No description available.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H2O2 treatment and compared to that of FeSOD. It’s found that the responses to H2O2 treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe3+ reduction was not observed in Fe(Mn)SOD even H2O2 is believed to oxidize proteins via highly reactive intermediates including Fe and formed via Fe2+, which is true in FeSOD. What’s more, the activities of Fe(Mn)SOD and FeSOD were totally different in the ABTS assay or Amplex Red assay. These results indicated that the mechanism of peroxidase reaction of Fe(Mn)SOD is not identical to that of FeSOD.

Microbial chlorate respiration plays an important role in the turnover of oxochlorates in nature and industrial waste management. This thesis deals with the characterization of the molecular components of chlorate respiration in Ideonella dechloratans. Chlorate respiration utilizes two soluble periplasmic enzymes, chlorate reductase and chlorite dismutase, to convert chlorate to chloride and oxygen. The genes encoding the enzymes participating in the chlorate degradation have been sequenced, and are found in close proximity, forming a gene cluster for chlorate metabolism. This work also includes the successful recombinant expression of three genes from Ideonella dechloratans. Two of the gene products, chlorite dismutase and the C subunit of chlorate reductase, participate in the chlorate respiration. The third gene, which is found close to the gene cluster for chlorate metabolism, encodes a soluble c-type cytochrome. The localization of the gene suggests the corresponding protein as a candidate for a role as electron donor to chlorate reductase. Also, the role of soluble periplasmic c cytochromes of Ideonella dechloratans in chlorate respiration was studied. At least one of the soluble c cytochromes was found capable of serving as electron donor for chlorate reduction. This c cytochrome, and several others, can also donate electrons to a terminal oxidase for subsequent reduction of oxygen, as required for the branched electron flow during chlorate respiration.

Maintainence of appropriate redox homeostasis is crucial for processes such as protein folding in the endoplasmic reticulum (ER) and to minimise genesis of oxidative stress. Previous studies have indicated a possible link between ER stress and production of reactive oxygen species (ROS) although the cellular mechanisms involved were not fully elucidated. To investigate the cellular mechanisms involved in tolerance to oxidative stress and ER stress, genome-wide screens were performed to identify mutants sensitive to chronic ER stress induced by dithiothreitol and tunicamycin. These screens identified the Cu,Zn superoxide dismutase (SOD1) and genes involved in NADPH generation (RPE1, TKL1) as important for chronic ER stress tolerance. Superoxide anion has been identified as one of the ROS generated during ER stress. The ER oxidoreductase Ero1p, previously implicated in ROS production in vitro, did not appear to be a source of superoxide when the protein was over-expressed. It was also found that cellular NADP(H) levels affected induction of the unfolded protein response (UPR), since cells lacking TKL1 or RPE1 exhibited decreased UPR induction. These data indicate an important role for superoxide dismutase and cellular NADP(H) in survival of cells during ER stress. Subsequent analysis determined that NADPH generation was also required for adaptation to H2O2. Mutants affected in NADPH production were chronically sensitive to H2O2 but resistant to an acute dose. These mutants over-accumulated reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This over- production of GSH was not regulated at the transcriptional level of GSH1 encoding ??- glutamyl cysteine synthetase. These data raise the important question as to how cells maintain cellular glutathione redox balance. To better understand how cells respond to perturbations in glutathione redox homeostasis, cells deleted for GLR1, encoding GSSG reductase, were exposed to extracellular oxidised glutathione (GSSG) and intracellular GSH and GSSG were monitored over time. Intriguingly cells lacking GLR1 showed increased levels of GSH accumulation upon GSSG treatment in a manner independent of GSH synthesis. It was subsequently found that the cytosolic thioredoxin-thioredoxin reductase system contributes to the reduction of GSSG in vivo.

Thèse (Ph. D.)--Université Laval, 2009. / Bibliogr.: f. 182-212. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Made available in DSpace on 2014-06-11T19:23:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-22Bitstream added on 2014-06-13T20:10:55Z : No. of bitstreams: 1 corniani_n_me_botfca.pdf: 1146521 bytes, checksum: fa6a3da1e11828289d5c9cb1e7208258 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As espécies do gênero Eucalyptus apresentam notável capacidade de extração de água e nutrientes, assimilação fotossintética e crescimento, permitindo seu cultivo em ambientes considerados impróprios para muitas outras espécies. Além dos fatores ambientais atuando adversamente no desempenho das plantas, atualmente estas estão sujeitas aos efeitos das mudanças ambientais globais causadas pelas atividades antrópicas, tais como o aumento na incidência da radiação ultravioleta-B (RUV-B) em conseqüência da destruição da camada de ozônio. Estudos recentes têm mostrado que a RUV-B promove aumento no nível de espécies reativas de oxigênio (ERO), ocasionando estresse oxidativo nas plantas. Entretanto, as plantas apresentam um sistema de defesa contra o estresse oxidativo, constituído de enzimas antioxidantes, tais como superóxido dismutase (SOD, EC 1.15.1.1) e peroxidase (POD, EC 1.11.1.7), além de outros compostos, como antocianinas, carotenóides e compostos fenólicos, que impedem a formação de ERO ou eliminam as já existentes. Há algum tempo, tem sido verificada em plantas a presença de óxido nítrico (NO), radical livre endógeno que possui a capacidade de controlar o nível e a toxicidade das ERO. Portanto, o objetivo deste trabalho foi investigar o possível efeito citoprotetor do NO em plantas de eucalipto (Eucalyptus urograndis) expostas à RUV-B. Assim, foi realizado um primeiro experimento no qual plantas de eucalipto foram expostas à RUV-B em baixa (controle) e elevada incidência, com o intuito de verificar se a radiação promove estresse oxidativo nesta espécie. Foram realizadas coletas de lâminas foliares aos cinco, dez e quinze dias após o início da exposição à RUV-B para posterior determinação do nível de peroxidação lipídica. A exposição por 15 dias à RUV-B... / The species of the genus Eucalyptus present notable capacity to extract water and nutrients, photosynthetic assimilation and growth, allowing cultivation in ambient considered inappropriate for many other species. Besides the environmental factors acting adversely in the performance of the plants, nowadays they are subject to the effects of the global environmental changes caused by human activities, such as the increase in the incidence of the ultraviolet-B radiation (UV-B) in consequence of the ozone layer destruction. Recent studies show that UV- B radiation promotes increase in the level of reactive oxygen species (ROS), causing oxidative stress in plants. However, plants have a defence system against oxidative stress, constituted of antioxidant enzymes, such as, superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), and others compounds, such as anthocyanins, carotenoids and phenolic compounds, that avoid ROS formation or eliminate the already existent. There has been long, it was noticed the presence of nitric oxide (NO) in plants, endogenous free radical able to control the ROS level and toxicity. Therefore, the aim of this work was to investigate the possible citoprotector effect of the NO in eucalyptus (Eucalyptus urograndis) plants subjected to UV-B radiation. So, a first experiment was setup in which plants of eucalyptus were subjected to radiation in low (control) and high incidence of UV-B, to verify if UV-B radiation promotes oxidative stress in this specie. Leaves were collected at five, ten and fifteen days after the beginning of exposure to UV-B for subsequent evaluation of the level of lipid peroxidation. Exposure for 15 days to RUV-B increased the lipoperoxide content in the eucalyptus... (Complete abstract click electronic access below)

Submitted by Eduardo Barros de Almeida Silva (eduardo.philippe@ufpe.br) on 2015-04-17T12:44:08Z No. of bitstreams: 2 Tese Henriqueta Dias Cardoso.pdf: 5265290 bytes, checksum: e2e8c3ba450de08f6e92ede26a203fc0 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-17T12:44:08Z (GMT). No. of bitstreams: 2 Tese Henriqueta Dias Cardoso.pdf: 5265290 bytes, checksum: e2e8c3ba450de08f6e92ede26a203fc0 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02-26 / FACEPE / Os ácidos graxos essenciais (AGEs) têm sido indicados como potenciais agentes preventivos e terapêuticos em uma grande variedade de doenças neurodegenerativas assim como indispensáveis para o desenvolvimento cerebral. O principal objetivo deste trabalho foi investigar os mecanismos relacionados com a perda de neurônios dopaminérgicos induzida pela deficiência crônica em AGEs previamente detectada na substância negra (SN) de ratos Wistar jovens (J), estendendo a análise também a animais adultos (A). Para isso, foram utilizadas dietas balanceadas e que diferiram apenas na fonte lipídica, sendo óleo de soja para os grupos controles (C) e óleo de coco para os grupos experimentais (E). As dietas foram fornecidas às mães a partir do acasalamento e mantidas por uma (F1) ou duas (F2) gerações. Marcadores de insulto oxidativo: lipoperoxidação (LP), atividade das enzimas superóxido dismutase total (SOD-t), catalase (CAT) na SN e corpo estriado (CE) foram avaliados em animais AF1 e em JF2 e AF2. Indicadores de neurodegeneração na SN e CE destes animais foram avaliados utilizando a técnica de marcação com o fluoróforo, Fluoro Jade C. Análise quantitativa do tamanho e nº de neurônios dopaminérgicos e da distribuição de células imunorreativas ao fator neurotrófico derivado do cérebro (BDNF) foi realizada em animais AF2. Os níveis de nitrito, como indicador da produção de óxido nítrico na SN e CE, foram analisados em animais JF2 e AF2. A dieta experimental reduziu em ~28%, ~50% e ~60% os níveis de ácido docosahexaenóico (DHA) na SN dos grupos experimentais AF1, JF2 e AF2 respectivamente, comparado aos seus controles. Nos animais EAF1 um aumento em ~17% e ~45% na atividade da SOD-t foi observado na SN e CE comparado ao grupo controle, o evitou níveis danosos de lipoperoxidação. Por outro lado, um aumento nos níveis de lipoperoxidação (~34%) foi detectado na SN de animais EJF2, acompanhados de não reatividade da SOD-t e de uma redução em 4,8 vezes na atividade da CAT. Sinais de neurodegeneração foram evidenciados em neurônios dopaminérgicos e não dopaminérgicos da SN do grupo EJF2. No CE, o aumento da LP em ~39% foi acompanhado de redução em 3,8 vezes e 2,8 vezes da atividade da SOD-t e CAT, respectivamente, só foram observados nos animais do grupo EAF2. A dieta experimental não alterou os níveis de nitrito na SN, mas aumentou de forma significativa estes níveis no CE de animais jovens (30%) e adultos (1,8 vezes). A deficiência crônica em DHA até a idade adulta comprometeu o crescimento do corpo celular e aumentou a perda de neurônios dopaminérgicas na SN rostro-dorso-medial (~35%) afetando também aqueles localizados na região caudo-ventro-lateral deste núcleo. Uma redução de ~22% no número de células BDNF+ foi também observada na SN. Os resultados mostram que a restrição dietética em AGEs por duas gerações até a idade adulta é capaz de induzir lipoperoxidação na SN e CE devido a comprometimento na atividade das enzimas anti-oxidantes, perda de células BDNF+ na SN e aumentados níveis de óxido nítrico no CE. Tidos em conjunto, tais mecanismos podem estar atuando de forma sinérgica na degeneração de neurônios dopaminérgicos induzida pela deficiência em DHA.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-18T17:08:25Z No. of bitstreams: 1 Maria Shirlei Barros.pdf: 885582 bytes, checksum: 12e5ba4dbe4dbc6811c0b54fef726658 (MD5) / Made available in DSpace on 2016-10-18T17:08:25Z (GMT). No. of bitstreams: 1 Maria Shirlei Barros.pdf: 885582 bytes, checksum: 12e5ba4dbe4dbc6811c0b54fef726658 (MD5) Previous issue date: 2010-02-24 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / With the objective of to evaluate mitochondrial membrane potential (MMP), kinetic, the structural and ultrastructural of goat sperm submitted to freezing in skimmed-milk and glycerol, superoxide dismutase and catalase. It was used five bucks of Boer race, submitted to semen collect by artificial vagina. Semen samples were diluted in skimm-milk plus glycerol (7%), aiming have 320x106 sperm/mL, supplemented with antioxidants according to the experimental groups: G1) extender (control), G2) extender SOD + 25 IU/mL; G3) extender + SOD 50 IU/mL; G4) extender + SOD 100 IU/mL; G5) extender + CAT 25 IU/mL; G6) extender + CAT 50 IU/mL; G7) extender + CAT 100 IU/mL and G8) extender + SOD 100 IU/mL + CAT 25 IU/mL. Then the samples were packed in straws (0.25 mL), frozen and stored in cold storage cylinder (-196 oC). After thawing (37 ºC/30 seconds), aliquots of semen frozen/thawed of each group were evaluated to PMM, kinetic (CASA), structure and ultrastructure of spermatozoa, where did not observe significant difference (P>0.05) among experimental groups in parameters PMM, kinetic, iMP and iAc. In ultrastructural evaluation, fresh sperm was morphologically preserved mainly on mitochondrial membrane. Analyzes of thawed cells showed greater quantity of damages on acrosome; however, in lower percentage on G8 cells. G7 cells showed ultrastructural damages in acrosomes of 92.31%ofspermatozoas. In contrast, great percentage of spermatozoa have plasma and acrossomal membranes intacts, mainly on G2, G4 and G8 groups. Based on the ultrastructural results, it can be recommended addition of SOD (100 U/mL) and CAT (25 U/mL) on the freezing goat semen diluents with skimmed-milk and glycerol; as well as other studies should be realized using higher concentrations than 100U/mL of SOD in this diluent to freezing goat semen, associated to in vivo evaluation of these antioxidant action. / Objetivou-se com esse estudo avaliar o potencial de membrana mitocondrial (PMM), cinética, estrutura e ultraestrutura de espermatozoides caprinos, submetidos à criopreservação com diluente à base de leite desnatado e glicerol (7%), suplementado com superóxido dismutase (SOD) e catalase (CAT), em diferentes concentrações. Foram utilizados cinco reprodutores caprinos da raça Boer, submetidos à colheita de sêmen com vagina artificial. As amostras de sêmen foram diluídas em leite desnatado acrescido de glicerol (7%), de forma a apresentar 320x106 espermatozoides/mL, e suplementados com antioxidantes de acordo com os grupos experimentais: G1) diluente (Controle); G2) diluente + SOD 25 U/mL; G3) diluente + SOD 50 U/mL; G4) diluente + SOD 100 U/mL; G5) diluente + CAT 25 U/mL; G6) diluente + CAT 50 U/mL; G7) diluente + CAT 100 U/mL e G8) diluente + SOD 100 U/mL + CAT 25 U/mL. Em seguida, as amostras foram acondicionadas em palhetas (0,25 mL), congeladas e armazenadas em botijão criobiológico (-196o C). Após descongelação (37 oC/30 segundos), alíquotas de sêmen de cada grupo foram avaliadas quanto a PMM, cinética (CASA), iMP, iAc e ultraestrutura (microscopia eletrônica de transmissão). Não se constatou diferença significativa (P>0,05) entre os grupos experimentais dos parâmetros PMM, iMP, iAc e cinética espermática. Na avaliação ultraestrutural, espermatozoides in natura apresentaram-se morfologicamente preservados, principalmente na membrana mitocondrial. A análise das células pós-descongelação evidenciou maior quantidade de danos no acrossoma, todavia emmenor porcentual nas células do G8. O G7 apresentou dano ultraestrutural no acrossoma de 92,31% dos espermatozoides avaliados. Em contrapartida, grande porcentagem de espermatozóides apresentaram membrana plasmática e acrossomal intactas, principalmente nos grupos G2, G4, e G8. Com base nos resultados de ultraestrutura, é possível recomendar a adição de SOD (100U/mL) e CAT (25 U/mL) ao diluente de congelação do sêmen caprino à base de leite desnatado e glicerol; assim como outros estudos devem ser realizados utilizando concentrações maiores do que 100U/mL de SOD neste diluidor de congelação de sêmen caprino, associado à avaliação in vivo da ação deste antioxidante.

Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins,various flavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine flavonoids in C. mellei and C. quadrifidus. 8 flavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect flavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants.UV-visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 μmol L-1 and 1.44 μA(μmol L-1)-1, respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively. May 2009