In 2007, Bangladesh’s national strategy to reduce anemia included adolescents and newly married women as target groups for iron and folic acid (IFA) supplementation. This thesis is comprised of a pilot study and a double-blinded randomized controlled trial (RCT) aimed at providing evidence-based research to inform decision-making on periconceptional IFA programs in rural Bangladesh. Results from the pilot study indicate that women who marry during adolescence had significantly longer intervals to first pregnancy compared to adult brides; however, the time interval was not long enough to delay family formation beyond adolescence. Education, age at marriage and contraceptive use were significant factors for delaying time to first pregnancy. The RCT examined the effect of daily periconceptional iron (60 mg) and folic acid (400 μg) vs. folic acid (FA) on iron and folate indictors. Of 272 women, 37% were anemic (Hb <120 g/L), 13% had low plasma folate levels (≤10 nmol), 15% were iron deficient (plasma ferritin <12 μg/L or TfR>4.4 mg/L), 11% were iron deficient and anemic and 81% were estimated to have <500 mg of iron stores. Adolescents had significantly lower plasma ferritin and body iron stores vs. adults. Among 88 pregnant women (PW), an interaction between treatment and adherence was significantly associated with change in Hb (p=0.04) and anemia (p=0.05). During pregnancy, group differences for iron status were not significant. Among NPW (n=146), IFA reduced anemia by 80.0% (95% CI: 0.04, 1.00, p=0.05) and significantly improved change in plasma ferritin concentrations by level of adherence in a dose-response relationship (p=0.01). In both groups, mean plasma folate concentrations increased from 16.9 to31.8 nmol/L and the prevalence of low plasma folate concentrations (<10 nmol/L) was reduced from 14% to 8%. Results suggest that periconceptional iron supplementation along with adequate folic acid intake is efficacious in reducing the prevalence of anemia during pregnancy and improving body iron stores before pregnancy when adherence is high. The effectiveness of periconceptional IFA supplementation remains unclear. Further research needs to examine maternal and infant functional and health outcomes, ways to increase adherence and cost-effectiveness.

In 2007, Bangladesh’s national strategy to reduce anemia included adolescents and newly married women as target groups for iron and folic acid (IFA) supplementation. This thesis is comprised of a pilot study and a double-blinded randomized controlled trial (RCT) aimed at providing evidence-based research to inform decision-making on periconceptional IFA programs in rural Bangladesh. Results from the pilot study indicate that women who marry during adolescence had significantly longer intervals to first pregnancy compared to adult brides; however, the time interval was not long enough to delay family formation beyond adolescence. Education, age at marriage and contraceptive use were significant factors for delaying time to first pregnancy. The RCT examined the effect of daily periconceptional iron (60 mg) and folic acid (400 μg) vs. folic acid (FA) on iron and folate indictors. Of 272 women, 37% were anemic (Hb <120 g/L), 13% had low plasma folate levels (≤10 nmol), 15% were iron deficient (plasma ferritin <12 μg/L or TfR>4.4 mg/L), 11% were iron deficient and anemic and 81% were estimated to have <500 mg of iron stores. Adolescents had significantly lower plasma ferritin and body iron stores vs. adults. Among 88 pregnant women (PW), an interaction between treatment and adherence was significantly associated with change in Hb (p=0.04) and anemia (p=0.05). During pregnancy, group differences for iron status were not significant. Among NPW (n=146), IFA reduced anemia by 80.0% (95% CI: 0.04, 1.00, p=0.05) and significantly improved change in plasma ferritin concentrations by level of adherence in a dose-response relationship (p=0.01). In both groups, mean plasma folate concentrations increased from 16.9 to31.8 nmol/L and the prevalence of low plasma folate concentrations (<10 nmol/L) was reduced from 14% to 8%. Results suggest that periconceptional iron supplementation along with adequate folic acid intake is efficacious in reducing the prevalence of anemia during pregnancy and improving body iron stores before pregnancy when adherence is high. The effectiveness of periconceptional IFA supplementation remains unclear. Further research needs to examine maternal and infant functional and health outcomes, ways to increase adherence and cost-effectiveness.

Dry beans (Phaseolus vulgaris L.) contain high levels of folates. These compounds are essential vitamins and folate deficiencies may lead to a number of health problems. The objectives of this study were to examine the mode of inheritance of folate content and identify quantitative trait loci (QTL) associated with folate content in dry beans. Inheritance of folate content was studied in the F1 hybrids of one-way diallel crosses among Othello, AC Elk, Redhawk and Taylor, and an F2 population of the cross between Redhawk and Othello. Total folate content and 5 methyltetrahydrofolate (5MTHF) were measured twice within a one hour interval. Significant variation in folate content was observed among the parental genotypes, their F1 hybrids, and the F2 individuals of a cross between Redhawk and Othello, ranging from 147 to 345 µg/100g. Reductions in the 5MTHF content and total folate content values in the second measurement from samples were highly variable for all four parental lines ranging from 5 to 30% and 7 to 33%, respectively. A single marker QTL analysis identified at least three QTL for folate content in the F2 population. For the majority of identified QTL, dominance effects appeared to be the major genetic effect.

Maternal consumption of alcohol during pregnancy is associated with alterations in fetal development that negatively impact the offspring causing neurochemical and neurobehavioural dysfunction termed fetal alcohol spectrum disorders; the most severe outcome is fetal alcohol syndrome. Changes in maternal and fetal hypothalamic-pituitary-adrenal (HPA) axis activation, induction of cytochrome P450 2E1 (CYP2E1) enzyme activity and alterations in micronutrient status, including folate, following chronic ethanol exposure (CEE) are key contributors to the neuroendocrine and neurobehavioural effects observed in offspring. This study tested the following hypotheses: Maternal consumption of ethanol throughout pregnancy can: alter maternal and fetal HPA axis function and induce CYP2E1 enzyme activity in the third-trimester-equivalent; decrease folate status in the maternal-fetal unit, which can be mitigated by folic acid supplementation; and cause neurobehavioural deficits in offspring at low-moderate dose of maternal ethanol consumption. These hypotheses were tested in the guinea pig, a well established model of ethanol neurobehavioural teratogenicity. CEE had no effect on maternal HPA axis function at any gestational day (GD). Fetal cortisol was unaffected by CEE, but did increase with gestational age in both CEE and control. CEE increased maternal and GD 65 fetal liver CYP2E1 enzyme activity. Maternal supplementation with folic acid did not mitigate CEE fetal growth restriction, but did increase maternal red blood cell (RBC) folate at term. At term, maternal supplementation prevented the CEE-induced decrease in fetal liver folate, did not affect fetal RBC folate, and did not mitigate the nutritional-deficit-induced decrease in fetal hippocampal folate. Maternal consumption of 5% (v/v) ethanol decreased offspring birth weight, increased spontaneous locomotor activity, increased preference for ethanol, and delayed learning on day two of Morris water maze testing in young adult offspring. These data indicate that, in the guinea pig: there is a threshold blood ethanol concentration for HPA axis activation; CEE can induce CYP2E1 in the GD 65 fetus; folic acid supplementation is not protective in this model of CEE; and low-moderate CEE can cause neurobehavioural perturbations in offspring. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2012-05-31 14:38:44.391

Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA replication. The only known enzyme performing de novo dTMP synthesis is thymidylate synthase (TS). The goals of this thesis were to provide biochemical evidence for the existence of TS in chlamydiae, to investigate the mechanism by which the parasite obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts dUMP into dTXP. In vitro experiments conclusively establish these findings by the demonstration of TS activity in extracts prepared from host-free chlamydial reticulate bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial strains can synthesize folates de novo; however strains vary significantly in their ability to transport preformed folates from the host cell. A C. trachomatis gene which is capable of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned. Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open reading frame shows absence of sequence homology to known TS. Unique in vitro characteristics shared by the enzyme activities from both chlamydial extract and recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.

Since host cell-derived thymidine is not incorporated into Chlamydia trachomatis DNA, we hypothesized that chlamydiae must synthesize dTMF de novo for DNA replication. The only known enzyme performing de novo dTMP synthesis is thymidylate synthase (TS). The goals of this thesis were to provide biochemical evidence for the existence of TS in chlamydiae, to investigate the mechanism by which the parasite obtains folate, a necessary cofactor for TS, and to provisionally characterize chlamydial TS. Results of a series of in situ experiments using a mutant cell line as chlamydial host which is incapable of de novo dTMP synthesis suggest that C. trachomatis converts dUMP into dTXP. In vitro experiments conclusively establish these findings by the demonstration of TS activity in extracts prepared from host-free chlamydial reticulate bodies. Furthermore it was found that both sulfa-sensitive and sulfa-resistant chlamydial strains can synthesize folates de novo; however strains vary significantly in their ability to transport preformed folates from the host cell. A C. trachomatis gene which is capable of complementing thymidine auxotrophy in Escherichia coli deficient in TS was cloned. Auxotrophic E. coli containing the complementing chlamydial DNA sequence converts dUMP to dTMP, using methylene tetrahydrofolate as the cofactor. The complementing DNA fragment contains an open reading frame of 1587 bp. Surprisingly this open reading frame shows absence of sequence homology to known TS. Unique in vitro characteristics shared by the enzyme activities from both chlamydial extract and recombinant E. coli extract suggest that C. trachomatis might encode a novel TS.

Sugars have a profound impact on plant biology, acting as structural components, signalling molecules, and sources of energy. As such, the availability of sugars has important implications for plant growth and development. Sugar levels rise and fall as part of a daily cycle, while cues from other environmental stimuli are also in flux. As sessile organisms in an ever-changing environment, plants must integrate signals from multiple pathways in order to promote the appropriate developmental responses. To uncover pathways that interact with sugars during seedling development, a chemical screen was performed in search of compounds that modify responses to sucrose. This screen identified an interaction between the folate inhibitor sulfamethoxazole (SMX) and sucrose that resulted in changes to auxin signalling and distribution. Synergy between sucrose and SMX was used to explore the effect of metabolic cues on auxin signalling during hypocotyl elongation. A second line of investigation explored whether sucrose and folates influence root meristem activity. Sucrose induced hormone signalling pathways that promote cell division and differentiation. Treatment with SMX perturbed the effect of sucrose on hormone networks that mediate growth, and resulted in a loss of meristem integrity. This study highlights the influence of metabolism on hormone signalling at the root apex, and its role in maintaining balance of a complex signalling network that drives root growth. These studies characterise an interaction between metabolic pathways that is integrated with hormone signalling during plant development. Taken together, they highlight a mechanism through which plant growth might be regulated by metabolism.

Sugars have a profound impact on plant biology, acting as structural components, signalling molecules, and sources of energy. As such, the availability of sugars has important implications for plant growth and development. Sugar levels rise and fall as part of a daily cycle, while cues from other environmental stimuli are also in flux. As sessile organisms in an ever-changing environment, plants must integrate signals from multiple pathways in order to promote the appropriate developmental responses. To uncover pathways that interact with sugars during seedling development, a chemical screen was performed in search of compounds that modify responses to sucrose. This screen identified an interaction between the folate inhibitor sulfamethoxazole (SMX) and sucrose that resulted in changes to auxin signalling and distribution. Synergy between sucrose and SMX was used to explore the effect of metabolic cues on auxin signalling during hypocotyl elongation. A second line of investigation explored whether sucrose and folates influence root meristem activity. Sucrose induced hormone signalling pathways that promote cell division and differentiation. Treatment with SMX perturbed the effect of sucrose on hormone networks that mediate growth, and resulted in a loss of meristem integrity. This study highlights the influence of metabolism on hormone signalling at the root apex, and its role in maintaining balance of a complex signalling network that drives root growth. These studies characterise an interaction between metabolic pathways that is integrated with hormone signalling during plant development. Taken together, they highlight a mechanism through which plant growth might be regulated by metabolism.

O ácido fólico, a forma mais estável entre os folatos, é indicado como forma de prevenção sendo encontrado em forma de suplementação medicamentosa e alimentos fortificados, como leite e derivados. O folato pode reagir com a riboflavina singlete excitada, 1kq = 4,8&middot;1010 L&middot;mol-1&middot;s-1, como determinado por desativação da fluorescência do estado estacionário, e com a riboflavina triplete excitada, com uma reação um pouco mais lenta, 3kq= 4,8&middot;108 L&middot;mol-1&middot;s-1, como determinado por fotólise de pulso de laser e espectroscopia de absorção de transientes verificando-se que ambos os processos são competitivos e próximos ao limite de difusão. A preferência cinética de um a outro depende da matriz alimentar. O rendimento quântico para solução de riboflavina e de folato preparado em solvente aquoso e deuterado e em meio anaeróbico e aeróbico mostrou a prevalência do mecanismo fotoreacional do Tipo I. A voltametria cíclica apresentou um processo irreversível anódico de dois elétrons para o ácido fólico (E= 1,14 V vs. NHE). Os principais produtos da fotodegradação do folato sensibilizado pela riboflavina foram identificados por LC-IT-MS/MS como: 6-carboxipterina,p-aminobenzoil-L-ácido glutâmico e oxaziridina derivada do ácido fólico, como confirmando a desativação química do estado triplete excitado da riboflavina por transferência de elétrons com subseqüente clivagem oxidativa entre N(10) e C(9) no ácido fólico. / Folic acid, the most stable among folate, is recommended as prevention and it is found in supplementation and fortified foods such as milk and dairy products. The folate can react with singlet-excited state of riboflavin, 1kq= 4.8&middot;1010 L&middot;mol-1&middot;s-1, as determined by steady-state fluorescence quenching, and with triplet-excited state of riboflavin in a slower reaction with 3kq= 4.8&middot;108 L&middot;mol-1&middot;s-1, as determined by laser flash photolysis and transient absorption spectroscopy, verifying that both the processes are competitive and they are near limited diffusion. The kinetic preference depends on the matrix food. The quantum yield for the solution of riboflavin and folate prepared in aqueous and deuterated solvents and in anaerobic and aerobic medium showed the prevalence of the mechanism Type I. The cyclic voltammetry showed an irreversible two-electron anodic process for folate (E = 1.14 V vs. NHE). The main products of folate photodegradation sensitized by riboflavin were identified by LC-IT-MS/MS as: pterin-6-carboxylic acid, p-aminobenzoyl-L-glutamic acid and oxaziridine derivative of folic acid, as confirming chemical quenching of the triplet-excited state of riboflavin by electron transfer with subsequent oxidative cleavage between N(10) and C(9) in folic acid.

Brain cerebrospinal fluid (CSF) bulk flow is maintained thanks to a balance between CSF secretion from the choroid plexus and CSF absorption by arachnoid villi, where it drains into nearby blood vessels, thereby reaching the general blood circulation. Congenital hydrocephalus starts during the first trimester of pregnancy with impeded CSF flow, and consequent CSF build-up within the brain ventricles. This event is followed by CSF compositional changes, increased intracranial pressure, and, if untreated, brain damage and fetal death. Previous research has revealed a unique folate delivery system which serves the developing cerebral cortex. Abnormal folate provision due to impairment of this system was directly connected to a decrease in a CSF folate enzyme: 10-Formyl-Tetrahydrofolate dehydrogenase (FDH). In light of these findings, low FDH was linked with folate deficiency and the poor cortical development found in congenital hydrocephalus. In this context, investigations were carried out to ascertain whether folates in the presence and absence of the folate enzyme FDH are beneficial for fetal brain development. The current study also aims to investigate the FDH -folate delivery system in the fetal brain in order to understand its role in CNS development and its relationship to currently known folate transport mechanisms (FRα). Furthermore, we hypothesize that folates may prevent congenital hydrocephalus through a re-establishment of CSF drainage and flow circulation at the level of the arachnoid membrane/villi. This assumption implies that the leptomeninge arachnoid may also be dysfunctional in the hydrocephalic brain due to a variation in hydrocephalic CSF composition (folates). Finally, an overall metabolic pathway analysis of the constituents uniquely present in abnormal CSF, hence missing in normal CSF, and vice versa, was carried out to establish associations with suggested activated and inactivated biological processes during congenital hydrocephalus.