Made available in DSpace on 2014-07-29T15:29:16Z (GMT). No. of bitstreams: 1 Tatiana Moreria da Silva DISSERTACAO DE MESTRADO.pdf: 4157039 bytes, checksum: 141730648f501822185cd3dc86bb7823 (MD5) Previous issue date: 2012-07-06 / Research shows that oral supplementation with antioxidants can reduce the damage caused by reactive oxygen species and increase the fertilizing capacity of sperm due to improvement in semen parameters. Semen analysis was assessed according to the criteria of the World Health Organization (1999), subfertile men who received folic acid at a dose of 5 mg/day or placebo, comparing results before and after treatment. A total of 49 patients aged 23-56 years (mean 35.3 years) and diagnosis of subfertility were randomized into two groups. The treated group (n= 23) receiving folic acid and the control group (n= 26) received placebo, each for 3 months. Before the intervention, the mean sperm concentration (x106/mL) in the treated group was 25.91 and 26.08 after treatment. In the control group was: 20.85 pre-treatment and 20.04 after treatment (p= 0.92). Means of progressive motile sperm obtained from the treated and placebo groups before treatment were, respectively: 46.48% and 49.23%. After the intervention, the mean values found were 48.26% in the treated group and 49.65% in the control group (p= 0.99). The mean normal sperm morphology was 23.26% in the treated group pre-intervention, and later was 23.91%. The control group had an average of 22.3% and 24.23%, respectively (p= 0.83). Mean vitality found in the treated group was 66.73% before the treatment and 69.56% after the treatment with folic acid. In the control group was 65.77% after pre-treatment and 65.38% was not significant (p= 0.95). Numerous studies have evaluated the use of antioxidants for improving sperm quality and thus fertility. These substances are used individually or in combination, and differ in the type, the target population, dosage and duration of therapy. This study showed that folic acid at a concentration of 5 mg / day did not improve sperm parameters in subfertile men compared to the control group. / Acredita-se que a suplementação oral com antioxidantes possa reduzir os danos causados por espécies reativas de oxigênio e aumentar a capacidade de fertilização dos espermatozoides devido à melhora dos parâmetros seminais. Avaliou-se o espermograma, segundo os critérios da Organização Mundial de Saúde (1999), de homens subférteis que receberam ácido fólico na dosagem de 5 mg/dia ou placebo, comparando os resultados pré e pós tratamento. Um total de 49 pacientes com idade entre 23 a 56 anos (média 35,3 anos) e diagnóstico de subfertilidade foi randomizado em dois grupos. O grupo tratado (n= 23) recebeu ácido fólico e o grupo controle (n= 26) recebeu placebo, ambos durante 3 meses. Antes da intervenção, a concentração média de espermatozóides (x106/mL) no grupo tratado foi 25,91 e após o tratamento 26,08. No grupo controle foi: 20,85 pré-tratamento e 20,04 pós-tratamento (p=0,92). Quanto à motilidade, as médias de espermatozóides móveis progressivos obtidas nos grupos tratado e placebo, antes da terapêutica foram, respectivamente: 46,48% e 49,23%. Após a intervenção, as médias encontradas foram 48,26% no grupo tratado e 49,65% no grupo controle (p=0,99). A média de morfologia normal dos espermatozóides foi 23,26% no grupo tratado pré-intervenção, e posteriormente foi de 23,91%. O grupo controle apresentou média de 22,3% e 24,23%, respectivamente (p= 0,83). A média de vitalidade encontrada no grupo tratado foi de 66,73% antes do tratamento e 69,56% após o tratamento com ácido fólico. Já no grupo controle foi de 65,77% pré-tratamento e após foi de 65,38% não sendo significante (p= 0,95). Inúmeras pesquisas têm avaliado o uso de antioxidantes para melhorar a qualidade do esperma e assim, a fertilidade. Essas substâncias foram utilizadas individualmente ou em combinação, e se diferem quanto ao tipo, à população-alvo, dose e duração da terapia. Esse estudo mostrou que o ácido fólico na concentração de 5 mg/dia não melhorou os parâmetros espermáticos de homens subférteis em comparação ao grupo controle.

Durante o processamento industrial ocorrem modificaÃÃes nos componentes dos frutos que afetam sensivelmente suas propriedades, tais como sabor, aroma e tambÃm o valor nutritivo. Deste modo, torna-se importante verificar suas variaÃÃes sob influÃncia das operaÃÃes durante o processamento de sucos. Com o objetivo de determinar estas alteraÃÃes, foi realizada uma caracterizaÃÃo quÃmica e fÃsico-quÃmica do suco de caju com alto teor de polpa, em vÃrias etapas do seu processamento industrial. Foram analisados trÃs lotes de suco de caju apÃs as etapas de formulaÃÃo, homogeneizaÃÃo e pasteurizaÃÃo, realizando-se em cada uma delas as determinaÃÃes de acidez, pH, sÃlidos solÃveis totais, aÃÃcares redutores, nÃo-redutores e totais, atividade de Ãgua (Aw), carotenÃides, antocianinas, Ãcido ascÃrbico e Ãcido fÃlico, sendo que, para este Ãltimo, foi necessÃrio desenvolver uma metodologia analÃtica especÃfica, tendo em vista sua ausÃncia na literatura para o suco em estudo. Ao final do experimento, constatou-se que os parÃmetros de sÃlidos solÃveis totais, acidez, atividade de Ãgua, vitaminas e pigmentos apresentaram variaÃÃes significativas durante as etapas do processamento. As perdas de Ãcido ascÃrbico, carotenÃides e antocianinas foram mais elevadas na etapa de pasteurizaÃÃo. O Ãcido fÃlico apresentou um comportamento diferenciado, apresentando variaÃÃo significativa entre as etapas de formulaÃÃo e homogeneizaÃÃo. Estes resultados demonstram que as vitaminas e os pigmentos presentes nos frutos podem ser comprometidos durante a fabricaÃÃo de sucos, naturalmente devido à sua instabilidade frente a altas temperaturas e interaÃÃo com o oxigÃnio, de modo que devem ser determinadas quais as melhores condiÃÃes a serem adotadas pela indÃstria de sucos a fim de minimizar estas perdas, tendo em vista que se tratam de substÃncias que resultam em benefÃcios à saÃde humana e, portanto, devem ser preservadas sempre que possÃvel / During the industrial processing modifications in the components of the fruits that affect sensitively their property, like taste, smell and nutritionâs value usually occurs. Thus it is important to verify their variations under the influence of the operating conditions during the juiceâs processing. The objective of the present work was to determine these changes carrying out a chemical and physics- chemistry characterization of cashewâs apple juice with high pulp content, in some stages of industrial processing. Three samples of cashewâs juice, collected after the stages of formulation, homogenization and pasteurization, were analyzed I order quantify pH, total soluble solids, reducing sugars, total sugars, non reducing sugars, water activity (Aw), carotenoids, anthocyanins, ascorbic acid and folic acid. To folic acid determination it was necessary to develop a specific analytical methodology due to the absence of literature about this juice. According to the results total soluble solids water activity, vitamins and pigments showed significant variations during the processing. The losses of ascorbic acid, carotenoids and anthocyanins were higher in the pasteurization stage. The folic acid showed a different behavior, significant variations were observed only between the stages of formulation and homogenization. These results shows that the vitamins and the pigments present in the fruits are changed during the manufacture of the juices due to their instability at high temperatures in presence of oxygen. The presented results can be useful to improve the industrial processing of fruits juices in order to minimize these losses since the substances related to the human health benefits that should be preserved always it is possible

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-06-09T20:26:25Z No. of bitstreams: 2 Tese - Marco Júnio Peres Filho - 2014.pdf: 2890867 bytes, checksum: 94c9616394d6a389aadabf3d9a0b128c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-12T12:31:24Z (GMT) No. of bitstreams: 2 Tese - Marco Júnio Peres Filho - 2014.pdf: 2890867 bytes, checksum: 94c9616394d6a389aadabf3d9a0b128c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-12T12:31:24Z (GMT). No. of bitstreams: 2 Tese - Marco Júnio Peres Filho - 2014.pdf: 2890867 bytes, checksum: 94c9616394d6a389aadabf3d9a0b128c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-04-30 / The impact of Nanotechnology is constantly raising in different areas of science, with the development of new products that bring benefits in comparison with the alternatives available in the market. When encapsulated in nanoparticles, anticancer drugs can achieve several advantages, most importantly the possibility of reducing the amount of drug administered through targeting strategies, which are not accomplished by conventional medication. Passive targeting is related to leaky vasculature in pathological sites, and active targeting comprehends the attachment of specific ligands, anchored in nanoparticles surface, to recognize and bind receptors overexpressed in cancer cells. Coencapsulation of anticancer drugs in the same pharmaceutical carrier can coordinate pharmacokinetics of encapsulated drugs. In the present work, liposomal formulations targeted to folate receptor with paclitaxel (PTX) and imatinib (IB) coencapsulated were obtained, aiming to combine cytotoxic and antiangiogenic effects of the drugs, respectively. New analytical method was developed and validated for simultaneous quantification of IB and PTX. Soy phosphatidylcholine liposomes were prepared, with cholesterol and DSPEmPEG( 2000), to obtain long circulation particles. DSPE-PEG(2000)-FA was obtained by an unpublished method of synthesis, and this product was further used in the formulation by post-insertion technique. Cytotoxic effect and VEGF gene suppression were studied in vitro in two different cell lines, MCF7 (breast adenocarcnioma) and PC3 (prostatic adenocarcinoma), after treatment with liposomal vesicles. Analytical procedures were developed with isocratic elution, 6,5 minutes runs, with linearity, specificity, precision and accuracy. Quantification limit was 750 Ng/mL and 1000 Ng/mL for IB and PTX, respectively. After extrusion, liposomes had mean diameter close to 100 nm and low polidispersion index. Post-insertion of folic acid attached to lipid anchor procedure increased polidispersion, because the procedure lasted 24h. Drug to lipid ratios were 1:26 and 1:27 (IB and PTX respectively). Lyophilized formulations containing trehalose remained stable after 60 days of storage in terms of %EE. Synthesis of DSPE-PEG(2000)-FA was confirmed by RMN, FT-IR and ESIMS techniques. Liposomal PTX was more cytotoxic (p<0,05) than free drug in MCF7 cell line, after both 24h and 48h of exposion, for all tested concentrations. Targeted formulation containing folic acid ligand, had more impact on cell viability reduction (p<0,05) than non targeted liposomes (LPIP), also after 24h. On PC3 cell line cell viability reduction was greater (p<0,01) when the cells were exposed to targeted vesicles loaded with 1 and 10 Ng/mL of IB and PTX, after 24 and 48h. VEGF gene expression was reduced in MCF7 and PC3 (p<0,05), and once more targeted vesicles showed better results than non-targeted liposomes. It is, thus, plausible to conclude, through in vitro experiments results, that the attachment of folic acid to liposomal formulations, resulting in multi-functional liposomes, is an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells. This was observed by the enhancement of cytotoxic and antiangiogenic effects in breast and prostate cell lines. / A nanotecnologia tem ganhado cada vez mais destaque em várias áreas da ciência, no sentido de desenvolver novos produtos que possam trazer benefícios em comparação com alternativas já existentes no mercado. Uma série de vantagens emergem da nanoencapsulação de fármacos quimioterápicos, principalmente, a possibilidade de diminuição da dose administrada através de estratégias de direcionamento. Dentre elas, podem ser citadas a vetorização passiva, relacionada ao aumento da permeabilidade vascular em regiões tumorais, e a vetorização ativa, que significa o uso de ligantes específicos, cujos receptores são superexpressos em células tumorais, e que ficam ancorados na superfície de nanoestruturas. A coencapsulação de fármacos antitumorais é altamente relevante, uma vez que permite a utilização de diferentes abordagens para eliminação de tumores e a sincronização da farmacocinética dos agentes coencapsulados. No caso deste trabalho, o objetivo foi a associação do efeito citotóxico do paclitaxel (PTX) ao efeito antiangiogênico do imatinibe (IB), encapsulados no mesmo lipossoma direcionado para o receptor folato, superexpresso em vários tumores sólidos. Foi desenvolvido e validado novo método analítico para quantificação simultânea do IB e do PTX. Os lipossomas foram desenvolvidos usando fosfatidilcolina de soja (PC) como lipídio estrutural, colesterol e DSPE-mPEG(2000), este último para conferir efeito de longa circulação. Foi desenvolvido método inédito de síntese da molécula DSPEPEG( 2000)-AF, âncora lipídica ligada ao ácido fólico destinada a biorreconhecimento. Essa molécula foi adicionada à formulação por pós-inserção. Os nanossistemas obtidos foram testados in vitro quanto à citotoxicidade e quanto à inibição da expressão gênica de VEGF em células de adenocarcinoma mamário (MCF7) e de próstata (PC3). O método analítico foi isocrático, com corrida de apenas 6,5 minutos, linear, seletivo, preciso e exato, com limite de quantificação 750 Ng/mL para o IB e 1000 Ng/mL para o PTX. Os lipossomas tiveram diâmetro médio próximo de 100 nm e baixo índice de polidispersão. Apenas após a inserção da âncora ligada ao ácido fólico as amostras ficaram mais polidispersas, devido ao tempo de agitação desse processo (24 h). O IB foi encapsulado na razão molar fármaco:lipídio 1:26 e o PTX na razão 1:27. As formulações liofilizadas, usando o crioprotetor trealose, mantiveram a estabilidade em termos de %EE após 60 dias de armazenamento. O método desenvolvido para síntese do componente DSPEPEG( 2000)-AF foi bem sucedido, o que pôde ser verificado através dos espectros de RMN, FT-IR e IES-EM, que evidenciam a formação de uma nova substância a partir dos reagentes, que tem as características químicas esperadas. O paclitaxel lipossomal foi mais citotóxico (p<0,05) para a linhagem MCF7, tanto em 24h como em 48h, em comparação com o fármaco livre, em todas as concentrações testadas. A formulação vetorizada com a âncora ligada ao ácido fólico, com IB e PTX coencapsulados, foi mais citotóxica (p<0,05) que a não vetorizada (LPIP) nas concentrações testadas, no tempo 24 h. Na linhagem PC3 a redução da viabilidade celular causada pela vetorização ativa, em comparação com o resultado da formulação não vetorizada, foi ainda maior (p<0,01) tanto em 24 como em 48h, nas concentrações 1 e 10 Ng/mL. Houve redução, nas linhagens MCF7 e PC3, da expressão gênica de VEGF, e também nesse caso o tratamento com a formulação vetorizada causou maior efeito em comparação com a não vetorizada (p<0,05). É razoável chegar-se à conclusão, através dos testes in vitro, de que o direcionamento de fármacos nanoencapsulados ao receptor folato, através do emprego de lipossomas multi-funcionais, é uma estratégia interessante no sentido de aumentar a quantidade de fármaco que entra nas células, o que pôde ser observado através do aumento dos efeitos citotóxico e anti-angiogênico em linhagens tumorais de mama e próstata.

A Terapia Fotodinâmica (TFD) é um tratamento usado principalmente na terapia anticâncer e que depende da retenção de um composto fotossensibilizante nas células tumorais e posterior irradiação dessas células com luz visível. Após ativação, o fármaco pode gerar espécies reativas de oxigênio (EROs), como o oxigênio singlete (1O2) e radicais (O2-, OH), que são capazes de danificar membranas, DNA e outras estruturas celulares, induzindo a apoptose ou necrose das células tumorais. O ácido fólico, por ser super expressado na superfície das células de alguns tipos de cânceres (principalmente os ginecológicos), pode desempenhar o papel de vetorizador, tornando-se um importante aliado aos sistemas de liberação de fármacos. Neste trabalho foi avaliado o uso de ácido fólico como aditivo aos lipossomas contendo ftalocianina de cloro-alumínio como fármaco fotossensibilizante, aplicados em TFD com células MCF7. Os estudos demonstraram que os sistemas lipossomais desenvolvidos apresentam tamanho nanométrico (menor que 200 nm) e possuem biocompatibilidade quando avaliados em cultura celular de monocamada. Além disso, foi possível observar o efeito fototóxico satisfatório das formulações e o aumento da internalização do fármaco, quando utilizado o ácido fólico como vetorizador. / Photodynamic Therapy (PDT) is mainly used in anticancer therapy. The efficiency of this treatment is dependent on retention of photosensitizer compound at the tumor cells and posterior irradiation of these cells with visible light. After activation, the drug may generate reactive oxygen species (ROS) such as singlet oxygen (1O2) and radicals (O2-, OH), which are capable of damaging membranes, DNA and other cell structures, inducing apoptosis or necrosis of tumor cells. Folic acid is super-expressed on the surface of some cancers cells (especially gynecological cancer cells) and can play the role of target system, becoming an important ally for drug delivery systems. This study evaluated the use of folic acid as an additive to liposomes containing chloro-aluminum phthalocyanine as a photosensitizer drug, applied in PDT with MCF7 cells. These studies showed that liposomal systems have nanometer size (less than 200 nm) and have biocompatibility when evaluated in monolayer cell culture. Moreover, it was possible to observe satisfactory phototoxic effect of the formulations and increased internalization of the drug when folic acid is used.

Une des limitations de la thérapie photodynamique est la faible sélectivité des photosensibilisateurs (PS) pour les tissus tumoraux. La recherche de nouveaux PS plus sélectifs s’est orientée vers la synthèse de PS couplés à des motifs afins pour des récepteurs membranaires surexprimés dans certains cancers. Le récepteur à l’acide folique est surexprimé dans les carcinomes ovariens et des PS ont été conjugués à de l’acide folique. Des PS ont aussi été couplés à un peptide spécifique de neuropiline 1 surexprimé au niveau des cellules tumorales de médulloblastome. Une autre stratégie pour augmenter la sélectivité du traitement vise à produire les espèces réactives de l’oxygène spécifiquement au niveau du site tumoral. L’activité de clivage enzymatique de marqueurs biologiques surexprimés dans les zones tumorales est utilisée. Les gélatinases (MMP-2 et -9) et leur activateur MMP-14 sont connues pour jouer un rôle primordial dans l'angiogenèse tumorale et la croissance du glioblastome multiforme. Différents photodynamic molecular beacons (PMB), associant un PS et un quencher lié par un peptide substrat des gélatinases et MMP-14, ont été développés / One limitation of photodynamic therapy is the low selectivity of photosensitizers (PS) to tumour tissue. The search of new PS more selective began to focus on the synthesis of PS coupled with substrate specific of the membrane receptors overexpressed in certain cancers. The acid folic receptor is overexpressed in ovarian carcinomas and PS were conjugated with folic acid. PS were also coupled with a specific peptide of neuropilin 1 overexpressed in tumoral cells of medulloblastoma. Another strategy for increasing the selectivity of the treatment is to produce reactive oxygen species specifically at the tumor site. The activity of enzymatic cleavage of biomarkers overexpressed in tumour areas is used. The gelatinases (MMP-2 and MMP-9) and their activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of glioblastoma multiform. Different photodynamic molecular beacons (PMB), composed of a photosensitizer and a quencher linked together by a peptide substrate of gelatinases or MMP-14, were designed.

Folate sind als Überträger von C1-Fragmenten verschiedener Oxidationsstufen direkt an der Nukleinsäuresynthese beteiligt und spielen somit eine entscheidende Rolle im Zellstoffwechsel. So kann ein suboptimaler Folatstatus zur Ausprägung einer klinischen Symptomatik führen, die sich primär in Zellen mit hoher Teilungsrate manifestiert. Dabei gilt die megaloblastäre Anämie als Leitsymptom für einen akuten Folatmangel. Herausragende Bedeutung besitzen Folate in der Schwangerschaft, wo ihnen bei der Entwicklung des fetalen Nervensystems eine besondere Rolle zukommt. Bei einem zu niedrigen Serumfolatspiegel der Mutter steigt das Risiko für embryonale Missbildungen wie Neuralrohrdefekte stark an. Die weitere medizinische Relevanz der Folate steht momentan im Fokus der Forschung, wie z. B. der protektive Effekt in Bezug auf bestimmte Krebsformen, die Risikoreduktion für verschiedene kardiovaskuläre Erkrankungen sowie Zusammenhänge zwischen dem Folatstatus und neurologischen Störungen (Alzheimer, Depressionen). Allerdings kann Folat als essentielles Vitamin von Säugetieren nicht selbst synthetisiert werden, sondern muss aus externen Quellen über den Verdauungstrakt aufgenommen werden. Die Resorption aus dem Darm erfolgt nach aktuellem Kenntnisstand insbesondere über den Reduced Folate Carrier (RFC, SLC19A1) und den Proton-Coupled Folate Transporter (PCFT, SLC46A1). Trotz der Existenz dieser spezifischen Transporter ist der Folatmangel des Menschen der häufigste Vitaminmangel in Mitteleuropa. Er lässt sich durch eine ausschließlich ernährungsphysiologische Problematik nur unzureichend erklären. Interessanterweise besitzen sowohl das PCFT-Gen als auch das RFC-Gen in ihrer Promoterregion verschiedene Regulationselemente, unter anderem auch funktionell aktive DREs (Dioxin Response Element), die als Bindungsstelle für einen ligandenaktivierten Transkriptionsfaktor, den nukleären Arylhydrocarbon-Rezeptor (AhR), dienen. DREs wurden bisher hauptsächlich bei fremdstoffmetabolisierenden Enzymen wie z. B. Cytochrom P450-Isoenzymen gefunden und vermitteln bekanntermaßen die toxischen und karzinogenen Effekte von AhR-Liganden. Von besonderem Interesse sind in diesem Zusammenhang ubiquitär verbreitete Umweltkontaminanten wie die polyzyklischen und die halogenierten aromatischen Kohlenwasserstoffe (PAK, HAK), da sie in der Umwelt sehr persistent sind und sich dadurch in der Lebensmittelkette anreichern. Infolgedessen wurde im Rahmen dieser Studie der mögliche Einfluss von AhR-Liganden wie TCDD (2,3,7,8-tetrachlordibenzo-p-dioxin) und B[a]P (Benzo[a]pyren) auf die carriervermittelte intestinale Folatresorption beim Menschen untersucht sowie die Regulation der Transportproteine RFC und PCFT auf transkriptioneller Ebene experimentell überprüft. Als adäquates in vitro Modell diente dabei die humane Kolonzelllinie LS180, für die zunächst eine Charakterisierung erfolgte. Mittels RT-PCR wurde der Nachweis erbracht, dass alle am Folattransport beteiligten Import-und Exportcarrier auf mRNA-Ebene exprimiert werden. Für die Transportproteine RFC und PCFT erfolgte über den Western Blot auch der Nachweis auf Proteinebene. Die Funktionalität der AhR-Signalkaskade, die über methylcholanthrenartige Induktoren wie TCDD zur Induktion von Cytochrom P450 führt, konnte im Folgenden mittels Ethoxyresorufin-O-deethylase-Assay (EROD) überprüft werden. Es zeigte sich nach Induktion mit TCDD (0,01 - 10 nM) oder B[a]P (0,01 - 1 μM) über 12 – 96 h ein hochsignifikanter, dosis- und zeitabhängiger Effekt auf die Cyp1A1 vermittelte Enzymaktivität der Ethoxyresorufin-O-deethylase in den LS180-Zellen. Dabei konnten ligandenabhängige Unterschiede im Induktionsmuster ermittelt werden. Im Anschluss wurde im intestinalen Zellmodell die initiale konzentrations- und zeitabhängige Folataufnahme bei pH 5.5 über funktionelle Aufnahmeversuche mit Tritium-markierter Folsäure untersucht und charakterisiert. Sie stellte sich als aktiver, sättigbarer Prozess dar, wobei in den unbehandelten LS180-Zellen ca. eine Verdreifachung der intrazellulären Radioaktivität über einen Zeitraum von 2,5 min beobachtet werden konnte. Als kinetische Parameter wurden ein Km-Wert von 27,91 μM sowie ein Vmax-Wert von 281,2 pmol/min berechnet. Nachfolgende Untersuchungen mit den spezifischen Inhibitoren Raltitrexed (RTX) und Pemetrexed (PMX) konnten zeigen, dass sowohl PCFT als auch RFC an der funktionellen Folsäureaufnahme bei einem pH-Wert von 5.5 in dem gewählten Versuchsaufbau beteiligt sind. Der RFC scheint jedoch einen etwas höheren Anteil an der Gesamtaufnahme zu haben als der PCFT. In LS180-Zellen, die vorher mit den Modellsubstanzen TCDD (1 bzw. 10 nM) oder B[a]P (0,1 bzw. 1 μM) über 24 - 120 h inkubiert wurden, konnte eine zeit- und dosisabhängige, statistisch signifikante Reduktion der carriervermittelten Folatresorption beobachtet werden, wobei die maximale Verminderung der Aufnahmerate ca. 75 % betrug. Da sich dieser Effekt durch die AhR-Antagonisten Salicylamid (SAL) und CH-223191 (CH) dosisabhängig umkehren ließ, erfolgt die Regulation vermutlich über den AhR-Signalweg. Um den Mechanismus der Regulation zu klären, wurde mit Hilfe der quantitativen qRT-PCR unter Verwendung von TaqMan-Sonden die Genexpression von RFC und PCFT nach Vorbehandlung mit 1 - 10 nM TCDD oder 0,1 – 1 μM B[a]P über 12 - 120 h untersucht. Analog zu den Aufnahmeversuchen konnte hier eine zeit- und dosisabhängige Reduktion beider Transporter auf transkriptioneller Ebene beobachtet werden. Auch führte eine Vorbehandlung mit CH und SAL wiederum zu einer Umkehr des Effektes, wobei CH wahrscheinlich einen ligandenselektiven AhR-Antagonisten darstellt. Zusammenfassend konnte durch die vorliegende Arbeit nachgewiesen werden, dass Cytochrom P450-Induktoren über die AhR-Signalkaskade die carriervermittelte Folatresorption in humanen Kolonzellen herabregulieren. Es erscheint anhand der gewonnen Erkenntnisse möglich, dass die Folathomoöstase durch verschiedene Umweltkontaminanten wie z. B. TCDD negativ beeinflusst werden kann und als Folge daraus auch eventuell der Folatmangel der Bevölkerung in Industrienationen zumindest teilweise erklärbar wird. Es ist darüber hinaus denkbar, dass auch andere Fremdstoffe die Folataufnahme beeinflussen, da große planare Strukturen häufig über den AhR-Weg wirken. Weiterführende Studien sind notwendig, um das Verständnis für den Einfluss von Umweltkontaminanten auf die intestinale Folataufnahme zu verbessern.

Background: Preeclampsia, small for gestational age (SGA), placental abruption, and fetal death are pregnancy complications linked to the utero-placental vasculature with serious consequences for maternal and infant well-being. Elevated homocysteine, a marker of cardiovascular disease risk, is postulated to play a role in placenta-mediated complications, but epidemiologic studies have reported inconsistent findings. The two primary objectives of this thesis were to 1: comprehensively investigate the association of homocysteine with placenta-mediated complications and examine modifying effects of pre-specified factors on this association, and 2: comprehensively investigate determinants of maternal homocysteine during pregnancy. Methods: A systematic review and meta-analysis of prospective studies was conducted to address thesis objective 1. The Ottawa and Kingston (OaK) Birth Cohort, a prospective cohort study that recruited pregnant women between 2002 and 2009, was used to address thesis objectives 1 and 2. Homocysteine concentration was measured between 12 and 20 weeks gestation. Analyses based on the OaK Birth Cohort consisted of multivariable regressions using restricted cubic splines to model associations with continuously distributed variables. Results: Objective 1: In an analysis of 7587 participants, a significant association between homocysteine concentration and a composite outcome of any placenta-mediated complication was observed (odds ratio (OR) for a 5 µmol/L increase: 1.63, 95% Confidence Interval (CI) 1.23-2.16) and SGA (OR 1.76, 95% CI 1.25-2.46), with potential modifying effects of the methylene tetrahydrofolate reductase (MTHFR) 677C>T variant (SGA) and high-risk pregnancy (preeclampsia). In the systematic review identifying 30 prospective cohort or nested case-control studies, a random effects meta-analysis of pooled mean differences in homocysteine between cases and controls in 28 studies revealed significantly higher means for SGA: 0.35 µmol/L (95% CI 0.19 to 0.51, I2=33%); and preeclampsia: 0.87 µmol/L (95% CI 0.52 to 1.21, I2=92%). Significant sources of heterogeneity were study region (SGA and preeclampsia), adjusting for covariates (preeclampsia), folate status (preeclampsia), and severity (preeclampsia). Objective 2: In 7587 OaK participants, factors related to favourable health status were associated with lower maternal homocysteine concentrations. Folic acid supplementation during pregnancy of >1 mg/day did not substantially increase serum folate concentration. Conclusion: This thesis suggests an independent effect of slightly higher homocysteine concentration in the early to mid-second trimester on the risk of any placenta-mediated complication, SGA, and preeclampsia. Modifying effects explain some of the variability in previous studies. Favourable preconception health status was associated with lower maternal homocysteine.

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Thesis (MPath)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Major Depressive Disorder (MDD) is a severe debilitating medical condition that may lead to suicide. Due to a poor understanding of the biological mechanisms underlying the disease process therapeutic decisions are usually taken using a ‘trial and error’ approach. This is not ideal since many treatments do not work as expected for all individuals. Studies have shown that only half of MDD patients receive the appropriate treatment, whereas many patients have adverse response to anti-depressants. These may include weight gain and raised homocysteine levels that may further compromise the health status of MDD patients and may partly explain the link with cardiovascular disease. The objective of the study was to identify genetic risk factors interacting with environmental factors implicated in MDD that may be of relevance to the South African population. Polymorphisms in the MTHFR (677 C>T, rs1801133 and 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) and SLC6A4 (43 bp ins/del, rs4795541) genes were genotyped in 86 MDD patients and 97 population-matched controls. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MDD. A total of 183 unrelated Caucasians participated in the study, including 69 females and 17 males with MDD and 57 female and 40 male controls without a personal and family medical history of overlapping stress/anxiety and depressive disorders. All study participants were genotyped for the six selected SNPs considered clinically useful based on international data. The allelic distribution of the SNPs, single or combined into a genotype risk score after counting their minor alleles, did not differ between MDD patients and controls. Homocysteine levels were determined and correlated with body mass index (BMI) and other variables known to influence these phenotypes. The folate score assessed with use of the study questionnaire was significantly lower in the patient group compared with controls (p=0.003) and correlated significantly with BMI, particularly in females (p=0.009). BMI was on average 8% higher in the MDD patients compared with controls (p=0.015) after adjustment for age and sex. The MTHFR rs1801133 677 T-allele was associated with a 14% increase in BMI in MDD patients but not controls (p=0.032), which in turn was associated with significantly increased homocysteine levels (p<0.05). The aims of the study were successfully achieved. Identification of the MTHFR rs1801133 677 T-allele reinforces the importance of adequate folate intake in the diet due to increased risk of obesity and depression found to be associated with low dietary intake. Evidence of shared genetic vulnerability for many chronic diseases and drug response mediated by the MTHFR 677 T-allele support the clinical relevance of this low-penetrance mutation. / AFRIKAANSE OPSOMMING: Major depressie (MD) is ‘n aftakelende siektetoestand wat tot selfdood kan lei. Onkunde oor die siekte se onderliggende biologiese meganismes lei dikwels tot ‘n lukrake terapeutiese benadering. Dit is ‘n onbevredigende situasie aangesien indiwidue verskillend reageer op die middels wat voorgeskryf word. Navorsing toon dat slegs ongeveer die helfte van MD pasiënte toepaslike behandeling kry, terwyl anti-depressante ‘n nadelige uitwerking het op baie pasiënte. Dit sluit massatoename en verhoogde homosisteïenvlakke in wat MD pasiënte se gesondheid bykomend nadelig kan beïnvloed en die verband met kardiovaskulêre siekte gedeeltelik kan verklaar. Hierdie studie poog om MD verwante genetiese risikofaktore en omgewingsfaktore wat mekaar beïnvloed en moontlik op die Suid Afrikaanse bevolking betrekking het, te identifiseer. Polimorfismes in die MTHFR (677 C>T, rs1801133 en 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) en SLC6A4 (43 bp ins/del, rs4795541) gene is geanaliseer in 86 MD pasiënte en 97 kontroles geselekteer van dieselfde populasie. Die spesifieke doelwitte was om 1) hoë deurset direkte polimerase kettingreaksie (RT-PCR) genotiperingstoetse vir die 6 gekose polimorfismes met direkte volgordebepaling as maatstaf analities te valideer vir 2) moontlike insluiting in ‘n patologie-ondersteunde genetiese toetsstrategie met die oog op beter kliniese hantering van MD. Altesaam 183 Kaukasiërs het aan die studie deelgeneem. Die MD pasiënte het uit 69 vroue en 17 mans bestaan. Die kontroles (57 vroue en 40 mans) het geen mediese geskiedenis (persoonlik of familie) van oorvleuelende stress/angstigheid of depressie gehad nie. Gebaseer op internasionale data, is al die deelnemers vir die 6 gekose, potensieel klinies-bruikbare polimorfismes getoets. Die alleliese verspreiding van die polimorfismes enkel of gekombineer (uitgedruk as ‘n genotipe-risiko-syfer nadat minor allele getel is), was dieselfde in MD-pasiënte en kontroles. Homosisteïenvlakke is bepaal en gekorreleer met die liggaamsmassa-indeks (BMI) en ander veranderlikes wat bekend is vir hulle invloed op hierdie fenotipes. In teenstelling met die kontroles, was die folaat telling, soos bepaal met die studievraelys, betekenisvol laer in die pasiënte (p=0.003). Die korrelasie met die liggaamsmassa-indeks, spesifiek by vroue, was ook betekenisvol (p=0.009). Na aanpassings vir ouderdom en geslag, is gevind dat die liggaamsmassa-indeks gemiddeld 8% hoër was in die die MD pasiënte teenoor die kontroles. By MD-pasiënte, maar nie by die kontroles nie, is die MTHFR rs1801133 677 T-alleel geassosieer met ‘n 14% toename in liggaamsmassa-indeks (p=0.032), wat ook geassosieer was met betekenisvolle verhoogde homosisteïenvlakke (p<0.05). Die doelwitte van die studie is bereik. Identifisering van die MTHFR rs1801133 677 T-alleel beklemtoon hoe belangrik dit is om voldoende folaat in te neem, veral omdat ‘n verhoogde risiko vir vetsug en depressie met ‘n lae folaatinname in die diet geassosieer word. Die kliniese belang van die MTHFR 677 T-alleel word beklemtoon deur toenemende bewyse wat daarop dui dat gedeelde genetiese vatbaarheid vir ‘n verskeidenheid van kroniese siektes asook middelrespons aan bemiddeling deur hierdie lae penetrasie mutasie toegeskryf kan word. / Winetech / Technology for Human Resources and Industry Program (THRIP).