Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 205-218).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 186-200).

Thesis (M.S.)--Universidad de Puerto Rico, Recinto Universitario de Mayagüez, 2005. / Tables. Printout. Includes bibliographical references (leaves 68-73)

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

The adverse effects of maternal smoking on infant mortality and morbidity has been well documented in the literature. Maternal tobacco use is causally associated with fetal growth restriction and correlates negatively with folate intake and metabolism. Studies have examined the association between smoking and folate levels during pregnancy, but very few have assessed this relationship using objective and accurate measures of both variables. Furthermore, despite evidence of a causal association between smoking in pregnancy and intrauterine growth restriction, and a plausible relationship between tobacco use and low maternal folate which is required for optimal fetal growth, no experimental study has investigated the potential benefit of folic acid in mitigating the adverse effects of maternal smoking on fetal outcomes. The objectives of this study were to investigate the relationship between maternal smoking and folate levels and examine the efficacy of higher-strength folic acid supplementation, in combination with enrollment in a smoking cessation program, in promoting fetal body and brain growth. Our hypothesis was that women who smoke during pregnancy have lower peri-conceptional folic acid reserves than non-smoker pregnant women and that folic acid reserves will decrease with increasing cotinine level. Additionally, smoker pregnant women on higher-strength folic acid (4mg daily) in combination with smoking cessation programs will experience faster fetal brain growth and have infants with larger body size at birth compared to smokers on the standard dose of folic acid (0.8mg daily). Participants were pregnant women (smokers and non-smokers) who received antenatal care between 2010-2014 at the Genesis Clinic of Tampa, a community health center affiliated with the Department of Obstetrics and Gynecology of the University of South Florida (USF). They were aged 18-44 years and had a gestational age of less than 21 weeks at study enrollment. To determine the peri-conceptional folic acid reserves in smoking versus nonsmoking women during pregnancy and associated sociodemographic factors, baseline (crosssectional) data from a double-blinded randomized controlled trial were analyzed using Tobit regression models (n=496). Smoking information was assessed using salivary cotinine, a sensitive and specific tobacco use biomarker. Folate reserve was measured using red blood cell folate. To investigate the efficacy of higher-strength folic acid on fetal body and brain size, baseline and follow-up data from pregnant smokers enrolled in the randomized controlled trial were utilized (n=345). All primary analyses of the clinical trial data were conducted on a modified intention-to-treat basis and included participants who completed the trial with an observed endpoint, irrespective of compliance to protocol. Multilevel modeling, linear regression, and log-binomial regression analyses were conducted. A significant inverse association between salivary cotinine level and periconceptional red blood cell folate concentration was found among pregnant women in the early to midpregnancy period. Smokers on high-dose folate during pregnancy had infants with a 140.38g higher birth weight than infants of their counterparts on standard dose folate (P =0.047). Mothers who received higher strength folate had a 31.0% lower risk of having babies with SGA compared to their mothers on the standard-dose (adjusted relative risk-ARR=0.69, 95% CI: 0.46–1.03; (P =0.073)). High-dose folate had no significant effect on the intrauterine rate of growth in head circumference, and head circumference and brain weight at birth in our trial sample. However, the brain-body ratio of infants of mothers who received high-dose treatment was 0.33 percentage-point lower than that for infants of mothers who received the standard dose of folate (P =0.044). Higher strength folic acid supplementation in pregnant women who smoke might be a cost-effective and safe option to improve birth outcomes and reduce low birth weight and SGA associated infant morbidity and mortality. Future studies with larger sample sizes and diverse populations are indicated to confirm or refute the results of this study. Randomized controlled trials starting during the preconception period and with follow-up until delivery are warranted, to identify the most folate-sensitive period of fetal growth and determine the optimal dose of folic acid supplement. Further research investigating several pathways through which the effects of prenatal smoking on adverse birth outcomes can be mitigated is needed.

A Fissura Oral (FO) é uma malformação craniofacial comum na espécie humana e sua etiologia é complexa com aspectos genéticos e ambientais envolvidos na sua formação. Por ser uma malformação de prevalência variável, estudos de associação em populações distintas são necessários, principalmente em populações heterogêneas como no Brasil. A suplementação com ácido fólico está envolvida na redução do risco de recorrência para algumas malformações, mas a natureza da reação entre a ingestão do ácido fólico, a interação entre os genes da rota metabólica e o seu efeito nas concentrações de folato é pouco caracterizada. Além disso, existem poucos estudos envolvendo um grande número de genes e a suplementação com ácido fólico a longo prazo. O objetivo desse trabalho foi estudar o papel dos genes MSX1 e IRF6 e região 8q24 em indivíduos com fissuras orais não sindrômicas de diferentes regiões do Brasil e analisar o efeito da suplementação com ácido fólico e dos polimorfismos nos genes da rota metabólica do folato nos níveis de folato séricos e eritrocitários. Nossos resultados mostram associação positiva entre o alelo 4 do polimorfismo de repetição CA (MSX1) e FO, alelo A da variante rs987525 (8q24) foi associado com FL/P e o haplótipo G/A (rs2235371/rs642961) do gene IRF6 associado com o aumento do risco para FL/P. Dos 23 genes da rota metabólica do ácido fólico estudados, 5 (FPGS, FOLR1, FOLR2, SHMTI e MTHFR) foram relacionados com os níveis de folato sérico e eritrocitário. As variantes rs7033913 (FPGS), rs11235462 (FOLR1) e rs2276048 (FOLR2) foram associadas com os níveis de folato sérico após suplementação. Os polimorfismos rs2168781 e rs2461837 (SHMT1) foram relacionados com os níveis de folato eritrocitário basal e o rs1801131(MTHFR) com os níveis de folato eritrocitário durante a suplementação. Conhecer a etiologia das fissuras orais e entender os efeitos da suplementação e de variantes dos genes da rota do folato nos níveis basais de folato é essencial tanto para auxiliar no manejo clínico através de uma medicina personalizada quanto para aconselhamento genético. / Oral cleft (OC) is a common craniofacial malformation. The etiology is complex and involves genetic and environmental factors. OC have a variable prevalence and association studies are needed in different populations, especially in heterogeneous populations as the Brazilian. Folic acid supplementation reduce the recurrence risk for some malformation, but the reaction between folic acid intake, the interaction between genes of metabolic pathway and effect on folate concentrations is poorly characterized. Furthermore, there are few studies with a large number of genes and long-term folic acid supplementation. The aim was to analyze the role of MSX1 and IRF6 gene and 8q24 region in individuals with non-syndromic oral clefts in different regions of Brazil and to analyze the effect of folic acid supplementation in folate pathway genes and correlate to levels of serum and red blood cell (RBC) folate. Our results have shown a positive association between the CA repeat polymorphism 4 allele (MSX1) and OC, between rs987525 A allele (8q24) and CL/P and the G/A haplotype (rs2235371 / rs642961) of IRF6 associated with increased risk of CL/P. The 23 folate pathway genes studied, 5 (FPGS, FOLR1, FOLR2, SHMTI and MTHFR) were correlated to serum and red blood cell (RBC) folate levels. The variants rs7033913 (FPGS), rs11235462 (FOLR1) and rs2276048 (FOLR2) were associated to serum folate levels after supplementation. Polymorphisms in SHMT1 (rs2168781 and rs2461837) were associated with basal RBC folate while MTHFR (rs1801131) were associated with RBC folate levels during supplementation. Understanding of oral cleft etiology and folate gene pathway will assist clinic management and genetic counseling since folate is involved in important biologic processes.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-03T18:51:56Z No. of bitstreams: 1 RenatoLeal Associação de polimorfismos nos genes....pdf: 1146092 bytes, checksum: dc3d04e8dbf3baaa5becc9279b8cc89f (MD5) / Made available in DSpace on 2012-08-03T18:51:56Z (GMT). No. of bitstreams: 1 RenatoLeal Associação de polimorfismos nos genes....pdf: 1146092 bytes, checksum: dc3d04e8dbf3baaa5becc9279b8cc89f (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Níveis elevados de homocisteína (Hcy) no plasma têm sido associados à presença de polimorfismo no gene da Metilenotetrahidrofolato Redutase (MTHFR), Metionina Sintase (MS) e Cistationina beta Sintetase (CBS) ou relacionados com quadros nutricionais, que podem alterar uma das duas vias metabólicas que envolvem os precursores do metabolismo da Hcy, remetilação ou trans-sulfuração. Desta forma, os polimorfismos gênicos associados ao metabolismo da vitamina B12, folato podem conferir níveis elevados de homocisteína, assim como diminuição nos níveis de folato e vitamina B12 plasmático, produzindo complicações durante a gravidez bem como, alterações na metilação do DNA e conseqüentemente evoluir para doenças cardíacas, vasculares, câncer, síndrome de Down, defeitos de fechamento de tubo neural e baixo peso ao nascer. Considerando o metabolismo anormal de folato, vitamina B12 e homocisteína às mutações do gene de enzimas envolvidas no metabolismo destes podem ser marcadores de fatores de risco materno. O objetivo deste trabalho foi investigar em 240 mães e 240 recém-nascidos (RNs) associações entre os polimorfismos presentes em genes responsáveis no metabolismo da homocisteína e seus substratos caracterizando de forma hematológica, bioquímica e molecular. Foram investigados os polimorfismos C677T e A1298C no gene da MTHFR; A2756G no gene da metionina sintase (MS) e a inserção 844ins68 no gene da enzima cistationina beta sintetase (CBS), utilizando a técnica de PCR – RFLP. Os níveis séricos de homocisteína, vitamina B12 e folatos foram determinados pela técnica de quimioluminescência. As freqüências alélicas dos polimorfismos entre 191 mães e 226 recém-nascidos foram 27,7%. e 25,6%, respectivamente, para o polimorfismo C677T da MTHFR; 14,0% e 14,6%, respectivamente, para o polimorfismo A1298C da MTHFR; 23,9% e 24,5%, respectivamente, para o polimorfismo A2756G da MS; e 22,3% e 20,2%, respectivamente, para a inserção 844ins68 da CBS. Os níveis de Folatos e Vitamina B12 entre mães normais e portadores para os diversos genótipos estudados não demonstraram diferenças estatisticamente significativas. Porém, para os RNs diferenças estatisticamente significantes foram encontradas para os portadores do polimorfismo A2756G e níveis de vitamina B12 (p=0,0023). Diferenças estatísticas significantes foram encontradas entre mães normais e portadores para o polimorfismo A1298C e níveis de homocisteína (p=0,0002). Também foram encontradas diferenças estatisticamente significantes para a ocorrência dos polimorfismos C667T da MTHFR e valores de leucócitos (p=0,0482); A2756G da MS e valores de leucócitos (p=0,0168) e de Ht (p=0,0103); e 844ins68 da CBS e valores de Hb (p=0,0432) no grupo de mães.Para o grupo de RNs os dados hematológicos apresentaram significância estatística apenas entre os valores de plaquetas (p=0,0247) e HCM (p=0,0278) e o polimorfismo A1298C. Os resultados deste estudo contribuem para o entendimento de fatores genéticos relacionados com nutrientes indicando a grande importância de apontar os fatores de risco que possam ser prevenidos e definir estratégias intervencionistas que reduzam os riscos maternos, bem como os cuidados que devem ser tomados durante o acompanhamento pediátrico relacionado aos níveis séricos de Vitamina B12 e folatos e a ocorrência de alterações genéticas de enzimas, uma vez que a presença destas pode conduzir a associação de riscos relacionados a doenças genéticas. / High levels of homocysteine (Hcy) in plasma have been associated with the presence of the polymorphism in the gene MTHFR, Methionine Synthase (MS) and Cystathionine beta Synthase (CBS) or related to nutrition tables, which can alter one of two metabolic pathways involving the precursors of metabolism of Hcy, remethylation or trans-sulphuration. Thus the gene polymorphisms associated with the metabolism of vitamin B12, folate can give high levels of homocysteine, as well as reduction in the levels of folate and vitamin B12 plasma, producing complications during pregnancy as well as changes in methylation of DNA and thus evolve into heart disease, vascular, cancer, Down syndrome, defects in the neural tube closure and low birth weight. Considering the abnormal metabolism of folate, vitamin B12 and homocysteine to mutations of the gene of enzymes involved in the metabolism of these can be markers of risk factors mother. The research aimed to investigate in 240 mothers and 240 newborns (NBs) associations between polymorphisms in these genes responsible for the metabolism of homocysteine and their way of characterizing substrates hematological, biochemical and molecular. We investigated the C677T and A1298C polymorphisms in the gene MTHFR; A2756G in the gene of methionine synthase (MS) and 844ins68 insertion in the gene of the enzyme cystathionine beta synthase (CBS), using the technique of PCR - RFLP. Serum levels of homocysteine, vitamin B12 and folate were determined by the technique of chemiluminescences. Allelic frequencies of polymorphisms between 191 mothers and 226 newborns were 27.7%. and 25.6% respectively, for the MTHFR C677T polymorphism, 14.0% and 14.6% respectively in the A1298C of MTHFR polymorphism, 23.9% and 24.5% respectively in the A2756G polymorphism of MS, and 22.3% and 20.2% respectively in the integration of CBS 844ins68. The levels of Folate and Vitamin B12 between mothers and normal carriers for the various genotypes studied showed no statistically significant differences. But for NBs statistically significant differences were found to carry the A2756G polymorphism and levels of vitamin B12 (p = 0.0023). Significant statistical differences were found between normal and mothers bearers for the polymorphism A1298C and levels of homocysteine (p=0.0002).Were also found statistically significant differences in the occurrence of the MTHFR C667T polymorphism and values of white blood cells (p = 0.0482); A2756G of MS and values of white blood cells (p = 0.0168) and Ht (p = 0.0103) and 844ins68 of CBS and values of Hb (p = 0.0432) in the group of mothers. For the group of NBs data hematological statistically significant only between the values of platelets (p = 0.0247) and HCM (p = 0.0278) and A1298C polymorphism. The results of this study contribute to the understanding of genetic factors related nutrients indicating the great importance of pointing the risk factors that can be prevented and interventionist strategies that reduce risks maternal, and the care that must be taken during the pediatric related to the serum levels of vitamin B12 and folate and the occurrence of genetic modification of enzymes, since the presence of these can lead to association of risks related to genetic diseases.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In order to eliminate the problems related to deficient intake of vitamins folate class, many food products are being subjected to the enrichment process. Among them, all types of wheat flour must be enriched with folic acid. Therefore, it is evident the need of analytical methods for routine able to effectively determine the levels of folic acid in enriched matrix. The objective of this work was the development and validation of a rapid method for determination of folic acid added to wheat flour, using the technique of high performance liquid chromatography with UV detection. Folic acid was separated using a C8 column. Was used for elution gradient mobile phase with water acidified with glacial acetic acid (pH 2.8) and acetonitrile. Detection was performed with a diode array detector employing a wavelength of 290 nm. External standard was used for quantification. Vitamin matrix was extracted with aqueous potassium hydroxide, 0.1 mol L-1 and aqueous sodium tetraborate 0.04 mol L-1 pH 8.5, followed by cleaning step of the extract of the sample through SPE using SAX cartridges. The method was efficient, with recovery values between 96 and 99%, RSD from 1.3 to 3.8% and LOD and LOQ of 0.28 and 0.937 mg kg-1, respectively. In the analysis of real samples of five brands tested, only one brand presented concentration of folic acid above the minimum required by law. / Com o objetivo de suprimir os problemas relacionados à deficiência de ingesta de vitaminas da classe dos folatos, muitos produtos alimentícios estão sendo submetidos ao processo de enriquecimento. Entre eles, todos tipos de farinha de trigo devem ser enriquecidos com ácido fólico. Portanto, fica evidenciada a necessidade de métodos analíticos para a rotina capazes de determinar com eficácia os níveis de ácido fólico na matriz enriquecida. O objetivo deste trabalho foi o desenvolvimento e validação de um método rápido para determinação de ácido fólico adicionado a farinha de trigo, através da técnica de cromatografia líquida de alta eficiência com detector espectrofotométrico. O ácido fólico foi separado utilizando-se coluna de C8. Utilizou-se eluição por gradiente na fase móvel, com água acidificada com ácido acético glacial (pH 2,8) e acetonitrila. A detecção foi efetuada com detector por arranjo de diodos empregando o comprimento de onda de 290 nm. Foi utilizada padronização externa para quantificação. A vitamina foi extraída da matriz com solução aquosa de hidróxido de potássio 0,1 mol L-1 e solução aquosa de tetraborato de sódio 0,04 mol L-1 de pH 8,5; seguida por etapa de limpeza do extrato da amostra através de SPE, utilizando cartuchos de SAX. O método mostrou-se eficiente, com valores de recuperação entre 96 e 99%, RSD de 1,3 a 3,8% e LOD e LOQ de 0,28 e 0,937 mg kg-1, respectivamente. Nas análises das amostras reais, das cinco marcas analisadas, somente uma marca apresentou concentração de ácido fólico acima do mínimo exigido pela legislação.

Nanoparticulas magnéticas (NPMs) de óxido de ferro tem sido amplamente utilizadas em diversas áreas da biotecnologia e biomedicina, tais como no tratamento de câncer, na entrega controlada de fármacos e como agentes de contraste em imagem por ressonância magnética. O intuito deste trabalho foi sintetizar nanopartículas magnéticas com magnetização de saturação intensificadas via processo do poliol modificado e modificar sua superfície afim de promover a biocompatibilização dos sistemas. Além da funcionalização de nanopartículas previamente biocompatibilizadas utilizando dois métodos: via ligação amida com moléculas de ácido fólico (AF) e com a encapsulação das nanopartículas com quitosana. A modificação de superfície deu-se via procedimentos de lavagem de superfícies, adição e/ou troca de ligantes utilizando moléculas de ácido ?- aminocapróico (EACA), aminopropiltrimetoxisilano (APTMS) e ácido dimercaptosuccínico (DMSA). Através da microscopia eletrônica de transmissão (TEM), foi obtido que as nanopartículas magnéticas de magnetita obtiveram um diâmetro médio de 8 nm, em uma estreita distribuição de tamanho. A difração de raios-X (DRX) indicou a formação de magnetita em todos os sistemas em que o método do poliol modificado foi utilizado. As medidas de espectroscopia vibracional na região do infravermelho (FTIR) evidenciaram a presença de modos de vibração relacionados às macromoléculas e compostos inorgânicos utilizados na modificação de superfície das nanopartículas magnéticas e/ou funcionalização. A TEM das diferentes modificações de superfície mostram a formação de aglomerados dependendo da molécula utilizada. Os estudos de estabilidade coloidal foram necessários para que o meio biológico fosse simulado para uma possível aplicação destes sistemas como carreadores para tratamento via magnetohipertermia ou entrega controlada de NPMs para tratamento de câncer. A nanopartícula recoberta com DMSA apresentou melhores resultados de estabilidade coloidal. Com os sistemas funcionalizados com ácido fólico, o procedimento via ligação com carbodiimida na presença de NHS demonstrou ser eficaz na formação de ligação amida, confirmada por FTIR e quantificação de ligantes. A funcionalização com quitosana necessita de alguns ajustes, visto ser um novo procedimento, porém alguns sistemas em que foi utilizado o método da gelificação iônica possuíram bons resultados de nanocápsulas de quitosana formadas com nanopartículas biocompatibilizadas em seu interior. / Magnetite, iron oxide, is a type of magnetic nanoparticles (NPMs) that is a widely adopted in several areas of biotechnology and biomedicine, such as in the treatment of cancer, controlled delivery of drugs and as contrast agents in magnetic resonance imaging. The purpose of this work is to synthesize magnetic nanoparticles with enhanced saturation magnetization via modified polyol process and modify its surface to promote a biocompatibilization in these systems. In addition, there was the aim to functionalize nanoparticles with modificate surfaces, using two methods: via amide bonding with folic acid molecules (AF) and encapsulation of nanoparticles with chitosan. Surface modification was done via surface washing, addition and / or exchange ligands using ?-aminocaproic acid (EACA), aminopropyltrimethoxysilane (APTMS) and dimercaptosuccinic acid (DMSA) molecules. By transmission electron microscopy (TEM), it was obtained that the magnetite nanoparticles had an average diameter of 8 nm, in a narrow size distribution. X-ray diffraction (XRD) indicated formation of magnetite in all systems where modified polyol method was used. Infrared spectroscopy (FTIR) showed the presence of vibration modes related to macromolecules and inorganic compounds used in the surface modification of magnetic nanoparticles and / or functionalization. The TEM of different surface modifications showed the formation of agglomerates, depending on the molecule used. Colloidal stability studies were necessary to simulate a biological medium for a possible application of these systems as carriers for treatment via magnetohyperthermia or controlled delivery of NPMs for cancer treatment. Nanoparticles coated with DMSA showed better colloidal stability results. With folic acid functionalized systems, the procedure via carbodiimide linkage in the presence of NHS had been shown to be effective in FTIR-confirmed amide bond formation and ligand quantification. The functionalization with chitosan requires some adjustments, since it was a new procedure, however some systems using the ionic gelation method had good results of chitosan nanocapsules formed with biocompatibilized nanoparticles in structure.

Made available in DSpace on 2014-10-09T12:34:45Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:15Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP