Os consumos inadequados de cobalamina (Cbl) e de folato associados aos polimorfismos em genes de enzimas chaves do metabolismo da homocisteína podem agravar as comorbidades relacionadas a deficiências destas vitaminas. Os objetivos deste estudo foram avaliar o consumo de cobalamina, de folato e de vitamina 86 por gestantes através de três inquéritos recordatórios de 24 horas (IR24h); determinar as correlações entre as concentrações séricas de folato, de cobalamina, de metionina, de .S-adenosilmetionina (SAM), de Sadenosilhomocisteína (SAH), de homocisteína total (tHcy) e de ácido metilmalônico (MMA) com as vitaminas e as proteínas ingeridas na dieta pelas mulheres; analisar as associações entre os polimorfismos MTHFR C677T, MTHFR A1298C, MTR A2756G e MTRR A66G e alterações nas concentrações das vitaminas e dos metabólitos, além de avaliar os determinantes nutricionais e genéticos das concentrações dos metabólitos tHcy\" SAM, MMA e SAM/SAH durante a gestação. Participaram do estudo 73 mulheres com idades gestacionais de 16, 28 e 36 semanas, no qual foram aplicados três IR24h para cada gestante, um em cada idade gestacional. Foram realizadas as dosagens séricas de cobalamina sérica, folato sérico e eritrocitário, homocisteína total, metionina, MMA, SAM e SAH. As genotipagens dos polimorfismos MTHFR C677T, MTHFR A1298C, MTR A2756G e MTRR A66G foram feitas por PCR-RFLP. Observou-se que a ingestão de folato foi baixa em relação ao valor de 600 µg/dia recomendado para gestantes, a ingestão de cobalamina foi menor que a encontrada na literatura, a ingestão de vitamina 86 e a de proteínas totais foram semelhantes às da literatura. As concentrações de Cbl séricas foram diretamente relacionadas com a ingestão de proteínas. As concentrações de MMA estavam relacionadas inversamente com a ingestão de cobalamina, com a ingestão das proteínas totais e com a renda per capita. Houve redução das concentrações de cobalamina sérica e aumento de MMA no decorrer das idades gestacionais, sendo observada maior concentração sérica de MMA entre as mulheres que ingeriram menos cobalamina. Não houve associação entre os polimorfismos MTHFR A1298C e MTRR A66G e as variações nas concentrações de vitaminas e de metabólitos. Com relação ao polimorfismo MTHFR C677T, as mulheres portadoras do alelo 677T possuíam menores concentrações médias de foiato eritrocitário, e para o polimorfismo MTR A2756G, as mulheres com genótipos AG e GG apresentaram menores concentrações de metionina. As concentrações aumentadas de tHcy foram explicadas pelas menores concentrações de folato eritrocitário, pela menor ingestão de proteínas totais, pelas maiores concentrações de creatinina sérica e pela ingestão de vitamina 86. A creatinina sérica foi a responsável pela variabilidade das concentrações da SAM. Maiores concentrações de folato sérico e menores concentrações de creatinina sérica foram os responsáveis pelo aumento dos valores da SAM/SAH. As maiores concentrações de MMA foram atribuídas à menor ingestão de cobalamina, à menor renda per capita e à menor concentração sérica de cobalamina. Conclusões: a ingestão de folato foi menor que a metade do recomendado. A ingestão de cobalamina atingiu o recomendado, no entanto não foi suficiente para manter o metabolismo materno. Os polimorfismos não foram associados a baixa ingestão de vitaminas para explicar as alterações nas concentrações de tHcy, da SAM, da SAM/SAH e do MMA. / The inadequate intake of cobalamin and folate associated to polymorphisms in key-enzyme genes of homocysteine can worsen comorbidities related to the deficiency of these vitamins. The aims of this study were to evaluate the intake of cobalamin, foiate and vitamin 86 by pregnant women through three 24-hour dietary recaIl (IR24h); to assess the correlation between serum concentrations of folate, cobalamin, methionine, S-adenosilmethionine (SAM), S-adenosilhomocysteine (SAH), total homocysteine (tHcy) and methylmalohic acid (MMA)towards vitamins and proteins intaked by women, to analyze association between MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRR A66G polymorphisms and changes on vitamin and metabolites concentration, and to determine the nutritional and genetic determinants during a gestational period. 73 pregnant women participated in this study, with gestational ages of 16,28 and 36 weeks, onto whom were applied three IR24h to each woman, being one in each gestational age. Serum concentrations of cobalamin, foiate and red blood cell foiate, total homocysteine, methionine, MMA, SAM and SAH were evaluated. The polymorphisms of MTHFR C677T, MTHFR A1298C, MTR A2756G and MTRRA66G were performed by PCR-RFLP. It was observed that folate intake was low in relationship to recommended value for pregnant women of 600 µg/day. Cobalamin intake was less than the one found in literature. Vitamin 86 and total protein intake was similar to the one found in literature. Serum Cbl concentrations were directly related to protein intake. MMA concentrations were in,verselycorrelated to cobalamin intake, to total proteins intakes and mounth per capita income. There were reduction of serum cobalamin concentration and enhance of MMAthroughout pregnancy weeks, being observed higher serum concentration of MMAamongst women that intaked less cobalamin. There was no association between MTHFRA1298Cand RRA66Gpolymorphism and the change in vitamins and metabolites concentrations. The women carrying allele 2756G for MTR polymorphism presented lesser serum methionine concentrations and alieie 677T for MTHFR C677T had less concentration of red blood cell folate. The red blood cell folate concentrations, intake of total proteins, serum creatinine concentrations and intake of vitamin 86 were eterminant of tHcy concentration. Serum creatinine was responsible for SAMconcentrations variability. Higher concentrations of serum foiate and lesse r concentrations of serum creatinine were responsible for the enhance of SAM/SAHvalues. The higher concentrations of MMAwere attributed to lesse r cobalamin intake, to lower per capita income and to lesser seruro concentration Df coba}amin. ConcJusions: lo/ate intake was less than ha(f of recommended amount, and cobalamina intake reached the recommended amount, however this amount was not enough to keeping the maternal metabolism. The poly~orphisms were not associated to low vitamin intake for explaining the changes Inserum concentrationsof tHcy,SAM,SAM/SAHand MMA.

O aborto espontâneo recorrente (AER) caracteriza-se pela ocorrência de três ou mais abortos consecutivos espontâneos até a 20ª semana de gestação. É uma condição patológica multifatorial, em que alterações morfológicas uterinas, distúrbios endócrinos, alterações no cariótipo, polimorfismos genéticos relacionados aos genes envolvidos no metabolismo da homocisteína, hemostasia, infecções, autoanticorpos e o processo inflamatório podem contribuir para a ocorrência de AER. O estado fisiológico do endométrio é essencial para a implantação do embrião no útero durante a gestação. Na interface materno-fetal, há uma modulação de citocinas, necessária para o estabelecimento da angiogênese e desenvolvimento da placenta. Um desequilíbrio entre as citocinas pode diminuir a tolerância ao feto e ocasionar rejeição fetal. A concentração de citocinas pode ser modificada por conta de uma diminuição na expressão de alguns genes, e esta pode ser regulada pelo seu estado de metilação sítio-específica. A metilação do DNA é um mecanismo epigenético de regulação gênica, e que corresponde à incorporação de grupos metila em ilhas CpG localizadas próximas às regiões promotoras de genes humanos, e isso pode ser importante na avaliação do risco de complicações gestacionais. Além disso, o estado nutricional de vitaminas foi relacionado a alterações no padrão de metilação de alguns genes. Os objetivos deste trabalho foram avaliar as concentrações dos mediadores inflamatórios em mulheres com aborto e em grupo controle, verificar correlações entre as concentrações de vitaminas, homocisteína total e taxa de metilação do DNA, verificar correlações entre concentração de citocinas e taxa de metilação do DNA e determinar odds ratio (IC 95%) de ter aborto em modelos multivariados. Foram incluídas 253 mulheres com história de aborto recorrente e 264 mulheres saudáveis (controle). O DNA foi extraído de leucócitos de sangue periférico para o estudo de metilação. Foram separadas alíquotas de soro e plasma para dosagem de vitaminas, metabólitos e citocinas. Não foram encontradas diferenças nas taxas de metilação do DNA entre os grupos aborto e controle. A citocina TNFα está aumentada nos grupos de aborto em comparação ao controle. A taxa de metilação do DNA no gene IFNG foi correlacionada inversamente às concentrações de folato sérico e citocina IFNγ no grupo controle. E as concentrações de IL10 foram inversamente correlacionadas à taxa de metilação do DNA nos grupos de aborto secundário e controle. Neste trabalho, verificou-se que as vitaminas e as citocinas influenciam na taxa de metilação do DNA do gene IFNG e a citocina pró-inflamatória TNFα apresenta-se aumentada em mulheres com história de aborto. / Recurrent spontaneous abortion (RSA) is characterized by the occurrence of three or more consecutive spontaneous abortions until the 20th week of gestation. It is a multifactorial pathological condition in which morphological uterine, endocrine disorders, changes in the karyotype genetic polymorphisms related to genes involved in homocysteine metabolism, infection, autoimmunity and inflammatory processes may contribute to the occurrence of RSA. The physiological state of the endometrium is essential for embryo implantation in the uterus during pregnancy. In maternal-fetal interface, there is a modulation of cytokines necessary for the establishment and development of placental angiogenesis. An imbalance between cytokines can decrease tolerance to fetus and cause fetal rejection. Concentration of cytokines may be modified due to a decrease in the expression of genes related to some of these cytokines that can be regulated by DNA methylation, which is an epigenetic mechanism of gene regulation and which corresponds to the incorporation of groups Methyl CpG islands located near the promoter regions of human genes, and this may be important in assessing the risk of pregnancy complications. In addition, the nutritional status of vitamins was associated with changes in the methylation pattern of certain genes. The aims of this study were to determine the concentrations of inflammatory mediators in women with abortion and the control group, examine correlations between concentrations of vitamins, total homocysteine and DNA methylation rate, examine correlations between cytokine concentration and DNA methylation and determine odds ratio (95% CI) of having abortion in multivariate models. We included 253 women with a history of recurrent miscarriage and 264 healthy women (control). DNA was extracted from peripheral blood leukocytes for the study of methylation. Serum and plasma aliquots were used for determination of vitamins, metabolites and cytokines. There were no differences in rates of DNA methylation between control and abortion groups. The cytokine TNFα is increased in abortion groups compared to the control. DNA methylation rate in gene IFNG was inversely correlated with serum folate and serum cytokine IFNγ in the control group. Also IL10 concentrations were inversely correlated to DNA methylation rate in groups of miscarriage and secondary control. In this work, it was found that vitamins and cytokines influence DNA methylation rate in the promoter region and are different in the study and control groups.

O aborto espontâneo recorrente (AER) é definido pela ocorrência de três ou mais abortos espontâneos consecutivos com idade gestacional de até 20 semanas. O AER é um evento multifatorial, tem um índice de elucidação da causa na ordem de 50% e, mesmo com os avanços da medicina diagnóstica ainda assim 40% dos casos permanecem com sua causa desconhecida. O crescimento fetal é totalmente dependente do aporte de nutrientes oirundos da mãe, dentre esses nutrientes a cobalamina e o ácido fólico desempenham um papel fundamental para a viabilidade fetal. O objetivo geral deste estudo foi investigar se existe associação entre polimorfismos em genes relacionados ao metabolismo da cobalamina (MTHFR c.677C>T, TCN2 c. 776C>G e TCN2 c. 67A>G), e o aborto espontâneo recorrente. Os objetivos específicos deste estudo foram: 1 determinar se os polimorfismos MTHFR c. 677C>T, TCN2 c. 776C>G e TCN2 c. 67A>G estão associados ao aborto primário e secundário. 2 - Avaliar se os genótipos dos polimorfismos estudados estão relacionados com as concentrações séricas de cobalamina, folato e homocisteína total em mulheres com aborto espontâneo recorrente. Foram incluídas 256 mulheres com história de abortos espontâneos recorrentes, provenientes do Ambulatório de Obstetrícia da Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da USP e 264 mulheres saudáveis, sem história de aborto espontâneo e que tenham tido pelo menos duas gestações normais (grupo controle), pareadas segundo as idades. Foram colhidas amostras de sangue para a realização das dosagens bioquímicas, hormonais e das vitaminas e também para a realização das genotipagens dos polimorfismos por meio de PCR-RPFL (MTHFR c.677C>T , TCN c.776C>G e c. 67A>G). As dosagens bioquímicas e hormonais apresentaram resultados dentro dos limites de variação do normal. Quanto as concentrações de folato e cobalamina, houve diferença estatística significante entre os grupos p<0,05. As frequências dos genótipos e alelos para os polimorfismos estudados comparadas entre os grupos abortos primário, aborto secundário e grupo controle não apresentaram diferença estatística significante. Optamos a seguir por dividir o grupo de estudo entre abortos primários, onde não existe história de feto viável, e secundário neste caso onde há história de feto viável. Desta forma foram refeitas as análises estatísticas entre os grupos e encontramos diferença estatísticamente significante p<0,05 quando confrontamos os genótipos do polimorfismo TCN c.776C>G entre o grupo primário e o grupo controle. Em conclusão, quando comparamos as frequência dos genótipos e alelos em conjunto não apresentaram associação com o AER. Quando comparado separadamente o grupo de aborto primário e grupo controle houve diferença estatística significante associando o polimorfismo TCN2 c.776C>G ao AER primário. / The recurrent spontaneous abortion (RSA) is defined by the occurrence of three or more consecutive miscarriages with gestational age up to 20 weeks. The AER is a multifactorial event, has an index of elucidating the cause of around 50% and, even with advances in diagnostic medicine still remain 40% of cases with a known cause. Fetal growth is totally dependent on the supply of nutrients from the mother oirundos among these nutrients cobalamin and folic acid play a key role in fetal viability. The aim of this study was to investigate whether there is an association between polymorphisms in genes related to metabolism of cobalamin (MTHFR c.677C> T, c TCN2. 776c> G and c TCN2. 67A> G), and recurrent miscarriage. The specific objectives of this study were: 1 determine whether MTHFR c. 677C> T, TCN2 c. 776c> G and c TCN2. 67A> G are associated with abortion primary and secondary. 2 - Assess whether the genotypes studied polymorphisms are associated with serum concentrations of cobalamin, folate and total homocysteine in women with recurrent spontaneous abortion. We included 256 women with a history of recurrent miscarriages, from the Clinic of Gynecology, Obstetrics, Hospital das Clinicas, Faculty of Medicine, USP and 264 healthy women with no history of miscarriage and have had at least two normal pregnancies (group control), matched according to age. Blood samples were collected to perform the biochemical, hormonal and vitamins and also to perform the genotyping of polymorphisms by PCR-RPFL (MTHFR c.677C> T, TCN c.776C> G and c. 67A> G). The biochemical and hormonal results presented within the limits of normal variation. As the concentrations of folate and cobalamin, statistically significant difference between groups p <0.05. The frequencies of genotypes and alleles for the polymorphisms studied compared between groups abortions primary, secondary and abortion control group showed no statistically significant difference. We chose then to divide the study group between primary abortions where there is a history of viable fetus, and secondary in this case where there is a history of viable fetus. Thus were repeated statistical analyzes between groups and found statistically significant difference p <0.05 when confronted TCN genotypes of polymorphism c.776C> G between the primary group and the control group. In conclusion, when comparing the frequency of genotypes and alleles together apresntaram no association with AER. When compared separately the group of abortion primary and control group was statistically significant associated polymorphism TCN2 c.776C> G the primary AER.

This study involves data from the Cache County Study, which began in 1994 with joined efforts by Duke University, Utah State University, and Johns Hopkins University. It consisted of 5,092 participants from Cache County, Utah, located in the northern part of the state. Characteristics of the population include high participation rates (~ 90%), a majority of participants are members of The Church of Jesus Christ of Latter-day Saints, longer life expectancy than the general US population, a greater than 80% rate of at least a high school education, and low rates of migration. Subjects cognitive status was screened using the Modified Mini-Mental State Examination or rated by knowledgeable informants using an Informant Questionnaire for Cognitive Decline. Low scoring subjects were examined using the Dementia Questionnaire, an inventory of cognitive symptoms, functional impairments, and medical conditions relevant to dementia. The clinical data were reviewed by a geropsychiatrist and neuropsychologist. Those suspected of dementia underwent further testing and final dementia diagnoses were decided by a consensus conference of experts. Clinical assessment at the baseline interview identified 368 individuals out of the original 5,092 subjects as having dementia. These individuals were removed from the present analysis. Prevalent cases of dementia were excluded in our analyses of risk associated with incident AD. Dietary data were collected using a food frequency questionnaire at baseline in 1995. A list of 142 foods was provided and participants noted frequency they consume the food or food group. To calculate intake of a specific nutrient, the nutrient content of each food is multiplied by the frequency of consumption for each food. This number is summed over all food items. Cox Proportional hazards modeling was used to assess risk of incident AD in relationship to folate and B-vitamin intake over eleven subsequent years of data collection. Cox modeling assists in analysis of censored cases (drop-outs and deaths). No relationship was found between folate from food, supplement, or combined sources with dementia or with AD. Similar results were observed for B-12 and B-6.

Research Doctorate - Doctor of Philosophy (PhD) / Maternal factors prior to conception and during pregnancy may influence the development of the metabolic, cardiovascular and endocrine systems of the offspring and subsequent disease pathogenesis. This thesis explores the concept of the developmental origins of health and disease. Human observational research studies were undertaken to test the relationships amongst maternal dietary intake, weight gain during pregnancy and changes in biochemical markers between pregnancy and postpartum for the mother and infant. This thesis presents three chapters of original research related to maternal and fetal nutrition, and one chapter of empirical data concerning the methodological challenges faced when recruiting for research purposes. An analysis of dietary intake data from the young cohort of the Australian Longitudinal Study on Women’s Health was used to determine the overall diet quality in a contemporary cohort, and to assess whether those who are pregnant eat differently to those who are not. Only small differences in diet quality and nutrient intakes were detected between pregnancy groups, and diet quality scores were consistently low. When the intake data were compared to Australian recommendations it appears that many young women fail to reach key nutrient targets, including those set for folate, fibre, calcium, iron, potassium and vitamin E. The research focus then shifted to prospective longitudinal data collection for women and their children during pregnancy and after birth. Low recruitment to this component of the studies threatened the potential to achieve the research aims. Rather than jeopardising the power of the investigations efforts were made to understand what had gone wrong and how the situation could be rectified. An investigation of the relationship between fetal adiposity and maternal weight changes in pregnancy was performed. Pre-pregnancy body mass index (BMI) and weight changes during pregnancy were taken as broad markers of maternal nutritional status and energy regulation. Intrauterine growth, including the accumulation of adipose tissue, was assessed using serial ultrasounds. Fetal size was positively related to maternal pre-pregnancy weight (and BMI) and weight gain (change in BMI) during pregnancy. Maternal pre-pregnancy weight was positively associated with adiposity at the fetal abdomen, but not the thigh. However, overall maternal weight gain was not associated with fetal adiposity. To determine whether maternal vitamin B12 and folate (methyl donors) in pregnancy could influence the offspring’s homocysteine metabolism at birth, changes in plasma vitamin B12, plasma folate and red cell folate were characterised for the cohort of more than 100 women during pregnancy and up to six months after birth. A small sub-sample of infants also had blood collected at six months postpartum. Average maternal plasma folate during pregnancy was significantly predictive of infant plasma homocysteine. In conclusion, the research outlined herein demonstrates important interactions between the mother and her offspring during the critical windows of early development. The research is multidisciplinary in its application and contributes to our understanding of some of the nutritional influences in pregnancy and postpartum for women and their children.

This thesis investigates the use of scientific evidence in the process of making public health policy. A case study located within a food regulation setting is used. The aim is to test theory against this case study. The outcome is a theoretical understanding of the use of scientific evidence in the policy-making process in a food regulation setting. Food regulation can influence food composition and food labelling and thereby affect the population's dietary intake. Frequently there are contested values, beliefs, ideologies and interests among stakeholders regarding the use of food regulation as a policy instrument to effect public health outcomes. The protection of public health and safety, taking into account evidence based practice, is generally employed by food regulators as the priority objective during the policy-making process to adjudicate among the competing expectations of stakeholders. However, this policy objective has not been clearly defined and is vulnerable to interpretation and application. The process by which folate fortification policy was made in Australia, in response to epidemiological evidence of a relationship between folate intake during the periconceptional period and reduced risk of neural tube defects, was analysed as a case study of the policy-making process. The folate fortification policy created a precedent for both food fortification and subsequently health claims policy in Australia. A social constructivist method was used to analyse the case study. The method involved deconstructing the food regulatory system into three levels; decision-making process; procedural; and political environment. Data aligned with each level of analysis was collected from 22 key informant interviews, documentary sources, field notes and surveys of both a random sample of the Australian population's knowledge of folate and use of folic acid-containing supplements (n = 5422), and the implementation of folate fortified food products into stores (n = 60). The insights that emerged from each of the three levels of analysis were assessed iteratively to identify a pattern of interrelationships associated with the policy-making process within the food regulatory system. The identified pattern was interpreted against existing theory to gain a theoretical understanding of the public health policy-making process in this political setting. The central argument of this thesis extends Sabatier and Jenkins-Smith's Advocacy Coalition Framework theory to a food regulation setting. The argument is that within the contemporary political climates of neoliberalism and globalisation, a coalition between corporate interests and the values of scientists with a positivist-reductionist approach to public health research is privileged so as to invoke certain scientific evidence to, in turn, legitimise food regulation policy decisions. The theory will help to inform policy-makers about how and why the public health policy objective in a food regulation setting is interpreted and applied. This will contribute to improving policy practice intended to effect public health outcomes. It is concluded that irrespective of the quantity and quality of the scientific evidence that is being made available, scientific evidence cannot be assumed to speak for itself Policy-making is an inherently political and value-laden process and the potential for politically motivated interpretation and application of otherwise value-neutral scientific evidence can undermine the investment in its generation. From this perspective, evidence based practice, far from liberating policy-making from political influence, can itself become part of the problem rather than the solution. Nevertheless, rational evidence based practice is an ideal to strive for and a series of recommendations is proposed to help make the use of evidence in current food regulation policy processes more transparent and democratic.

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Tumours of the central nervous system (CNS) are the second most common cancers diagnosed in children, yet the cause of this disease remains largely unknown. This thesis examines whether polymorphisms in folate-metabolising and glutathione S transferase (GST) genes influence the risk and disease outcome of childhood CNS tumours. 204 children aged ≤18 years diagnosed with a CNS tumour at the Sydney Children??s Hospital between 1989 and 2004 were included in the study. DNA samples were isolated from archival frozen and formalin-fixed paraffin-embedded tumour tissue. Polymorphisms in GST and folate pathway genes were examined using real-time PCR. Genotype distributions in children with CNS tumours were compared to those observed in a control panel of cord blood samples from 363 healthy newborns. Children carrying at least one variant allele for each of MTHFR 677 C>T, MTHFR 1298 A>C, MTR 2756 A>G, MTRR 66 A>G, and RFC 80 G>A were found to have a 2.8-fold greater risk of developing a CNS tumour than non-carriers (OR=2.80; 95%CI: 1.08-7.56, P=0.022), an association which was even more apparent in those children with an embryonal tumour (OR=4.54; 95%CI: 1.13-15.85, P=0.016). Results also showed that children with the GSTP1 105 Val/Val genotype were three times more likely to develop a CNS tumour of embryonal cell origin than children with the GSTP1 105 Ile/Ile or Ile/Val genotypes (OR=3.02; 95%CI: 1.34-6.46, P=0.005). No such association was observed for CNS tumours of glial cell origin. The GSTM1, GSTT1, and GSTP1 Ala114Val polymorphisms did not appear to be associated with the development of a childhood CNS tumour. In addition, children with the MTHFR 677 TT or RFC 80 AA genotypes were found to have a higher risk of death within 5 years of diagnosis compared to children with one or more MTHFR 677 C or RFC 80 G alleles, respectively (HR=5.52, 95%CI: 1.00-30.37, P=0.049 and HR=5.69, 95%CI: 1.38-23.51, P=0.016, respectively), after adjusting for other prognostic factors such as sex, age at diagnosis, period of diagnosis, and tumour grade. Conversely, children with the MTR 2756 AG or GG genotypes, or MTRR 66 AG or GG genotypes, were more likely to survive compared to those with the MTR 2756 AA or MTRR 66 AA genotypes, respectively (HR=0.21, 95%CI: 0.05-0.93, P=0.040 and HR=0.11, 95%CI: 0.02-0.53, P=0.006). Results presented in this thesis indicate that polymorphisms in folate-metabolising and GST genes may play a role in the aetiology and survival of childhood CNS tumours, and that this may vary depending on the histological sub-type of tumour.

La conversion par les thymidylate synthases du désoxyuridine 5'-monophosphate (dUMP) en désoxythymidine 5'-monophosphate (dTMP) est une des étapes clés de la biosynthèse des désoxyribonucléotides pyrimidiques. Chez les procaryotes (bactéries et archées), il existe deux familles de thymidylate synthases non homologues : ThyA et ThyX. La distribution phylogénétique quasi-exclusive des deux familles est complexe et ne suit pas les relations phylogéniques entre les organismes. Les protéines de ces deux familles ne présentent aucune similarité de séquence, de structure et leurs mécanismes et efficacités catalytiques diffèrent. Les travaux présentés dans cette thèse ont permis de montrer que l'activité de ThyX est essentielle à la survie de la bactérie Rhodobacter capsulatus en absence de thymidine exogène. A partir des résultats de tests de complémentation génétique et de la modélisation mathématique du métabolisme des folates nous avons pu mettre en évidence que seulement une faible activité dihydrofolate réductase était nécessaire à la survie des organismes utilisant ThyX. D'autre part, j'ai pu montrer par des approches génétiques et par l'étude statistique de plus de 400 génomes procaryotes que les organismes utilisant ThyX ont une réplication et un taux de croissance lents, et possèdent majoritairement un petit génome. Je propose un modèle dans lequel la faible activité catalytique de ThyX limite la réplication de l'ADN e! t par conséquent l'expansion du génome. Enfin, j'ai étudié le métabolisme des pyrimidines chez la bactérie pathogène humaine, Helicobacter pylori. J'ai pu en particulier mettre en évidence qu'en absence de voie de récupération de l'uracile, cette bactérie est capable de métaboliser un analogue toxique le 5-fluorouracile (5FU), utilisé comme anticancéreux. J'ai par ailleurs montré que l'orotate phosphoribosyltransférase de H. pylori était capable de restaurer l'auxotrophie pour l'uracile d'une souche d'Escherichia coli indiquant que cette enzyme pourrait être responsable de la sensibilité de H. pylori au 5FU. Les travaux présentés dans cette thèse ont permis une meilleure compréhension des conséquences physiologiques de l'utilisation de la thymidylate synthase ThyX. Ils ont notamment permis d'expliquer la distribution apparemment sporadique des deux familles de thymidylate synthases dans le monde procaryote.

<p>Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations.</p><p>The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. </p><p>Another example of stable resistance can also be seen in sulfonamide resistant <i>Streptococcus pyogenes</i>. Sequence determinations of the dihydropteroate synthase (<i>dhps</i>) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213.</p><p>The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (<i>hppk</i>). In the malaria parasite <i>Plasmodium falciparum</i> this enzyme is part of a polyfunctional entity, also encoding <i>dhps</i>. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to <i>P. falciparum</i>. Both are crucial for enzyme activity.</p><p>To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.</p>