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No. of bitstreams: 1 pereira_fcm_dr_jabo.pdf: 2652760 bytes, checksum: fe30a505f009d928b1772bbb1ba24ca9 (MD5) Previous issue date: 2016-06-10 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cultura do eucalipto apresenta uma grande importância no cenário econômico nacional e mundial, oferecendo uma vasta gama de produtos florestais. No entanto, a disponibilidade de fósforo nos solos raramente é adequada para o crescimento, desenvolvimento e produtividade das plantas de eucalipto, bem como para diversas outras culturas. A presença de plantas daninhas em eucaliptais é outro fator limitante, e para o manejo da comunidade infestante, glyphosate é o herbicida mais utilizado. As plantas de eucalipto, quando cultivadas em diferentes concentrações de fósforo, podem alterar a expressão gênica de transportadores de fosfato. Em algumas condições, a síntese de transportadores de fosfato de alta afinidade pode ser incrementada. Esses transportadores podem ter afinidade pelo grupo fosfonato do glyphosate, transportando esse herbicida via membrana plasmática. Assim, podem ocorrer alterações no comportamento do glyphosate em função das concentrações de fósforo. Estudos do efeito diferencial de glyphosate em função de concentrações de fósforo e do transporte de glyphosate mediado por transportadores de fosfato ainda são escassos, principalmente em uma espécie perene, como o eucalipto. Com essa pesquisa objetivou-se (1) avaliar, em clones de Eucalyptus urophylla (GG100 e I144) a cinética de absorção de fósforo, as eficiências nutricionais de macronutrientes e seus reflexos no crescimento das plantas; (2) verificar as alterações fotossintéticas, metabólicas e nutricionais de clones de E. urophylla (GG100 e I144) cultivados em concentrações de fósforo e submetidos à aplicação de glyphosate; e (3) estudar, em E. grandis, a coordenação da expressão gênica de transportadores de fosfato, a absorção e a translocação de 14C-glyphosate, e o transporte de 14C-glyphosate através da membrana plasmática em protoplastos. Os experimentos com E. urophylla foram realizados na Universidade Estadual Paulista, Câmpus de Jaboticabal, SP, enquanto aqueles com E. grandis foram desenvolvidos na University of Maryland, Maryland, EUA. Após a avaliação dos parâmetros cinéticos de absorção de fósforo, o clone I144 foi superior, apresentando menores Cmín e Km (concentração limite abaixo da qual a planta é incapaz de absorver um elemento na solução e concentração do nutriente na solução onde foi atingida metade da Vmáx, respectivamente) e maior Vmáx (velocidade máxima de absorção). As eficiências de absorção, translocação e utilização de macronutrientes também foram maiores no clone I144, com exceção da eficiência de utilização de potássio, superior no clone GG100. Não houve diferenças na eficiência nutricional dos clones em relação ao boro. Para a avaliação das alterações fotossintéticas, metabólicas e nutricionais ocasionadas por glyphosate em função de concentrações de fósforo, experimentos foram conduzidos em casa de vegetação utilizando areia como substrato (pulverização foliar de glyphosate) e em câmara de crescimento utilizando sistema hidropônico (aplicação radicular de glyphosate). Para ambos os experimentos, os nutrientes foram oferecidos por meio da solução nutritiva, que foi ajustada para as doses de fósforo 0,5X; 1X e 1,5X de fósforo (em que X correspondeu à concentração de fósforo recomendada para o preparo da solução nutritiva). Interações entre as concentrações de fósforo e as doses de glyphosate ocorreram nas características que avaliaram o processo fotossintético (teores de clorofila e eficiência quântica do fotossistema II), nos teores de glyphosate, AMPA e ácido chiquímico, nos teores nutricionais e massas secas. Essas características ou teores foram menores ou mais intensamente reduzidos, após glyphosate, nas plantas cultivadas com menores concentrações de fósforo (0,5X e, em alguns casos, 1X). Por fim, experimentos foram conduzidos em E. grandis, e a expressão gênica dos transportadores de fosfato (mensurada por meio de RT-qPCR) nas folhas e nas raízes dessas plantas foram alteradas em resposta a deficiência de fosfato. A expressão de transportadores de alta afinidade foi incrementada pela deficiência de fosfato. A absorção de 14C-glyphosate aplicado via foliar ou radicular foi maior nas plantas submetidas à deficiência de fosfato, e a translocação deste herbicida também foi mais rápida nessas plantas. Nos protoplastos, o transporte de 14C-glyphosate via membrana plasmática ocorreu rapidamente na ausência de fosfato, e de modo mais lento quando NaH2PO4 foi adicionado ao meio. Possivelmente, o transporte de fosfato através da membrana plasmática foi realizado em um primeiro momento, e com a redução do fosfato disponível no meio, o transporte de glyphosate foi iniciado, provavelmente, por transportadores de fosfato de alta afinidade. / Eucalypt plantation have great importance in the national and global economic environment, offering a wide range of forest products. However, the phosphorus availability in soils is rarely appropriate for growth, development and yield of eucalypt plants, as well as several other crops. The presence of weeds in eucalypt plantations is another limiting factor, and for weed management glyphosate is the most used herbicide. Eucalypt plants grown under different phosphorus concentrations can change phosphate transporters gene expression. Under specific conditions, high-affinity phosphate transporter synthesis can be increased. Such transporters can have affinity for glyphosate phosphonate group, transporting this herbicide through plasma membrane. So, changes may occur in glyphosate behavior depending on phosphorus concentrations. Studies of glyphosate effect as a function of phosphorus concentrations and glyphosate transport mediated by phosphate transporters are still scarce, especially in a perennial species, such as eucalypt. This study aimed (1) to evaluate, in Eucalyptus urophylla clones (GG100 and I144), the phosphorus absorption kinetics, the nutritional efficiency of macronutrients and their effects on plant growth; (2) verify the photosynthetic, nutritional and metabolic changes in E. urophylla clones (GG100 and I144) grown under phosphorus concentrations and submitted to glyphosate application; and (3) study in E. grandis the phosphate transporters gene expression coordination, the 14C-glyphosate uptake and translocation, and the 14C-glyphosate transport through the plasma membrane in protoplasts. The experiments with E. urophylla were carried out at São Paulo State University, Campus of Jaboticabal, SP, while the experiment with E. grandis were developed at University of Maryland, Maryland, USA. After the evaluation of phosphorus absorption kinetic parameters, I144 clone was considered superior, presenting lower Cmin and Km (respectively, the limiting concentration for absorbing an element in solution and the nutrient concentration in solution which was reached half of Vmax,) and higher Vmax (maximum rate of absorption). The efficiency of absorption, translocation and utilization of macronutrients were also higher in I144 clone, with the exception of potassium utilization efficiency, higher in GG100 clone. No differences in boron nutritional efficiency were found. For the evaluation of photosynthetic, nutritional and metabolic changes caused by glyphosate according phosphorus concentrations, experiments were performed in a greenhouse using sand (glyphosate sprayed in leaves) and in a growth chamber using hydroponic system (glyphosate applied in roots). For both experiments, nutrients were provided by nutrient solution, which was adjusted to phosphorus levels 0.5X, 1X and 1.5X (where X corresponded to the phosphorus concentration recommended for nutrient solution preparation). Interactions between phosphorus concentration and glyphosate doses occurred in the characteristics that evaluated the photosynthetic process (chlorophyll content and quantum efficiency of photosystem II), in the glyphosate, AMPA and shikimic acid content, and in the nutritional content and dry mass. These characteristics or contents were smaller or more strongly reduced after glyphosate in plants grown with lower phosphorus concentration (0.5X, and in some cases, 1X). Finally, experiments were conducted in E. grandis, and the gene expression of phosphate transporters (measured by RT-qPCR) in leaves and roots of these plants have been modified as a phosphate deficiency response. High-affinity transporters expression was increased by phosphate deficiency. The absorption of 14C-glyphosate applied in leaves or roots was higher in plants subjected to phosphate deficiency, and glyphosate translocation was also faster in these plants. In protoplasts, 14Cglyphosate transport via plasma membrane was fast in phosphate absence and slower when NaH2PO4 was added to the medium. Possibly, the phosphate transport through the plasma membrane was performed initially, and with the phosphate reduction in the medium, the glyphosate transport was started probably for highaffinity phosphate transporters. / CNPq: 156323/2012-5 / CAPES: BEX-9553/14-2 / FAPESP: 2011/201705-3

Filmes de Langmuir são usados como modelos simplificados de membranas celulares, cujas propriedades podem ser correlacionadas com efeitos fisiológicos de moléculas de interesse biológico, como os peptídeos antimicrobianos (PAMs). Nesta dissertação investigamos a interação do peptídeo Aureína 1.2, na forma de monômero (AU), dímero ((AU)2K) e com variações na porção N-terminal (KAU e DAU), com filmes de Langmuir obtidos do extrato lipídico da bactéria Escherichia coli. Todos os peptídeos injetados em concentrações de 20 a 200nM se incorporaram ao filme de Langmuir, causando expansão nas isotermas de pressão superficial, que foi significativamente maior para o dímero. O módulo de compressibilidade do filme de E. coli à pressão superficial correspondente à de uma membrana real praticamente dobrou, de cerca de 40mN/m para 80nM/m para o dímero, ao passo que para os outros peptídeos a alteração não foi significativa. Dos espectros de reflexão e absorção no infravermelho com modulação de polarização (PM-IRRAS), observou-se que todos os peptídeos interagiram tanto com as caudas quanto com as cabeças polares das moléculas do extrato de E. coli no filme de Langmuir. Diferentemente dos resultados de pressão e compressibilidade, não há tendência de um peptídeo ter interação mais relevante do que os outros. O maior efeito do dímero na expansão e compressibilidade do filme de Langmuir não se refletiu numa maior atividade bactericida contra E. coli, pois sabe-se da literatura que a atividade é maior para a Aureína 1.2 (AU). Provavelmente porque essa atividade deve depender da camada externa de lipopolissacarídeos de uma bactéria Gram-negativa. / Langmuir films are used as simplified cell membrane models whose properties can be correlated with physiological effects of molecules of biological interest, such as antimicrobial peptides (AMPs). In this dissertation we report on the interaction of Aurein 1.2 peptide as monomer (AU), dimer ((AU)2K) and modified peptide in the N-terminal portion (KAU and SAD), with Langmuir films obtained from a lipid extract of Escherichia coli. All peptides injected at concentrations from 20 to 200nM were incorporated into the Langmuir film, causing the surface pressure isotherm to expand, particularly for the dimer. The compressibility modulus of the E. coli Langmuir film at the surface pressure corresponding to an actual membrane nearly doubled, from about 40mN/m to 80nM/m for the dimer, whereas for the other peptides the change was not significant. From the polarization-modulated infrared reflection - absorption spectra (PM-IRRAS), we observed that all peptides interacted with both tails and polar heads of the molecules of E. coli extract in the Langmuir film. Unlike the results of pressure and compressibility, there was no tendency of a peptide having more relevant interaction than the others. The larger effect of the dimer in the expansion and compressibility of the Langmuir film was not reflected in a higher bactericidal activity against E. coli, since it is known from literature that the activity is higher for Aurein 1.2 (AU). Probably because this activity should depend on the outer layer of lipopolysaccharides of Gram-negative bacteria.

Orientador: Claudia Regina Baptista Haddad / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T00:53:28Z (GMT). No. of bitstreams: 1 Ferreira_MariaCristinadaCosta_M.pdf: 561166 bytes, checksum: 57a597ae699902d2f7063c9a77a30325 (MD5) Previous issue date: 2005 / Resumo: O excesso de sais no solo afetao metabolismo geral da planta, causando alterações fisiológicas e morfológicas. Visando avaliar os efeitos do sal no crescimento e metabolismo sob estresse salino, foram comparadas as respostas de Vigna luteola (halófita) e Vigna unguiculata (sensível). As plantas foram cultivadas na presença de NaCl nas concentrações de: 100mM, 250mM e 500mM. V. unguiculata apresentou reduções significativas de massas fresca e seca, área foliar e número de folhas sob salinidade. Estes parâmetros não foram afetados em V. luteola. Há dados na literatura que mostram que plantas adaptadas à salinidade apresentam a razão raiz/parte aérea maior que plantas sensíveis. Contudo, este aumento foi evidenciado na espécie mais sensível ao sal, sob salinidade, mantendo-se constante em V. luteola. O aumento de suculência, que é considerado uma adaptação ao estresse salino, não foi verificado em nenhuma das espécies estudadas. A resistência ao estresse salino tem sido relacionada com a atividade de enzimas antioxidantes, que removem espécies ativas de oxigênio. O aumento da atividade de peroxidases totais foi observado apenas em V. luteola, associado ao estresse salino. Sob estresse salino, ocorreu o aumento da atividade de siringaldazina oxidase somente em V. unguiculata. Não foi observada relação entre crescimento de raízes e atividade desta enzima, pois nesta espécie a salinidade provocou maior crescimento da raiz. A atividade de peroxidases totais em Vigna luteola está localizada na epiderme, córtex e cilindro central e, em V. unguiculata na epiderme e cilindro central. Sob estresse salino, a atividade foi mais intensa nas duas espécies. Em V. luteola foi observada em toda raiz, em V. unguiculata, além da epiderme e do cilindro central, foi possível observa-la em porções do córtex. A respeito da localização da enzima siringaldazina oxidase, em V. unguiculata restringiu-se à epiderme, expandindo - se para o córtex e cilindro central sob estresse salino. Já em V. luteola estava presente em todas as regiões da raiz, intensificando - se com a aplicação de NaCl no meio de cultivo. A alteração da permeabilidade das membranas é um dos resultados da salinidade, que está relacionado ao aumento do teor de espécies ativas de oxigênio. Foi observado aumento de liberação de eletrólitos em ambas as espécies de Vigna. Entretanto, em V. unguiculata, o acréscimo neste parâmetro foi altamente significativo na presença de NaCl e com resultados sempre maiores aos mesmos tratamentos de V. luteola. A manutenção da razão Na+/K+ nas células é um fator relevante para a tolerância à salinidade. O acúmulo de Na+ foi observado em raízes e folhas das duas espécies de Vigna. Em raízes, obteve-se valores maiores em V. luteola, em decorrência a alta concentração de Na+ neste órgão. Em folhas, a maior razão Na+/K+ foi obtida em V. unguiculata. As análises de parâmetros bioquímicos sob estresse salino, indicaram que não houve variação no teor de proteínas (folhas), sacarose (folhas) e açúcares solúveis totais (raiz e folhas) em Vigna luteola. Sob salinidade, o teor de proteínas foi reduzido em folhas de V. unguiculata. Já as concentrações de açúcares solúveis totais (raízes e folhas), sacarose (folhas) e malondialdeído (raiz) ficaram inalterados. Mediante salinidade, não ocorreram alterações significativas nos parâmetros bioquímicos em V. luteola, o que permitiu seu crescimento e desenvolvimento normal. As mudanças de indicadores bioquímicos e ausência de resposta antioxidante em V. unguiculata sob salinidade, podem estar relacionadas com o comprometimento do seu crescimento sob estresse salino / Abstract: The excess of salt in the soil affects all the general metabolism of the plant causing physiologic and morphologic alterations. With the objective of evaluating the effects of salt in the growth and metabolism under saline stress, this study compares the responses of two Vigna species, with distinct sensitivity to salt, cultivated with the presence of NaCl in the following concentrations: 100mM, 250mM e 500mM. V. unguiculata presented significant reductions in fresh and dry mass, leaf area and number of leaves when subjected to salinity. However, these parameters were not affected in V. luteola. There is data in the literature which shows that plants that have adapted to salinity present the root/shoot ratio higher than sensitive plants. On the other hand, this increase was evidenced in the species which is most sensitive to salt, under salinity, keeping itself constant in V. luteola. The increase in juiciness, which is considered an adaptation to saline stress, was not present in either of the species studied. The resistance to saline stress has been associated to the activity of antioxidant enzymes, which remove reactive oxygen species. The increase in activity of total peroxidases was observed only in V. luteola. Under saline stress, only V. unguiculata presented an increase in siringaldazine oxidase activity. There was no evidence of relation between the growth of roots and the presence of this enzyme, since this species presented larger root growth due to salinity. The histochemical location of total peroxidases activity indicated that the distribution of the activity of these enzymes is similar in both species of Vigna (epidermis, cortex and central cylinder), however, there is stronger activity in V. luteola, especially under stress. In relation to the location of the seringaldazine oxidase enzyme, in V. unguiculatat was restricted to the epidermis, expanding to the cortex and central cylinder under saline stress. In V. luteola it was present in all parts of the root, intensifying when NaCl was applied during cultivation. The change in the membrane permeability is one of the results of salinity which is connected to the increase of reactive oxygen species concentration. An increase in eletrolites release was noticed in both Vigna species. Meanwhile, in V. unguiculata, the rise in this parameter was highly significative in the presence of NaCl and with results constantly higher than the results of the same treatments in V. luteola. The maintenance of the Na+/K+ ratio in the cells is a relevant factor to the tolerance of salinity. Na+ accumulation was observed in both Vigna species¿ roots and leaves. In roots, higher values were obtained in V. luteola, due to the higher Na+ concentration in this organ. In leaves, the highest Na+/K+ ratio change was obtained in V. unguiculata. The analysis of biochemical parameters under saline stress, show that there was no change in protein content (leaves), sucrose (leaves) and total soluble sugars (root and leaves) in Vigna luteola. Protein content was reduced in V. unguiculata leaves, while concentrations of total soluble sugars (root and leaves), sucrose (leaves) e malondialdeid (root) remained the same. Under salinity, there were no significant changes in the biochemical parameters of V. luteola, which allowed for its normal growth and development. The changes in biochemical indicators and lack of antioxidant response in V. unguiculata under salinity may be connected to its growth and development compromise under saline stress / Mestrado / Mestre em Biologia Vegetal

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The protective role of dimethylsulfoxide (DMSO), lipoic acid (LA) and ternatin (TN), known free radical scavengers and cell membrane protectants were evaluated in an experimental model of testis ischemia/reperfusion. One hundred and twenty young male Wistar rats, mean weight 172.5Â15g, were randomized in five equal groups (GS - Sham, GC â Control, GD â Dimethylsulfoxide, GA â Lipoic acid and GT/D â Ternatin/DMSO). Rats of each group were distributed into four subgroups (T-0, T-1, T-3 and T-6), each comprising six animals. All surgical procedures were performed under inhalatory ether anesthesia. Experimental rats received intraperitoneal (i.p.) injections of 3% aqueous solution of DMSO (10 ml/kg, GD), LA (10 mg/kg, GA) or TN/DMSO (12 mg/kg, suspended in 3% aqueous solution of DMSO), GT/D) 24, 12 and 4 hours before cord detorsion. Last dose was given 1 hour before ischemia induction or second Sham operation. Sham (GS) and Control (GC) rats received NaCl 0.9% 10 ml/Kg i.p. Thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) levels (Âmol/g wet tissue) were assayed in testis. Total Antioxidant Power (TAP) (ÂM) was measured in blood plasma. Data were first analyzed by Kolmorogov-Smirnov test to assess differences in the general shapes of distribution. Comparisons between groups were made using the t test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. P values of <0.05 were considered to indicate statistical significance. Comparisons between antioxidant-treated and saline-treated rats (Control group, GC) were made using Dunnettâs test. Spermatic cord torsion induced significant decrease in testis MDA levels during ischemia and reperfusion in DMSO (GD) and LA (GA) and in TN/DMSO (GT/D) treated rats during reperfusion, compared with respective controls. MDA levels were significantly increased in GC compared with Sham (GS) rats at the end of the ischemia and during reperfusion as well as one hour after detorsion (T-1) in GC rats compared with T-0, in the same group, indicating additional damage imposed by the afflux of oxygenated blood (reperfusion) to the ischemic tissues. GSH levels decreased significantly in saline-treated (GC) compared with Sham-operated rats and increased significantly in DMSO and LA treated rats at the end of ischemia and during reperfusion. The model utilized in this study did not induce systemic effects. TAP was significantly increased during reperfusion (T-1) in LA (GA) and TN/DMSO (GT/D) pretreated rats, compared with respective controls. The results of the present study reinforce the hypothesis that ischemia and reperfusion are free radicals generating processes. The different antioxidants studied here have demonstrated great efficacy in cell membrane protection by decreasing lipid peroxidation in all experiments. Increased reduced glutathione levels in DMSO and LA treated rats support the view that these substances can exert a protective effect against testis oxidative stress injury caused by ischemia/reperfusion in rats subjected to torsion of the spermatic cord lasting three hours. TN protective effects in reducing lipoperoxidation were more pronounced in ischemic tissues. / O efeito protetor do dimetilsulfÃxido (DMSO), do Ãcido lipÃico (LA) e da ternatina (TN), conhecidos seqÃestradores de radicais livres e protetores da membrana celular foram avaliados utilizando um modelo experimental de isquemia/reperfusÃo do testÃculo. Cento e vinte ratos Wistar jovens com peso mÃdio de 174 g foram distribuÃdos ao acaso em cinco grupos (GS - Simulado, GC â Controle, GD â DimetilsulfÃxido. GA â Ãcido lipÃico e GT/D â Ternatina/DMSO) numericamente iguais e posteriormente redistribuÃdos em quatro subgrupos (T-0, T-1, T-3 e T-6), com seis animais cada. Todos os procedimentos cirÃrgicos foram realizados sob os efeitos da anestesia geral inalatÃria. Os animais receberam injeÃÃes intraperitoniais (i.p.) de soluÃÃo aquosa de DMSO a 3% (10 ml/kg, GD), LA (10 mg/kg, GA) ou TN+DMSO (12 mg/kg, GT/D, em soluÃÃo aquosa de DMSO a 3%) 24, 12 e 4 horas antes da destorÃÃo do cordÃo espermÃtico. A Ãltima dose foi administrada uma hora antes da induÃÃo da isquemia ou da segunda operaÃÃo simulada (Sham). Os ratos do grupo Simulado (GS) e do grupo Controle (GC) receberam de NaCl 0,9% (10 ml/Kg) i.p. Foram determinadas as concentraÃÃes de substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS) e glutationa reduzida (GSH) no tecido (testÃculo), e a capacidade antioxidante total (CAP) do plasma. Para determinaÃÃo do padrÃo de distribuiÃÃo das amostras utilizou-se o teste de Kolmorogov-Smirnov. As comparaÃÃes entre os grupos foram feitas utilizando-se o t teste ou o teste U de Mann-Whittney quando assim indicado. Para as comparaÃÃes dos grupos tratados com antioxidantes ao grupo Controle (GC) utilizou-se o teste de Dunnett. Valores de p<0,05 foram considerados significantes. A torÃÃo do cordÃo espermÃtico induziu uma reduÃÃo significante das concentraÃÃes de MDA, no tempo mÃximo de isquemia e na reperfusÃo, em tratos prÃ-tratados com DMSO (GD) e LA (GA) e durante a reperfusÃo naqueles prÃ-tratados com TN/DMSO (GT/D), comparados com seus respectivos controles. Os nÃveis de MDA estavam significantemente elevados nos ratos do grupo GC comparados aos animais do grupo GS, no tempo mÃximo de isquemia e durante a reperfusÃo, bem como uma hora apÃs a destorÃÃo (T-1) comparada ao T-0, demonstrando o dano adicional decorrente do afluxo de sangue oxigenado (reperfusÃo) ao tecido isquÃmico. As concentraÃÃes de GSH diminuÃram nos ratos prÃ-tratados com soluÃÃo salina e aumentaram significativamente nos ratos prÃ-tratados com DMSO ou LA, no tempo mÃximo de isquemia e durante a reperfusÃo. O modelo isquÃmico utilizado nÃo foi capaz de gerar efeitos sistÃmicos. A capacidade antioxidante total aumentou significantemente durante a reperfusÃo (T-1) nos ratos prÃ-tratados com LA (GA) e TN/DMSO (GT/D) comparados aos respectivos controles. Esses resultados fortalecem a hipÃtese de que a isquemia e reperfusÃo sÃo processos geradores de radicais livres. Os diferentes antioxidantes estudados mostraram grande eficiÃncia na proteÃÃo da membrana celular, reduzindo a peroxidaÃÃo lipÃdica, em todos os experimentos. O aumento dos nÃveis de glutationa reduzida, nos ratos prÃ-tratados com DMSO e LA, mostra que estes antioxidantes exercem uma proteÃÃo eficaz contra o estresse oxidativo induzido pela isquemia/reperfusÃo em ratos submetidos à torÃÃo do cordÃo espermÃtico por trÃs horas. Os efeitos protetores da ternatina se manifestam com maior intensidade no tecido isquÃmico, no controle da peroxidaÃÃo lipÃdica.

Orientador: Marta Cristina Teixeira Duarte / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T04:57:52Z (GMT). No. of bitstreams: 1 Matsumura_LauraYumeRodrigues_M.pdf: 1312693 bytes, checksum: 7afc440e71f8e63ffdb4e0f57d78115f (MD5) Previous issue date: 2014 / Resumo: As leveduras causam deterioração de uma grande variedade de produtos alimentícios e nas indústrias de bebidas, além de serem resistentes a muito conservantes químicos. Os óleos essenciais e extratos de plantas têm surgido como alternativas seguras para substituir conservantes sintéticos. Contudo, os mecanismos de ação de óleos essenciais sobre micro-organismos são complexos e não estão completamente elucidados. Neste trabalho, foi investigada a ação de 14 óleos essenciais e 2 extratos diclorometânicos provenientes de plantas medicinais e aromáticas pertencentes à CPMA (Coleção de Plantas Medicinais e Aromáticas) do CPQBA/UNICAMP sobre leveduras do gênero Candida e sobre Pichia guilliermondii de importância em alimentos. Foi determinada a concentração mínima inibitória (MIC) e os efeitos sobre a lise da membrana celular, sobre os carboidratos de reserva trealose e glicogênio, sobre a depleção de ATP e sobre a biossíntese de ergosterol. Os resultados demonstraram que o óleo essencial de Cinnamomum burmanni foi o que apresentou melhor potencial para controle das leveduras, sendo o cinamaldeído e o acetato de cinamila os compostos majoritários presentes neste óleo. A investigação dos possíveis mecanismos de ação do óleo essencial de C. burmanni sobre Candida albicans ATCC 10231 demonstrou que este afetou a viabilidade da levedura a partir da concentração de 0,5 mg/mL (5MIC), e ocasionou a lise da membrana celular, havendo liberação de proteínas e lipídios para o meio extracelular, além de depleção de ATP. No caso dos carboidratos de reserva, os resultados demonstraram que o óleo essencial de C. burmanni ocasionou acúmulo de trealose, possivelmente pelo estresse ocasionado às células. Nenhum efeito foi observado sobre a reserva de glicogênio e sobre a inibição da síntese de ergosterol. Os resultados indicam a ação inibitória do óleo essencial de C. burmanni e mostram que este apresenta potencial para controle de leveduras de importância em alimentos / Abstract: Yeasts cause deterioration of a wide variety of food and drink industries, besides being very resistant to chemical preservatives. Essential oils and plant extracts have emerged as safe alternatives to synthetic preservatives. However, the mechanisms of action of essential oils on microorganisms are complex and not fully elucidated. In this work, was investigated the action of 14 essential oils and 2 dichloromethane extracts from medicinal and aromatic plants belonging to the Collection of Medicinal and Aromatic Plants - CPMA at CPQBA/ UNICAMP on Candida species and Pichia guilliermondii of importance in foods. The minimum inhibitory concentration (MIC) was determined and the effects of the essential oil on the cell membrane lysis, the carbohydrate reserves trehalose and glycogen, depletion of ATP and ergosterol biosynthesis were evaluated. The results showed that the essential oil from Cinnamomum burmanni presented the best potential to control the Candida spp. being the cinnamaldehyde and cinnamyl acetate the major compounds present in this oil. The investigation of the possible mechanisms of action of the C. burmanni essential oil on Candida albicans ATCC 10231 showed that the oil affected the viability of the yeast from 0.5 mg/mL (5MIC), and caused lysis of the cell membrane, with release of proteins and lipids into the extracellular environment, as well as ATP depletion. In the case of carbohydrate reserves, the results showed that the essential oil of C. burmanni caused accumulation of trehalose, possibly due to the cellular stress. No effect was observed on the synthesis of glycogen and ergosterol. The results indicate the inhibitory action of the essential oil from C. burmanni and show its potential to control yeasts of importance in foods / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Electric field mediated gene delivery modalities have preferable safety profiles with the ability to rapidly transfect cells in vitro and in vivo with high efficiency. However, the current state of the art has relied on trial and error studies that target the average cell within a population present in treated tissue to derive electric pulse parameters. This results in fixed gene electrotransfer (GET) parameters that are not universally optimum. Slow progress towards the validation of a mechanism that explains this phenomena has also hindered its advancement in the clinic. To date, GET methods utilizing feedback control as a means to optimize doses of electric field stimulation have not been investigated. However, with modern electric components the electric characteristics of tissue exposed to electric pulses can be measured in very short time scales allowing for a near instantaneous assessment of the effect these pulses have on cells and tissue. This information is ideal for use in optimizing GET parameters to ensure the conditions necessary for gene delivery can be created regardless of anisotropic tissue architecture and electrode geometry. Bioimpedance theory draws parallels between cell structures and circuit components in an attempt to use circuit theory to describe changes occurring at a cellular and tissue level. In short, a reduction in tissue impedance indicates a reduction to the opposition of current flow in a volume conductor indicating new pathways for current. It has been purported these new pathways exist in the cell membrane and indicate a degree of membrane permeability/destabilization that either indicates or facilitates the uptake of exogenous molecules, such as nucleic acids or plasmid DNA. This study evaluated the use of relative impedance changes from 10 Hz – 10 kHz that occur in tissue before and after GET to indicate relative increase in tissue and membrane permeability. An optimum reduction in impedance was then identified as an indicator of the degree of membrane permeability required to significantly enhance exogenous DNA uptake into cells. This study showed the use of impedance-based feedback control to optimize GET pulse number in real time to target 80% or 95% reduction in tissue impedance resulted in an 12 and 14 fold increase in transgene expression over controls and a 6 and 7 fold increase in transgene expression over fixed pulse open loop protocols.

Drosophila are used extensively for genetic, developmental and now molecular biology research. At present, germline transformation of these organisms can only be achieved by microinjection of P-element vectors into the pole cells of young embryos. The technique of microinjection however, requires a delicate touch and is quite laborious. Therefore, the development of a rapid and simple technique was investigated. Electroporation, like microinjection, is a physical means of introducing DNA into a cell and is therefore potentially applicable to all cell types. Electroporation involves the use of an electrical current to create pores in the membrane of a cell. Macromolecules, such as DNA may enter a cell via these pores. Electroporation is a quick, reproducible, and efficient method for transforming cells. Through studies of the survival and permeability of Drosophila melanogaster embryos exposed to electrical currents, it was discovered that although the survival of the embryos decreased steadily as field strength increased, the embryos did not become permeable to a water soluble dye unless a pulse of 10 kV/cm was applied. Few embryos survived this extreme voltage required for dye uptake. Attempts to introduce DNA into dechorionated Drosophila embryos utilizing this technique however, produced no transformants. These results suggested that the remaining protective coatings of the dechorionated embryo were obstructing efficient pore formation, thus preventing DNA penetration. In view of these results, methods to eliminate the wax layer, present between the chorion and vitelline membrane of laid eggs, were examined. Wax removal by detergent solubilization, solvent extraction and melting by heating were investigated, yet did not produce a satisfactory procedure. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Cumulative clinical and experimental evidence indicates that volatile sulphur compounds (VSC) the principal components of oral malodour, may play an important role in the pathogenesis of periodontal disease. As their (H₂S and CH₃SH) concentrations in gingival sulci increase with the severity of periodontal involvement, the objective of this investigation is to ascertain if they exert an effect on the permeability of oral mucosa. Permeability determinations were performed on excised porcine sublingual mucosal specimens which consisted of non-keratinized epithelium, basal membrane and connective tissue layers mounted in a two compartment perfusion apparatus. Using radioactive and fluorescent-labelled penetrants, it was found that exposure of the epithelial surface to an atmosphere containing physiological concentrations of both thiols (15 ng H₂S or CH₃SH / ml of 95% air - 5% C0₂) increased the permeability of the mucosa to (³⁵S)-S0₄⁻², (³H)-prostaglandin E₂ (PGE₂) and fluorescein isothiocyanate labelled E. coli lipopolysaccharide (F-LPS). A three hour exposure of the mucosa to H₂S and CH₃SH resulted in a 75% and 103% increase respectively in permeability to (³⁵S)-labelled sulphate ion. Similarly, the mercaptan induced up to a 70% increase in permeability of the mucosa to (³H)-prostaglandin E₂. The magnitude of changes in the permeability were found to depend on duration of exposure to the thiols and to their concentration. Studies using (³⁵S)-H₂S suggest that the observed changes in the tissue permeability are related to the reaction of the thiols with tissue components. In addition, the (³⁵S)-H₂S is capable of perfusing through all three layers of the mucosa at 12.3 ng / cm². In contrast to H₂S , the CH₃SH effect was irreversible in control air / C0₂ environment. This infers that CH₃SH is potentially a more deleterious agent to the tissue barrier. However, its effect can also be reversed by treatment of tissues with 0.22% ZnCl₂ either prior to or after exposure to mercaptan. This suggests that Zn⁺² ion may be useful in preventing the potentially harmful effects of VSC. Fluorescent studies with F-LPS indicate that thiols can also potentiate the penetration of endotoxin. Whereas the fluorescence of the F-LPS in control systems was confined to the superficial epithelial layer in contact with the endotoxin, the CH₃SH- exposed mucosa exhibited fluorescence throughout the epithelial and connective tissue layers. Fluorescent staining of the mucosal specimens with fluorescein diacetate followed by counter staining with ethidium bromide provides evidence of membrane impairment to some cells by CH₃SH. Collectively these observations provide strong experimental evidence that the VSC, products of putrefaction produced in the gingival sulcus by oral microflora, may adversely affect the integrity of the crevicular barrier to deleterious agents and thus contribute to the etiology of periodontal disease. / Dentistry, Faculty of / Graduate

Fuel molecules are organic solvents that have disruptive effects on the bacterial membrane. This is a significant barrier in biofuel production, as it limits the fuel concentration that can be achieved through fermentation. One potential way of overcoming this barrier is to identify lipid compositions that can better withstand solvent stress, for which it is important to understand how organic solvents disrupt the membrane. Use of biophysical characterization techniques to quantify physical properties like fluidity and thickness will enable us to understand the mechanism by which solvents disrupt membranes. Native membranes are very complex, and we sought to develop in-vitro models for the membrane of the bacterium Bacillus subtilis that use pure phospholipids. Toward this goal, a number of the unusual B. subtilis fatty acids were synthesized, partial synthesis of the membrane phospholipids was achieved, and preliminary assessment of solvent effects on standard lipids was performed using a fluorescence technique.

Concerns about global warming have resulted in a surge of research into alternatives to fossil fuels. In recent years, biofuels have gained traction due to their low environmental impact. Biofuel production most commonly employs microorganisms to convert biomass to fuel for industrial and transportation applications. Compounds made in biofuel production, however, are toxic to cell membranes and disrupt their integrity, harming the microorganisms and limiting biofuel yield. A key to overcoming this challenge is understanding how fuels interact with microorganisms’ cell membranes, which perform a host of functions, including transport, cell recognition, transduction, and movement. Phospholipids are the cell membrane’s building blocks and provide the critical matrix to support these vital functions. This research sought to make in-vitro membrane phospholipid models of the bacterium Bacillus subtilis (a biofuel producer candidate), subject them to fuel stress and employ fluorescence techniques to understand how fuels affect membrane integrity.