Análise da Expressão de APE1 após Estresse Oxidativo Induzido em Células Proficientes e Deficientes na Via de Reparo por Excisão de Nucleotídeos

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

Proteômica comparativa de linhagens celulares humanas expostas a estresse oxidativo induzido por riboflavina fotossensibilizada

Reactive oxygen species (ROS) are continuously generated and can be derived from cellular metabolism or induced by exogenous factors, in addition, have the capacity to damage molecules like DNA and proteins. BER is considered the main route of DNA damage oxidative repair, however, several studies have demonstrated the importance of the proteins participation of other ways to correct these injuries. NER enzymes deficiency, such as CSB and XPC, acting in the damage recognition step in the two subways this system influences the effectiveness of oxidative damage repair. However, the mechanisms by which cells deficient in these enzymes respond to oxidative stress and its consequences still need to be better understood. Thus, the aim of this study was to perform a proteomic analysis of cell lines proficient and deficient in NER, exposed to oxidative stress, in order to identify proteins involved, directly or not, in response to oxidative stress and DNA repair. For this, three strains of human fibroblasts, MRC5-SV, CS1AN (CSBdeficient) and XP4PA (XPC-deficient) were treated with photosensitized riboflavin and then carried out the differentially expressed proteins identification by mass spectrometry. From the results, it was observed in MRC5-SV increase expression in most of the proteins involved in cellular defense, an expected response to a normal cell line subjected to stress. CS1AN showed a response disjointed, it is not possible to establish many interactions between the proteins identified, may be one explanation for their sensitivity to treatment with riboflavin and other oxidants and increased cell death probably by induction of pro-apoptotic pathways. Already XP4PA showed higher expression of apoptosis-blocking proteins, as there was inhibition or reduced expression of others involved with the activation of this process, suggesting the activation of an anti-apoptotic mechanism in this lineage, which may help explain the high susceptibility to develop cancers in XPC individuals. These results also contribute to elucidate action mechanisms of NER in oxidative damage and the understanding of important routes in the oxidative stress correlation, repair and malignant tumors formation

Avaliação de parâmetros de estresse oxidativo em plantas de cana-de-açúcar tratadas com peróxico de hidrogênio

The genus Saccharum belongs to Poaceae family. Sugarcane has become important monocultures in Brazil due to their products: ethanol and sugar. The production may change between different regions from Brazil. This difference is related to soil, climatic conditions and temperature that promotes oxidative stress that may induce an early flowering. The aim of this work was to identify the effects of oxidative stress. In order to analyse this, sugarcane plants were submitted to oxidative stress using hydrogen peroxide. After this treatment, the oxidative stress were analyzed Then, the plant responses were analyzed under different approaches, using morphophysiological, biochemical and molecular tools. Thus, sugarcane plants were grown under controlled conditions and until two months they were subjected first to a hydroponics condition for 24 hours in order to acclimation. After this period, these plants were submitted to oxidative stresse using 0 mM, 10 mM, 20 mM and 30 mM hydrogen peroxide during 8 hours. The histomorphometric analysis allowed us to verify that both root and leaf tissues had a structural changes as it was observed by the increased in cell volume, lignin accumulation in cell walls. Besides, this observation suggested that there was a change in redox balance. Also, it was analyzed the activity of the SOD, CAT and APX enzymes. It was observed an increase in the SOD activity in roots and it was also observed a lipid peroxidation in leaves and roots. Then, in order to identify proteins that were differently expressed in this conditions it was used the proteomic tool either by bidimensional gel or by direct sequencing using the Q-TOF EZI. The results obtained with this approach identified more than 3.000 proteins with the score ranging from 100-5000 ions. Some of the proteins identified were: light Harvesting; oxygenevolving; Thioredoxin; Ftsh-like protein Pftf precusor; Luminal-binding protein; 2 cys peroxiredoxin e Lipoxygenase. All these proteins are involved in oxidative stress response, photsynthetic pathways, and some were classified hypothetical proteins and/or unknown (30% of total). Thus, our data allows us to propose that this treatment induced an oxidative stress and the plant in response changed its physiological process, it made changes in tissue, changed the redox response in order to survival to this new condition

Mutagênese induzida por flavonóides presentes do decocto das cascas da aroeira (Schinus terebinthifolius, Raddi)

The decoction of Brazilian pepper tree barks (Schinus terebinthifolius, Raddi), is used in medicine as wound healing and antiinflamatory. Once extracts from this plant are used for acceleration of scar s process, it is important to study their mutagenic and genotoxic potential. In previous works in our laboratory, it was observed mutagenicity caused by the decoction when in high concentrations. Among the chemical compounds of this plant that could be able to induce mutation, the flavonoids were the only group that was referred to have either an oxidant or antioxidant potential. The flavonoids were isolated, purified and quantified by adsorptive column chromatography under silica gel, bacterial and in vitro genotoxic tests were realized to determine if the flavonoids were the responsible agents for this mutagenicity found. The tests realized with plasmidial DNA were indicative that the flavonoids are probably genotoxic, due to the presence of correlation between increase of the flavonoid concentration and in plasmidial DNA double strand breakage visualized in agarose gel, as well as they were capable to generated abasic sites shown by the in vitro treatment with exonuclease III. The same tests with plasmidial DNA in the presence of copper [10 µM] and of a Tris-HCl pH 7.5 [10 µM] buffer were realized with the isolated flavonoids to determine if there would be or not participation of reactive oxygen species (ROS). The transformation of plasmidial DNA in different bacterial strains proficient and deficient in DNA repair enzymes in the presence or not of a Tris-HCl buffer, suggests that the enzymes that repair oxidative lesions are necessary to repair the lesions generated by the flavonoids and that ROS are generated and are necessary to promote the lesions. Bacterial tests with Escherichia coli strains of the CC collection (deficient or not for DNA repair enzymes), showed that the flavonoids are able to increase the frequency of mutations, mainly in strains mutated in repair enzymes (MutM, MutY-glicosylases and double mutant), suggesting that these agents are responsible for the enhancement in the mutation rate. In order to determine the mutation spectrum caused by the flavonoids of the Brazilian pepper tree stem bark, plasmidial DNA previously treated with the flavonoids were transformed in bacterial strains deficient and proficient in the DNA repair enzymes, followed by a blue-white selection with X-gal, DNA amplification by PCR and sequencing the positive mutant clones. Analysis of the mutants obtained from strains CC104, CC104mutM, CC104mutY, CC104mutMmutY, BW9101, BW9109 indicated a predominance of some mutations like G:C to C:G that can be correlated with the origin of 8-oxoG, due to oxidative lesions caused by the flavonoids. So it can concluded that the flavonoid isolated or in fractions enriched on them are genotoxic and mutagenic, and their mutations are predominantly oxidative, mediated by ROS, and the lesions are recognized by the BER system. In this way it is proposed that the flavonoids can act in two different ways to generate the DNA lesion: 1. in a Fenton-like reaction, when the flavonoid are in the presence of metal ions and that together with the water generate ROS that promotes the DNA lesions; 2. in another way the lesions can be generated by the formation of ROS due to the internal chemical structure of the flavonoid molecule due to the quantity and location of hydroxyl groups, and so producing the DNA lesions, those lesions can be directly (suggested by the in vitro experiments) or indirectly done (supported by the experiments using the CC bacterial strains)

Albuns fotográficos de/por Enoque Neves: uma poética visual

La imagen fotográfica la imagen despierta pulsiones, en una experiencia provocativa que acciona el campo mítico-fenomenológico descíframe o te devoro . El texto fotográfico no constituye sólo un momento del real , es mucho más que un recorte gélido del tiempo pasado. Sobretodo, porque la comprensión del lenguaje subyacente a la imagen fotográfica se coloca dentro y más allá de lo meramente dado y/o objetivado . En la comprensión de la imagen se disponen regímenes de subjetivación horadado por códigos culturales, dominios del lenguaje, símbolos, experiencias estéticas, creación simbólica, valores, memorias, imaginarios. En ese sentido, surge el estudio, el levantamiento y la sistematización del acervo fotográfico dejado por el fotógrafo autodidacta: Enoque Pereira das Neves (1918-2002), que produjo formas visuales reveladoras de orientaciones culturales colectivas y universos fotográficos imaginarios. Él fotografió por medio siglo la vida cotidiana del campesino, en tierras potiguares y paraibanas. En la comprensión del acervo opte por subdividirlo en álbumes los cuales revelan las intimidades del fotógrafo con el lugar, una escrita de si, un estado del alma, del sujeto que observa la relación sociedad y naturaleza, en una especie de simbiosis en que accionan potencias resonantes de creación en el devaneo de la materia, sobresaliéndose a la pulsión terrestre, o enraizamiento, os rizomas del hombre unido a la tierra, las tradiciones, a rusticidad, laboral, bien como el apelo estético direccionado al agua: pulsión de vida en tierras áridas. La comprensión del inmenso acervo, evaluado en más de cincuenta mil fotografías es profundamente representativa de predilección temática circunstanciada por la niñez, la narrativa enfoca imágenes míticas como la del Eros nudo, la madona y el niño, y prácticas culturales del universo infantil, como el juguete y el juego. Mismo el acervo siendo un apelo estético a la vida surgió la imagen fotográfica de la muerte, especialmente, la muerte en la niñez o ángel y la muerte de ancianos como o deseo de la última imagen de aquel entre los suyos. La investigación es transversal por los siguientes objetivos: propongo un reconocimiento del acervo de Enoque Pereira das Neves en la intención de comprender el universo imaginario presente en su obra fotográfica. Invisto también, en la posibilidad de hacer la lectura del acervo como álbum imaginario.

Ergonomia, sustentabilidade sócio-ambiental e atividade de pesca artesanal com jangadas: estudo de caso na Praia de Ponta Negra, Natal-RN

The fishing is made possible by the handiwork of a fisherman, not using appliances that achieve the same levels of industrial fishing productivity and not causing environmental impacts on fish stocks with the same intensity. The adoption of sustainability practices in this type of fishing has been requested for the achievement of environmental and socioeconomic improvements, since its production has been decreasing over the years and overfishing has led to reduced stocks also stand out environmental aggressions such as pollution. Thus, this research aims to analyze the interference of environmental aspects/impacts that permeate the activity performed by fisherman on the beach of Ponta Negra, Natal-RN, in order to propose recommendations for environmental sustainability. We used a methodology based on the ergonomic work analysis (WISNER, 1987; GUERIN, 2001; VIDAL, 2008) using techniques such as observational and interactional observation protocols, photographs, videos, conversational action, and listening to verbalizations. To understand the vulnerability of the environment in which the activity is carried fisherman and the interferences that it suffers, we used the study of environmental aspects/impacts (SÁNCHEZ 2008). As a result, it appears that from the observations located and reports raftsmen, changes in the sea, the search for new fishing grounds farther from the coast, reducing fishery production, waste generation and pollution of the workplace, and the difficulty in docking rafts, factors arising from the pressures both for society as Seen by fisherman, denoting the necessity of drawing up recommendations and actions proposed by management activity by promoting discussion among raftsmen and accountable institutions, and training through workshops on environmental education, aiming to positive changes in the activity fisherman from Ponta Negra and the environment in which raftsmen are inserted in order to seek a sustainable activity, preserving its characteristics

Expressão do gene da leptina e seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras

Objetivou-se com este trabalho avaliar os efeitos de uma dieta de alto nível de energia e proteína combinada com a aplicação de bST no perfil de expressão dos genes da leptina e de seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras. Foram utilizadas amostras de parênquima mamário de 32 novilhas holandesas distribuídas aleatoriamente em quatro tratamentos (n=8): dieta com alto ou baixo teor de energia e proteína combinada ou não com a aplicação de bST. O delineamento utilizado foi em blocos casualizados com arranjo de tratamentos em esquema fatorial 2 × 2. A extração do RNA total das amostras de tecido foi feita e o nível de expressão gênica foi analisado por qRT-PCR utilizando-se o gene da glicuronidase β como controle, pelo método 2-ΔΔCt. Animais que receberam a dieta com alto conteúdo de energia e proteína apresentaram maior expressão de mRNA de leptina, com aumento de 56%, e menor expressão de mRNA do receptor Ob-Rb, com redução de 18%. Por outro lado, a aplicação de bST resultou em diminuição da expressão do mRNA de leptina e do receptor Ob-Rb em 74% e 23%, respectivamente. Não houve interação entre dieta e aplicação de bST. O aumento na expressão de leptina pode explicar, ao menos em parte, os efeitos negativos da dieta de alta energia e proteína, oferecida no período pré-púbere, sobre a produção de leite de novilhas leiteiras.

Plantas de cobertura e calagem na implantação do sistema plantio direto

O objetivo deste trabalho foi avaliar o efeito de plantas de cobertura e da calagem sobre a produtividade da soja e as características químicas do perfil do solo, após implantação do plantio direto. O experimento foi realizado em campo por dois anos, em Latossolo Vermelho‑Amarelo. A área era explorada como pastagem há cinco anos. Avaliaram-se três sistemas de cultivo: soja/pousio/soja, soja/Pennisetum glaucum/soja e soja/Urochloa ruziziensis/soja, com aplicação de calcário à dosagem de 0, 0,5, 1,0 e 2,0 vezes a quantidade necessária para elevar a saturação de bases (V) a 50%, na camada de 0-20 cm. O calcário foi incorporado ao solo, a 20 cm de profundidade, previamente à implantação dos sistemas de cultivo. Aos 490 dias após a calagem, foram coletadas amostras de solo das profundidades de 0-­10, 10-­20, 20-­40 e 40­-60 cm. Avaliaram-se o estado nutricional das plantas, as alterações das características químicas do solo e a produtividade da soja. As plantas de cobertura associadas à calagem promovem melhorias nos parâmetros de acidez do solo - sobretudo quando a dose aplicada é igual ou mais elevada do que a recomendada para V 50% -, e aumentam o teor foliar de P e a produtividade da soja, independentemente da calagem. Quanto maior a dose de calcário, maior é o avanço da frente alcalina no solo e a produtividade de grãos.

Murilo Rubião: enlaces eróticos, fantásticos e absurdos

The present research intends to propose a reading in a new dimension of the fantastic (weird) literature and, especially, of its Brazilian more representative author, Murilo Rubião. At the beginning with concepts and associations with the erotic, starting from the myth of Eros and Psique and developing relations with Georges Bataille's theory on the theme. Afterwards, other associations are made, now with relation to the own fantastic literature. With the essence of this literature, examples of the traditional close to the contemporary. A last, association is made with the absurd literature. The contemporary man as being object and its configuration represented in the field of the dreams and its possibilities. At the end of the work, a "post scriptum" suggests a more imersive reading of tales in the entire that appeared broken into fragments along the research

A paz tensa da chama fugaz: a configuração do amor no romance contemporâneo, Lygia Fagundes Telles e Lídia Jorge

This thesis A paz tensa da chama fugaz: a configuração do amor no romance contemporâneo, Lygia Fagundes Telles e Lídia Jorge aims to study the configuration of Love in contemporary novels. Their corpuses of study are the novels As horas nuas (1989), by the Brazilian writer Lygia Fagundes Telles; and O vento assobiando nas gruas (2002), by the Portuguese writer Lídia Jorge. The following research tries to understand how the characters from those texts deal with some love questions on the context of the contemporary narrative; it also tries to comprehend the love expression which is formed as a contradictorily fluid feeling and intensely wanted, which are important points concerning the love discourse at the present day. As a critic-comparative study, this research focuses on the problematic concerned love as a synonym of Eros, in other words, the relation between lovers, analyzed through social and philosophical perspective. Through that, this work broadens the study of two contemporary novels, and it also establish connections between the characters from those novels and the literary environment where they are placed, once it focuses on the human and social context presented in this books. The thesis ends calling attention to the dichotomy of love and death, to the image of love which grows through absence and through the longing of completeness of the human being; that is why this work sustains that the Lygia Fagundes Telles and Lídia Jorge update and materialized in their texts the plurality of contemporary love conception, which continues to be contradictory, fragmented and problematic

Heteroplasmy in hair: Differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair hafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples. (C) 2007 Elsevier B.V.. All rights reserved.

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

No relationship found between point heteroplasmy in mitochondrial DNA control region and age range, sex and haplogroup in human hairs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise das mudanças no perfil protéico durante o estresse oxidativo in vivo e atuação da MutY-Glicosilase em respostas celulares

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior