Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Efeito da salinidade, densidade de estocagem e da infecção hipodermal e necrose hematopoiética (IHHN) na imunidade do camarão Litopenaeus vannamei cultivados em fazendas do Rio Grande do Norte

The main problem faced by the shrimp industry are the infectious diseases. The hypodermal and hematopoietic necrosis infection (IHHN) is one of the major cause of disease in the cultured shrimp, Litopenaeus vannamei. Environmental changes involving water quality, oxygen concentration, salinity, temperature, stocking density, presence of pathogens, among others, triggering a stressing condition for the cultured shrimp, weakening them and allowing the outbreak of diseases. The stress on the animal leads to a change in the molecules immune response components, which can be used as indicators of shrimp health. Thus, the objective of the present study was to evaluate the effect of salinity, stocking density and IHHNV infection on the L. vannamei shrimp. The immune parameters used to check the shrimp health were the total hemocytes counts (THC), the agglutinating activity (AA) and the clotting time (CT) of the serum of shrimp. These parameters were analyzed in healthy and IHHNV-infected shrimp, grown in low (0-0.5 ), medium (19-24 ) and high (> 38 ) salinity, and extensive (7-12 cam.m-2), semi-intensive (15-25 cam.m-2) and intensive (33-45 cam.m -2) stocking density. The IHHNV infection rate was significantly higher in low salinity (P<0.005) and intensive density (P<0.005), both stressful conditions for L. vannamei. Low salinity significantly increased THC (P<0.05) and decreased and CT (P<0.05) in healthy and infected shrimp, but AA (P<0.05) significantly decreased in healthy shrimp at medium salinity. Culture intensification did not affect the THC, AA and CT of healthy and infected shrimp (P>0.05). The IHHNV infection did not affect any immune parameters of shrimp cultured at different salinities and stocking densities. It is necessary to emphasize that this study was conducted in shrimp grown in ponds, where several environmental factors are acting simultaneously. Thus, further studies are needed about the influence of other environmental factors on the immune parameters of shrimp cultured in pond

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

Method of numerical correction of errors occasioned by delay of records during the monitoring of environmental variables of interest for animal production

As condições de ambiente térmico e aéreo, no interior de instalações para animais, alteram-se durante o dia, devido à influência do ambiente externo. Para que análises estatísticas e geoestatísticas sejam representativas, uma grande quantidade de pontos distribuídos espacialmente na área da instalação deve ser monitorada. Este trabalho propõe que a variação no tempo das variáveis ambientais de interesse para a produção animal, monitoradas no interior de instalações para animais, pode ser modelada com precisão a partir de registros discretos no tempo. O objetivo deste trabalho foi desenvolver um método numérico para corrigir as variações temporais dessas variáveis ambientais, transformando os dados para que tais observações independam do tempo gasto durante a aferição. O método proposto aproximou os valores registrados com retardos de tempo aos esperados no exato momento de interesse, caso os dados fossem medidos simultaneamente neste momento em todos os pontos distribuídos espacialmente. O modelo de correção numérica para variáveis ambientais foi validado para o parâmetro ambiental temperatura do ar, sendo que os valores corrigidos pelo método não diferiram pelo teste Tukey, a 5% de probabilidade dos valores reais registrados por meio de dataloggers.

Relação entre estresse oxidativo e oesteopenia diabética em ratas pré-menopausadas

The relationship between lipid peroxidation, antioxidant defense and diabetic osteopenia remains unclear. This study evaluated the relationship between lipid peroxidation index, antioxidant defense parameter and bone metabolism in a premenopausal diabetic model by measurements such as thiobarbituric acid-reactive substances concentration (TBARS) and reduced glutathione (GSH) content in brain homogenates, histomorphometric analysis, biomechanical testing and bone mineral density (BMD). Female Wistar rats with regular estrous cycle were divided into two groups: Group 1: control rats (n = 15) and Group 2: diabetic rats (n = 15). Diabetes mellitus was induced by alloxan and confirmed by glycemia 250 mg/dL. The experimental period understood 1 and 5 after days induction and 45, 75 and 120 days after the installation of diabetes mellitus.The lipid peroxidation index, measured by TBARS concentration, showed a significant increase (p<0.05) in diabetic animals in comparison to animals control. However, the antioxidant parameter, measured by GSH content, was significantly decrease (p<0.05) in diabetic animals. Histomorphometric analysis showed a significant increase (p<0.05) in femoral trabecular separation together with a significant decrease (p<0.05) in trabecular thickness and reduced trabecular bone volume in diabetic rats. Moreover, biomechanical testing and BMD values were significant decrease (p<0.05) in diabetic group. Thus, our results demonstrated that increased lipid peroxidation and altered antioxidant defense could be related to the development of oxidative stress and diabetic osteopenia in premenopausal rats

Play behaviour in nonhuman animals and the animal welfare issue

The mission of defining animal welfare indicators is methodologically difficult, limited, and possibly impossible. A promising alternative, however, to evaluate suitable environmental conditions is the assessment of play behaviour. In the present review, we summarise the general aspects of play behaviour in nonhuman animals and propose its use as a potential indicator of animal welfare. Play behaviour probably occurs in most vertebrates and some invertebrates, but predominately in mammals. It is also more frequent in young males and is associated with the environmental context in which animals find themselves. Animals play if they are healthy and well-fed, but not if they are under stressful conditions or if they are in a stressful state. We can therefore use the prevalence of play behaviour as an indicator of suitable environmental conditions, considering the specificity associated with the above-mentioned modifying factors.

Representações sociais de enfermeiros brasileiros e portugueses sobre o estresse em serviço de urgência

This study aimed to analyze the social representations of Brazilian and Portuguese nurses on stress in the emergency service. A semi-structured interview and the free word association test, with "stress" as the inductive stimulus, were used as research instruments. Data were collected from 120 nurses, being 60 from an emergency hospital in the city of Natal, Brazil and 60 from an urgency hospital in the city of Aveiro, Portugal. Data from the word association test were analyzed with the EVOC 2002 program, after thematic categorical content analysis, enabling construction of a data bank. Data gathered from the interview were analyzed by ALCESTE 4.8 software. Nurse represent the stress in the urgency department as a generation of physical and mental detrition where adaptation is unsatisfactorily, resulting in the onset of fatigne, irritability, lack of concentration, lack of motivation, pessimism, impaired interpersonal relationship and low productivity. The solution is part of a complex whole, which demands an integrated way of acting that has demanded increasingly professional attitudes based on multidisciplinarity

Desenpenho funcional, carga alostática e biomarcadores do estresse em idosos residentes na comunidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Aspectos socioeconômicos e demográficos relacionados ao estresse e a síndrome de Burnout em fisioterapeutas no Brasil

Dos problemas de saúde existentes no ambiente de trabalho, 18% afetam o aparelho psíquico, a exemplo do estresse, atingindo cerca de 30% dos trabalhadores em geral. A persistência e intensidade do estresse, associada às sucessivas tentativas de lidar com os mesmos, tornam os indivíduos vulneráveis ao surgimento da Síndrome deBurnout. O objetivo deste trabalho foi identificar a relação dos aspectos socioeconômicos e demográficos com o estresse e a Síndrome de Burnout em fisioterapeutas do Brasil. Para isso, este estudo teve uma abordagem do tipo transversal, com 1040 fisioterapeutas do Brasil, através de uma amostragem do tipo snowbolle não probabilística. Utilizou-se um questionário socioeconômico, demográfico e profissional, a Escala de Estresse no Trabalho (EET) e a adaptação do Cuestionario para la Evaluación del Síndrome de Quemarse por el Trabajo (CESQT). Na análise dos dados, foram utilizadas a estatística descritiva e inferencial. Dentre os principais resultados obtidos, percebeu-se uma maior representatividade da região Nordeste (48,7%), com idade média de 31anos, sexo feminino (75,7%), carga horária semanal de 35,4 horas, com 3-5 anos de atuação profissional. Observou-se que 37,0% apresentavam estresse relacionado estatisticamente com a idade (p=0,008),atividade física (p=0,039) e satisfação com a saúde (r=-0,322; p<0,001). Não foi observado nenhum caso de Burnout, porém houve uma média elevada nas dimensões, desgaste psíquico, indolência e culpa, totalizando 49,0% comtendência ao desenvolvimento da síndrome. Portanto, as variáveis, idade, prática da atividade física e satisfação com a saúde obtiveramrelação com o estresse. Para o Burnout, destacaram-se a região de moradia (centro-oeste), satisfação com a saúde, local de trabalho (clínicas e hospitais), além do maior número de locais de trabalho. Diante desse contexto, os estudos sobre o estresse e a Síndrome de Burnout se apresentam como elementos derelevância dentro do contexto da prevenção dos riscos laborais e da análise das condições de trabalho

Physiological response of American bullfrog tadpoles to stressor conditions of capture and hypoxia

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

Avaliação dos efeitos da atividade física na prevenção do estresse oxidativo e do câncer de mama

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors

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