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The stabilisation of the extracellular matrix of bone on biomaterial surfacesHeath, Deborah Jane January 2000 (has links)
No description available.
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Effects of Atrazine Exposure on Aromatase Expression in Male Zebrafish (<i>danio rerio</i>)Schmaus, Carrie January 2015 (has links)
No description available.
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Avaliação dos efeitos tóxicos de novas substâncias bioativas: detecção de estresse oxidativo e mutagenicidade / Evaluation of the toxic effects of new bioactive substances: oxidative stress detection and mutagenicityRocha, Camila de Melo Romero 22 March 2018 (has links)
A produção de espécies reativas de oxigênio (ROS) nos sistemas biológicos é contrabalanceada pelos sistemas antioxidantes enzimáticos e não-enzimáticos. Quando há um desequilíbrio entre a geração de ROS e esses sistemas, ocorre um aumento dessas espécies reativas causando estresse oxidativo, que pode levar a danos a macromoléculas, como lipídios, proteínas e o DNA. Os fármacos doxorrubicina (antineoplásico) e benzonidazol (antiparasitário) são conhecidos por induzir efeitos colaterais que podem estar relacionados ao aumento de ROS. Além disso, ensaios de mutagenicidade demonstram que esses fármacos apresentam atividade mutagênica por danos oxidativos. O Grupo NEQUIMED desenvolve substâncias com potencial atividade antineoplásica e antiparasitária, as quais ainda não foram avaliadas em relação às propriedades tóxicas e genotóxicas. Dessa forma, o objetivo deste trabalho foi analisar as propriedades mutagênicas e de estresse oxidativo dessas novas substâncias, comparando aos fármacos benzonidazol e doxorrubicina. Para detecção de ROS foi realizado o ensaio fluorimétrico utilizando o marcador 2,7-diacetato de diclorofluoresceína (DCFH-DA) em linhagens celulares de hepatocarcinoma humano (HepG2) e fibroblasto de camundongo (Balb/C 3T3 clone A31). O estado redox destas células foi avaliado através da quantificação da expressão gênica e do conteúdo proteico das enzimas antioxidantes através das técnicas de qRT-PCR e Western blot, respectivamente. A atividade mutagênica foi analisada com o ensaio Ames miniaturizado Salmonella/microssoma utilizando a linhagem TA102 de Salmonella typhimurium que detecta agentes mutagênicos que causam danos por oxidação. Os resultados mostraram que as substâncias estudadas pelo Grupo não induzem aumento na produção de ROS ou induzem em menores níveis do que doxorrubicina e benzonidazol, além de levar a alterações menos proeminentes que os fármacos para a expressão das proteínas antioxidantes. No ensaio mutagênico, o benzonidazol apresentou o pior perfil, doxorrubicina e Neq0438 somente foram mutagênicos com ativação enzimática, enquanto Neq0551 foi inativo. Assim, as novas substâncias (Neq0438 e Neq0551) apresentaram um perfil melhor do que os fármacos de referência, tornando-os candidatos promissores para estudos in vitro e in vivo subsequentes. / The production of reactive oxygen species (ROS) in biological systems is compensated by the enzymatic and non-enzymatic antioxidant systems. The excessive ROS production causes oxidative stress, which can damage important cellular macromolecules such as lipids, proteins and DNA. The drugs doxorubicin (antineoplastic) and benzonidazole (antiparasitic) are both known for their side effects, which can be related to the increase of ROS. Besides, mutagenicity assays show that these drugs have a mutagenic activity via oxidative damages. The research group NEQUIMED studies new substances with potential antineoplastic and antiparasitic activities, but their toxic and genotoxic properties have not been fully evaluated yet. Thus, the aim of this work is to assess the mutagenic potential and the oxidative stress generated by these substances, comparing them to benzonidazole and doxorubicin. The fluorimetric assay using the probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for ROS detection in human hepatocarcinoma (HepG2) and mouse fibroblast (Balb/C 3T3 clone A31) cell lines. The redox state of these cells was evaluate by qRT-PCR and Western blot methods to quantifying gene expression and protein content of the antioxidant enzymes. The mutagenic potential was assessed by the miniaturized Ames text with the Salmonella/microssome mutagenicity assay, using the TA102 strain of Salmonella typhimurium, which detects oxidation damages to the DNA. The new substances did not induce an increase on ROS production, or did in lower levels when compared to doxorubicin and benzonidazole. Moreover, reference drugs also induced greater changes on the expression of the antioxidant enzymes. Benznidazole had a higher mutagenic activity, while Neq0438 and doxorubicin were mutagenic only when incubated with enzymatic activation. Neq0551 was inactive for Ames assay. Therefore, these new substances (Neq0438 and Neq0551) had a better overall profile than the reference drugs, turning out to be promising candidates for further in vitro and in vivo studies.
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Avaliação dos efeitos tóxicos de novas substâncias bioativas: detecção de estresse oxidativo e mutagenicidade / Evaluation of the toxic effects of new bioactive substances: oxidative stress detection and mutagenicityCamila de Melo Romero Rocha 22 March 2018 (has links)
A produção de espécies reativas de oxigênio (ROS) nos sistemas biológicos é contrabalanceada pelos sistemas antioxidantes enzimáticos e não-enzimáticos. Quando há um desequilíbrio entre a geração de ROS e esses sistemas, ocorre um aumento dessas espécies reativas causando estresse oxidativo, que pode levar a danos a macromoléculas, como lipídios, proteínas e o DNA. Os fármacos doxorrubicina (antineoplásico) e benzonidazol (antiparasitário) são conhecidos por induzir efeitos colaterais que podem estar relacionados ao aumento de ROS. Além disso, ensaios de mutagenicidade demonstram que esses fármacos apresentam atividade mutagênica por danos oxidativos. O Grupo NEQUIMED desenvolve substâncias com potencial atividade antineoplásica e antiparasitária, as quais ainda não foram avaliadas em relação às propriedades tóxicas e genotóxicas. Dessa forma, o objetivo deste trabalho foi analisar as propriedades mutagênicas e de estresse oxidativo dessas novas substâncias, comparando aos fármacos benzonidazol e doxorrubicina. Para detecção de ROS foi realizado o ensaio fluorimétrico utilizando o marcador 2,7-diacetato de diclorofluoresceína (DCFH-DA) em linhagens celulares de hepatocarcinoma humano (HepG2) e fibroblasto de camundongo (Balb/C 3T3 clone A31). O estado redox destas células foi avaliado através da quantificação da expressão gênica e do conteúdo proteico das enzimas antioxidantes através das técnicas de qRT-PCR e Western blot, respectivamente. A atividade mutagênica foi analisada com o ensaio Ames miniaturizado Salmonella/microssoma utilizando a linhagem TA102 de Salmonella typhimurium que detecta agentes mutagênicos que causam danos por oxidação. Os resultados mostraram que as substâncias estudadas pelo Grupo não induzem aumento na produção de ROS ou induzem em menores níveis do que doxorrubicina e benzonidazol, além de levar a alterações menos proeminentes que os fármacos para a expressão das proteínas antioxidantes. No ensaio mutagênico, o benzonidazol apresentou o pior perfil, doxorrubicina e Neq0438 somente foram mutagênicos com ativação enzimática, enquanto Neq0551 foi inativo. Assim, as novas substâncias (Neq0438 e Neq0551) apresentaram um perfil melhor do que os fármacos de referência, tornando-os candidatos promissores para estudos in vitro e in vivo subsequentes. / The production of reactive oxygen species (ROS) in biological systems is compensated by the enzymatic and non-enzymatic antioxidant systems. The excessive ROS production causes oxidative stress, which can damage important cellular macromolecules such as lipids, proteins and DNA. The drugs doxorubicin (antineoplastic) and benzonidazole (antiparasitic) are both known for their side effects, which can be related to the increase of ROS. Besides, mutagenicity assays show that these drugs have a mutagenic activity via oxidative damages. The research group NEQUIMED studies new substances with potential antineoplastic and antiparasitic activities, but their toxic and genotoxic properties have not been fully evaluated yet. Thus, the aim of this work is to assess the mutagenic potential and the oxidative stress generated by these substances, comparing them to benzonidazole and doxorubicin. The fluorimetric assay using the probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for ROS detection in human hepatocarcinoma (HepG2) and mouse fibroblast (Balb/C 3T3 clone A31) cell lines. The redox state of these cells was evaluate by qRT-PCR and Western blot methods to quantifying gene expression and protein content of the antioxidant enzymes. The mutagenic potential was assessed by the miniaturized Ames text with the Salmonella/microssome mutagenicity assay, using the TA102 strain of Salmonella typhimurium, which detects oxidation damages to the DNA. The new substances did not induce an increase on ROS production, or did in lower levels when compared to doxorubicin and benzonidazole. Moreover, reference drugs also induced greater changes on the expression of the antioxidant enzymes. Benznidazole had a higher mutagenic activity, while Neq0438 and doxorubicin were mutagenic only when incubated with enzymatic activation. Neq0551 was inactive for Ames assay. Therefore, these new substances (Neq0438 and Neq0551) had a better overall profile than the reference drugs, turning out to be promising candidates for further in vitro and in vivo studies.
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The Effect of N-Methyl-N'-Nitro-N-Nitrosoguanidine on Herpes Simplex Virus Replication and Gene ExpressionArshoff, Larry Usher 12 1900 (has links)
Experiments were carried out to determine if pretreatment of cells with N-Methyl-N'-Nitro-N-Nitrosoguanidine (a potent alkylating agent known to induce DNA repair) would affect Herpes Simplex Virus Replication. The data demonstrated a 1.5 fold increase in virus yield, a 2 fold increase in HSV specific TK activity and no change in HSV specific DNA polymerase activity in MNNG treated cultures. The effects of MNNG treatment on virus replication and enzyme expression are discussed. / Thesis / Master of Science (MSc)
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Atividade enzimática e expressão diferencial da Superóxido Dismutase (SOD) em plantas de arroz de terras altas sob deficiência hídrica / Enzymatic activity and differential expression of Dismutase Superoxide (SOD) in rice plants of high terrains under water deficiencyDeus, Karinne Evaristo de 30 June 2014 (has links)
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Previous issue date: 2014-06-30 / Drought is a major cause for reduced productivity in the cultivation of upland rice farming in many regions of the world. One of the consequences of the drought is the production in excess of reactive oxygen species (ROS), causing a series of oxidative damage to various biomolecules with subsequent cell death. With the function of protecting structures and functioning of cells from the damaging effects of ROS, a complex antioxidant system is activated in plants. This system consists of: (1) lipid soluble membrane-associated and tocopherols; (2) reducing water soluble compounds such as ascorbate (ASA) and glutathione (GSH) and (3) antioxidative enzymes, and superoxide dismutase (SOD) considered as a major enzymes of the antioxidant defense system. The present study aimed to evaluate the SOD in activity level via spectrophotometric method and level of gene expression via qPCR in two genotypes of upland rice (Oryza sativa japonica), Douradão and BRS Primavera, with contrasting for drought tolerance characteristics, watching the leaf and root tissue, two stages of plant development (vegetative and reproductive), grown under optimal water conditions and water deficit (100% and 50% water in the vessels), respectively. The results revealed a differential pattern of SOD activity in different tissues and developmental stages in tolerant and sensitive genotypes, and for the tolerant genotype that activity was increased only in leaf / root and vegetative/reproductive tissue, as was sensitive the leaf / reproductive and reproductive/ root. Regarding gene expression, we also observed a very different pattern of regulation in tolerant and sensitive genotypes. The CuZnSOD1, CuZnSOD4, and MnSOD genes, expression was significantly (p≤ 0.05) increased in the tolerant, the first in leaves and roots off the reproductive stage, the second vegetative stage only in leaves and the third gene in the two tissues and plant developmental stages. As for the sensitive only FeSOD1 gene had highlighted with increased expression in roots at the reproductive stage. Certainly, the different patterns of induction level of activity and / or gene expression of SOD in plants of upland rice, should be strongly considered to elucidate the cellular mechanisms of drought tolerance, aiming to support improvement programs to develop cultivars more efficient and better suited to prone areas with water deficiency. / A seca é uma das principais causas para redução da produtividade na cultura do arroz de terras altas em muitas regiões agrícolas do mundo. Uma das consequências da seca é a produção, em excesso, de espécies reativas de oxigênio (EROs), podendo causar uma série de danos oxidativos a diversas biomoléculas com consequente morte celular. Com a função de proteger estruturas e funcionamento das células dos efeitos prejudiciais das EROs, um complexo sistema antioxidativo é ativado nas plantas. Esse sistema é constituído de: (1) lipídeos solúveis e tocoferóis associados à membrana; (2) compostos redutores solúveis em água, tais como ascorbato (ASA) e glutationa (GSH), e (3) enzimas antioxidativas, sendo a superóxido dismutase (SOD) considerada como uma das principais enzimas do sistema de defesa antioxidativo. O presente estudo teve como objetivo avaliar a SOD, em nível de atividade via método espectrofotométrico e em nível de expressão gênica via qPCR, em dois genótipos de arroz de terras altas (Oryza sativa japonica), Douradão e BRS Primavera, com características contrastantes para tolerância à deficiência hídrica, contemplando parte aérea e tecido radicular, dois estádios de desenvolvimento das plantas (vegetativo e reprodutivo), cultivadas sob condição hídrica ótima e de deficiência hídrica (100 % e 50 % de água nos vasos), respectivamente. Os resultados revelaram um padrão diferencial de atividade da SOD nos diferentes tecidos e estádios de desenvolvimento nos genótipos tolerante e sensível, sendo que para o genótipo tolerante essa atividade foi aumentada somente em tecido foliar fase vegetativa e radicular fase reprodutiva, enquanto no sensível foi foliar e radicular estádio reprodutivo. Quanto à expressão gênica, também observou um padrão bastante diferenciado de regulação nos genótipos tolerante e sensível. Os genes Cu/ZnSOD1, Cu/ZnSOD4 e MnSOD apresentaram expressão significativamente (p ≤ 0,05) aumentada no tolerante, sendo o primeiro em folhas e raízes do estádio reprodutivo, o segundo estádio vegetativo somente em folhas e para o terceiro gene nos dois tecidos e estádios de desenvolvimento da planta. Já para o genótipo sensível somente o gene FeSOD1 apresentou destaque com aumento da expressão em raízes no estádio reprodutivo. Certamente, os diferentes padrões de indução em nível de atividade e/ou expressão gênica da SOD, em plantas de arroz de terras altas, devem ser fortemente considerados para elucidar os mecanismos celulares de tolerância à seca, objetivando subsidiar programas de melhoramento para desenvolvimento de cultivares mais eficiente e mais bem adaptada às áreas propensas à deficiência hídrica.
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Flavonoid glucodiversification with engineered sucrose-active enzymes / Glucodiversification des flavonoïdes par ingénierie d’enzymes actives sur saccharoseMalbert, Yannick 10 July 2014 (has links)
Les flavonoïdes glycosylés sont des métabolites secondaires d’origine végétale, qui présentent de nombreuses propriétés physico-chimiques et biologiques intéressantes pour des applications industrielles. La glycosylation accroît généralement la solubilité de ces flavonoïdes mais leurs faibles niveaux de production dans les plantes limitent leur disponibilité. Ces travaux de thèse portent donc sur le développement de nouvelles voies de gluco-diversification des flavonoïdes naturels, en mettant à profit l’ingénierie des protéines. Deux transglucosylases recombinantes, structurellement et biochimiquement caractérisées, l'amylosaccharase de Neisseria polysaccharea et la glucane-saccharase de branchement α-(1→2), forme tronquée de la dextran-saccharase de L. Mesenteroides NRRL B-1299, ont été sélectionnées pour la biosynthèse de nouveaux flavonoïdes, possédant des motifs originaux d’α-glycosylation, et potentiellement une solubilité accrue dans l'eau. Dans un premier temps, une librairie de petite taille de mutants de l’amylosaccharase, ciblée sur le site de liaison à l’accepteur, à été criblée en présence de saccharose (donneur d’unité glycosyl) et de lutéoline comme accepteur. Une méthode de screening a donc été développée, et a permis d’isoler des mutants améliorés pour la synthèse de nouveaux glucosides de lutéoline, jusqu’à 17000 fois plus soluble dans l’eau que la lutéoline aglycon. Afin de glucosyler d’autres flavonoïdes, la glucane-saccharase de branchement α-(1→2), a été préférentiellement sélectionnée. Des plans expérimentaux alliés à une méthodologie en surface de réponse ont été réalisés pour optimiser la production de l’enzyme sous forme soluble et éviter la formation de corps d’inclusion. Cinq paramètres ont été ainsi analysés : le temps de culture, la température, et les concentrations en glycérol, lactose (inducteur) et glucose (répresseur). En appliquant les conditions optimales prédites, 5740 U.L-1 de culture d’enzyme soluble ont été produites en microplaques, alors qu’aucune activité n’était retrouvée dans la fraction soluble, lors de l’utilisation de la méthode de production précédemment utilisée. Finalement, Une approche de modélisation moléculaire, structurellement guidés par l’arrimage de flavonoïdes monoglucosylés dans le site actif de l’enzyme, a permis d’identifier des cibles de mutagenèse et de générer des libraries de quelques milliers de variants. Une méthode rapide de criblage sur milieu solide, basée sur la visualisation colorimétrique d’un changement de pH, a été mise au point. Les mutants encore actifs sur saccharose ont été sélectionnés puis analysés sur leur capacités à glucosyler la quercétine et la diosmétine. Une petite série de 23 mutants a ainsi été retenue comme plate-forme d’enzymes améliorées dédiées à la glucosylation de flavonoïdes et a été évalués pour la glycosylation de six flavonoïdes distincts. La promiscuité, remarquablement générée dans cette plateforme, à permis d’isoler quelques mutants beaucoup plus efficaces que l’enzyme sauvage, produisant des motifs de glucosylation différents et fournissant des informations intéressante pour le design et l’amélioration des outils enzymatiques de glucosylation des flavonoïdes. / Flavonoid glycosides are natural plant secondary metabolites exhibiting many physicochemical and biological properties. Glycosylation usually improves flavonoid solubility but access to flavonoid glycosides is limited by their low production levels in plants. In this thesis work, the focus was placed on the development of new glucodiversification routes of natural flavonoids by taking advantage of protein engineering. Two biochemically and structurally characterized recombinant transglucosylases, the amylosucrase from Neisseria polysaccharea and the α-(1→2) branching sucrase, a truncated form of the dextransucrase from L. Mesenteroides NRRL B-1299, were selected to attempt glucosylation of different flavonoids, synthesize new α-glucoside derivatives with original patterns of glucosylation and hopefully improved their water-solubility. First, a small-size library of amylosucrase variants showing mutations in their acceptor binding site was screened in the presence of sucrose (glucosyl donor) and luteolin acceptor. A screening procedure was developed. It allowed isolating several mutants improved for luteolin glucosylation and synthesizing of novel luteolin glucosides, which exhibited up to a 17,000-fold increase of solubility in water. To attempt glucosylation of other types of flavonoids, the α-(1→2) branching sucrase, naturally designed for acceptor reaction, was preferred. Experimental design and Response Surface Methodology were first used to optimize the production of soluble enzyme and avoid inclusion body formation. Five parameters were included in the design: culture duration, temperature and concentrations of glycerol, lactose inducer and glucose repressor. Using the predicted optimal conditions, 5740 U. L-1of culture of soluble enzyme were obtained in microtiter plates, while no activity was obtained in the soluble fraction when using the previously reported method of production. A structurally-guided approach, based on flavonoids monoglucosides docking in the enzyme active site, was then applied to identify mutagenesis targets and generate libraries of several thousand variants. They were screened using a rapid pH-based screening assay, implemented for this purpose. This allowed sorting out mutants still active on sucrose that were subsequently assayed for both quercetin and diosmetin glucosylation. A small set of 23 variants, constituting a platform of enzymes improved for the glucosylation of these two flavonoids was retained and evaluated for the glucosylation of a six distinct flavonoids. Remarkably, the promiscuity generated in this platform allowed isolating several variants much more efficient than the wild-type enzyme. They produced different glucosylation patterns, and provided valuable information to further design and improve flavonoid glucosylation enzymatic tools.
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