Elevated total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and neurocognitive disease such as dementia. The B vitamins folate and B12 are the main de terminants of tHcy. tHcy concentration can also be affected by mutations in genes coding for receptors, enzymes and transporters important in the metabolism of Hcy. This thesis focuses on mutations in the genes for folate receptor-alpha and methylenetetrahydrofolate reductase (MTHFR) and the effect they have on tHcy concentrations. Six novel mutations in the gene for folate receptor-alpha were described in Paper I. Taken together they exist in a population with a prevalence of approximately 1% and thus are not unusual. There may be an association of –69dupA and –18C>T to tHcy but for the 25-bp deletion, –856C>T, –921T>C and –1043G>A there is probably no association to tHcy. Mutation screening was continued and four additional mutations, 1314G>A, 1816delC, 1841G>A and 1928C>T, were described in Paper II. The prevalences for the heterozygotes were between 0.5% and 13% in an elderly population. There was no significant difference in prevalence between the elderly subjects and patients with dementia. The 1816(–)-allele and the 1841A-allele were in complete linkage and the haplotype 1816(–)-1841A may possibly have a tHcy raising effect. The 1314G>A and 1928C>T mutations had no association to tHcy. The genotype prevalences and haplotype frequencies of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms were determined in a population sample of Swedish children and adolescents (Paper III). The MTHFR 677T-allele was associated with increased tHcy concentrations in both children and adolescents. A small elevating effect of the 1298C-allele and a small lowering effect of the 1793A-allele could be shown. In an epidemiological sample of adults from the Canary Islands, Spain, data for serum folate and vitamin B12 were used for a broader study of the nutrigenetic impact on tHcy (Paper IV). The 677T-allele had a significant tHcy increasing effect in men but not in women. The 1298C-allele had a minor elevating effect on tHcy in men with the 677CT genotype. It was not possible to document any effect of the 1793A-allele on tHcy due to its low prevalence. A slightly superior explanatory power for the genetic impact was obtained using the MTHFR haplotypes in the analysis compared to the MTHFR 677C>T genotype-based approach in both the Swedish children and adolescents and in the Spanish adults. Therefore MTHFR haplotypes should be considered when analysing the impact of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms on tHcy. Notwithstanding the large geographical distance between our study populations the haplotype composition is quite similar. The MTHFR 677T-allele is slightly more prevalent in Spain compared to Sweden but it has only an effect on tHcy in the Spanish men. Age, gender and factors linked to the ethnicity of the studied subjects, seem to be able to override the nutrigenetic impact of tHcy-raising genotypes or haplotypes in particular settings, such as in the Spanish women in our study. Gene-nutrient interactions on plasma tHcy levels thus may or may not exist in a certain population. The transferability of nutrigenetic findings may therefore be limited, and must be re-evaluated for each particular setting of age-gender-ethnicity.

Thesis addresses the physiological role of formyl tetrahydrofolate synthetase (Fhs) and bifunctional folate dehydrogenase (FolD) in folate mediated one carbon metabolism in bacteria. Thesis consists of 5 chapters. First chapter provides the details of the literature on folate metabolism, enzymes involved the synthesis and physiological roles various folate co-factors. Second chapter discusses the study of Clostridium perfringens Fhs generation of folD deletion in the support of fhs. Third chapter explores the characterization of the folD deletion strain. Fourth chapter presents the characterization of monofunctional versions of FolD from Clostridium perfringens. Fifth chapters talks about anti-correlation existence of Fhs and PurT (phosphoribosyl glycinamide formyl transferase II) The detailed experimental study is discussed below i. Characterization of Clostridium perfringens Formyl Tetrahydrofolate Synthetase (Fhs) In this chapter we have characterized Fhs from pathogenic Clostridium perfringens. Fhs catalyzes the formation of N10-formyl THF from THF and formate. Previously Fhs has been characterized from various non-pathogenic species of Clostridium. In addition, the detailed kinetic parameters are not known. In this report we have characterized the Fhs Clostridium perfringens and detailed kinetic parameters were determined. We have also shown the biological function by rescue of UV photorepair sensitive strain. ii. One-carbon metabolic pathway rewiring in Escherichia coli reveals an evolutionary advantage of 10-formyltetrahydrofolate synthetase (Fhs) in survival under hypoxia In cells, N10-formyltetrahydrofolate (N10-formyl THF) required for formylation of eubacterial/organeller initiator tRNA and purine biosynthesis is produced by methylene- tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). folD is present in all organisms, where as fhs shows mixed distribution. We show that in E. coli, which naturally lacks fhs, essential function of folD could be replaced with fhs of Clostridium perfringens when provided on a medium copy plasmid or integrated as single copy gene in the chromosome of the ∆folD strains, for their growth in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N10-formyl THF in the ∆folD strains (harboring fhs) were limiting despite their high enzymatic capacity to synthesize the same. Auxotrophy for purines could be alleviated by adding formate to the medium, and that for glycine by engineering THF import into the cells. The ∆folD strains showed high NADP+/NADPH ratio and were hypersensitive to trimethoprim (TMP). Further, the presence of fhs was disadvantageous to E. coli under aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. And, the computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria. We also propose that inhibitors aimed at folD could potentiate the effect TMP drugs. iii. 5, 10-methylene-THF dehydrogenase (DH) and 5, 10-methenyl-THF cyclohydrolase (CH) activities of FolD are essential to maintain folate homeostasis and anti-folate resistance While E. coli and many other organisms have folD alone or folD and fhs, Clostridium species possess an annotated bi-functional FolD and an annotated methenyl tetrahydrofolate cyclohydrolase (FchA). Simultaneous presence of 3 enzymes for the synthesis of N10-formyl THF was intriguing. To understand this unusual feature we have cloned Clostridium perfringens CpeFolD and CpeFchA, over expressed and purified to near homogeneity. Biochemical analyses revealed that CpeFolD possess only dehydrogenase activity as opposed to in silico prediction, while CpeFchA possess cyclohydrolase activity as expected. We also show that expression of both proteins together allowed folD deletion in E. coli. From this study we found that presence of dehydrogenase and cyclohydrolase functions are very important in the maintenance of folate homeostasis and anti-folate resistance. iv. Analysis of distribution of fhs and purT genes in the organisms While analysing distribution of fhs across genomes, serendipitously we also found that large number of organism which have fhs lack purT(phosphoribosyl glycinamide formyl transferase II), in short where ever purT was present fhs was absent. This kind of anti-correlation was strictly conserved in Bacillus genes as well. Growth competition experiments were done to address anti-correlation between fhs and purT. Growth competition experiments revealed that simultaneous presence of both purT and fhs is disadvantageous, when compared to presence of either one gene.

Foram estudadas 363 gestantes de primeira consulta, que não faziam uso de medicamentos que continham ferro, ácido fólico, vitamina B12 ou associação destes, na ocasião da coleta do material, em Centros de Saúde do Estado, no Subdistrito de Paz do Butantã, município de São Paulo. A prevalência de anemia (concentração de hemoglobina inferior a 11,6 g/dl) foi de 12,4%. As médias das concentrações de hemoglobina e as prevalências de anemia, segundo o trimestre de gestação, foram respectivamente: 13,47 g/dl e 3,57% no primeiro, 12,47 g/dl e 20,86% no segundo e 12,25 g/dl e 32,14% no terceiro trimestre. Não encontramos diferença. estatisticamente significativa entre as médias das concentrações de hemoglobina e entre as prevalências de anemia nas gestantes primigestas e multigestas. O mesmo aconteceu com as médias e as prevalências das gestantes multigestas com intervalo do último parto até dois anos e maior que dois anos. Na amostra estudada, encontramos maior prevalência de anemia naquelas gestantes que pertenciam às famílias que tinham renda per capita até 0,5 SMPC (salário mínimo per capita). As prevalências de verminose e de ancilostomídeos nas 300 gestantes foram respectivamente: 64,7% e 14,0%. Nas gestantes anêmicas foram respectivamente: 73,7% e 7,9%. Nas gestantes anêmicas, a prevalência de deficiência de ferro foi de 42,2% (concentração de ferro sérico <50 &#181;g/dl) ou 46,7% (saturação da transferrina <15% ou 40,0% (concentração de ferro sérico <50 &#181;g/dl e saturação de transferrina <15%). A prevalência de deficiência de ácido fólico foi 44,4%. A prevalência de deficiência de ferro e ácido fólico foi de 17,8%. Não encontramos deficiência de vitamina B<SUB<12 nas gestantes anêmicas. / Abstract not available

O adenocarcinoma ocorre em 90% dos casos de tumores malignos dos ovários e apresenta-se bilateralmente em 30 a 50% das pacientes. Devido à falta de sintomas iniciais da doença e à baixa especificidade dos marcadores tumorais existentes, quando detectado, o câncer encontra-se em estadios III e IV da doença, os quais indicam disseminação na cavidade peritoneal. O paclitaxel (PCX) é o fármaco de primeira escolha para o tratamento do câncer de ovário, entretanto a sua baixa solubilidade aquosa reduz sua biodisponibilidade. Dessa forma, no medicamento comercial, Taxol®, o PCX está solubilizado em uma mistura de tensoativos tóxicos. O desenvolvimento de nanocarreadores de fármacos tem sido investigado para promover a redução dos efeitos tóxicos e aumentar a segurança e a eficiência terapêutica com PCX. A funcionalização dos nanocarreadores é uma das estratégias utilizadas para aumentar a seletividade às células tumorais que superexpressam receptores de folato. O objetivo do presente trabalho foi desenvolver e caracterizar nanopartículas poliméricas contendo paclitaxel, funcionalizadas e não funcionalizadas com ácido fólico, visando a otimização do tratamento do câncer de ovário. Os sistemas foram obtidos com sucesso, utilizando técnica de nanoprecipitação. Os resultados obtidos mostraram que as formulações NPPCX e NPPCX-AF apresentaram distribuição de tamanhos de partículas de 140 e 154,6 nm, respectivamente, e índices de polidispersão menores que 0,1, com alta eficiência de encapsulação do PCX. Os resultados obtidos foram adequados para a via de administração endovenosa e promoção do direcionamento ativo no ambiente tumoral. Os resultados in vitro em testes de citotoxicidade em linhagens celulares SKOV-3 e OVCAR-3 demonstraram que as nanopartículas contendo PCX (NPPCX e NPPCX-AF) foram capazes de disponibilizar o PCX e reduzir a viabilidade celular. Os ensaios de citometria de fluxo e de microscopia confocal demonstraram a capacidade de ambas as nanopartículas apresentaram um uptake celular tempo dependente, mostrando a capacidade dos nanocarreadores serem internalizados. Além disso, comparando as duas linhagens celulares, a SKOV-3 apresentou maior uptake por apresentar maior número de receptores de folato. Dessa forma, as análises in vitro sugerem que os nanocarreadores, NPPCX e NPPCX-AF, apresentam potencial para a otimização da terapia do câncer de ovário. No entanto, estudos in vivo são necessários para a obtenção de resultados adicionais relativos à eficiência e à segurança para o tratamento de câncer de ovário. / Adenocarcinoma occurs in 90% of cases of malignant ovarian cancer and is present bilaterally in 30 to 50% of patients. Due to lack of initial symptoms and the low specificity of the existing tumor markers, the cancer is detected when it\'s in stages III and IV, which indicate spread into the peritoneal cavity. Paclitaxel (PCX) is the drug of first choice for ovarian cancer treatment, but it has low aqueous solubility, which reduces its bioavailability. Thus, in the commercial drug, Taxol®, PCX is solubilized in a mixture of toxic surfactants. The development of drug nanocarriers has been investigated to promote the reduction of toxic effects and increase the safety and therapeutic efficacy of PCX. Functionalization of nanocarriers is one of the strategies used to increase selectivity to tumor cells that overexpress folate receptors. The aim of the present work was the development and characterization of folate-modified nanoparticles (NPPCX-AF) and unmodified nanoparticles (NPPCX) and evaluation of in vitro efficacy of developed systems using adenocarcinoma cell lines. The systems were successfully obtained using nanoprecipitation technique. The results showed that the NPPCX and NPPCX-AF formulations had a particle size distribution of 140 and 154.6 nm, respectively, and polydispersity indexes smaller than 0.1, with high PCX encapsulation efficiency. The results obtained were suitable for the intravenous administration route and promotion of active targeting in the tumor microenvironment. The in vitro cellular cytotoxicity assays of SKOV-3 and OVCAR-3 cell lines demonstrated that NPPCX and NPPCX-AF were able to release PCX and reduce cell viability. The flow cytometry assays demonstrated that both nanoparticles presented a time dependent cellular uptake, showing the ability of nanocarriers to be uptake. In addition, comparing both cell lines, SKOV-3 showed a higher uptake due to its greater amount of folate receptors. Thus, in vitro results suggested that the nanocarriers, NPPCX and NPPCX-AF, present a distinguish potential for ovarian cancer therapy optimization. In vivo studies are needed to confirm the in vitro results and provide additional data regarding safety and efficacy of ovarian cancer treatment.

Em 2015, de acordo com a Organização Mundial da Saúde, o total de mortes na população humana foi de 52,5 milhões de pessoas, em que as doenças infecciosas mais especificamente, infecções respiratórias - aparecem em terceiro lugar no ranking de causas de morte. No total foram 3,5 milhões de mortes ou 6,7% do total, demonstrando um aumento significativo quando comparado com a pesquisa anterior, realizada em 2011, na qual as doenças infecciosas foram responsáveis por 3,2 milhões ou 5,9% do total. Das doenças infecciosas, podemos destacar a Malária, HIV/AIDS, Hepatite, Tuberculose - que aparece em 8º lugar neste mesmo ranking, e a Hanseníase - que é uma importante preocupação no Brasil. A tuberculose, causada pelo Mycobacterium tuberculosis sendo uma das principais causas de morte por doenças infecciosas na população humana, já apresenta cepas resistentes ao tratamento denominadas Cepas multirresistentes (MDR) e Cepas extremamente resistentes (XDR). Enquanto que a hanseníase, causada pelo Mycobacterium leprae, apesar de não ser letal, causa a invalidez dos portadores e o Brasil possui o segundo maior número de casos desta doença. Outro patógeno de importância médica é a Pseudomonas aeruginosa que causa sérias doenças em pacientes com baixa imunidade, principalmente aqueles em hospitais em especial, pacientes com queimaduras. A via do Folato tem despertado a atenção das indústrias farmacêuticas para o desenvolvimento de novos fármacos, uma vez que está via oferece seletividade para estes novos inibidores. Isso se deve ao fato de que o tetrahidrofolato é um componente essencial para os organismos procariotos, e apenas eles, precisam realizar a produção de novo desta substância, enquanto que os eucariotos, o adquirem através da alimentação. Entretanto, o surgimento de cepas resistentes não só reforça a necessidade do desenvolvimento de novos fármacos, mas também a compreensão dos mecanismos de resistências destas cepas. O presente trabalho tem como objetivo obter informações biofísicas e estruturais de três enzimas da via do folato, a 7,8 dihidroneopterina aldolase, dihidropteroato sintase e dihidrofolato redutase. Além disso, esse trabalho teve como objetivo a compreensão do mecanismo de resistência através da indução de mutações nos códons 53 e 55 na enzima dihidropteroato sintase (DHPS), caracterização estrutural preliminar da enzima DHNA e estudos estruturais da enzima DHFR em complexo com cicloguanil. Assim, neste trabalho foi realizado a clonagem gênica da região codificando do gene para a enzima DHNA e expressão da proteína recombinante, purificada e utilizada em ensaios biofísicos. Além disso a mutagênese sítio dirigida foi feita com o intuito de se produzir DHPSs mutantes (T70S e P72R), na qual foram utilizadas nos ensaios de ITC, fluorescência, e cristalização, o que possibilitou demonstrar que a resistência de M. leprae às sulfas pode ser devido a troca de uma prolina por uma arginina na posição 72. Por fim, para DHFR, foi possível obter sua estrutura em complexo com cicloguanil que possibilitou avaliar as diferenças estruturais entre a forma aberta e fechada desta enzima. Além disso, confirmamos que o grupo nicotidamida tem essencial importância para a interação com os ligantes. / In 2015, according to the World Health Organization, the total number of deaths in the human population was 52.5 million people, in which infectious diseases - specifically, respiratory infections - appear third in the ranking of causes of death. In total, 3.5 million deaths or 6.7% of the total occurred, showing a significant increase when compared to the previous survey conducted in 2011, in which infectious diseases accounted for 3.2 million or 5.9% of the total total. Of the infectious diseases, we can highlight Malaria, HIV / AIDS, Hepatitis, Tuberculosis - which appears in 8th place in this same ranking, and Leprosy - which is an important concern in Brazil. Tuberculosis, caused by Mycobacterium tuberculosis is one of the main causes of death due to infectious diseases in the human population, and already presents resistant strains to the treatment - denominated multiresistant strains (MDR) and extremely resistant strains (XDR). While leprosy, caused by Mycobacterium leprae, despite not lethal, causes the invalidity of patients and Brazil has the second largest number of cases for this disease. Another pathogen of medical importance is Pseudomonas aeruginosa, which causes serious illness in patients with low immunity, especially those in hospitals - especially patients with burns. The Folate pathway has brought the attention of the pharmaceutical industries to the development of new drugs, since this pathway offers selectivity for these new inhibitors. This is since Tetrahydrofolate is an essential component for prokaryote organisms, and only they, need to perform the new production of this cofactor, while Eukaryotes acquire it through food. However, the emergence of resistant strains, not only reinforces the need for the development of new drugs, but also the understanding of the mechanisms of resistance of these strains. The aim of the present work was to obtain biophysical and structural information of three folate pathway enzymes, 7,8 dihydroneopterin aldolase, Dihydropteroate synthase and Dihydrofolate reductase. In addition, this work aimed to understand the mechanism of resistance through the induction of mutations at the codons 53 and 55 in the enzyme dihydropteroate synthase (DHPS), preliminary structurally characterize the DHNA enzyme and structurally study the enzyme DHFR in complex with cycloguanil. Thus, the cloning of the coding region of the gene for DHNA and the expression of the recombinant protein, purification and biophysical characterization were performed in this work. In addition, site-directed mutagenesis was performed aiming of producing mutant DHPSs (T70S and P72R), which were used in ITC, fluorescence, and crystallization tests to demonstrate that M. lepreae resistance to sulfas may be due to the exchange of a proline to an arginine at position 72. Finally, for DHFR, it was possible to obtain its structure in complex with cycloguanil which made it possible to evaluate the structural differences between the open and closed conformation of this enzyme. In addition, we have confirmed that the nicotidamide group has a crucial importance for interaction with the ligands.

abstract: According to a 2016 census, eight million adults conform to a vegetarian diet within the United States, and about 50% of these adults follow a vegan diet. The census determined that plant-based diets are quickly growing in popularity particularly in young adults between the ages of 18 to 34 years. Many Americans are aware of the health benefits of a plant-based diet, however, the dietary risks associated with these diets are not well emphasized. Health concerns such as vitamin deficiencies and altered metabolism are heightened in vegetarian populations. One Particular nutrient that is commonly lacking in the vegetarian diet is vitamin B12. Vitamin B12 is found mainly in animal-derived food sources such as meat, poultry, fish, dairy, and eggs. Although some vegetarians, called lacto-ovo vegetarians, consume dairy and eggs, vegans do not consume any animal products at all. Vitamin B12 deficiency can have devastating consequences on the human body due to its role as a methylation cofactor. Metabolism, DNA replication, and cancer formation all involve methylation processes. This cross-sectional, differential study aimed to further understand the relationship between vegetarianism, vitamin B12 status, and methylation capacity in healthy adults. A group of 34 healthy adults (18 vegetarians and 16 omnivores) was recruited to analyze serum B12, homocysteine, methylmalonic acid, serum total folate, and transcobalamin II status. It was hypothesized that (1) vegetarians would have a lower vitamin B12 status, and thus, a lower methylation capacity than omnivores and that (2) low vitamin B12 status would be correlated with low methylation capacity. The data show that vegetarians did not have significantly lower vitamin B12 methylation capacity status than omnivores. Nor was vitamin B12 status correlated with methylation capacity. However, the data revealed that diet quality had a positive influence on folate status. There was also a statistical trend (p=0.08) for homocysteine reduction in participants consuming high-quality diets. The data herein suggest that methylation capacity may be impacted by the quality of diet rather than the type of diet. / Dissertation/Thesis / Masters Thesis Nutrition 2019

Neural Tube Defects (NTDs) are a group of common congenital malformations that result from incomplete neural tube closure leading to abnormalities of the brain and/or spinal cord. Unfortunately, their etiology remains unknown, probably due to complex multifactorial interactions. The protective effect of dietary folates in preventing NTDs is well known, but this beneficial effect is limited to the 60 to 70% of cases; leaving 30% of the population without any known option for improving pregnancy outcomes. The mechanism by which folates rescue NTD-affected embryos is poorly understood, but the ability of folate supplementation to overcome a significant percentage of NTDs and the critical role of 5-methyltetrahydrofolate in the remethylation of homocysteine (Hcy) to methionine in the placenta suggests that folate binding and/or transport might play a critical role during development. We hypothesized that maternal autoantibodies (AB) targeting placental folate receptor alpha (FRα) are blocking the receptor and limiting the ability of the FRα to bind folates, reducing intraembryonic folate levels. Furthermore, we hypothesized that AB binding to other relevant proteins required for trophoblastic growth and placentation can be involved in activating pathologic inflammatory pathways that can result in suboptimal uptake of nutrients and contribute to an abnormal closure of the neural tube. We developed a high throughput ELISA to evaluate whether mothers experiencing pregnancies complicated with NTDs are more likely to have placental AB to FRα than are mothers experiencing normal pregnancies. We optimized and simplified a protocol for AB elution from placental tissues and determined whether these antibodies were blocking the FRα from binding with available folates. Although anti-FRα IgG antibodies were not associated to the blocking activity in this study, we found that the blocking activity was higher in the placentas from NTD-affected pregnancies compared to controls, that FRα IgM antibodies are most likely the type of antibody produced during gestation that is most relevant to the blocking activity and that it is unlikely that autoimmunity against other developmental proteins associated with NTDs is generating the NTDs. / text

Objectives Prenatal alcohol exposure can lead to fetal alcohol spectrum disorders (FASD), which include a broad range of cognitive, growth, behavior, and physical abnormalities. Early detection of the teratogenic effects of prenatal alcohol exposure is necessary to identify early interventions. The aim of this study is to identify the effects of prenatal alcohol exposure on growth and infant development, to determine if ultrasound imaging can be used as an early identification tool, and to determine if maternal folate supplementation can mitigate the detrimental effects on growth and infant development. Methods A prospective cohort study and randomized trial from 2008 to 2014 conducted in two sites of Western Ukraine was analyzed. A sample of pregnant women who reported moderate-to-heavy alcohol consumption during pregnancy, and a sample reporting little-to-no alcohol use during pregnancy participated in a comprehensive maternal interview and screening process. Women were further randomized into micronutrient supplementation groups. Standard ultrasound examinations during pregnancy including study specific brain growth measurements, along with blood and urine samples were obtained during follow-up visits. A biometric screening was conducted at birth, along with Bayley Scales of Infant Development-II Mental Development Index (MDI) and Psychomotor Development Index (PDI) assessments at 6 and 12 months of age. Results Estimated fetal weight (EFW), abdominal circumference (AC), biparietal diameter (BPD), transverse cerebellar diameter (TCD), occipitofrontal diameter (OFD), caval-calvarial distance (CCD), and orbital diameter (OD) were significantly reduced by alcohol exposure at third trimester ultrasound (p2(7)=18.044, p=0.012), AC at third trimester (X2(5)=17.955, p=0.003), and birth weight (X2(6)=75.058, p2(11)=63.051, p Conclusions Significant reductions in fetal growth measurements during third trimester ultrasound suggest that the effects of prenatal alcohol exposure may be detectable in late pregnancy. Significant associations between multivitamin supplementation and specific growth measurements suggest that micronutrient supplementation during pregnancy, including high doses of folate, may be an early intervention to reduce the harmful effects of prenatal alcohol exposure. Further studies are needed to identify the specific micronutrients producing these effects, and to assess the appropriate level of these micronutrients necessary to provide the greatest benefit without exceeding the safe limit.

Trichloroethylene (TCE) is among the most common water contaminant in the United States and around the world. It is estimated that between 9% and 34% of all drinking water sources contain some TCE. The EPA set a drinking water standard for TCE at 5 parts per billion (ppb) in 1989, however since this date, many studies have shown TCE is dangerous to the health of adults and unborn children, even at low-level exposures. These studies reveal exposure to TCE can cause multi-organ damage, especially for the kidney, liver, reproductive and development systems. We investigated how TCE can effect embryonic heart development by identifing possible target mechanisms changing after exposure. Acute and chronic exposure to rat cardiomyocytes produced altered calcium flow and significant changes with TCE doses as low as 10ppb. Embryonic carcinoma cells, rat cardiomyocytes and fetal heart tissue all showed global changes in gene expression after low-dose TCE exposure, including critical ion channels that drive calcium flux. High levels of folic acid supplementation in combination with 10ppb TCE exposure in maternal diets caused significant genetic modifications in mRNA expression levels of Day 10 embryonic mouse cardiac tissue. We also found both high and low folate maternal diets leads to similar phenotypic outcomes in embryo development.

In der vorliegenden experimentell durchgeführten Dissertation wurden neue Mutationen im FOLR1-Gen molekular charakterisiert. Die untersuchten Mutationen führten zu Veränderungen im Folatrezeptor α, einem von Zellen des Plexus choroideus exprimierten Protein, welches den Haupttransporter von  5-MTHF über die Blut-Liquor-Schranke darstellt. Mutationen im FOLR1-Gen sind kürzlich als Ursache einer zerebralen Folattransport-Defizienz identifiziert worden. Diese Erkrankung gehört zu einer Gruppe neurologischer Störungen, die sich durch isoliert niedrige Liquorfolatwerte  auszeichnen und zusammenfassend als zerebrale Folatdefizienz bezeichnet werden. Die zerebrale Folattransportdefizienz manifestiert sich typischerweise im frühen Kleinkindesalter und äußert sich klinisch in Form einer chronisch-progredienten psychomotorischen Regression mit zerebralen Krampfanfällen und einer durch MRT nachweisbaren Myelinisierungsstörung. Eine Substitution mit  5‘-Formyltetrahydrofolat konnte bei der Mehrzahl der identifizierten Patienten eine partielle Remission der Symptome bewirken.  Im Rahmen dieser Arbeit wurden vier Mutationen im FOLR1-Gen hinsichtlich ihrer molekularen Auswirkungen auf die Proteinxpression, die Funktionalität bzw. Rezeptor-Bindungsfähigkeit und die Lokalisation des Proteins untersucht. Bei den Mutationen handelt es sich um zwei neue, aus Patienten-DNS identifizierte Punktmutationen, p.C169Y und p.N222S, sowie die bereits beschriebenen FRα-Mutanten p.C105R und FRα p.K44_P49dup.  Die heterologe Expression der mutanten Folatrezeptoren zeigte in Westernblot-Analysen keine signifikanten Veränderungen der Protein-Expressionsrate, verglichen mit dem Wildtyp-Protein. Allerdings bestand eine stark verminderte Rezeptor-Folsäurebindung in radioaktiven Bindungsassays. Ein funktioneller Unterschied zwischen den einzelnen Mutanten konnte im Verlauf der Experimente identifiziert werden. Die FRα-Proteinmutanten p.N222S und p.K44_P49dup zeigten verglichen mit den anderen Mutanten eine höhere Folsäure-Restbindung von ca. 20 % des Wildtypproteins. Im Rahmen von Immunfluoreszenzmikroskopien konnte gezeigt werden, dass die FRα-Mutante p.K44_P49dup partiell zellmembranständig, entsprechend dem FRα-Wildtyp, exprimiert wurde. Die übrigen untersuchten Proteinmutanten zeigten in intrazellulären Kompartimenten zumindest teilweise eine Kolokalisation mit dem Marker des endoplasmatischen Retikulums. Alle Untersuchungen wurden mit transfizierten CHO-K1-Zellen durchgeführt und konnten in zwei polaren Zelllinien, immortalisierten Epithelzellen des Plexus choroideus (Z310) und humanen Leberzellkarzinomzellen (HepG2) bestätigt werden. Die Ergebnisse dieser Dissertation sind Bestandteil einer kürzlich in BRAIN veröffentlichten Arbeit (Grapp et al. 2012) und tragen zum besseren Verständnis der molekularen Grundlagen der zerebralen Folattransport-Defizienz, einer neuerkannten, behandelbaren neuropädiatrischen Erkrankung, bei. Die  Pathogenität der untersuchten FOLR1-Mutationen wird auf molekularer Ebene belegt. Die Bedeutung dieser molekulargenetischen Untersuchungen besteht darin, dass eine frühzeitige Folat-Behandlung erkrankter Kinder zu einer deutlichen Verbesserung der Symptome führt.