, we showed that methyl donor deficiency was associated with a post-translational modification corresponding to an irreversible N- homocysteinylation of neuronal proteins, especially those associated with the cytoskeleton. Such a process leads to protein aggregation, a phenomenon involved in many neurodegenerative diseases. The combination of these different mechanisms provides new insights into developmental defects and cognitive impairment associated with an early MDD, highlighting the importance of "fetal programming" in the occurrence of some neurological diseases