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.

O paclitaxel (PCX) é o fármaco de primeira escolha para o tratamento do câncer de ovário. Contudo, a baixa solubilidade do PCX torna necessária a adição de adjuvantes tóxicos à formulação comercial (Taxol®). Assim, o desenvolvimento de sistemas de liberação nanoestruturados têm sido estudados para promover a redução dos efeitos tóxicos e aumentar a segurança e a eficiência terapêutica do PCX. Uma das estratégias utilizadas para aumentar a seletividade dos nanocarreadores às células tumorais é a funcionalização de suas superfícies com folato, devido a superexpressão dos receptores de folato nas células tumorais. O objetivo do presente trabalho foi desenvolver e caracterizar nanopartículas de PLGA funcionalizadas (NP-PCX-AF) e não funcionalizadas (NP-PCX) para a veiculação do PCX, bem como a avaliação in vitro da eficácia dos sistemas desenvolvidos em linhagens celulares de adenocarcinoma de ovário. Os nanocarreadores foram produzidos pela técnica de nanoprecipitação e avaliados por meio das análises físico-químicas de diâmetro médio de partícula, índice de polidispersão, potencial zeta, eficiência de encapsulação, estabilidade coloidal e espectro de infravermelho. Além disso, foi avaliada a eficácia antitumoral e o uptake dos sistemas desenvolvidos por meio dos ensaios de citotoxicidade celular por resazurina e internalização por microscopia confocal e citometria de fluxo. Os resultados obtidos mostraram que as formulações funcionalizadas e não funcionalizadas apresentaram reduzidos valores de diâmetros médios de partículas (menor que 150 nm) e índices de polidispersão reduzidos (menor que 0,2) com elevada eficiência de encapsulação do PCX, próximo a 100%, o que é adequado para a via de administração endovenosa e promoção do direcionamento ativo e passivo no ambiente tumoral. Os resultados de citotoxicidade celular in vitro em linhagens OVCAR-3 e SKOV-3 demostraram a elevada capacidade das formulações desenvolvidas em liberar o PCX em tempo hábil para ocasionar dano celular. Além disso, a formulação NPPCX- AF apresentou maior capacidade citotóxica que a formulação NP-PCX nas menores concentrações de PCX avaliadas, com redução máxima da viabilidade celular, aproximadamente 22 % para a linhagem celular SKOV-3 e 11% para OVCAR-3, utilizando a concentração de 1000 nM no tempo de 72 horas. Os ensaios de citometria de fluxo e de microscopia confocal mostraram a elevada capacidade de ambos os carreadores serem internalizados através do processo de endocitose, com valores de internalização celular superiores a 80% após 1 hora de tratamento. Estudos in vivo subsequentes serão necessários para confirmar o potencial da formulação NP-PCX-AF no tratamento do câncer de ovário / Paclitaxel (PCX) is a first-line medicinal treatment for ovarian cancer. However, the low PCX water solubility makes necessary the addition of toxic adjuvants to the commercial formulation (Taxol®). Thus, the development of nanostructured delivery systems has been studied to promote a reduction on side effects and an increase on safety and PCX therapeutic efficacy. One of strategies used to increase nanocarriers selectivity to tumor cells is the modification of their surfaces with folate due to folate receptor overexpression on tumor cells membrane. The aim of this work was the development and characterization of folate-modified PLGA nanoparticles (NP-PCXAF) and unmodified nanoparticles (NP-PCX) to PCX delivery, as well as the evaluation of in vitro efficacy of developed systems using human ovarian cancer cell lines. The nanocarriers were produced through nanoprecipitation technique and evaluated through physico-chemical parameters like particle size, polydispersity index, zeta potential, encapsulation efficiency, colloidal stability and infrared spectroscopy. Besides that, antitumoral efficacy and cellular uptake were evaluated using resazurin cytotoxicity assay, confocal microscopy and flow cytometry. The results obtained for folate-modified nanoparticles and unmodified nanoparticles showed low particle size distribution (less than 150 nm) and polydispersity index (less than 0.2) with high PCX encapsulation efficiency (about 100%) which is suitable for intravenous administration and for promoting active and passive targeting to tumor microenvironment. The in vitro cellular cytotoxicity assays of OVCAR-3 and SKOV-3 cell lines demonstrated the high ability of both formulations to release PCX and consequently to cause cellular damage. Furthermore, in lower concentrations NP-PCX-AF formulation showed higher cytotoxicity capacity than NP-PCX with higher cell viability reduction, about 22% for SKOV-3 cell line and 11% for OVCAR-3, using concentration of 1000 nM at 72 hours. Finally, flow cytometry and confocal microscopy assays indicated a high capacity of both nanocarriers to be internalized through endocytosis process. Therefore, subsequent in vivo studies are necessary to confirm the NP-PCX-AF potential for ovarian cancer treatment

Neural tube defects (NTDs) occur at higher rates in Hispanic populations in the USA. Such populations would benefit from folic acid fortification of corn masa flour (CMF). This study evaluated folate stability in fortified CMFs and products made from the flours, tortillas and tortilla chips. There was no significant loss of folate during the six-month shelf-life of fortified tortilla CMF and tortilla chip CMF. There was a 13% loss (P < 0.05) of folate during tortilla baking and no loss during tortilla chip frying. Both tortillas and tortilla chips showed significant folate losses over the two-month shelf-life for these products, with a 17% loss in fortified tortillas and 9% loss in tortilla chips. Folate in fortified CMFs, tortillas and tortilla chips is relatively stable and comparable to the stability of folate in wheat flour and breads.

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.

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This thesis investigates the use of scientific evidence in the process of making public health policy. A case study located within a food regulation setting is used. The aim is to test theory against this case study. The outcome is a theoretical understanding of the use of scientific evidence in the policy-making process in a food regulation setting. Food regulation can influence food composition and food labelling and thereby affect the population's dietary intake. Frequently there are contested values, beliefs, ideologies and interests among stakeholders regarding the use of food regulation as a policy instrument to effect public health outcomes. The protection of public health and safety, taking into account evidence based practice, is generally employed by food regulators as the priority objective during the policy-making process to adjudicate among the competing expectations of stakeholders. However, this policy objective has not been clearly defined and is vulnerable to interpretation and application. The process by which folate fortification policy was made in Australia, in response to epidemiological evidence of a relationship between folate intake during the periconceptional period and reduced risk of neural tube defects, was analysed as a case study of the policy-making process. The folate fortification policy created a precedent for both food fortification and subsequently health claims policy in Australia. A social constructivist method was used to analyse the case study. The method involved deconstructing the food regulatory system into three levels; decision-making process; procedural; and political environment. Data aligned with each level of analysis was collected from 22 key informant interviews, documentary sources, field notes and surveys of both a random sample of the Australian population's knowledge of folate and use of folic acid-containing supplements (n = 5422), and the implementation of folate fortified food products into stores (n = 60). The insights that emerged from each of the three levels of analysis were assessed iteratively to identify a pattern of interrelationships associated with the policy-making process within the food regulatory system. The identified pattern was interpreted against existing theory to gain a theoretical understanding of the public health policy-making process in this political setting. The central argument of this thesis extends Sabatier and Jenkins-Smith's Advocacy Coalition Framework theory to a food regulation setting. The argument is that within the contemporary political climates of neoliberalism and globalisation, a coalition between corporate interests and the values of scientists with a positivist-reductionist approach to public health research is privileged so as to invoke certain scientific evidence to, in turn, legitimise food regulation policy decisions. The theory will help to inform policy-makers about how and why the public health policy objective in a food regulation setting is interpreted and applied. This will contribute to improving policy practice intended to effect public health outcomes. It is concluded that irrespective of the quantity and quality of the scientific evidence that is being made available, scientific evidence cannot be assumed to speak for itself Policy-making is an inherently political and value-laden process and the potential for politically motivated interpretation and application of otherwise value-neutral scientific evidence can undermine the investment in its generation. From this perspective, evidence based practice, far from liberating policy-making from political influence, can itself become part of the problem rather than the solution. Nevertheless, rational evidence based practice is an ideal to strive for and a series of recommendations is proposed to help make the use of evidence in current food regulation policy processes more transparent and democratic.

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

When food is heated, a vast number of compounds are formed. Some of these are known to be toxic. Among these are furan, HMF, PhIP, IQ, and MeIQx, the subjects of this thesis. All these compounds are known or suspected carcinogens but the detailed mechanisms behind their carcinogenicity have not yet been fully examined. The aim of this thesis was to study genotoxic properties of these compounds using both in vitro and in vivo methods. Clastogenic effects of all five compounds were assessed with the flow cytometer-based micronucleus assay in vivo and for furan also with the micronucleus assay in vitro. DNA-damaging effects of HMF were studied using the comet assay. No induction of micronuclei was obtained after exposure to IQ, MeIQx or furan. Hence, it can be argued that non-genotoxic mechanisms are partly responsible for the carcinogenic properties of these compounds. PhIP, on the other hand, generated a clear response in the in vivo test. Comparing these result with previous results on acrylamide indicates that PhIP is much more potent. However, acrylamide probably poses a higher risk for humans as the intake is considerably higher. For HMF no effects were seen using the in vivo setup. To further investigate the influence of bioactivation of HMF by sulfotransferases (SULTs) the comet assay was performed in cell lines expressing different levels of SULT. However, no correlation between SULT-expression and DNA-damage was observed. Thus, the DNA-damaging effects found in our experimental setup is probably due to other factors than SULT mediated effects. Furthermore, in this thesis the effects of folic acid on chromosomal stability in healthy people were studied. A negative correlation was found between micronucleus frequency and folate status. The results gained within this thesis will hopefully contribute to the risk assessment of compounds present in our diet.

Maternal diabetes is associated with increased risk of growth disturbances and congenital malformations. The malformations rate in the offspring of diabetic mothers is 2-3 fold higher compared to infants of nondiabetic mothers. In this thesis we have investigated the role of the protein kinase C (PKC) pathway and the apoptotic machinery in embryopathy. We investigated the involvement of PKC isoforms in the embryopathy of diabetic rat pregnancy. Embryos of diabetic rats showed altered activity and protein distribution of several PKC isoforms compared with embryos of normal rats. Using whole embryo culture we found increased activity of PKC-delta and PKC-zeta after 24h of culture and increased rate of malformations and growth retardation in embryos cultured in high glucose concentration compared to embryos cultured in low glucose concentration. Addition of α-cyano-4-cinnamic acid and N-acetylcysteine to the culture medium normalized malformations and growth retardations whereas specific PKC-inhibitors abolished malformations and partly restored the growth retardations. All treatment normalized glucose-induced increase of PKC activity. Estimated occurrence of apoptosis in embryos of diabetic rats and in embryonic cells exposed to high glucose concentration showed increased rate of pro-apoptotic markers. The increased apoptosis in the high glucose exposed embryonic cells was normalized by supplementation of N-acetylcysteine or apoptosis inhibitor. Treatment with vitamin E and folic acid to diabetic pregnant rats decreased diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. These results suggest that oxidative stress is augmented in embryos of diabetic rats and that it also plays a role in the activation of PKC and apoptosis. We used antioxidative treatment with beneficial effect although we could not completely abolish the embryonic demise; this may indicate that other mechanisms are involved in diabetic embryopathy. Further studies are needed to develop multi-nutrient dietary supplement to eliminate embryonic abnormalities induced by maternal diabetes.