Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil

The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river

Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil

The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river

Impactos do estresse salino e da cobertura morta nas características químicas do solo e no desenvolvimento do amaranto

Availability of good quality water has been reduced vertiginously, over the last decade, in the world. In some regions, the water resources have high concentration of the dissolved salts, these characteristics of the water make it s use impossible. Water quality can be a limitation for irrigated agriculture, principally in regions of arid or semiarid climate where the water resources are generally saline and are exposed at high evaporation ratio. For that reason, precipitation of the salts occurs near the soil surface and those salts themselves cumulate in the vegetal tissue, reducing the soil fertility and crop production. The adoption of tolerant crop to the water salinity and soil salinity, adaptable to the climatic conditions is other emergent necessity. This work had the goal of studying the effects of four salinity levels of the irrigation water salinity and use of mulch, dried leaves of Forest mangrove (Acacia mangiumWilld), in cultivated soil with amaranth (Amaranthus cruentus, BRS Alegria variety), in greenhouse. It was utilized the transplant of plants to PVC columns, containing 30 kg of silty loam soil, 10 days after emerging, with space of 50 x 50 cm between lines. Treatments were composed by combination of four levels of salinity (0.147; 1.500; 3.000 e 4.500 dS m-1), obtained by addition NaCl (commercial) to irrigation water and soil with and without protection, by mulch. A factorial system 4 x 2 was used with four repetitions, totalizing 32 parcels. The concentrations of nutrients in soil solution have been evaluated, in the dry matter of the vegetal tissue (roots, stem, leaves and raceme residue), at the end of the vegetative cycle. The use of soil protection reduced time for the beginning inflorescence of plants, at the same time, the increase of the salinity delayed this phase of amaranth development. The use of the mulch effectively increased the height, stem diameter, area of the larger leaf, humidity and dry matter content and amaranth grain production. The vegetal species showed salinity tolerance to experimented levels. The adopted treatments did not affect the pH values, exchangeable cation contents, electrical conductivity of soil solution (EC1:5) and saturated extract (ECSE), and Ca+2, Mg+, Fe+2 and Mn+2 contents, in the soil solution. The increase of the salinity concentration in the irrigation water inhibited the mineralization process of the organic matter (OM) and, consequently, the efficiency in the it´s utilization by plants, at the same time, produced increase in the values of the exchangeable sodium percentage (ESP), sodium adsorption ratio (SAR) and potassium adsorption ratio (PAR), in the soil solution. Therefore, the use of the mulch did not affect the first three parameters. The protein and nutrient contents: K+, Ca+2, P, Mg+2 e Cu+2, in amaranth grains, were improved by tillage condition. The raceme residues showed chemical/nutritional composition that makes advantageous its application in animal ration. In this context, it follows that amaranth tolerate the saline stress, of the irrigation water, until 4.500 dS m-1, temperature and relative humidity of the air predominant in the experimental environment

Qualidade fisiológica de sementes de genótipos de algodoeiro sob estresse salino

The germination of cotton seeds and the seedlings emergency are generally delayed and reduced by the salinity. Although the cotton is considered a tolerant culture, it can suffer substantial reductions in regarding its growth and production when exposed to salinity condition. The aims of this study went evaluate the effect of the saline stress in the germination phase to four cotton genotypes (BRS Rubi, BRS Safira, BRS 201 and CNPA 187 8H), using different osmotic potentials generated with increment of sodium chloride (NaCl). The saline stress was simulated using NaCl aqueous solutions in the potentials: 0.0 (Control); -0.2; -0.4; -0.6; -0.8 and -1.0 MPa. The treatments were monitored by means of tests for analysis of seeds, germination, first counting, speed germination index, length of shoot, radicle length, dry weigth of embrionic axis and shoot/radicle ratio. The tests for germination, first counting and index of germination speed were accomplished using 50 seeds for repetition and for the study of length of shoot, radicle length, dry weigth of embrionic axis and shoot/radicle ratio were used 20 seeds by repetition. For both tests four repetitions were accomplished by genotype for each one of the potentials. The seeds of each repetition were involved in papers Germitest humidified with NaCl solution corresponding to the potential. The repetitions of both tests were maintained in a germinator with saturated humidity. The analysis were initiate four days after the induction of the saline stress. The evaluations of the first three variables analyzed were accomplished daily; the seeds were remove and counted when its germinated. For the length tests just the repetitions corresponding to the potential of NaCl 0,0 MPa were analysis 4 days after the beginning of the induction of the saline stress. The analysis of the repetitions of the potentials -0,2 and -0,4 and of the potentials -0,6, -0,8 and -1,0 MPa they were accomplished with 12 and 20 days, respectively. For accomplishment of the analisis of this test the shoot of the 20 plantules of each repetition was separate from the radicle and both parts were measured. The statistical analyses were performed using the GENMOD and GLM procedures of the SAS. For the variable germination, the cultivates CNPA 187 8H and BRS Safira stood out for the potential -0.8 MPa, with averages of 89% and 81%, respectively. The test of speed germination index to cultivate BRS Safira presented the largest averages for the two higher saline potentials. It was observed that the increase of the saline potential reduces the germination percentage and speed germination index. For each day of evaluation it was verified that the increase of the saline potential causes a reduction of the length both of the shoot and of the radicle. The radicle tends to grow more than the shoot until the potential -0,4 MPa

Crescimento inicial e morfologia foliar em plantas de Mimosa caesalpiniaefolia Benth. Em função do manejo microbiano, sob estresse salino

The sabiá (Mimosa caesalpiniaefolia Benth.) is an endemic species of the Caatinga biome, considered tolerant to salt and water stress. The process of salinization of soil and groundwater and surface water is one of the most important problems of environmental degradation, with its harmful effects being more pronounced in the areas of arid and semiarid regions, and rapidly growing in many parts of the globe, causing problems of the major crop yield. Organic conditioners as barnyard manure, and rice hulls can contribute to reducing the PST, possibly due to the release of CO2 and the production of organic acids during the decomposition of organic matter, and act as sources of calcium and magnesium and inhibit the availability sodium. The intimate association of mycorrhizae and beneficial to plants results in increased uptake of water and nutrients by plants, especially phosphorus, due to their low mobility in soil. The objective of this study was to evaluate the initial growth of thrush seedlings under inoculation with mycorrhizal fungi and fertilized with manure corral and irrigated with water of different salinity levels. The experiment was conducted in greenhouse conditions of vegetation on the premises of the Agricultural School of Jundiaí - UFRN, Campus Macaíba. The adopted statistical design was randomized composed of twelve treatments - three substrates (sterile soil, manure and FMA), four salinity levels (0.2, 1.5, 3.0 and 4.5 dS m-1 ) and five repetitions, totaling sixty experimental units. The results indicate that inoculation with mycorrhizal fungi own contributions to the growth of plants, especially in roots and shoots, which suggests that its application is beneficial in establishing thrush plants in natural conditions, with poor soil in P. Levels salinity caused no effects with statistical significance in plant development, indicating Sabia resistance to it.

Alterações morfofisiológicas e moleculares em resposta a estresses abióticos em mamona (Ricinus communis L.)

The use of fossil fuels has been considered one of reason for the increase of pollution in the atmosphere and it may be related to the climate changes. Then, the research of the new sources of fuels will be important. Considering this, the use of biodiesel has been considered not as bad as petrol. The castor bean (Ricinus communis L.) is an important oilseed, which belongs to Euphorbiaceae family, and the oil found in the seed has important characteristics for biodiesel. This plant is considered as “rustic” as it does not need so much water for its development and oil production. Due to this, this plant has been considered to be ideal in semi-arid regions, such as the Northeast of Brazil. The aim of his study is to better understand the responses to abiotic stresses (drought and salinity) from castor bean plants using morphological, physiological and molecular tools. In order to do this, the castor bean plants were subjected to salt stress (50, 100, 150 and 200 mM NaCl) in a controlled environment and drought stress (5, 10, 15 days and 10 days cyclic). After these treatments, these plants were subjected to different analyzes: a) the expansion and retention of water from leaves; b) anatomy using leaves and roots. Based on these results, we found that castor suffered decrease in leaf area with increase drought stress, however restricted water loss, probably by accumulation of compatible solutes in the leaves. The anatomy data showed modifications in the vascular system. These modifications observed suggested that castor bean plant may be resistant to stress as it was verified in 5 days of drought as well as in 100 mM NaCl. In both conditions, these plants were fine. Probably these plants keep some solutes in the cell and then maintain the cell tugor. The data obtained in this study gave a better idea how castor bean plant responds to abiotic stress conditions - drought and salt stress

Influência do estresse nutricional programado na composição da microalga isochrysis galbana

Global warming due to Greenhouse Gases (GHG) emissions, especially CO2, has been identified as one of the major problems of the twenty-first century, considering the consequences that could represent to planet. Currently, biological processes have been mentioned as a possible solution, especially CO2 biofixation due to association microalgae growth. This strategy has been emphasized as in addition to CO2 mitigation, occurs the production of biomass rich in compounds of high added value. The Microalgae show high photosynthetic capacity and growth rate higher than the superior plants, doubling its biomass in one day. Its culture does not show seasons, they grow in salt water and do not require irrigation, herbicides or pesticides. The lipid content of these microorganisms, depending on the species, may range from 10 to 70% of its dry weight, reaching 90% under certain culture conditions. Studies indicate that the most effective method to promote increased production of lipids in microalgae is to induce stress by limiting nitrogen content in the culture medium. These evidences justify research continuing the production of biofuels from microalgae. In this paper, it was studied the strategy of increasing the production of lipids in microalgae I. galbana with programmed nutritional stress, due to nitrogen limitation. The physiological responses of microalgae, grown in f / 2 with different concentrations of nitrogen (N: P 15,0-control, N: 5,0 P and N: P 2,5) were monitored. During exponential phase, results showed invariability in the studied conditions. However the cultures subjected to stress in stationary phase, showed lower biomass yields. There was an increase of 32,5% in carbohydrate content and 87.68% in lipids content at N: P ratio of 5,0 and an average decrease of 65% in protein content at N: P ratios of 5, 0 and 2.5. There were no significant variations in ash content, independently of cultivation and growth phase. Despite the limitation of biomass production in cultures with N: P smaller ratios, the increase of lipid accumulation highest lipids yields were observed as compared to the control culture. Given the increased concentration of lipids associated to stress, this study suggests the use of microalgae Isochrysis galbana as an alternative raw material for biofuel production

Metagenoma de comunidades microbianas expostas à radiação natural e a metais

Many species have specialized to live in the most varied existing environments showing the remarkable adaptability of the microbial world the most diverse physicochemical conditions. Environments exposed to natural radiation and metals are scarce around the world, presenting a microbiota still unknown. With a total number estimated between 4 and 6 x 1030 microrganisms on earth, they constitute an enormous biological and genetic pool to be explored. Metagenomic approach independent of cultivation, provides a new form to access to the potential genomic environmental samples becoming a powerful tool for the elucidation of ecological functions, metabolic profiles, as well as to identify new biomolecules. In this context, the genetic material of environmental soil and water samples from Açude Boqueirao Parelhas-RN, under the influence of natural radiation and the presence of metals, was extracted, pirosequencing and the generated sequences were analyzed by bioinformatics programs (MG-RAST and STAMP). Taxonomic comparative profiles of both samples showed high abundance of Domain Bacteria, followed by a small portion attributable to Eucaryota Domains, Archaea and Viruses. Proteobacteria, Actinobacteria and Bacterioidetes phyla showed the greater dominance in both samples. Important genera and species associated with resistance to various stressors found in region were observed. Sequences related to oxidative and heat stress, DNA replication and repair, and resistance to toxic compounds were observed, suggesting a significant relationship between the microbiota and their metabolic profile, influenced by regional environmental variables. The results of this study add valuable and unpublished data on the composition of microbial communities in these regions

Avaliação do estresse oxidativo induzido por superfícies de titânio

Commercially pure Titanium (cp Ti) is a material largely used in orthopedic and dental implants due to its biocompatibility properties. Changes in the surface of cp Ti can determine the functional response of the cells such as facilitating implant fixation and stabilization, and increased roughness of the surface has been shown to improve adhesion and cellular proliferation. Various surface modification methods have been developed to increase roughness, such as mechanical, chemical, electrochemical and plasma treatment. An argon plasma treatment generates a surface that has good mechanical proprieties without chemical composition modification. Besides the topography, biological responses to the implant contribute significantly to its success. Oxidative stress induced by the biomaterials is considered one of the major causes of implant failure. For this reason the oxidative potential of titanium surfaces subjected to plasma treatment was evaluated on this work. CHO-k1 cells were cultivated on smooth or roughed Ti disks, and after three days, the redox balance was investigated measuring reactive oxygen species (ROS) generation, total antioxidant capacity and biomarkers of ROS attack. The results showed cells grown on titanium surfaces are subjected to intracellular oxidative stress due to hydrogen peroxide generation. Titanium discs subjected to the plasma treatment induced less oxidative stress than the untreated ones, which resulted in improved cellular ability. Our data suggest that plasma treated titanium may be a more biocompatible biomaterial.

Identificação de genes em Chromobacterium violaceum relacionados à resposta ao estresse

The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III δ subunit). In this way, we propose that the C. violaceum δ subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., σS subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress.

Proteômica diferencial da Chromobacterium violaceum em resposta ao estresse oxidativo induzido por peróxido de hidrogênio

The β-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response

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

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