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Confocal laser scanning microscopy (CLSM) is one of the most prevalent fluorescence microscopy techniques for assessing the progression of cancer cells in three-dimensional structures, such as vasculogenic mimicry (VM). We show a basic approach for using DAPI and phalloidin dyes to detect the early stages of progression and VM of melanoma tumor cells grown in a 3D environment, as well as demonstrating how to acquire images and improve them by changing the software acquisition parameters.

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Phytomelanin is a brown to black pigment found in plant tissues, mainly in Asparagales and Asteraceae species. However, few studies deal with the processes of its synthesis, and there are still many questions to be answered regarding the organelles involved in this process and their functions, especially in vegetative organs. In a previous study with stems and leaves of 77 Vernonieae (Asteraceae) species, phytomelanin was demonstrated to always be associated with sclereids, which suggests the involvement of these cells in the pigment synthesis. Thus, we selected another species of tribe Vernonieae, Piptocarpha axillaris (Less.) Baker, which produces abundant phytomelanin secretion in stem tissues, to investigate which cells and organelles are involved in the synthesis and release of this pigment, as well as its distribution in the tissues. To achieve this goal, stems in different developmental phases were analyzed under light and transmission electron microscopy. Anatomical analysis showed that the polymerization of phytomelanin in P. axillaris starts at the second stem node, in the pith region, and occurs simultaneously with sclereid differentiation. The plastids of cells that will differentiate into sclereids actively participate in the phenolic material synthesis, following the "tannosome" and the "pearl necklace" models, giving rise to the main precursor of phytomelanin, which is then polymerized in the intercellular spaces during the sclerification process of sclereids. In stems with an established secondary structure, the pigment can be observed more frequently in the cortex, pericycle, primary phloem, secondary phloem, and pith.

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BACKGROUND: Staphylococcus aureus is a human pathogen of clinical importance related to a variety of infections. AIM: The objective of this study was to analyze the molecular and epidemiological characteristics of S. aureus obtained from healthcare professionals (HCP) of a hospital in southwestern Bahia, Brazil. METHODS: Samples were collected from hands, nasal cavity, and laboratory coats of 80 HCP. The bacterial isolates recovered from 240 samples were identified as S. aureus, and then analyzed for their antimicrobial resistance profile, genotypic characterization, and pathogenicity. FINDINGS: 178 isolates were identified as S. aureus, being mostly isolated from the nasal cavity. Thirty isolates (16.8%) were characterized as MRSA. The virulence gene frequency varied according to isolate source. All virulence genes were identified in at least one hand isolate. Isolates from laboratory coats did not show seb and pvl. Isolates from the nasal cavity did not exhibit pvl. The SCCmec type I was identified in 56.7% of MRSA isolates. Among MRSA isolates, 14 PFGE pulsotypes were characterized, with profile A being predominant (nine isolates). Clonal complexes CC5, CC45, and CC398 were found. MRSA isolates induced cytokine gene expression in macrophages, with IL-10 and IL-17 being expressed more often. CONCLUSION: We found a high colonization rate for S. aureus among HCP. Moreover, we observed that MRSA strains presented different virulence factors and could induce cytokine gene expression, indicating an urgent need to control colonization rates of HCP by MRSA isolates in order to protect hospital patients and the general public.

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In a previous study, we described a series of 28 aryl- and alkyl-substituted isothiouronium salts with antitumor activity and selectivity toward a leukemia cell line. Among the synthesized compounds, methyl (Z)-2-(isothioureidomethyl)-2-pentenoate hydrobromide (IS-MF08) showed conspicuous activity. In the present study, we investigated the mechanism of action of IS-MF08. Our results showed that its mechanism most likely is related with the membrane receptor Fas and subsequent activation of the extrinsic cell death pathway, triggered by a decrease in the levels of the anti-apoptotic protein Bcl-2 and caspase-8 and -3 cascade activation, causing DNA damage and mitotic arrest. IS-MF08 also caused an increase in intracellular ROS, endoplasmic reticulum (ER) stress, and mitochondrial membrane permeabilization, resulting in organelle degradation as an attempt to reestablish cell homeostasis. Furthermore, cells exposed to IS-MF08 combined to an autophagy inhibitor were less susceptible to compound's cytotoxicity, suggesting that autophagy makes part of its mechanism of action. These data support the hypothesis that IS-MF08 acts by the apoptosis extrinsic pathway and possibly by autophagy as mechanisms of cell death.

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Atrazine is one of the most used herbicides and has been associated with persistent surface and groundwater contamination, and novel formulations derived from nanotechnology can be a potential solution. We used poly(ε-caprolactone) nanoencapsulation of atrazine (NC+ATZ) to develop a highly effective herbicidal formulation. Detailed structural study of interaction between the formulation and Brassica juncea plants was carried out with evaluation of the foliar uptake of nanoatrazine and structural alterations induced in the leaves. Following postemergent treatment, NC+ATZ adhered to the leaf and penetrated mesophyll tissue mainly through the hydathode regions. NC+ATZ was transported directly through the vascular tissue of the leaves and into the cells where it degraded the chloroplasts resulting in herbicidal activity. Nanocarrier systems, such as the one used in this study, have great potential for agricultural applications in terms of maintenance of herbicidal activity at low concentrations and a substantial increase in the herbicidal efficacy.

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Scanning electron microscopy was used to examine and describe the egg of Psorophora ciliata, which was not previous adequately described, and the egg of Ps. ferox, which exhibited regional variation. Eggs of Ps. ciliata have long tubercles (length 26.7±1.72 µm) in the outer chorion, surrounded by a hexagonal reticulation. The micropyle (external diameter, to the outer edge of the collar, 26.59 µm; internal diameter 20.8 µm) is surrounded by an irregular collar with a thickness of 14.97 ±0.63 µm. Eggs of Ps. ferox exhibit some differences in the size and shape of tubercles compared to those from other more northern regions.

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Seaweeds living at their temperature limits of distribution are naturally exposed to physiological stressors, facing additional stress when exposed to coastal pollution. The physiological responses of seaweeds to environmental conditions combining natural and anthropogenic stressors provide important information on their vulnerability. We assessed the physiological effects and ultrastructural alterations of trace metals enrichment at concentrations observed in polluted regions within the temperature ranges of distribution of the endemic seaweed Halimeda jolyana, an important component of tropical southwestern Atlantic reefs. Biomass yield and photosynthetic performance declined substantially in samples exposed to metal, although photosynthesis recovered partially at the highest temperature when metal enrichment was ceased. Metal enrichment caused substantial ultrastructural alterations to chloroplasts regardless of temperatures. The lack of photosynthetic recovery at the lower temperatures indicates a higher vulnerability of the species at its temperature limits of distribution in the southwestern Atlantic.

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Spatial patterns of genetic variation can help understand how environmental factors either permit or restrict gene flow and create opportunities for regional adaptations. Organisms from harsh environments such as the Brazilian semiarid Caatinga biome may reveal how severe climate conditions may affect patterns of genetic variation. Herein we combine information from mitochondrial DNA with physical and environmental features to study the association between different aspects of the Caatinga landscape and spatial genetic variation in the whiptail lizard Ameivula ocellifera. We investigated which of the climatic, environmental, geographical and/or historical components best predict: (1) the spatial distribution of genetic diversity, and (2) the genetic differentiation among populations. We found that genetic variation in A. ocellifera has been influenced mainly by temperature variability, which modulates connectivity among populations. Past climate conditions were important for shaping current genetic diversity, suggesting a time lag in genetic responses. Population structure in A. ocellifera was best explained by both isolation by distance and isolation by resistance (main rivers). Our findings indicate that both physical and climatic features are important for explaining the observed patterns of genetic variation across the xeric Caatinga biome.

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Many studies propose that Quaternary climatic cycles contracted and/or expanded the ranges of species and biomes. Strong expansion-contraction dynamics of biomes presume concerted demographic changes of associated fauna. The analysis of temporal concordance of demographic changes can be used to test the influence of Quaternary climate on diversification processes. Hierarchical approximate Bayesian computation (hABC) is a powerful and flexible approach that models genetic data from multiple species, and can be used to estimate the temporal concordance of demographic processes. Using available single-locus data, we can now perform large-scale analyses, both in terms of number of species and geographic scope. Here, we first compared the power of four alternative hABC models for a collection of single-locus data. We found that the model incorporating an a priori hypothesis about the timing of simultaneous demographic change had the best performance. Second, we applied the hABC models to a data set of seven squamate and four amphibian species occurring in the Seasonally Dry Tropical Forests (Caatinga) in northeastern Brazil, which, according to paleoclimatic evidence, experienced an increase in aridity during the Pleistocene. If this increase was important for the diversification of associated xeric-adapted species, simultaneous population expansions should be evident at the community level. We found a strong signal of synchronous population expansion in the Late Pleistocene, supporting the increase of the Caatinga during this time. This expansion likely enhanced the formation of communities adapted to high aridity and seasonality and caused regional extirpation of taxa adapted to wet forest.

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The Atlantic Forest (AF) of Brazil has long been recognized as a biodiversity conservation hotspot. Despite decades of studies the species inventory of this biome continues to increase with the discovery of cryptic diversity and the description of new species. Different diversification mechanisms have been proposed to explain the diversity in the region, including models of forest dynamics, barriers to gene flow and dispersal. Also, sea level change is thought to have influenced coastal diversification and isolated populations on continental islands. However, the timing and mode of diversification of insular populations in the AF region were rarely investigated. Here, we analyze the phylogeography and species diversity of the small-sized direct-developing frog Ischnocnema parva. These frogs are independent from water bodies but dependent on forest cover and high humidity, and provide good models to understand forest dynamics and insular diversification. Our analysis was based on DNA sequences for one mitochondrial and four nuclear genes of 71 samples from 18 localities including two islands, São Sebastião, municipality of Ilhabela, and Mar Virado, municipality of Ubatuba, both in the state of São Paulo. We use molecular taxonomic methods to show that I. parva is composed of six independently evolving lineages, with the nominal I. parva likely endemic to the type locality. The time-calibrated species tree shows that these lineages have diverged in the Pliocene and Pleistocene, suggesting the persistence of micro-refuges of forest in the AF. For the two insular populations we used approximate Bayesian computation to test different diversification hypotheses. Our findings support isolation with migration for São Sebastião population, with ∼1Mya divergence time, and isolation without migration for Mar Virado population, with ∼13Kya divergence time, suggesting a combination of different processes for diversification on AF islands.

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Studies have clearly demonstrated the damaging effects of UV-B exposure on macroalgae, but few have reported the impact of UV-B on spore germination and development at juvenile stages. Therefore, this work aimed to analyze the effects of UV-B radiation on germlings of Nemalion helminthoides at the tetrasporophytic phase. To accomplish this, germlings of N. helminthoides were cultivated in the laboratory and separated into two groups. The control group was exposed onlyto photosynthetic radiation, while the treatment group was exposed to photosynthetic radiation + UV-B for 2 hours during a period of 12 days. Control germlings showed increasing cellular proliferation and accumulation of reserve substances, as well as intense ramification in the last observed stages between 9 days and 12 days of development. Moreover, the chloroplasts presented a typical globular pyrenoid, profusely traversed by thylakoid membranes. Treated germlings, by contrast, showed intracellular damage, such as cell wall thickness, loss of chloroplast organization, changes in mitochondrial cristae, and increasing atrophy of the Golgi bodies. Additionally, changes in developmental patterns were observed, including loss of polarity in the first divisions of carpospores and abnormal stem ramification. The quantification of autofluorescence data coincided with the ultrastructural changes observed in the chloroplasts of cells exposed to UV-B. It can be concluded that exposure to radiation changed the developmental pattern and morphology of the germlings of N. helminthoides.

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O ciúme romântico é caracterizado como reações exibidas diante da ameaça de perda de um parceiro romântico para um possível rival. Este estudo teve como objetivo revisar os itens do Inventário de Ciúme Romântico (ICR), visando melhorar sua compreensão e testar seu funcionamento em uma amostra da Bahia. Buscou-se verificar evidências de validade da versão modificada do ICR com base na estrutura interna via análise fatorial confirmatória e modelo de Rasch, a partir de dados de 400 participantes de 18 a 76 anos (M=29,96; DP=11,73), sendo 62% do sexo feminino. Verificou-se o impacto de características individuais na severidade do ciúme romântico. Os dados encontrados corroboram a literatura na área, indicando que o ciúme romântico é composto por diversos fatores, além de um fator geral, o que é refletido também na versão presentemente revisada do ICR. Além disso, outras questões específicas do construto são discutidas no artigo.(AU)

Romantic jealousy relates to a series of reactions when facing the threat of loss of a romantic partner to a possible rival. This study aimed to review the items of the Romantic Jealousy Inventory (ICR), in order to improve the their understandability and test its functioning in a sample from Bahia. It sought to verify internal validity evidences via confirmatory factor analysis and the Rasch model. We also looked for the impact of individual features on the severity of romantic jealousy. Therefore, the ICR was applied to 400 participants aged between 18 and 76 years (M=29.96; SD=11.73), and 62% were female. The data corroborate the literature on the matter, indicating that romantic jealousy is composed of several factors, which is also reflected in the currently revised version of the ICR. In addition, other specific construct questions are discussed in the article.(AU)

Celos románticos se refiere a una serie de reacciones cuando se enfrentan a la amenaza de la pérdida de una pareja romántica para un posible rival. Este estudio tuvo como objetivo revisar los artículos del Inventario Romántica los Celos (ICR), con el fin de mejorar la comprensibilidad artículos y probar su funcionamiento en una muestra de Bahía. Se trató de verificar las evidencias de validez interna a través de análisis factorial confirmatorio y el modelo de Rasch. También nos fijamos en el impacto de las características individuales de la gravedad de celos románticos. Por lo tanto, el ICR se aplicó a 400 participantes de edades comprendidas entre 18 y 76 años (M=29,96; SD=11,73), y el 62% eran mujeres. Los datos corroboran la literatura en la zona, lo que indica que los celos románticos se compone de varios factores, que también se refleja en la versión revisada actualmente ICR. Además, otras preguntas constructo específico se discuten en el artículo.(AU)

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Two main hypotheses have been proposed to explain the diversification of the Caatinga biota. The riverine barrier hypothesis (RBH) claims that the São Francisco River (SFR) is a major biogeographic barrier to gene flow. The Pleistocene climatic fluctuation hypothesis (PCH) states that gene flow, geographic genetic structure and demographic signatures on endemic Caatinga taxa were influenced by Quaternary climate fluctuation cycles. Herein, we analyse genetic diversity and structure, phylogeographic history, and diversification of a widespread Caatinga lizard (Cnemidophorus ocellifer) based on large geographical sampling for multiple loci to test the predictions derived from the RBH and PCH. We inferred two well-delimited lineages (Northeast and Southwest) that have diverged along the Cerrado-Caatinga border during the Mid-Late Miocene (6-14 Ma) despite the presence of gene flow. We reject both major hypotheses proposed to explain diversification in the Caatinga. Surprisingly, our results revealed a striking complex diversification pattern where the Northeast lineage originated as a founder effect from a few individuals located along the edge of the Southwest lineage that eventually expanded throughout the Caatinga. The Southwest lineage is more diverse, older and associated with the Cerrado-Caatinga boundaries. Finally, we suggest that C. ocellifer from the Caatinga is composed of two distinct species. Our data support speciation in the presence of gene flow and highlight the role of environmental gradients in the diversification process.

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Seagrasses, which form an integral part of the worldwide coastal habitat, are considered highly relevant from an ecological point of view. Due to the scarcity of anatomical information, the present study analyzed the morphoanatomy, histochemistry, and ultrastructure of Halophila decipiens, Halodule wrightii, and Ruppia maritima leaves, discussing their adaptations to the marine environments observed throughout the southwestern tropical and subtropical Atlantic coast. The leaves of these three species feature a uniseriate epidermis with the presence of chloroplasts in large quantities and absence of stomata. The vascular system consists of a central vascular bundle with sieve tube elements of the phloem and protoxylem lacunae, as well as small vascular bundles near the leaf margins. The leaves of H. decipiens possess trichomes, but no mesophyll in the leaf margins. The mesophyll of H. wrightii and R. maritima is homogeneous with chlorenchyma cells and air lacunae scattered throughout the leaf. The histochemistry analysis revealed the absence of amyloplasts and the presence of proteins in the outer periclinal walls of ordinary epidermal cells of the three species. It was also possible to detect the presence of idioblasts containing phenolic compounds in H. decipiens and R. maritima. The ultrastructural analysis of the three species revealed many elliptical chloroplasts, with organized thylakoids, expansion of the epidermal cell wall into the cytoplasm, and a thin cuticle. Hydropoten were also observed in the three specimens. The results show that the species analyzed have important adaptations which enable their survival in the marine environment.

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Interest in biochemistry of organoselenium compound has increased in the last decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of diphenyl diselenide (PhSe)2 (5 µmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Swiss male mice were divided into four experimental groups: control, (PhSe)2 (5 µmol/kg, subcutaneous administration), MeHg (40 mg/L, in tap water), and MeHg + (PhSe)2. After the treatment (21 days), the animals were killed and the cerebral cortex was analyzed. Electron microscopy indicated an enlarged and fused mitochondria leading to a reduced number of organelles, in the MeHg-exposed mice. Furthermore, cortical creatine kinase activity, a sensitive mitochondrial oxidative stress sensor, was almost abolished by MeHg. Subcutaneous (PhSe)2 co-treatment rescued from MeHg-induced mitochondrial alterations. (PhSe)2 also behaved as an enhancer of mitochondrial biogenesis, by increasing cortical mitochondria content in mouse-receiving (PhSe)2 alone. Mechanistically, (PhSe)2 (1 µM; 24 h) would trigger the cytoprotective Nrf-2 pathway for activating target genes, since astroglial cells exposed to the chalcogen showed increased content of hemeoxygenase type 1, a sensitive marker of the activation of this via. Thus, it is proposed that the (PhSe)2-neuroprotective effect might be linked to its mitoprotective activity.

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Mosquitoes of the Neotropical genus Sabethes, some species of which are yellow fever vectors, most often develop through the immature stages in tree holes. Sabethes eggs have not been previously characterized using scanning electron microscopy. Eggs of Sabethes cyaneus (length: 349.6 +/- 2.7 microm; width: 172.6 +/- 1.14 microm; n = 10) are almost biconical when examined from the top. From a lateral perspective 2 surfaces can be seen. One surface is smooth and more convex, whereas the other is less convex and partially covered by a network from which many fungiform tubercles arise. The micropyle is situated on the smooth surface of the pointed anterior tip and is surrounded by an irregular row of tubercles, some of which are leaf shaped. No structures possibly involved in adhesion to surfaces are visible. When hatching, the egg splits dorsoventrally approximately two-thirds of the length from the anterior end. The tubercles appear to be water repellent, and the more convex/smoother surface is downturned, and this position on water was confirmed by direct observation. The eggs float free on the water surface.

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Neste trabalho, avaliou-se o rendimento em suco, arilo e sementes do maracujá-amarelo, para identificar o potencial de agregação de valor às sementes usadas na extração do óleo. Foram feitas medidas de massa, coloração da casca, tamanho e formato do fruto, rendimento em suco, sementes, arilo, número de sementes por fruto e umidade das sementes. Os dados foram interpretados por intervalo de confiança a P≤0,05 para amostras de uma população infinita de frutos. Verificou-se que o rendimento em suco aumentou com o amadurecimento, mas não foi influenciado pelo formato e tamanho. A proporção do resíduo total foi maior nos frutos maduros com o menor tamanho. O rendimento de sementes foi menor nos frutos grandes, mas não foi alterado pelo formato, apresentando média de 4,23 por cento de semente. O conteúdo de óleo estimado nas sementes com 25,7 por centobs indica um potencial de produção de 64,84t de óleo por ano no estado do Rio de Janeiro.

This research evaluated the juice yield, arils and seeds of yellow passion fruits aiming to identify the potential of adding values for the seeds used in the oil extraction. It was measured the weight, peel color, size and shape of the fruit, juice yield, seeds, arils, number of seeds per fruit and humidity of seeds. The data were interpreted by confidence intervals at P≤0.05 for samples of an infinite population of fruits. It was verified that the juice yield increased with the ripening, but it was not changed by the shape and fruit size. The proportion of arils and seeds was higher in ripe fruits with smaller size. The seed yield was lower in bigger fruits, but it was not changed by the fruit shape, presenting average of 4.23 percent of seeds with 14.8 percent humidity. The estimated content of oil in seeds with 25.7 percentdb indicates a production of 64.84t of oil per year at the Rio de Janeiro state.

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The fate of vicilins ingested by Callosobruchus maculatus and the physiological importance of these proteins in larvae and adults have been recently investigated. Vicilins have been demonstrated to be absorbed through the midgut epithelium, circulate in their trimeric form in the haemolymph and are deposited in the fat body. In fat body cells of both sexes, vicilins are partially hydrolyzed and the fragments are eventually deposited in the eggs. Tracking the fate of FITC-labelled vicilins in adult males revealed that the labelled vicilin fragments were also detected in oöcytes and eggs, when the males copulated with non-labelled females. Based on the results presented here, we propose that following absorption, vicilins accumulate in the fat body, where they are partially degraded. These peptides are retained throughout the development of the males and are eventually sequestered by the gonads and passed to the female gonads during copulation. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack, as these peptides are known to have antimicrobial activity. The contribution of vicilin-derived peptides from seminal fluids may be an investment that helps to increase the offspring survival. This study provides additional insights into the possible contributions of males to female fecundity following copulation in C. maculatus.

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This research evaluated the juice yield, arils and seeds of yellow passion fruits aiming to identify the potential of adding values for the seeds used in the oil extraction. It was measured the weight, peel color, size and shape of the fruit, juice yield, seeds, arils, number of seeds per fruit and humidity of seeds. The data were interpreted by confidence intervals at P0.05 for samples of an infinite population of fruits. It was verified that the juice yield increased with the ripening, but it was not changed by the shape and fruit size. The proportion of arils and seeds was higher in ripe fruits with smaller size. The seed yield was lower in bigger fruits, but it was not changed by the fruit shape, presenting average of 4.23% of seeds with 14.8% humidity. The estimated content of oil in seeds with 25.7 %db indicates a production of 64.84t of oil per year at the Rio de Janeiro state.

Neste trabalho, avaliou-se o rendimento em suco, arilo e sementes do maracujá-amarelo, para identificar o potencial de agregação de valor às sementes usadas na extração do óleo. Foram feitas medidas de massa, coloração da casca, tamanho e formato do fruto, rendimento em suco, sementes, arilo, número de sementes por fruto e umidade das sementes. Os dados foram interpretados por intervalo de confiança a P0,05 para amostras de uma população infinita de frutos. Verificou-se que o rendimento em suco aumentou com o amadurecimento, mas não foi influenciado pelo formato e tamanho. A proporção do resíduo total foi maior nos frutos maduros com o menor tamanho. O rendimento de sementes foi menor nos frutos grandes, mas não foi alterado pelo formato, apresentando média de 4,23% de semente. O conteúdo de óleo estimado nas sementes com 25,7%bs indica um potencial de produção de 64,84t de óleo por ano no estado do Rio de Janeiro.

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This research evaluated the juice yield, arils and seeds of yellow passion fruits aiming to identify the potential of adding values for the seeds used in the oil extraction. It was measured the weight, peel color, size and shape of the fruit, juice yield, seeds, arils, number of seeds per fruit and humidity of seeds. The data were interpreted by confidence intervals at P0.05 for samples of an infinite population of fruits. It was verified that the juice yield increased with the ripening, but it was not changed by the shape and fruit size. The proportion of arils and seeds was higher in ripe fruits with smaller size. The seed yield was lower in bigger fruits, but it was not changed by the fruit shape, presenting average of 4.23% of seeds with 14.8% humidity. The estimated content of oil in seeds with 25.7 %db indicates a production of 64.84t of oil per year at the Rio de Janeiro state.

Neste trabalho, avaliou-se o rendimento em suco, arilo e sementes do maracujá-amarelo, para identificar o potencial de agregação de valor às sementes usadas na extração do óleo. Foram feitas medidas de massa, coloração da casca, tamanho e formato do fruto, rendimento em suco, sementes, arilo, número de sementes por fruto e umidade das sementes. Os dados foram interpretados por intervalo de confiança a P0,05 para amostras de uma população infinita de frutos. Verificou-se que o rendimento em suco aumentou com o amadurecimento, mas não foi influenciado pelo formato e tamanho. A proporção do resíduo total foi maior nos frutos maduros com o menor tamanho. O rendimento de sementes foi menor nos frutos grandes, mas não foi alterado pelo formato, apresentando média de 4,23% de semente. O conteúdo de óleo estimado nas sementes com 25,7%bs indica um potencial de produção de 64,84t de óleo por ano no estado do Rio de Janeiro.