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.

Background: Inverse associations have been reported between homocysteine concentrations and poor cognitive performance in several cross-sectional studies of healthy elderly subjects. Folate supplementation with or without vitamins B-12 and B-6 is an effective means of lowering homocysteine concentrations. Mood disturbances, from mild mood changes to clinical depression, are common in older populations. Several studies have shown that depressed people have lower levels of folate and vitamin B-12 and higher levels of homocysteine than non-depressed people. Improvement of mood has been reported in depressed people following supplementation with folic acid. Clinical trials are required to determine if lowering homocysteine concentration with vitamins improves cognitive function and/or mood in healthy elderly participants. Objective: The primary aim of this research project was to carry-out a 2 year randomised, double-blind, placebo-controlled trial to determine if a supplement containing folate (1mg L-Mefolinic acid), vitamin B-12 (500(mu)g) and vitamin B-6 (10mg) improves scores or prevents decline on tests of cognition in a group of healthy older people ([greater than or equal to]̲ 65 years) with a plasma homocysteine concentration [greater than or equal to]̲13 (mu)mol/L. A second aim of this study was to determine if homocysteine lowering vitamins improved scores on tests of mood in this group. Methods: Four hundred and sixty-five individuals, aged 65 and over, were recruited from Dunedin and surrounds, and asked to attend a screening clinic and provide a fasting blood sample. Two-hundred and seventy-six volunteers with a plasma homocysteine concentration [greater than or equal to]13(mu)mol/L were randomised to take either a combination of 1mg L-Mefolinic acid, 500(mu)g vitamin B-12 and 10mg vitamin B-6 or placebo for 2 years. A battery of cognitive tests and indices of mood was administered at baseline, one year, and two years. A fasting blood sample was collected at baseline and every six months thereafter. Results: From baseline to 6 months of the intervention, homocysteine concentrations decreased by 37.5%, from 16.7 to 10.5 (mu)mol/L in the vitamin supplemented group and then plateaued. In the vitamin supplemented group there was a 181% increase in red blood cell folate concentration from a mean of 977 to 2752 nmol/L, and a 90.1% increase in plasma vitamin B-12 (from a mean 283 to 538 (mu)mol/L) over the study period of two years. In the vitamin supplemented group there was a trend to poorer performance on almost all tests of cognition compared to placebo group. The vitamin group was 8% slower on Part B of the Reitan Trail Making Test, a test of speeded attention, mental tracking, visual search and mental flexibility (p=0.009). The vitamin group scored significantly lower on tests of short-term recall, Weschler Paragraphs (p=0.03) after 2 years, and the Rey Auditory Verbal Learning Test ((p=0.04) after one year, than the placebo group. There was no difference in mood score by treatment in this largely non-depressed group. Conclusion: These results suggest a detrimental effect of high dose homocysteine lowering vitamin supplements on cognitive function in healthy older people. These results need to be confirmed in other randomised controlled trials.

From conception the dynamic balance between nutritional and activity factors play a role in the accumulation of risk for future disease. Maternal nutrient balance and the subsequent dietary pattern of the family set the path for the growth and development of the individual and therefore also for their offspring. There is strong evidence from studies in India that mothers who have a low vitamin B12 status, but high folate, will have children with higher adiposity and more cardiovascular risk factors than those with adequate B12. The B12 status is closely linked to the dietary pattern particularly the consumption of red meat which has a high B12 content. In New Zealand there are an increasing number of Indian migrants. Vegetarianism is also practiced by an increasing number including young women. In addition, there is a high rate (up to 60%) of unplanned pregnancies in New Zealand. In the 1997 New Zealand National Nutrition Survey (NNS97) report, vitamin B12 intake appeared adequate for the New Zealand population and breakfast cereals were reported as one major dietary source of B12. Cereals in New Zealand however, were not fortified with B12 and there was an error in the FOODfile™ data entries for B12 in some cereals. The raw data of reported B12 intakes in the 24-hour diet recall (24HDR) of NNS97 was reanalysed at the individual level by subtracting the B12 derived from breakfast cereals and applying the 2005 revised estimated average requirement (EAR) value. The possible prevalence of B12 insufficiency was 2.4 times that originally reported by the NNS97, translating into a prevalence of up to 27% of the population sampled. This analysis was limited as it was not adjusted for day-to-day variance or to the New Zealand population. This apparently high prevalence of risk for inadequate B12 intake in the surveyed individuals required confirmation that the B12 intake from 24HDR and also a 7-day diet diary (7DDD) was a valid assessment of B12 status. The group of particular interest is women of childbearing age (18-50y) with a range of eating patterns. Thirty eight women aged 19-48y; 12 non-red-meat-eaters (5 Indians vs. 7 non-Indians) and 26 red-meat-eaters (1 Indian vs. 25 non-Indians) participated in this validation study. Anthropometry and hand-to-foot bioelectrical impedance (BIA) were measured on the same day as a 24HDR was recorded. Fasting serum lipids, glucose, haematological parameters, and serum B12, holotranscobalamin II (holo-TC II, a specific B12 biomarker), and folate concentrations were measured. Foods eaten and time spent in physical activity during the following 7 days were extracted from 7DDD and 7-day physical activity diary (7DPAD). There was no significant correlation between dietary intake (24HDR or 7DDD) and biomarkers for B12 status. Indians reported lower mean daily B12 intakes in 7DDD than non-Indians (1.6 vs. 4.5 μg/day, p<0.001) and this was confirmed by Indians’ significantly low serum B12 (203 vs. 383 pmol/L, p=0.04) and holo-TC II (35 vs. 72 pmol/L, p=0.02) concentrations compared to non-Indians. A similar pattern was found between non-red-meat-eaters and red-meat-eaters in daily B12 intake in 7DDD (2.3 vs. 4.8 μg/day, p<0.001) and in B12 biomarkers (serum B12, 263 vs. 397 pmol/L, p=0.01; holo-TC II, 43 vs. 77 pmol/L, p<0.005). Non-red-meat-eaters reported significantly higher daily folate intake in 7DDD (359 vs. 260 μg/day, p=0.01) than red-meat-eaters but no significant difference was found in serum folate concentration between these groups (29 vs. 24 pmol/L, p=0.10). Indians/non-red-meat-eaters also reported lower daily protein intake and higher percentage of total energy from carbohydrate in 7DDD compared to non-Indians/red-meat-eaters but total reported energy intake tended to be under-reported and physical activity over-reported when assessed against estimated basal metabolic rate (BMR). Body composition varied by dietary pattern. Indians/non-red-meat-eaters had higher body fat percentage (BF %) and weaker grip strength than non-Indians/red-meat-eaters. In addition, Indians had a significantly higher waist-to-hip ratio (WHR) than non-Indians. Overall, the whole group reported that they were inactive. The median time spent in moderate, high and maximal intensity activities was only 19 minutes a day, which did not meet the NZ guideline for adults of 30 minutes a day. In this small study nutrient analysis of diet by 24HDR or 7DDD, was not a reliable or accurate way to assess B12 insufficiency. Questions about dietary patterns such as “do you eat red meat”, and taking ethnicity into account could more easily identify the at risk population. Supplementation and/or fortification of B12 should be considered before pregnancy.

<p>In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the <i>cob</i> operon and the <i>btuB</i> gene in <i>Salmonella typhimurium</i>. The <i>cob</i> operon encodes most of the 25 genes required for the <i>de novo</i> synthesis of vitamin B12, and the <i>butB</i> gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in <i>Salmonella typhimurium</i>. The regulation by vitamin B12 of the <i>cob</i> operon and the <i>btuB</i> gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.</p>

In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the cob operon and the btuB gene in Salmonella typhimurium. The cob operon encodes most of the 25 genes required for the de novo synthesis of vitamin B12, and the butB gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in Salmonella typhimurium. The regulation by vitamin B12 of the cob operon and the btuB gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.

La bacteria Myxococcus xanthus, responde a la luz azul produciendo carotenoides que la protegen del daño fotooxidativo. En oscuridad la transcripción de la mayoría de los genes implicados en la síntesis de estos pigmentos es reprimida por las proteínas CarA y CarH, parálogas y funcionalmente redundantes. Ambas contienen un dominio N-terminal y otro C-terminal de unión al DNA y a cobalaminas respectivamente. Sorprendentemente, CarH, pero no CarA, depende de B12 para llevar a cabo su función represora. En este trabajo se ha demostrado que CarH y su homólogo en Thermus thermophilus (TtCarH) son fotorreceptores que utilizan la adenosilcobalmina (forma de cobalamina) como grupo cromóforo. La luz desmantela la oligomerización de estas proteínas y su unión al DNA inducidas por la adenosilcobalamina, lo que activa la expresión de los genes implicados en la carotenogénesis. Este hallazgo ha sido publicado en la prestigiosa revista PNAS (Ortiz-Guerrero et al. 2011). / The bacteria Myxocccus xanthus responds to light by producing carotenoid, protecting itself against photooxidative damage. In the dark, most of the genes involved in carotenoid synthesis are repressed by the paralogous and functionally redundant proteins CarA and CarH. Both of them contain a DNA-binding N-terminal domain and a cobalamin-binding C-terminal domain. Surprisingly, CarH, but not CarA, repressive depends on B12. In this work we showed that CarH and its homologous in Thermus thermophilus (TtCarH) are photoreceptors in which adenosylcobalamin plays the role of a chromophore. Light dismantles CarH and TtCarH adenosylcobalamine-induced oligomerization and DNA binding, activating structural genes involved in carotenoid. This finding has been reported in the prestigious journal PNAS (Ortiz-Guerrero et al. 2011)

Thèse d'exercice : Médecine : Nancy 1 : 2006. / Titre provenant de l'écran-titre.

Thèse de doctorat : Médecine : Nancy 1 : 2000. / 2000NAN11315. Titre provenant de l'écran-titre.

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 112-114). Also available on the World Wide Web.

Thèse (Ph. D.)--Université Laval, 2009 . / Titre de l'écran-titre (visionné le 16 juin 2009). Bibliogr.