Made available in DSpace on 2014-06-12T23:16:16Z (GMT). No. of bitstreams: 2 arquivo958_1.pdf: 2790418 bytes, checksum: 41ba3566a5e13d0a2496b830fee53158 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os antibióticos são substâncias químicas que combatem ou inibem o crescimento de organismos, sendo muito utilizados para o controle de certas doenças de origem bacteriana. Faz-se necessário atestar a qualidade destes produtos, uma vez que se torna cada vez mais crescente o seu uso. Além disso, resíduos de antibióticos podem permanecer em alimentos de origem animal, acima de valores considerados seguros quando não são respeitadas as boas práticas veterinárias. Neste trabalho foram desenvolvidos e validados métodos para a determinação de antibióticos em formulações farmacêuticas e em leite. No que diz respeito às formulações farmacêuticas foi desenvolvido um procedimento de análises em fluxo com multicomutação baseado no conceito de fluxo-batelada para a determinação espectrofotométrica de amoxicilina. O procedimento foi baseado na reação da orto-nitroanilina diazotizada em meio alcalino resultando em um sal de diazônio e posterior acoplamento com a amoxicilina para a reação de azo acoplamento. O módulo de análises foi constituído por duas minibombas solenóide, uma válvula de estrangulamento e três válvulas solenóide de três vias, sendo os dispositivos ativos controlados por um microcomputador equipado com uma interface PCL-711S, empregando um programa escrito em linguagem QuickBASIC 4.5. As soluções de amostras e de reagentes foram introduzidas em uma câmara de reação onde foi fixado um LED (λ=435 nm) usado como fonte luminosa e um fototransistor (Til78) usado como detector. As soluções de referência foram estudadas na faixa de 25 a 400 mg L-1 de amoxicilina, com limite de detecção de 5,1 mg L-1, desvio padrão relativo de 3,9% (n=10) e frequência de amostragem de 50 determinações por hora. Foi também desenvolvido um método para determinação de amoxicilina, ampicilina, tetraciclina, oxitetraciclina e cloranfenicol em amostras de leite, empregando o procedimento QuEChERS para o preparo da amostra e de cromatografia líquida acoplada ao espectrômetro de massas no modo de ionização electrospray (LC-ESI-MS) para a identificação e quantificação. Foi utilizada uma coluna C18 e a fase móvel foi composta de água e metanol, com eluição por gradiente. O método foi validado e aplicado para análises de amostras de leites integral e in natura provenientes da bacia leiteira do estado de Pernambuco. A linearidade da curva analítica foi de 20,0 a 400,0 μg L-1 para a tetraciclina e oxitetraciclina. A partir de 2,0 μg L-1 até 12,0 μg L-1 para amoxicilina e ampicilina e entre 0,3 e 1,3 μg L-1 para o cloranfenicol. Os resultados do teste de recuperação variaram na faixa de 83 a 92 %. Pôde-se observar que das dez diferentes marcas de leite pasteurizado integral (tipo longa vida) analisados, em duas foram registradas a presença de amoxicilina e oxitetraciclina. Para as vinte e cinco amostras de leite in natura, pôde-se verificar a presença de amoxicilina e ampicilina em três amostras de leite e a presença de oxitetraciclina e tetraciclina em mais duas amostras. Os valores de resíduos de antibióticos encontrados, em todas as amostras, estavam abaixo do LMR permitido pela legislação vigente

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-08-04T13:34:48Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE - Waleska de Araújo Siqueira Bento - Versão final.pdf: 3392903 bytes, checksum: 633a749c72e4eb3f1d0e4f6b0f0c0b83 (MD5) / Made available in DSpace on 2017-08-04T13:34:48Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE - Waleska de Araújo Siqueira Bento - Versão final.pdf: 3392903 bytes, checksum: 633a749c72e4eb3f1d0e4f6b0f0c0b83 (MD5) Previous issue date: 2016-03-11 / O desenvolvimento de métodos de preparo de amostras é de suma importância para a análise química. Ao longo dos anos, cada vez mais os cientistas buscam técnicas para aprimorar metodologias e ferramentas matemáticas para a validação dos métodos desenvolvidos. No presente trabalho foram desenvolvidos dois diferentes métodos de preparo de amostras: um para a análise de corantes artificiais em iogurtes e posterior análise por HPLC-PAD e outro método utilizando a técnica de CE-UV/Vis para separar e fracionar amostras padrões de misturas de proteínas, seguido de um método para digestão destas e análise por LC-ESI-MS para posterior mapeamento dos peptídeos. O primeiro trabalho foi realizado para determinar a concentração de corantes artificiais em iogurtes e bebidas lácteas que são alimentos produzidos a partir da fermentação do leite sendo um alimento importante e indispensável a dieta de adultos e crianças. Para tanto foi desenvolvido um método de extração e determinação de corantes artificiais em iogurtes e bebidas lácteas por HPLC-PAD. O método foi devidamente validado e as amostras comerciais analisadas estavam de acordo com a legislação. Já o segundo trabalho visa o estudo da proteômica através do mapeamento de peptídeos. A eletroforese capilar (CE) por ser uma técnica de separação muito eficiente para a análise de proteínas e peptídeos foi utilizada na etapa inicial de preparo da amostra. A separação inicial foi seguida do fracionamento da amostra que é uma etapa muito importante e que a CE pode proporcionar, minimizando a complexidade da amostra. Posteriormente foi desenvolvido um método para a digestão das proteínas contidas em cada fração e o mapeamento dos peptídeos foi realizado com auxílio do LC-ESI-MS. Os resultados obtidos são animadores, visto que foi possível digerir as proteínas com quimotripsina em até duas horas. O que é um tempo curto se comparado a trabalhos da literatura que necessitaram de 12 a 24 horas para digestão de proteínas quando utilizadas enzimas livres em solução. / The development of sample preparation methods is of paramount importance for analytical chemistry. Over the years, more and more scientists are seeking techniques to improve methodologies and mathematical tools for the validation of the methods they have developed. In this study two different methods of sample preparation were developed: one for analysis of artificial colorants in yogurts and subsequent analysis by HPLC-PAD; and another method using the CE-UV/Vis technique to separate and fractionate standard samples of protein mixtures, followed by a digestion method for these and analysis by LCESI-MS for subsequent peptides mapping. The first study was conducted to determine the concentration of artificial colorant in yoghurts and milk drinks, foods produced from the fermentation of milk that are an important and indispensable part of the food diet for adults and children. To this end, a method was developed for the extraction and determination of artificial colorant in HPLC-PAD yoghurts and milk drinks. The method was validated and commercial samples were analyzed according to the legislation. The second work is aimed at the study of proteomics by peptide mapping. Capillary electrophoresis (CE) as an effective separation technique was used in the initial stage of sample preparation for the anaysis of proteins and peptides. The initial separation was followed by fractionation of the sample which is a very important step enabled by CE, thus minimizing sample complexity. Then, a method was developed to digest the proteins contained in each fraction and the mapping of the peptides was carried out with the aid of LC-ESI-MS. The results obtained are encouraging, since the study showed that it was possible to digest proteins with free chymotrypsin within two hours. This is a short time compared to that found in the published papers requiring 12 to 24 hours for protein digestion when free enzymes in solution are used.

Research was conducted on thirteen 100% cotton denim samples using an acid wash, established by Murray, to extract oligosaccharides from the cellulosic material. The oligosaccharide ion groups (+, +, and +) for molecules with degrees of polymerization between two and seven (DP2-DP7) were analyzed using liquid chromatography coupled to mass spectrometry with an electrospray ionization interface (LC-ESI-MS). The results were compared using the least-squares means in a Multivariate ANOVA (MANOVA) test followed by Univariate ANOVA and Tukey HSD tests and demonstrated that the method could correctly determine that two samples were statistically different 85.9% of the time when analyzing the amount (ng) of each of the oligosaccharide ion groups separately, and 82.0% when analyzing the total moles of monosaccharide units released. A dye extraction was performed on the denim materials and the extract analyzed using gas chromatography coupled with mass spectrometry (GC-MS). Indigo dye was present in all of the denim samples except one. When these results were combined with the two oligosaccharide statistical analyses, the discriminating power was increased to 88.5% and 85.9%, respectively. Additional cellulosic materials were also investigated including four white 100% cotton t-shirts as well as five raw cotton samples grown in Tajikistan, Uzbekistan, Egypt, Iran, and Benin West Africa. The analytical methodology gave results for the white cotton t-shirts and raw cotton samples that were inconsistent with those obtained from the denim samples.

no / Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI-MS/MS quantitative assays. The protocol we describe in this chapter outlines the steps we follow to a) extract prostanoids from solid or liquid samples, b) semi-purify the metabolites using solid phase extraction c) set-up the HPLC separation using reverse phase chromatography and d) set up the MS/MS assay using a triple quadrupole mass spectrometer. The experimental details and notes presented here are based on the detailed protocols followed in our group

Das übergeordnete Ziel dieser Promotion war die Untersuchung und Kompensation des Matrixeffekts für die Analytik von Arzneimitteln in komplexen Probenmatrices mit LC-ESI-MS/MS-Technik. Zunächst konnte eine einfache analytische Methode für eine breite analytische Anwendbarkeit entwickelt werden. Es zeigte sich jedoch, dass die Matrixeffektkompensation zu einem Mehraufwand bei der Probenvorbereitung führt. Deshalb wurde anschließend der Mechanismus des Matrixeffektes auf die LC-ESI-MS/MS-Technik genauer untersucht. Die gewonnenen Erkenntnisse wurden anschließend eingesetzt, um eine einfache alternative Quantifizierungsmethode mittels der PCI eines internen Standards zu entwickeln. Im ersten Teilprojekt wurde eine LC-ESI-MS/MS-Methode für die Analytik von häufig verschriebenen Antibiotika in Abwasserproben der Stadt Dresden entwickelt. Da weder Vergleichsmatrix für Abwasser zur Verfügung stand, noch für alle Zielanalyte isotopenmarkierte Standards erhältlich sind, wurde der stark variierende Matrixeffekt der Abwasserproben mittels der Standardaddition kompensiert. Die Ergebnisse der Methodenentwicklung zeigen, dass eine genaue und flexible Methode entwickelt werden konnte, die Matrixkompensation jedoch zu einem erhöhten Zeit- und Materialaufwand führt. Es wurde deutlich, dass neben bisher genutzten Kompensationsmethoden für den Matrixeffekt, wie Standardaddition und interner isotopenmarkierter Standards, neue alternative Strategien getestet werden müssen. In dem zweiten Teilprojekt wurde daher der Matrixeffektmechanismus von Urin-, Plasma- und verschiedenen Abwasserproben bei der Messung von verschiedenen Arzneimitteln mittels LC-ESI-MS/MS analysiert. Die Ergebnisse der Untersuchungen mittels „post-column infusion“ konnten bisherige Erkenntnisse zu Matrixeffektmechanismen bestätigen und das Verständnis vertiefen. Matrixeffekte sind von der jeweiligen Zusammensetzung der Probenmatrix abhängig, aber auch substanzspezifisch. Dabei kommt es zwischen Analyt und Begleitsubstanzen zu einer Konkurrenz um freie Ladungsträger oder zu einer veränderten Anordnung/Verteilung innerhalb der ESI-Spray-Tröpfchen. Gleichzeitig zeigten die Ergebnisse, dass es auch andere Mechanismen, wie z. B. Ladungstransfers zwischen Analyt und Begleitsubstanzen, geben muss. Schließlich wurden die Ergebnisse des zweiten Teilprojekts in einer innovativen Methodenentwicklung zur Matrixkompensation und zur Quantifizierung von 16 Arzneimitteln in Urinproben verwendet. Der Matrixeffekt der Substanzen mit vergleichbarer Signalsuppression konnte über einen einzelnen nachsäuleninfundierten internen Standard kompensiert werden. Die Ergebnisse zeigen einen deutlichen Vorteil der entwickelten Methode gegenüber Matrix-Kalibrierung in Präzision und Richtigkeit oder dem Einsatz von isotopenmarkierten internen Standards in Aufwand der Methodenentwicklung und Verbrauch von Standardsubstanzen. Die Ergebnisse der vorliegenden Arbeit zeigen die Bedeutung, Komplexität und den Einfluss der Matrixeffekte in der Anwendung der LC-ESI-MS/MS-Technik. Einerseits sind geeignete Methoden für die Minimierung von Matrixeffekten wie Probenvorbereitung und Chromatographie nötig, andererseits müssen Ionisierungsmechanismen, insbesondere die Wechselwirkungen von Zielanalyten und Begleitsubstanzen, zukünftig Gegenstand weiterer Untersuchungen sein. Die Ergebnisse dieser Arbeit liefern wichtige Beiträge zur Verbesserung der Analytik von komplexen Proben mittels der LC-ESI-MS/MS-Technik. / The overall goal of this Ph.D. thesis was to investigate and compensate the matrix effect of the analysis of drugs in complex sample matrices with LC-ESI-MS/MS technique. First, a simple analytical method for a broad analytical applicability was developed for wastewater analysis. However, the matrix effect compensation embraced the main part effort in sample preparation. Therefore, the mechanism of the matrix effect on the LC-ESI-MS/MS technique was examined in more detail. The findings were used to develop an alternative quantification method using post-column infusion of an internal standard substance. In the first project, a LC-ESI-MS/MS method was developed to analyze commonly prescribed antibiotics in wastewater samples of Dresden. Since neither comparison matrix for wastewater nor all isotopically-labeled analogs for the target analytes were available, the strongly varying matrix effect of the wastewater samples was compensated by standard addition. The results show that the developed method is precise and flexible, but the matrix effect compensation leads to an increased expenditure of time and materials. Besides previously used matrix effect compensation methods, such as standard addition and internal isotopically-labeled standard, new alternative strategies need to be tested. Therefore, the matrix effect mechanism of various drugs and sample matrix combinations was examined in the second project using post-column infusion. The results confirmed previous findings on matrix effect mechanisms and deepened our understanding that matrix effects not only depend on the composition of the sample matrix but are also substance-specific. This results to a competition of free charge carriers between analyte and accompanying substances or to an alternated distribution within the ESI spray droplets. Furthermore, the results indicate that there are other mechanisms, such as charge transfer between analyte and concomitant substances. The results of the second project were used to invent a method for matrix effect compensation and quantification of 16 drugs in urine samples. The matrix effects of the substances with comparable signal suppression were compensated by a single post-column infused internal standard. The developed method has a significant advantage over the matrix calibration regarding precision and accuracy as well as the use of isotopically-labeled internal standards in effort of method development and consumption of standard substances. Finally, the results of this work show the importance, complexity and influence of the matrix effects in the application of the LC-ESI-MS/MS-technique. Suitable methods for minimizing matrix effects such as sample preparation and chromatography are needed and ionization mechanisms, in particular the interactions of target analytes with accompanying substances, should be investigated in future studies. The work of this Ph.D. project contributes to the improvement of the analysis of complex samples using the LC-ESI-MS/MS-technique.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-07T12:58:36Z No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) / Made available in DSpace on 2016-04-07T12:58:36Z (GMT). No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) Previous issue date: 2013-01-24 / Sida tuberculata (Malvaceae), conhecida popularmente como “guanxuma”, é uma espécie vegetal de porte herbáceo, bem representada na região sul do Brasil. Na cultura popular é utilizada para tratamento de diversas enfermidades, em especial àquelas relacionadas ao diabetes e ao colesterol elevado. Para algumas espécies, existem relatos de eventual potencial antimicrobiano. Considerando a ausência de estudos sobre esta planta, o presente trabalho investigou a composição química dos extratos brutos de S. tuberculata e avaliou seu potencial antifúngico in vitro. Após a coleta e identificação, o material vegetal foi submetido aos processos de secagem e trituração. Submeteu-se a extração a frio por percolação, utilizando-se como solvente solução hidroetanólica a 40% para folhas e 70% para raízes. Para fins de comparação foram feitas extrações aquosas por infusão. Na sequência, foram determinados os teores de fenólicos totais e flavonóides totais. Posteriormente, as amostras foram analisadas através de método por CLAE-UV, com seleção das condições cromatográficas de melhor eficiência de separação. Sendo estas estabelecidas, efetuou-se análise por LC-MS em modo ESI positivo. Para os ensaios da atividade antifúngica foram utilizados os protocolos de microdiluição em ágar para determinação da concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM). Também foi aplicada a metodologia para avaliação do potencial de remoção de biofilme em Cateter Venoso Central (CVC). Os resultados obtidos demonstraram um maior teor de fenólicos e flavonóides totais nos extratos das folhas. As análises por LC-UV-MS permitiram a identificação e proposição de cinco compostos, entre ecdisteróides, flavonóides e alcalóides. Nos ensaios de atividade antifúngica os extratos aquosos apresentaram atividade contra linhagens de Candida krusei, com valores de CIM variando entre 3.9 - 62.5 μg/ml para folhas e 1.95 - 31.25 μg/ml para raízes. No teste de remoção de biofilme, os extratos aquosos das folhas demonstraram um maior potencial de remoção. Os dados de composição química obtidos, nas variantes de diferentes partes da planta, bem como a atividade antimicrobiana detectada, geram expectativas quanto a novos estudos de exploração do potencial biológico de S. tuberculata. / Sida tuberculata (Malvaceae), popularly known as "guanxuma", is an herbaceous plant species present in southern Brazil. In popular culture, it is used for the treatment of several diseases, such as those related to diabetes and high cholesterol level. For some species of Sida, there are also reports about the antimicrobial potential. Considering the lack of studies at this species, this study proposed an investigation about the chemical composition of Sida tuberculata extracts and their in vitro antifungal activity. After collection and identification, the plant material was submitted to dryness and powdered. Then, it was submitted to extraction by percolation using hydroethanolic solution at 40% and 70% as solvent, to leaves and roots respectively. For comparison, aqueous extracts were obtained by infusion. The total phenolic and flavonoid contents of extracts were determined. The samples were also evaluated by HPLC-UV, testing the chromatographic conditions that promote the better separation efficiency. After, for the identification procedure, the analysis by LC-ESI-MS in positive mode was conducted using previously established conditions. For the antifungal activity assay, the agar microdilution protocols were used for determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). In addition, the extracts were tested for potential biofilm removal in Central Venous Catheter (CVC). The results demonstrated a higher concentration of total phenolic and flavonoids compounds in the leaves extracts. LC-MS analysis allowed the identification of five components, between ecdysteroids, flavonoids and alkaloids. In the assay of antifungal activity, the aqueous extract had activity against Candida krusei strains, with the MIC values varying between 3.9 - 62.5 μg/ml for leaves and 1.95 - 31.25 μg/ml for roots. In the CVC biofilm removal testing, the aqueous leaves extracts presented a greater potential. The chemical composition data obtained in this work, considering the different parts of plant, as well as the antimicrobial activity detected, bring perspectives of exploring this species concerning its biological potential.

Sulforaphane is an isothiocyanate found in high concentrations in cruciferous vegetables like broccoli. Sulforaphane has received much attention due to the evidence that it inhibits phase I carcinogen-bioactivating enzymes and/or induces phase II antioxidant enzymes as well as metallothioneins (MTs) (Perocco et al., 2006; Clarke et al., 2008; Yeh & Yen, 2009). Since MTs and antioxidant enzymes are involved in the scavenging of reactive oxygen species (ROS), the question was raised whether sulforaphane can provide protection against increased oxidative stress and if sulforaphane exposure of a human hepatocellular carcinoma cell line, like HepaRG cells, will have a negative impact on phase I and II biotransformation in these cells. Oxidative stress was exogenously induced in HepaRG cells with tert- Butyl hydroperoxide (t-BHP). Phase I and phase II biotransformation pathways were assessed with caffeine, paracetamol, aspirin, sodium benzoate, and paraaminobenzoic acid, respectively, as probe substances. Through the use of a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) assay, the biotransformation of caffeine in phase I and the formation of paracetamol, aspirin, sodium benzoate and para-aminobenzoic acid conjugates in phase II were investigated. This involved elucidating the time it took for the whole probe to be completely biotransformed during phase I biotransformation and the unique conjugates formed during phase II biotransformation in HepaRG cells. The optimal t-BHP concentration and exposure time in HepaRG cells were standardized with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LC-ESI-MS/MS assays to monitor phase I and phase II biotransformation were optimized and validated. The optimal sulforaphane concentration and exposure time in HepaRG cells were standardized with a MTT assay. To evaluate the possible protective effect of sulforaphane against oxidative stress, HepaRG cells were pre-incubated with sulforaphane followed by the induction of oxidative stress with t-BHP and the quantification of the amount of viable cells with a MTT assay. To investigate the effect of sulforaphane on phase I and phase II biotransformation pathways, HepaRG cells were first pre-incubated with sulforaphane followed by the addition of a specific probe substance and the assessment of the biotransformation of the probe with a LC-ESI-MS/MS assay. The results partially supported the hypothesis of the study that sulforaphane will protect HepaRG cells against oxidative stress without negatively influencing phase I and phase II biotransformation. The results indicated that sulforaphane provided partial protection against t-BHP induced oxidative stress and had no effect on phase II paracetamol biotransformation in HepaRG cells. / Thesis, MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013.

Sulforaphane is an isothiocyanate found in high concentrations in cruciferous vegetables like broccoli. Sulforaphane has received much attention due to the evidence that it inhibits phase I carcinogen-bioactivating enzymes and/or induces phase II antioxidant enzymes as well as metallothioneins (MTs) (Perocco et al., 2006; Clarke et al., 2008; Yeh & Yen, 2009). Since MTs and antioxidant enzymes are involved in the scavenging of reactive oxygen species (ROS), the question was raised whether sulforaphane can provide protection against increased oxidative stress and if sulforaphane exposure of a human hepatocellular carcinoma cell line, like HepaRG cells, will have a negative impact on phase I and II biotransformation in these cells. Oxidative stress was exogenously induced in HepaRG cells with tert- Butyl hydroperoxide (t-BHP). Phase I and phase II biotransformation pathways were assessed with caffeine, paracetamol, aspirin, sodium benzoate, and paraaminobenzoic acid, respectively, as probe substances. Through the use of a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) assay, the biotransformation of caffeine in phase I and the formation of paracetamol, aspirin, sodium benzoate and para-aminobenzoic acid conjugates in phase II were investigated. This involved elucidating the time it took for the whole probe to be completely biotransformed during phase I biotransformation and the unique conjugates formed during phase II biotransformation in HepaRG cells. The optimal t-BHP concentration and exposure time in HepaRG cells were standardized with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LC-ESI-MS/MS assays to monitor phase I and phase II biotransformation were optimized and validated. The optimal sulforaphane concentration and exposure time in HepaRG cells were standardized with a MTT assay. To evaluate the possible protective effect of sulforaphane against oxidative stress, HepaRG cells were pre-incubated with sulforaphane followed by the induction of oxidative stress with t-BHP and the quantification of the amount of viable cells with a MTT assay. To investigate the effect of sulforaphane on phase I and phase II biotransformation pathways, HepaRG cells were first pre-incubated with sulforaphane followed by the addition of a specific probe substance and the assessment of the biotransformation of the probe with a LC-ESI-MS/MS assay. The results partially supported the hypothesis of the study that sulforaphane will protect HepaRG cells against oxidative stress without negatively influencing phase I and phase II biotransformation. The results indicated that sulforaphane provided partial protection against t-BHP induced oxidative stress and had no effect on phase II paracetamol biotransformation in HepaRG cells. / Thesis, MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013.

no / This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX-1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. ¿-Melanocyte stimulating hormone (¿-MSH), which had no effect on melanin production in FM55 cells, stimulated PGD2 production in these cells without affecting PGE2. While cAMP pathways may be involved in regulating PGD2 production, our results suggest that ¿-MSH acts independently of cAMP, possibly by regulating the activity of lipocalin-type PGD synthase. This ¿-MSH-mediated effect may be associated with its role as an immune modulator. / The Wellcome Trust

no / Prostanoids modulate the activity of human pregnant myometrium and their functional role can be appreciated through characterisation of prostanoid receptors and tissue concentration of prostanoids. We have applied a lipidomic approach to elucidate the profile of prostanoids in human non-labouring and labouring myometrium. We have identified a total of nineteen prostanoids including prostacyclin, thromboxanes, prostaglandins and dihydro-prostaglandins. Prostacyclin was the predominant prostanoid in both non-labouring and labouring myometria, with PGD2 and PGF2¿ being the second most abundant. Although the total amount of prostanoids was increased in the labouring tissue, PGE2 and 13,14-dihydro-15-keto-PGE2 were the only prostanoids to increase significantly at early and late labour (p¿0.001). Our data suggest that PGF2¿ plays an important role in parturition, whilst the increase in PGE2 could occur to facilitate cervical dilation and relaxation of the lower myometrium during labour. Although the elevation in TXA2 was less marked than expected, in terms of translation to function even a relatively small increase in the level of this potent spasmogen may have significant effects.