The ability of cells to respond to their microenvironment is controlled by a complex communication system. Cell signaling utilizes a series of post-translational events to regulate and coordinate cellular activities. Although phosphorylation is thought to be the key regulator of these events, recent findings implicate the O-GlcNAc modification as an additional control mechanism. Modulation of signal transduction requires compartmentalization of the kinases and phosphatases. Based on the evidence of subcellular localization of OGT isoforms, the diversity of O-GlcNAcylated proteins upon stimulation, and its role during insulin signaling, it can be hypothesized that O-GlcNAc is involved and regulates signal transduction in a compartmentalized manner. To investigate the spatio-temporal dynamics of O-GlcNAc in cell signaling, we have generated a series of genetically encoded O-GlcNAc reporters based on fluorescence resonance energy transfer (FRET). These reporters and localized variants have allowed compartment specific visualization of O-GlcNAc activity in the nucleus, cytoplasm and plasma membrane. Herein we describe these reporters and their use to examine O-GlcNAc dynamics in signaling using serum stimulation and environmentally relevant concentrations of arsenite. Acute exposure to arsenite through drinking water has become an environmental health concern worldwide. Our results imply a complex regulation of O-GlcNAc on a fast timescale that is tied to more canonical kinase pathways. / text

No description available.

The removal of arsenic toxicity from water is accomplished by a preliminary preoxidative step transforming the most toxic form, arsenite (As (III)), to the least toxic form, arsenate (As (V)). The potential of As (III) oxidation to As (V) was initially investigated in batch reactors using the chemoautotrophic Thiomonas arsenivorans strain b6 under varying initial As (III) and cell concentrations and at optimal pH and temperature conditions (pH 6.0 and temperature 30°C). The strain b6 completely oxidized As (III) to As (V) during exponential growth phase for lower levels of As (III) concentrations (≤ 100 mg/L) but continued into stationary phase of growth for higher levels (≥ 500 mg/L). Other important factors such as oxygen and carbon limitations during biological As (III) oxidation were also evaluated. The biokinetic parameters of the strain b6 were estimated using a Haldanesubstrate inhibition model with the aid of a non-linear estimation technique. Microbial As (III) oxidation was further investigated in continuous-flow bioreactors (CSTR and biofilm reactor) under varying As (III) loading rates. Both the reactors achieved As (III) oxidation efficiency exceeding 99% during the steady-state conditions. The reactors were also able to recover from an As (III) overloading phase establishing the resilient nature of the microorganism. The basic mass balance expressions on As (III) and biomass along with the Monod model were used to linearly estimate the biokinetic parameters in the CSTR study. However, in the biofilm study, a steady-state flux model was used to estimate the same parameters. The performance of the model was very good in simulating the transient and steady-state conditions. Finally, the potential application of one-stage and two-stage reactor systems was investigated for the near complete removal of arsenic. Activated alumina was used as the adsorbent for the As (V) produced by the biological oxidation of As (III). The two-stage reactor process performed better than the one-stage reactor system in lowering the arsenic level below the detection limit (1 mg/L) for at least eight days of operation. However, pH fluctuations and probable competition from ions such as PO43- , SO42-, and Cl- severely impacted the performance of the reactors. Further study is needed to improve the overall efficiency of the reactor systems for achieving complete removal of arsenic for a longer operating time.

Made available in DSpace on 2015-03-26T13:36:44Z (GMT). No. of bitstreams: 1 texto completo.pdf: 547989 bytes, checksum: cfc62c6982aac5d8709b56ed06b56ef4 (MD5) Previous issue date: 2014-01-24 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Pistia stratiotes plants were subjected to different concentrations of arsenite (AsIII) in order to assess the intensity of oxidative stress generated by this metalloid and antioxidant responses involved in tolerance in this species. After acclimatization (5 days) in Clark nutrient solution 1⁄4 ionic strength and pH 6.5, the plants were exposed to four treatments: control (nutrient solution), 5, 10 and 20 μM of AsIII, where remained for 24 hours to biochemical analyzes, and 4 days for arsenic (As) absorption analysis , visual symptoms and relative growth rate (RGR). Plants of P. stratiotes accumulated high concentrations of As, however, most of this element was retained in roots giving a low translocation factor. The presence of As promoted toxicity symptoms and changes in physiological and biochemical parameters, as evidenced by severe chlorosis, root loss, TCR reduction, reactive oxygen species (ROS) increase, membrane damages, changes in antioxidant enzymes activity and glutathione content. The results showed that plants of P. stratiotes exposed to lowest concentration of AsIII (5 μM) are tolerant to the metalloid, due to efficient performance of enzymatic and non-enzymatic antioxidants. However, exposure to higher AsIII concentration resulted in impairment of defense mechanisms, particularly in roots, due to the high accumulation of As in this organ. In this sense, the mechanism of AsIII toxicity involves increased ROS generation and, in parallel, inhibition of antioxidant mechanisms, resulting in high incidence of oxidative stress. Thus, it can be concluded that P. stratiotes is able to absorb and accumulate high concentrations of AsIII, however, under higher concentrations of this pollutant, plant metabolism is greatly affected, impacting their ability to tolerate stress viii and limiting their use in phytoremediation of environments where As III found in high concentrations. / Plantas de Pistia stratiotes foram submetidas a diferentes concentrações de arsenito (AsIII) com a finalidade de avaliar a intensidade do estresse oxidativo gerado por este metaloide e as respostas antioxidantes envolvidas na tolerância desta espécie ao estresse. Após a aclimatação (5 dias) em solução nutritiva de Clark 1⁄4 de força iônica e pH 6,5, as plantas foram expostas a quatro tratamentos: controle (apenas solução nutritiva), 5, 10 e 20 μM de AsIII, onde permaneceram por 24 horas para as análises bioquímicas e durante 4 dias para as análises de absorção de arsênio (As), sintomatologia visual e taxa de crescimento relativo (TCR). Plantas de P. stratiotes apresentaram altos teores de As, no entanto, a maior parte deste elemento ficou retida nas raízes, conferindo um baixo fator de translocação. A presença do As promoveu sintomas de toxidez e alterações nos parâmetros fisiológicos e bioquímicos, evidenciados por intensa clorose, perda radicular, redução da TCR, aumento das espécies reativas de oxigênio (ROS), danos de membrana, alterações na atividade de enzimas antioxidantes e no teor de glutationa total. Os resultados obtidos evidenciam que plantas de P. stratiotes expostas à concentração mais baixa de AsIII (5 μM) são tolerantes ao metaloide, devido a atuação eficiente dos antioxidantes enzimáticos e não enzimáticos. No entanto, a exposição à concentração mais elevada de As III, resultou em comprometimento dos mecanismos de defesa, principalmente nas raízes, em decorrência do elevado acúmulo de As neste órgão. Nesse sentido, o mecanismo de toxidez do AsIII envolve a maior formação de ROS e, paralelamente, a inibição dos mecanismos antioxidantes, resultando na alta incidência de estresse oxidativo. Assim, vi pode-se concluir que P. stratiotes é capaz de absorver e acumular altas concentrações de AsIII, no entanto, sob concentrações mais elevadas deste poluente, o metabolismo da planta é consideravelmente afetado, interferindo na sua capacidade de tolerar o estresse e limitando o seu uso na fitorremediação de ambientes em que o AsIII encontra-se em altas concentrações.

O arsênio inorgânico, um contaminante ambiental, produz uma série de respostas de estresse em células de mamíferos, incluindo o comprometimento da função mitocondrial, acompanhado por inibição do crescimento celular e carcinogênese. Como previamente identificamos efeitos deletérios do comprometimento da função mitocondrial e dos radicais livres do oxigênio na oogênese, investigamos os efeitos do arsenito na meiose, no desenvolvimento embrionário pré-implantação e na apoptose embrionária em camundongos. Camundongas com 6 semanas de idade foram tratadas com baixa (0,16 mg) ou média dose de arsenito (0,32 mg), por meio de 7 injeções intraperitoneais, 1 a cada 2 dias, durante 14 dias. Os controles foram injetados com solvente. A incidência de anomalias meióticas, caracterizadas por anormalidades do fuso celular e/ou mal alinhamento cromossômico, foi significantemente aumentada tanto nos oócitos in vivo ovulados, como nos in vitro maturados, oriundos dos animais tratados com arsenito. Foram detectadas reduções significativas das taxas de clivagem (24 horas de cultivo), de formação de mórula (72 h) e de desenvolvimento para blastocisto (96 h), nos embriões dos grupos tratados com arsenito. Apesar do número total de núcleos não ter diferido significativamente entre os blastocistos dos grupos controle e de tratamento, a percentagem de núcleos apoptóticos foi significantivamente maior nos blastocistos derivados dos animais tratados com a dose média de arsenito. Estes dados sugerem que o arsenito causa aberrações meióticas, que podem contribuir tanto para o comprometimento do desenvolvimento embrionário pré-implantação, como para a apoptose embrionária. / Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including mitochondrial uncoupling accompanied by growth inhibition and carcinogenesis. Because previously we identified detrimental effects of mitochondrial uncoupling, and reactive oxygen species (ROS) on oogenesis, we investigated effects of arsenite on meiosis, early embryo development, and apoptosis in mice. Six-week-old CD-1 mice were treated with either low (0.16mg) or medium (0.32mg) doses of arsenite every two days by 7 intraperitoneal injections for 14 days, and controls were injected with solvent. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment or spreading, was significantly increased in both in vivo and in vitro treated oocytes. Further, we found a significant decrease in cleavage rates at 24h, morula formation at 72h, and development to blastocyst at 96h in treated groups. Although the total number of nuclei in developed blastocysts did not significantly differ between the treated and control groups, the percentage of apoptotic nuclei was significantly increased in blastocysts derived from the medium dose treated group. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.

No description available.

The adsorption behavior of silicic acid and arsenite alone and competitively on goethite over a broad pH range (3-11) at environmentally relevant concentrations was investigated utilizing pH adsorption data and zeta potential measurements. Both addition scenarios (Si before As(III) and As(III) before Si) were examined. The results of the adsorption experiments and zeta potential measurements were then used to model the single ion and competitive ion adsorption on goethite with the CD-MUSIC model implemented in the FITEQL 4.0 computer program. Silicic acid adsorption was reduced by the presence of arsenite for all but one of the adsorption scenarios examined, while in contrast silicic acid had little effect upon arsenite adsorption. However, the presence of silicic acid, regardless of the addition scenario, dramatically increased the arsenite equilibrium solution concentration over the entire pH range investigated. The CD-MUSIC model was able to predict the single ion adsorption behavior of silicic acid and arsenite on goethite. The modeled zeta potential data provided further evidence of the CD-MUSIC model's ability to describe the single anion adsorption on goethite. Our model was also able to collectively describe adsorption and zeta potential data for the low Si-arsenite adsorption scenario quite well however, our model under-predicted silicic acid adsorption for the high Si-arsenite competitive scenario. / Master of Science

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Rice is the main source of inorganic arsenic (Asi), a carcinogenic substance, in the human diet. In this sense, there is great interest in reducing the accumulation of As in rice grains. To achive this, field management / rice processing and study of different cultivars have been explored. Globally, many countries that consume rice don t produce it, or even specifics regions of a producer country. As to maintain the rice consumption these regions have to import rice, the identifycation of regions with low As is an important option to ensure food security for the consumers. However, the location of As free areas seems to be an incomplete solution; as there is a large demand for rice and the already considerable number of areas described as contaminated. Thus, it is necessary to use a set of information able to describe As levels in different areas, comparing cultivars, including specific mechanisms of tolerance and low translocation, besides the description of side effects such as levels of other minerals present in the grain and effect of soil management on grain quality. Given the above, this study aimed to evaluate commercial samples of rice in Latin America, as well as samples collected in the field under different managements of water, fertilizer and land use; in addition it was used short-term experiments in greenhouse were performed to evaluate morphophysiological parameters in different cultivars. There is a large discrepancy regarding As concentration among the different regions tested. Although the genetic factor (cultivar) has an effect on As concentration in grain, environmental and management factors are decisive. Apparently the cultivar BR-IRGA 409 presents a large susceptibility to As, also presenting a lower translocation to grains while cultivars described as tolerant had a higher accumulation of this element. The phosphorus use efficiency and the concentration of non-protein thiols appear to be related to tolerance, being highly pronounced in tolerant cultivars. Finally, As has a high potential to genotoxic and oxidative stress, and the main abnormality observed was the presence of micro-nuclei. / O arroz é a principal fonte de arsênio inorgânico (Asi), uma substância cancerígena, na dieta humana. Neste sentido, existe um grande interesse em reduzir o acúmulo do mesmo em grãos. Para tal, a gestão a campo, criação/modificação no processamento de arroz e estudo com diferentes cultivares vêm sendo explorados. A nível global, muitos países que consomem arroz não produzem o mesmo, ou mesmo regiões dentro de um país produtor. Visto que para manter o consumo tais regiões importam arroz, a identificação de regiões com baixa concentração de As é uma importante opção para a salubridade dos consumidores. Entretanto, a localização de pequenas áreas livres ou com baixas concentrações de As não parece ser a solução completa para a contaminação observada em alimentos; visto a grande demanda de arroz e o número já considerável de áreas descritas como contaminadas. Desta forma, faz-se necessário o uso conjunto de informações como a descrição dos níveis de As em diferentes áreas, comparação entre cultivares, incluindo mecanismos específicos de tolerância e baixa translocação, além da descrição de efeitos secundários como nível dos demais minerais presentes no grão e efeito do manejo da cultura na qualidade de grãos. Visto o exposto, o presente estudo teve por objetivos avaliar amostras comerciais de arroz da América Latina, bem como amostras coletadas a campo sob diferentes manejos de água, fertilizantes e uso do solo; além de utilizar experimentos de curta duração em casa de vegetação para avaliar parâmetros morfofisiológicos em diferentes cultivares. Existe uma grande discrepância na concentração de As nas diferentes regiões testadas. Embora o fator genético (cultivar) tenha efeito sobre a concentração de As em grãos, os fatores ambiente e manejo são determinantes. Aparentemente, a cultivar BR-IRGA 409 apresenta uma maior suscetibilidade ao As, apresentando também uma menor translocação para os grãos, enquanto que cultivares com comportamentos tolerantes apresentaram um maior acúmulo deste elemento. A eficiência de uso de fósforo e a concentração de tióis não proteicos parecem estar relacionadas à tolerância ao As, sendo altamente pronunciadas em cultivares tolerantes. Finalmente, o As apresenta alto potencial genotóxico e oxidativo, sendo a principal anormalia observada a presença de micro-núcleos.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.