Rozprawy doktorskie na temat „Protein and folic acid”

MTHFD is a folate-dependent trifunctional protein comprised of three activities: N$ sp5$,N$ sp{10}$-methylenetetrahydrofolate dehydrogenase, N$ sp5$,N$ sp{10}$-methenyltetrahydrofolate cyclohydrolase and N$ sp{10}$-formyltetrahydrofolate synthetase. The enzymes catalyse the sequential interconversion of tetrahydrofolate derivatives required for purine, methionine and thymidylate synthesis. A Chinese hamster ovary cell line, reported to have reduced cyclohydrolase activity, was studied to characterize the nature of its mutation.
Enzymatic assays showed reduced activities of all three enzymes. Immunoblotting and immunoprecipitation of radiolabelled cell extracts indicated that the gene product was greatly reduced or absent in the mutant. Southern analysis showed no differences between normal and mutant cells, indicating that the defect was not due to a major gene rearrangement. RNA analysis, by Northern blotting and by RNA amplification using the polymerase chain reaction, showed that a mRNA for MTHFD of normal size was present in mutant cells. These results suggest that the mutation is post-transcriptional and that it disrupts the synthesis of MTHFD.

Thesis (M.S.)--West Virginia University, 2003.
Title from document title page. Document formatted into pages; contains vi, 91 p. : ill. Includes abstract. Includes bibliographical references (p. 87-91).

The objective of this study was to determine Thai women's nutrition awareness of folic acid and its role in the prevention of neural tube defects. Three hundred and two Thai women between 18-45 years completed a 30 itemized survey. There was a significant lack of nutrition awareness of folic acid. Many women (n= 274; 91.9%) had not heard of neural tube defects (NTD) and only 8.1 % (n=24) had heard of spina bifida/NTD. More than 50% (n=176) were unaware of NTD. Only 11.6% of the women (n=35) thought consuming vitamins during pregnancy would reduce the risk of birth defects. More than 80% of the women reported the best time to take folic acid supplement was during pregnancy. Less than 20% of the women indicated taking folic acid supplement before or after pregnancy. In this population, nutrition awareness of folic acid was minimal.
Department of Family and Consumer Sciences

La regulation de la voie de biosynthese de l'acide folique chez bacillus subtilis a ete analysee tant au niveau genetique que enzymatique. Tout d'abord, le premier enzyme de la voie de biosynthese, gtp cyclohydrolase i, a ete surexprime dans b. Subtilis et purifie. Nous avons determine les constantes cinetiques de cet enzyme et recherche de possibles inhibiteurs de l'activite enzymatique. Ces travaux nous ont permi de montrer que la dephosphorylation de l'intermediaire dihydroneopterine triphosphate en dihydroneopterine monophosphate pouvait se produire spontanement in vitro par une simple reaction chimique en presence de cations divalents. D'autre part, nous avons isole et sequence la partie 3' de l'operon acide folique chez bacillus subtilis. Trois nouveaux genes ont ainsi ete decouverts (orf3, orf4, et lyss), ils font parti integrante de l'unite de transcription acide folique. La presence des deux phases ouvertes de lecture, orf3 et orf4, suggere une fonction regulatrice pour ces deux genes a un niveau qui reste a determiner. Nous avons montre que l'operon acide folique est regule de deux facons differentes. D'une part, l'expression depend fortement de la phase de croissance, et d'autre part, nous avons montre l'existence d'un mecanisme de regulation par attenuation de transcription en aval du gene trpg. Nous avons aussi prepare des anticorps diriges contre tous les enzymes de la voie de biosynthese de l'acide folique. A l'aide de ces anticorps, nous avons demontre l'existence de la regulation de traduction du gene trpg par l'intermediaire de la proteine regulatrice trap (tryptophan rna-binding attenuation protein). Nous presentons aussi certaines caracteristiques pour les autres enzymes de la voie de biosynthese. Certains parametres genetiques et enzymatiques qui regulent la synthese d'acide folique dans bacillus subtilis sont decrits dans cette these

Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state.

We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy.

NAD-Dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a mitochondrial folate-dependent enzyme apparently expressed only in transformed mammalian cells and tissues containing undifferentiated cells. The cloning of both the cDNA and the gene encoding this bifunctional enzyme have led to a better understanding of the regulation of its expression in normal and transformed cells. Southern blot analyses revealed that the gene has a similar organization in normal and transformed cells and no major rearrangement or amplification were detected. The gene encodes two messenger RNAs which differ only by the length of their 3$ sp prime$-untranslated region and are derived by the use of alternative polyadenylation signals. The detection of transcripts in transformed cells and not in normal adult tissues suggests that the gene is primarily regulated at the transcriptional level. The promoter region of the gene contains several putative cis-regulatory elements which may have an important role in the differential expression of this folate-dependent enzyme.

The kinetic relationship between the activities of multifunctional enzymes from folate-mediated one-carbon metabolism were examined. Formyltetrahydrofolate dehydrogenase catalyzes the main disposal reaction for excess one-carbon units produced in the liver. The enzyme, which catalyzes also a hydrolytic reaction, was purified from porcine liver and a radioactive assay was developed to measure both activities simultaneously. These and other kinetic measurements established that the dehydrogenase and hydrolase are kinetically independent activities of a single type of polypeptide. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is found in all transformed mammalian cells and catalyzes sequential reactions with channeling of the metabolic intermediate. However, these activities are kinetically independent in contrast to similar activities of the NADP-dependent trifunctional enzyme found in all eukaryotic cells. These properties explain the observation that only the NAD-dependent enzyme conjugate can catalyze the conversion of formyl- to methylenetetrahydrofolate in vitro.

4 pp.
Before they may even know they are pregnant; women’s bodies and their level of folate play a critical role in preventing certain birth defects, specifically neural tube defects (NTDs). NTDs are birth defects in the brain, spinal cord, or spine. Considered ‘one of the most important public health discoveries of this century’ is that daily supplemental folic acid taken before becoming pregnant significantly reduces the risk of NTDs (1). In 1998, the United States made sweeping efforts that fortified cereal grains with folic acid to ensure all Americans consume adequate amounts of this vitamin. So what exactly is folate? What are the functions of this vitamin? What foods have high levels of folate and what is the recommended daily intake? This article will answer these questions and will go on to explain folic acid fortification and the impact fortification has had on the incidence of NTDs in Arizona.

Thesis (Ph. D.)--University of Texas at Austin, 2001.
"Fmet" after tRNA in title is superscript. Vita. Includes bibliographical references (leaves 102-119). Available also in a digital version from Dissertation Abstracts.

The main aim of this study is to synthesize theranostic nanoparticles (NPs) that will drastically increase the diagnostics and therapeutic efficacy for cancer. In this research, we had prepared the NPs which constitute carbon dots (CDs), the imaging agent, Folic acid, the targeting agent, and Doxorubicin (DOX) or Gemcitabine (GEM) as the chemotherapy agents. The prepared NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. The spectroscopy, ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy, and Fourier transform-infrared spectroscopy (FT-IR), were used to confirm the successful fabrication of these complexes. Through UV-vis analysis, the drug loading capacity (DLC) and drug loading efficiency (DLE) of the complexes were determined. The noncovalent series had a higher DLE of about 83% while the covalent series showed higher DLC, 70% on average indicating high drug content. The in-vitro pH-dependent drug release shows that the noncovalent FA-CDs-DOX and the covalent FA-CDs-GEM series release more drugs into the cancer cells (pH of 5.0) than into healthy normal (pH of 7.4). The sizes of NPs were measure around 2-5 nm with Dynamic light Scattering (DLS). The toxicity of CDs, CDs-drug, and FA-CDs-drug on MDA-MB468 breast cancer cell was tested through the methylthiazolytetrazolium (MTT) assay and found that the FA bonded NPs exhibited strong therapeutic efficacy. More pharmaceutical data towards the cancer cells are investigated by our research collaborators – the pharmaceutical department at ETSU and Xavier University at Louisiana.

This study aims to prepare theranostic nanoparticles (NPs) that are expected to increase cancer diagnostics and therapeutic efficacy. We prepared the NPs constituting carbon dots (CDs) as an imaging agent, folic acid as a targeting agent, doxorubicin (DOX), or gemcitabine (GEM) as chemotherapy agents. The NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. Through ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform-infrared spectroscopy, the fabrication of these NPs was confirmed. It was discovered that the high drug loading efficiency is the noncovalent series while the high drug loading capacity is the covalent series The in-vitro pH-dependent drug release data indicate the NPs release more drugs at around pH 5.0 than at pH 7.4. The NPs sizes are between 2-5 nm. The Cell viability was investigated using the Alamar Blue assay and the three NPs complexes exhibited strong therapeutic efficacy against MDA-MB-468 breast cancer cells as compared with CDs-drug.

Background Expert guidelines recommend low-risk women to consume a daily multivitamin supplement containing 400 µg of folic acid (FA) to prevent the occurrence of neural tube defects. This study assessed the knowledge, attitude and practice of physicians regarding FA recommendations, status and health outcomes during pregnancy since physicians play an essential role in promoting appropriate FA intake. Methods A cross-sectional survey of a sample of physicians practicing in the National Capital Region was self-administered in 2018-2019. Results Approximately 70% of physicians were not familiar with the most recent guidelines and 55% of them most often recommend a 1000 µg-FA supplement. A high level of willingness to recommend a supplement containing 400 µg-FA was reported by almost all physicians. Conclusion While most physicians would not feel comfortable recommending a supplement that is not in line with the most recent evidence-based guidelines, educational programs targeted to physicians are needed to improve their knowledge, attitude and practice.

Rice is an important dietary grain but may be difficult to fortify with water-soluble vitamins due to the losses incurred during processing and preparation. Edible coatings can offer reasonable protection against folate processing losses in fortified rice, in particular pectin (Shrestha, 2003). However, pectin, an indigestible fibre, may entrap or bind added folate, decreasing its absorption efficiency. Healthy volunteers (n=26, 18-39 yrs) received oral 400??g [13C5]PteGlu doses in three separate test meals in randomized cross-over trials as follows: 1) aqueous 2) 200g white rice and 3) 200g of pectin-coated rice premix. A plasma AUC0-8 was conducted (0, 1, 2, 5 and 8 hrs postprandial). Subjects followed a low folate basal diet (112??12 ??g/day) - verified using L.casei microbiological assay - during the AUC and for 24 hours prior. Optimisation of the pectin-coated rice premix gave folic acid coating and cooking losses of 33.5% and 15.5% respectively. The mean test dose error per 400??g folic acid was ?? 26 ??g. Single- or tri- enzyme extraction of fortified rice extracts did not significantly increase the mean assayable folate content compared to the mean folic acid content. The levels of plasma [13C5]5-methyl-THF, [13C5]PteGlu and 5-methyl-THF were quantified using a validated HPLC-tandem MS method. The calibration curves indicated good response linearity in the 0-100 ng/mL range (R2>0.9978). Inter- and intra-assay variation of 5-methyl-THF (100 ng/mL) was 6.9% (n=6) and 5.2% (n=4) respectively. The mean recovery of 5, 20 and 50 ng/mL 5-methyl-THF in spiked plasma extracts was 98.6 ?? 8.7%, 89.3 ?? 2.8% and 92.6 ?? 3.7% (n=3) respectively. Standard Reference Material-1846, infant formula (129??28 ??g/100g) was measured at 110 ?? 15 ??g folic acid/100g. The relative bioavailability of the folic acid in meals 2 and 3 was measured by comparing their [13C5]5-methyl-THF AUC???s relative to meal 1. The relative bioavailabilities (Mean % ?? CI) of meals 2 and 3 were 86.5 ?? 4.6 % and 68.7 ?? 5.4 % respectively. It appears the pectin coat moderately reduces short-term folic acid bioavailability. These studies define the basis for calculating the amount of folic acid to be added to rice so that an adequate amount can be absorbed after coating and cooking losses. Pectin coatings may be a useful means of increasing the folate status of populations that rely heavily on rice as a staple.

Carbon dots (CDs) have attracted much attention as an excellent gene/drug delivery and biological imaging agent for early cancer theranostics. In this study, we prepared two series of nanoparticles (NPs), which are composed of (CDs) with a targeting agent, folic acid (FA), and a chemotherapeutic agent Doxorubicin (Dox). All the NPs and their intermediates were characterized using ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy, and Fourier transform-infrared spectroscopy (FT-IR). The drug loading capacity (DLC) and drug loading efficiency (DLE) of two series of FA-CDs-Dox were assessed using UV-vis absorption spectroscopy at the wavelength of 485 nm. Both showed good DLE and DLC results when compared to literature data. In addition, the cumulative release property of Dox from the FA-CDs-Dox complexes were investigated in a pH solution of 7.4.

Neural tube defects (NTD) are among the most common birth defects and the leading cause of infant mortality. NTDs occur when the neural tube fails to close during early fetal development. The two most common types of NTD are spina bifida and anencephaly. NTDs result in lifelong complications like musculoskeletal deformities and loss of strength. The etiology of NTDs is complex and involves still unclear environmental and genetic factors. However, one of the well-established risk factors of NTDs is folic acid deficiency. The prevalence of NTDs can be lowered by an adequate intake of folic acid in the periconceptual period. In 1996, the Food and Drug Administration mandated that 140 micrograms of folic acid be added to 100 grams of bleached grain products with the goal of reducing the prevalence of NTDs. In the years following this fortification mandate, studies have shown that blood folate levels have more than doubled on average, that there are demographic and socioeconomic disparities in blood folate gains and that NTD rates have declined. However, no studies after the mandate have examined changes in blood folate distribution and differences in NTD prevalence by a wide range of theoretically and biologically relevant risk factors after the mandate. Using a nationally representative sample of non-institutionalized women of reproductive age, I investigated the relationship between the fortification mandate and blood folate levels. I also examined changes in the range/spread of blood folate distribution after the mandate. Using data on US live births from 45 states and the District of Colombia, the second study examined whether (1) the disparities in blood folate changes translate into differences in NTD prevalence and (2) NTD risk factors moderate the association between the mandate and NTD prevalence,. The final study explored potential unintended impacts of the mandate on birth weight, low birth weight, very low birth weight, high birth weight, and physician-diagnosed developmental delay, asthma and allergies. For this study, I employed samples from the Natality files and the National Survey for Children's Health. The cumulative results of my research suggested that the mandate was associated with increases in blood folate concentration, with greater increases in higher quantiles of the blood folate distribution and that the spread of blood folate distribution after the mandate widened. Additionally, the mandate was associated with a decrease in the prevalence of NTDs in the entire US population although the impact of the mandate was moderated by race/ethnicity, maternal educational attainment, acute illness during pregnancy and infant region of birth. Furthermore, the mandate was associated with other unintended infant and child health outcomes such as average birth weight increases in the population and increased risks of developmental delay among six year olds. This research is the first of its kind to examine changes in the spread of blood folate distribution after the mandate and whether NTD risk factors moderate the association between the mandate and NTD prevalence. It is also the first study to explore potential impacts of the actual mandate (not prenatal folic acid supplementation) on other unintended infant and child health outcomes. The results add significantly to our understanding of the effects of the mandate and have important implications for health care providers, women of reproductive age and policy makers because of the potentially increased risk of developmental delay among children and the increasing disparity in blood folate concentrations after the mandate.

Methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylene THF to 5-methyl THF, the carbon donor for the methylation of homocysteine to methionine. Patients with severe MTHFR deficiency (MRD) have neurologic abnormalities while a milder form (a thermolabile MTHFR variant) has been shown to be associated with coronary artery disease (CAD). Ten MRD patients, with reduced or non-detectable activity, were studied to characterize the nature of the mutation. Southern, Northern and Western analysis did not reveal any defects in the patients. These results suggest that the mutations may be minor insertions/deletions or single base substitutions that affect catalytic activity. Single strand conformation polymorphism (SSCP) analysis was used to detect base substitutions; 3 RFLPs were identified with this protocol. One was in the coding region (SphI) while the other two were in the 3$ sp prime$ untranslated region (MaeIII and MnlI). A difference in frequency of the SphI RFLP was found between control subjects and a small sample of CAD patients whose homocysteine levels were greater than the 99th percentile.

Folate is a B-vitamin required for cell growth and division, and its metabolism is linked to vitamin B12 (B12). Food fortification with folic acid (FA) has improved folate status but approximately 5% of Canadian adults, including pregnant women, are B12 deficient. This is concerning because an association between gestational exposure to high maternal folate and low B12 status and greater adiposity and insulin resistance in children has been reported. My thesis examined the effect of developmental exposure to maternal FA/B12 imbalance on programming of liver gene expression in adult offspring using an animal model. Female C57BL/6 mice were fed a high FA/adequate B12 (HFA+B12), high FA/no B12 (HFA-B12), or control diet 6 weeks prior to mating and through pregnancy and lactation. At weaning, offspring mice from each maternal diet group were randomly assigned to receive the control diet or a Western diet (45% fat, 35% carbohydrate) for 20 weeks (n=6 male mice/group) or for 40 weeks (n=6 female mice/group). Serum folate and B12 concentrations were quantified by microbiological assays. Relative mRNA expression of key enzymes in methyl metabolism in liver from adult offspring was quantified by real-time PCR. Male offspring mice from dams fed the HFA-B12 diet had lower Cbs and Mthfr mRNA expression and this was unaffected by post weaning diet. Male offspring mice fed the Western diet had higher Mtr mRNA expression compared to control-fed offspring mice, regardless of maternal diet. Female offspring from dams fed the HFA-B12 diet had lower Mtr mRNA expression and this was not affected by post weaning diet. Moreover, female offspring from dams fed the HFA-B12 diet had higher Mthfr mRNA expression when they were fed the Western diet. No effect of maternal and post weaning diets was observed for serum folate and B12 concentrations. In summary, developmental exposure to maternal FA/B12 imbalance was found to program expression of genes involved in folate and methionine metabolism in the liver of adult offspring mice. The functional consequences of this effect requires further investigation in order to consider B12 screening of pregnant women and to inform the debate on whether B12 fortification should be considered.
Land and Food Systems, Faculty of
Graduate

Background: Folic acid (FA) is added to grain products in Canada to reduce the incidence of neural tube defects. There have been concerns that FA fortification may have adverse effects on the population. L-5-methyltetrahydrofolate (L-5-methyl-THF) is an alternative form of folate and may be safer than FA. However, L-5-methyl-THF has poor stability relative to FA limiting its use as a food fortificant. Microencapsulation of L-5-methyl-THF in a polyglycerol monostearate coating could improve its stability in food matrices. Objective: To determine if microencapsulated FA and L-5-methyl-THF, either in capsule form or in skim milk powder, increases plasma folate concentrations over 8 hours, to the same extent, compared to free-form FA and L-5-methyl-THF. Methods: In a repeated-measures crossover design, participants (n=15) were randomly administered seven treatments as single doses. The doses were based on the molar equivalent of 400 μg of FA. Treatments included: placebo, FA, microencapsulated FA, L-5-methyl-THF, microencapsulated L-5-methyl-THF, microencapsulated FA in milk powder, and microencapsulated L-5-methyl-THF in milk powder. Blood was collected at baseline and at frequent intervals over the 8-hour test period. Plasma folate was quantified at each time point and corrected to baseline values. Area under the curve(AUC)was calculated for each treatment and differences between treatments were evaluated using repeated-measures ANOVA. Results: The AUC values (± SEM) for all six folate treatments were significantly greater than the placebo (P<0.05). AUC for L-5-methyl-THF was greater than FA (347±35 vs. 181±12 8h.nmol/L; P<0.001). The AUC for microencapsulated FA was not different than FA (222±12 vs. 181±14 8h.nmol/L; P=0.12). However, the AUC was less for microencapsulated L-5-methyl-THF than L-5-methyl-THF (147±17 vs 347±35 8h.nmol/L; P<0.001). The AUC for microencapsulated FA in milk powder was less than microencapsulated FA (120±11 vs. 222±12 8h.nmol/L; P<0.001) but microencapsulated L-5-methyl-THF in milk powder was not different than microencapsulated L-5-methyl-THF (178±14 vs 147±17 8h.nmol/L; P=0.284). Conclusions: L-5-methyl-THF may have greater short-term bioavailability than FA. Microencapsulation had no effect on FA but decreased the bioavailability of L-5-methyl-THF. Adding microencapsulated FA to a skim milk powder decreased the bioavailability however there was no effect on microencapsulated L-5-methyl-THF.

[EN] The present PhD thesis, entitled "Encapsulation of folic acid in silica porous supports: a nutritional and technological approach", focuses on the development of new smart systems for the controlled delivery of folic acid for nutritional applications. The first part of the thesis shows folic acid encapsulation in polyamines-functionalized silica porous matrices from a nutritional approach. The first part evaluates not only the influence of the loading method and the type of silica support employed (MCM-41, SBA-15, UVM-7 and Hollow Silica) on the efficacy of folic acid encapsulation, but also the influence of the morphology and porous system on the folic acid delivery profile from different supports. Folic acid release studies from different supports with various pH values have demonstrated that the designed systems are capable of smartly modulating the delivery of the folic acid dependent on the pH of the medium (inhibition of the release at an acidic pH -stomach-, controlled release at a neutral pH -intestine-). This capacity makes these developed delivery systems an excellent alternative to direct fortification to successfully modulate the bioaccessibility of folic acid along the gastrointestinal tract. The stability of the supports during an in vitro digestive process was evaluated, and demonstrated that not only small particles can be attacked during the digestion process, but also the functionalization with organic molecules, which act as molecular gates, prevents this attack. Finally, the cell viability studies carried out with four different cell lines revealed that neither the supports nor their degradation products caused any specific toxicity during the in vitro digestive process. The second part evaluates the influence of adding different silica supports to two food matrices: gelatin gels and yoghurts. This technological approach enabled us to know that the capacity of these smart systems to deliver folic acid in a controlled manner during an in vitro digestive process is mantained even after their incorporation in stirred yoghurt. The effect of the matrices on the gel's physical properties depends on the particle size, functionalization and concentration. Finally, this thesis tested that the optimization of folic acid loading, achieved in the first part of the thesis, allowed the fortification of yoghurt with 100% of the recommended daily allowance of folic acid with a very low amount of the system. This fortification affected neither the physico-chemical properties of the yoghurt, nor bacterial viability. In summary, it was concluded that the present thesis globally deals with folic acid encapsulation in silica porous matrices to be used in nutritional and food applications, which include the optimization of loading, release studies at diferent pH, in vitro digestions, stability studies of the employed matrixes, biocompatibility studies, and studies into the influence of their addition to food matrixes. The obtained results positively exhibit that the developed smart folic acid delivery systems open up a new way of fortifying food without endangering the properties of the food to which they are added.
[ES] La presente tesis doctoral que lleva por título "Encapsulación de ácido fólico en soportes porosos de óxido de silicio: una aproximación nutricional y tecnológica" está centrada en el desarrollo de nuevos sistemas inteligentes de liberación controlada de ácido fólico para aplicaciones nutricionales. La primera parte de la tesis muestra la encapsulación de ácido fólico en matrices porosas de óxido de silicio funcionalizadas con poliaminas desde una aproximación nutricional. En ella se ha evaluado la influencia del método de cargado y del tipo de soporte de óxido de silicio utilizado (MCM-41, SBA-15, UVM-7 y Hollow Silica) en la eficacia de encapsulación de ácido fólico. En esta primera parte, también se ha evaluado la influencia de la morfología y el sistema de poros de los diferentes soportes en el perfil de liberación del ácido fólico desde los mismos. Los estudios de liberación de ácido fólico desde los diferentes soportes a diferentes valores de pH han demostrado que los sistemas diseñados son capaces de modular inteligentemente la liberación de ácido fólico en función del pH del medio (inhibición de la liberación a pH ácido -estómago-, liberación controlada a pH neutro -intestino-). Esta capacidad convierte a los sistemas liberación desarrollados en una alternativa excelente a la fortificación directa para modular exitosamente la bioaccesibilidad del ácido fólico a lo largo del tracto gastrointestinal. Por otra parte, se ha evaluado la estabilidad de los soportes durante un proceso de digestión in vitro, demostrando que si bien algunos soportes pueden ser atacados durante la digestión, la funcionalización con moléculas que actúan como puertas moleculares previene este ataque. Por último, los estudios de viabilidad celular llevados a cabo en cuatro tipos de líneas celulares demuestran que ni los soportes, ni los productos de degradación de los mismos durante el proceso de digestión in vitro promueven ningún tipo de toxicidad inespecífica. En la segunda parte se ha evaluado la influencia de la adición de diferentes soportes de óxido de silicio a dos matrices alimentarias, geles de gelatina y yogures. Esta aproximación tecnológica ha permitido conocer que la capacidad de estos sistemas inteligentes para liberar controladamente el ácido fólico a lo largo de un proceso de digestión in vitro se mantiene incluso tras su incorporación en yogures batidos. Por otra parte, se ha comprobado que el efecto de las matrices sobre las propiedades físicas de los geles, es dependiente tanto del tamaño de las partículas, como de su funcionalización y concentración. Por último, se ha comprobado que debido a la optimización del cargado de ácido fólico alcanzada en la primera parte de la tesis, se puede lograr una fortificación de un yogur con el 100% de la cantidad diaria recomendada de ácido fólico con una cantidad tan pequeña de sistema que ni las propiedades físico-químicas de un yogur, ni la viabilidad bacteriana se ven comprometidas. En resumen, se puede concluir que la presente tesis ha abordado de una manera global la encapsulación de ácido fólico en matrices porosas de óxido de silicio para ser utilizados en aplicaciones nutricionales y alimentarias incluyendo estudios de optimización de cargado, estudios de liberación en función del pH, digestiones in vitro, estudios de estabilidad de las matrices utilizadas, estudios de biocompatibilidad, así como estudios de la influencia de la adición de estos sistemas inteligentes en matrices alimentarias. Los resultados obtenidos han puesto de manifiesto que los sistemas inteligentes de liberación de ácido fólico desarrollados abren la puerta a una nueva manera de fortificar los alimentos sin comprometer sus características.
[CAT] La present tesi doctoral, que porta per títol "Encapsulació d'àcid fòlic en suports porosos d'òxid de silici: una aproximació nutricional i tecnològica" està centrada en el desenvolupament de nous sistemes intel·ligents de lliberació controlada d'àcid fòlic per a aplicacions nutricionals. La primera part de la tesi mostra l'encapsulació d'àcid fòlic en matrius poroses d'òxid de silici funcionalitzades amb poliamines des d'una aproximació nutricional. En esta part, s'ha avaluat la influència del mètode de carrega i del tipus de suport d'òxid de silici que s'ha emprat (MCM-41, SBA-15, UVM-7 i Hollow Silica) en l'eficàcia de l'encapsulació d'àcid fòlic. En esta primera part, també s'ha avaluat la influència de la morfologia i el sistema de porus dels diferents suports en el perfil d'alliberament de l'àcid fòlic des dels mateixos. Els estudis d'alliberament d'àcid fòlic des dels diferents suports a diferents valors de pH han demostrat que els sistemes dissenyats són capaços de modular intel¿ligentment l'alliberament d'àcid fòlic en funció del pH del medi (inhibició de l'alliberament a pH àcid -estómac-, alliberament controlat a pH neutre -intestí-). Esta capacitat fa dels sistemes desenvolupats una excel·lent alternativa a la fortificació directa per a modular amb èxit la bioaccessibilitat de l'àcid fòlic a través del tracte gastrointestinal. D'altra banda, s'ha avaluat l'estabilitat dels suports en un procés de digestió in vitro, demostrant que mentre que les partícules menudes poden ser atacades durant la digestió, la funcionalització amb molècules orgàniques que actuen com a portes moleculars prevé aquest atac. Per últim, els estudis de viabilitat cel·llular duts a terme en quatre tipus de línies cel·lulars demostren que ni els soports, ni els productes de degradació dels mateixos durant el procés de digestió in vitro promouen cap tipus de toxicitat inespecífica. En la segona part, s'ha avaluat la influència de l'addició de diferents sopors d'òxid de silici a dos matrius alimentàries, gels de gelatina i iogurts. Esta aproximació tecnològica ha permés conéixer que la capacitat d'aquests sistemes intel·ligents per alliberar controladament àcid fòlic durant un procés de digestió in vitro es manté fins i tot després de ser incorporats en iogurts batuts. D'altra banda, s'ha comprovat que l'efecte de les matrius sobre les propietats físiques dels gels, és dependent tant de la grandària de les partícules, com de la seua funcionalització i concentració. Per últim, s'ha comprovat que a causa de l'optimització del carregat d'acid fòlic alcançada en la primera part de la tesi, es pot aconseguir una fortificació d'un iogurt amb el 100% de la quantitat diària recomanada d'àcid fòlic amb una quantitat tan baixa del sistema que ni les propietats físico-químiques d'un iogurt, ni la viabilitat bacteriana se'n veuen compromeses. En resum, es pot concluir que en la present tesi s'ha abordat d'una manera global l'encapsulació d'àcid fòlic en matrius poroses d'òxid de silici per a ser utilitzades en aplicacions nutricionals i alimentàries, que inclouen estudis d'optimització de carregat, estudis d'alliberament en funció del pH, digestions in vitro, estudis d'estabilitat de les matrius utilitzades, estudis de biocompatibilitat, així com estudis de la influència de l'addició d'aquests sistemes en matrius alimentàries. Els resultats obtesos han posat de manifest de forma positiva que els sistemes intel·ligents d'àcid fòlic que s'han desenvolupat obrin una porta a una nova manera de fortificar els aliments sense comprometre les seues caracterísitiques.
Pérez Esteve, E. (2015). ENCAPSULATION OF FOLIC ACID IN MESOPOROUS SILICA SUPPORTS: A NUTRITIONAL AND TECHNOLOGICAL APPROACH [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58613
TESIS
Premiado

Many Mexican women consume inadequate amounts of folic acid. Fortification of the corn tortilla could be an effective way to help increase the folic acid levels among the Mexican population. Previous studies have shown significant folic acid losses in fortified tortilla dough (masa) as it is held before baking. This loss in folic acid could be due to degradation by lactic acid bacteria naturally present in the masa. The microflora of traditionally made nixtamalized corn masa from six tortilla mills in Guadalajara, Mexico were isolated and characterized, and their effect on folic acid content was evaluated. Isolated bacteria were identified using whole cell fatty acid analysis via MIDI Inc.'s Microbial Identification System. Twenty-two unique bacterial species were identified, primarily belonging to the Streptococcus and Lactobacillus genera. Lactic acid bacteria were the predominant microorganisms, with counts ranging from 10^4 to 10^7 cfu/g. Aerobic mesophilic bacteria also ranged from 10^4 to 10^7 cfu/g. Coliforms and yeasts and mold were present at significantly lower levels. Masa samples, prepared from sterile fortified corn masa flour, were inoculated with a cocktail of bacteria isolated from the individual mills. Control samples were prepared using sterile media. Inoculated and uninoculated control samples were held at 56°C for 0, 3 and 6 hours, mimicking the elevated temperature of the masa as it is held before baking. The loss of folic acid in the sterile control was not different from the inoculated samples, indicating that the decline in folic acid is not due to bacteria present in the masa, but appears to be a chemical degradation related to time and temperature.

Proteins often depend on a-helices for binding to other biomacromolecules. Reversible control of a-helix stability was accomplished in previous studies by incorporating a photoisomerisable azobenzene cross-linker into peptides, subsequently enabling the optical control of DNA-protein interactions. This approach was extended in this study to include protein-protein and protein-RNA interactions. One of the primary regulatory components in apoptosis signalling is the antiapoptotic protein Bcl-xL which interacts with the a-helical BH3 domain of the Bak protein. The Rev/RRE interaction is crucially involved in the life cycle of Human Immunodeficiency Virus. These interactions were targeted by designing peptides based on the BH3 domain of Bak and on the RNA-binding domain of Rev these peptides are activated by external light pulses after the incorporation of the cross-linker. The ability to control cross-linker conformation and hence peptide secondary structure was demonstrated by CD and UV/Vis spectroscopy. The binding to the target structure and complex disruption was determined in the dark-adapted and irradiated states using fluorescence based assays. Structural studies using NMR spectroscopy demonstrated that the alkylated peptides bind to the same part of the target molecule as the wild-type peptide, regardless of their structure. Moreover, one of the BH3 domain-based peptides and the light-controllable transcription factor PhotoMyoD were modified with protein transduction domains to enable future in vivo studies. Overall, this work opens the possibility to interfere reversibly and specifically with protein-protein and protein-RNA interactions and to study and modulate cellular function by optical control.

Background: In addition to a diet including fortified dietary staples, the use of prenatal multivitamin supplements among women has been shown, in some cases, to lead to excessive micronutrient intake levels for nutrients such as folic acid (FA). It was therefore hypothesized that prenatal vitamin supplementation, in addition to a standard Canadian diet, would place pregnant Canadians at risk for excessive FA intake. With little available research on the potential negative impact of excess FA intake in pregnancy, it was further proposed that high concentrations of FA may adversely affect placental health and function. Thus, the aim of the current study was three-fold: 1) To determine micronutrient intake in a large Canadian cohort of pregnant women; 2) To determine the extent to which FA intake in this cohort may exceed the tolerable upper intake level (UL) after prenatal supplementation; and 3) To determine the effects of excessive FA exposure on placental health and function in vitro. Methodology: Second trimester 3-day food records of pregnant women (N=216) were analyzed for micronutrient intake using ESHA Food ProcessorTM. Nutrient intake values were compared to established Dietary Reference Intake (DRI) values. In a series of experiments, the effects of exogenous folic acid (2-4000 ng/ml) on placental health and function were examined in two placental cell lines [HTR-8/SVneo (N=3) and BeWo (N=3)], and a human placenta explant model (N=6). Following a 48-hour incubation period, the effects of excessive folic acid exposure on placenta cell proliferation, viability, and apoptosis were determined, along with evaluation of placenta cell function via cell invasion and B-hCG hormone release assays. Results: Through dietary sources alone, most pregnant women studied were consuming adequate levels of most micronutrients. However the majority of examined women (>50%) demonstrated a risk of dietary inadequacy for vitamin D, vitamin E, folate, and iron. In the examined cohort, 83% of study participants reported prenatal supplement usage. In vitro exposure of human placenta cells and explants to excessive FA concentrations resulted in no significant differences in cellular proliferation, apoptosis, invasion, or B-hCG hormone production. However, decreased cell viability was observed in BeWo cells at increased FA concentrations (200-2000 ng/mL). Conclusion: Food sources alone do not appear to provide women in Canada with adequate intake of all micronutrients recommended for a healthy pregnancy. Though a prenatal supplement containing FA may be necessary for most women, current FA levels in many prenatal supplements may lead to excessive FA intake above the established UL. Yet, as measured in this study, high FA concentrations do not seem to adversely affect most primary indicators of placental cell health or function.

Mestrado em Ciência e Engenharia de Materiais
In this work, biohybrid structures (BHS) based on streptavidin and pentavalent biotinylated dendritic glycopolymer were fabricated through non-covalent streptavidin-biotin conjugation and further functionalized with the folic acid for the selective targeting to tumor cells. Characterization of the hydrodynamic size and zeta potential of the structures together with the cytotoxicity studies were performed to assess the physical properties and biocompatibility of the structures. It was shown that stable BHS with defined sizes can be obtained due to a controlled polyassociation reaction. Furthermore, BHS are biocompatible and do not cause toxicity to the cells under the given experimental conditions. Next, the effect of the folic acid ligand on the targeting function towards folate receptor expressing cancer cells was studied in flow cytometry experiments. For this purpose the amount of folic acid, the length of the PEG-chain / spacer, and the type of protein used were evaluated, as well as the impact of purification strategies. Additionally, the cell uptake mechanism was also briefly looked into.
Neste trabalho foram fabricadas estruturas bio-híbridas (BHS) à base de glicopolímeros dendríticos biotinilados e estreptavidina as quais foram posteriormente funcionalizadas com o ácido fólico com vista ao reconhecimento preferencial por células tumorais. A estratégia de fabricação recorreu, em ambos os casos, a interações não-covalentes. A caracterização das estruturas no que respeita ao seu tamanho hidrodinâmico e potencial zeta, em conjunto com os estudos de citotoxicidade, foram realizados no sentido de avaliar as propriedades físicas, assim como a biocompatibilidade das estruturas. Os resultados obtidos demonstraram que BHS estáveis, com tamanhos definidos, podem ser obtidas através de poliassociação controlada. Além disso, nas condições experimentais consideradas, as BHS apresentam biocompatibilidade e não apresentam toxicidade para as células. Seguidamente, avaliou-se a influência do ligando do ácido fólico sobre a segmentação de células cancerígenas ricas em receptores de folato por citometria de fluxo. Para tal, foi avaliado o efeito da quantidade de ácido fólico, do comprimento da cadeia de PEG (espaçador) e da concentração de BHS, tal como o impacto das estratégias de purificação. Adicionalmente foi ainda estudado o mecanismo de absorção celular.

The aim of this study was to investigate the influence of folate on DNA stability and DNA methylation in three different systems. (i) Folate deficiency significantly increased DNA strand breakage and uracil misincorporation in human colon epithelial cells and lymphocytes in vitro. DNA methylation was decreased in both cell types when depleted of folate. (ii) Rats fed a folate-free diet for 10 weeks were moderately folate deficient measured as a 25-50% decrease in plasma, red blood cell or hepatic folate concentrations and a 20% rise in plasma homocysteine, a functional marker for folate status. Folate deficiency specifically increased DNA strand breakage in isolated rat lymphocytes and colonocytes and misincorporated uracil in lymphocytes. (iii) In a human colorectal cancer case-control study, no significant associations were observed between plasma or red blood cell folate, plasma vitamin B12 or homocysteine and lymphocyte DNA strand breakage, misincorporated uracil or DNA methylation. Homozygosity for the C677T polymorphism in the methylenetetrahydrofolate reductase gene was associated with a decreased risk of colorectal cancer, increased plasma homocysteine levels and decreased plasma folate levels compared with heterozygous or wild-type individuals. Plasma vitamin B12 level was associated with a reduction in cancer risk, whereas homocysteine was associated with increased risk of colorectal cancer. DNA strand breakage was associated with increased risk, whereas uracil misincorporation was protective. Red blood cell folate concentrations were not associated with risk. Old age (>65 years) and male gender were associated with increased risk of colorectal cancer.