There has been a nutritional transition based on diets composed of foods with a high glycemic index and a high glycemic load and ultraprocessed foods worldwide. This, associated with other factors, is crucial for increasing the obesity prevalence and various diseases, including the disease Non-Alcoholic Fatty Liver (NAFLD). NAFLD ranges from steatosis and steatohepatitis to severe conditions, such as cirrhosis and hepatocellular carcinoma. NAFLD treatment is a clinical necessity not answered. Thus, several studies have sought new therapies not only for NAFLD but also for obesity. Among them, we highlight the findings related to the trypsin inhibitor isolated from tamarind seeds (TTI) nanoencapsulated in chitosan and whey protein isolated (ECW). Therefore, the present study aimed to investigate the effect of ECW on the liver status of Wistar rats fed a high glycemic index and high glycemic load diet (HGLI diet). First, nanoparticles with and without TTI, respectively ECW and CW, were obtained by the technique of nanoprecipitation in an organic solvent and, characterized by morphology (SEM), particle size (Laser diffraction), chemical interactions (FTIR), and efficiency of encapsulation. The animals were divided into four groups, containing five animals each, being treated with 1. HGLI diet and water, 2. standard diet and water, 3. HGLI and ECW diet, and 4. HGLI and CW diet. The hematological biochemical parameters of renal and hepatic involvement were evaluated (liver enzymes, AST / ALT ratio), lipid profile, fasting glucose, insulin and insulin resistance, and β-cell homeostasis model (HOMA-IR and HOMA-β, respectively). Besides, liver scores FIB-4 (Fibrosis-4 Index for Liver Fibrosis) and APRI (AST to Platelet Ratio Index) were also investigated, and the liver histopathology of the animals was evaluated. Given the results, smooth, spherical nanoparticles were obtained in nanometric diameter (120.7 nm) containing TTI. In animals, ECW reduced (p < 0,05) fasting blood glucose levels by 17%, AST in 39%, and alkaline phosphatase in 24%, also significantly reduced (p < 0,05) the two scores of assessment of liver fibrosis, APRI, and FIB-4, and presented a better aspect of liver morphology when compared to the other groups. Thus, it was possible to show that the ECW promoted a new functionality for the isolated ITT, promoting benefits on the liver injury caused by the HGLI diet, considering the biochemical, morphological, and liver fibrosis evaluation parameters.

Microorganisms represent the largest biomass on Earth and are directly related to biogeochemical cycles. It has been described that microbial activity affects the production chain related to the oil and gas sector, being of interest the knowledge about the microbiology associated with reservoirs, aiming the optimization of the production chain, as well as the mitigation of environmental impacts due to environmental contamination by hydrocarbons. The Brazilian pre-salt reservoirs are extreme environments with high carbon dioxide (CO₂) concentration and high depth, whose microbiology is still poorly understood. The objective of this work was to characterize bacterial communities and isolates obtained from a sample of pre-salt oil submitted to cultivation at high CO₂ concentration. From an oil sample, six consortia were obtained and analyzed, organized in two groups: three undefined microbial consortia grown at high CO₂ concentration and the other three obtained from the same sample under atmospheric aeration and later submitted to high CO₂ concentrations. The microbial consortia were characterized for: i) biosurfactant (BS) production; ii) growth curve in the presence of hydrocarbons as carbon source; and iii) taxonomic and functional characterization by metagenomic sequencing. Functional assays of the consortia showed that three are able to produce BS molecules capable of generating emulsion, whereas none of the consortia is able to disperse oil. The growth curves showed that five consortia are able to grow in the absence of external carbon sources, indicating the occurrence of inorganic carbon fixation, which was corroborated by the metagenomic sequencing of the consortia via identification of sequences assigned to the genus Ochrobactrum, which is able to fix carbon. In the consortia, the most abundant phyla were Proteobacteria, Actinobacteria, and Firmicutes, and the consortia submitted to high CO₂ concentration, after growing in O₂, were more diverse than the consortia grown in high CO₂ concentration. As for the metabolic profile, the most enriched pathways in the consortia grown in CO₂ were the amino acid metabolism and carbohydrate metabolism pathways. Nineteen bacterial isolates were obtained from the consortia, and nine were characterized and tested. For the isolates the following assays were performed: i) BS production, ii) hydrocarbon degradation capacity, iii) cellular hydrophobicity, and iv) enzymatic activity of hydrolases. Of these, five were able to degrade between 50% and 80% of the xylene and toluene fractions, and eight others degraded 100% of the gasoline, diesel and kerosene fractions. It is concluded that the growth of undefined consortia in O₂ leads to a greater diversity of microorganisms, that CO₂ inhibits BS production, and that isolates and microbial consortia from the pre-salt show promise as a source of bioremediation products, as well as hydrolases with potential industrial application.

Resistance to antimicrobials and antineoplastic agents is considered a worldwide public health problem and the search for new molecules that are more effective or that supplement existing therapies is necessary. In this context, Stigmurin, a peptide native to the venom of scorpion Tityus stigmurus, showed antibacterial activity on gram-positive bacteria, with antiproliferative effect on different cancer cells, low toxicity in human erythrocytes in vitro and trypanocidal activity on Trypanossoma cruzi. In order to expand the potential of the native peptide, analogs to Stigmurin were designed and analyzed for structure and different biological activities. The structural analysis of the analogs peptides, called StigA8 and StigA18, was obtained by structural prediction, molecular modeling and circular dichroism. In vitro biocompatibility was evaluated in human red cells, murine fibroblasts and in vivo in Galleria mellonella moth larvae. The antibacterial activity against gram-positive and gram-negative strains was performed by microdilution in broth and the antibiofilm activity by the microtiter plate and analyzed by scanning electron microscopy, as well as the antibacterial effect in vivo on G. mellonella larvae infected with Staphylococcus aureus. The antiparasitic action of analogs peptides on the epimastigote and trypomastigote forms of T. cruzi was evaluated by counting viable cells and the antiproliferative effect in cancer cell lines was evaluated by the 3-(4,5-dimethylthiazole-2-yl)-2,5- diphenyl-tetrazolium (MTT) salt reduction method. StigA8 and StigA18 showed higher rates of cationicity (+4 and +5, respectively) and hydrophobic moment (0.652 and 0.708, respectively) in relation to the prototype molecule (+2 and 0.571, respectively) and a conformation mostly in α-helix in environments that mimic biological membranes. In addition, peptides showed hemolytic activity of 11.5-22% in a concentration range of 9.37-150 µM, with a cytotoxicity index in murine fibroblasts of 50% at a concentration of 20 µM and 12.3 µM for StigA8 and StigA18, respectively. In addition, no signs of toxicity were observed on G. mellonella larvae at a dose of 120 mg/kg. Both analog peptides showed significant antibacterial activity in strains of gram-positive and negative bacteria in concentrations ranging from 0.93-9.37 µM, with antibiofilm action against S. aureus in concentrations of 7.5-12.5 µM, causing damage such as matrix destruction, cell membrane invaginations, roughness and reduction of the cell diameter by 12.5 µM. StigA8 and StigA18 showed antibacterial effect in vivo, increasing larval survival at 120 mg/kg by 75% and 60%, respectively, at the peak of infection. Trypanocidal activity on the epimastigote and trypomastigote forms of T. cruzi was observed at 6.25 µM for both peptides and antiproliferative potential from the concentration of 2 µM for StigA8 and 4 µM for StigA18. Therefore, StigA8 and StigA18 are molecules with the potential for the development of new antimicrobial and anticancer drugs

Medicinal plants have been studied for centuries for their medicinal properties, including Malpighia emarginata D. C. (acerola) and Camellia sinensis L. (green tea) has been an alternative preventive and medicinal approach for various pathologies. In view of the potential anti-inflammatory and antioxidant uses, in this work we developed a mixture formulated in powder with Malpighia emarginata D. C. and Camellia sinensis L. which have a higher content of ascorbic acid and epigallatecatechin-3-gallate, respectively. The production of the blend was carried out through the spray drying process, using microencapsulation methods. Subsequently, the antioxidant and anti-inflammatory properties of the formulated blends were evaluated in an in vitro inflammatory model, using the RAW-264.7 macrophage cell line in two treatments, cells against extract and co-treatment, which are the blends against stimulated cells by lipopolysaccharide. We observed that co-treatment with blends was able to modulate redox parameters in cells during the in vitro inflammatory response. Furthermore, the co-treatment with the blends was able to modulate the inflammatory response, altering the secretion of IL-1β, IL-6, IL-10 and TNF-α cytokines. Thus, our results observed together demonstrate for the first time the synergistic antioxidant and anti-inflammatory effects of Malpighia emarginata DC and Camellia sinensis L. prevention of chronic diseases.

The increasing prevalence of obesity and chronic non-communicable diseases associated with it has increased, and the search for new strategies to control them. In this sense, vitamin A plays an important role in regulating body weight through the action of metabolites of this nutrient on different aspects in individuals with obesity. Based on this, this thesis is divided into two chapters. In the first chapter, a narrative review was performed to understand this relationship and the physiological effects of vitamin A on obesity. A search was carried out in the databases, selecting the studies that involved vitamin A or its active metabolites, encapsulated or not, and the role and relationship with obesity. Studies were found showing that there is an inverse relationship between adiposity and vitamin A concentration. In addition, it was demonstrated that encapsulating vitamin A or its precursors improves stability, bioavailability, and functionality and may favor better effects in the treatment of obesity. In the second chapter, a study was carried out to evaluate the impact of the crude extract rich in carotenoids from Cantaloupe melon (Cucumis melo L. var cantalupensis) nanoencapsulated in porcine gelatin (EPG) on the concentration of vitamin A in the liver of obese Wistar rats. The crude extract rich in carotenoids (CE) was obtained by processing, drying, and extracting bioactive compounds from the melon pulp, being used to synthesize the nanoformulation through the O/W emulsification technique. The nanoparticles were characterized in terms of physical and chemical aspects and evaluated the efficiency of incorporation of carotenoids. In the preclinical study, Wistar rats with obesity induced by a high glycemic index diet and a high glycemic load (HGLI diet) were divided into three groups (n = 5): 1. no treatment (HGLI diet + water), 2. treated with HGLI diet + CE (12.5 mg/kg), and 3. treated with HGLI diet + EPG (50 mg/kg) administered by gavage (1 mL) for ten days. These animals were evaluated for variation in dietary intake, retinol, and weight, hematological parameters, fasting glucose, and lipid profile. In addition, the investigation of hepatic retinol concentration and liver function was performed by determining the AST/ALT ratio and the FIB-4 (Fibrosis-4 Index for Liver Fibrosis) and APRI (AST to Platelet Ratio Index) scores. The results obtained for the characterization of the particles confirmed the presence of spherical particles with smooth surface, mean diameter of 80 (8.59) nm, polydispersion index of 0.47 (0.09), and the presence of new chemical interactions, indicating that CE was encapsulated in porcine gelatin. Furthermore, the CE incorporation efficiency was equal to 95.20% (0.74). For animals treated with EPG, lower dietary and retinol consumption was observed (p < 0.05). In addition, the same group of animals had significantly higher concentrations (p < 0.05) of hepatic retinol [266 (45) µg/g] when compared to the untreated group [186 (23.8) µg/g] and the one treated with CE [175 (8.08) µg/g]. The liver damage assessment scores did not show significant differences (p > 0.05), but the lowest means were observed in the group of animals treated with EPG. These findings suggest that nanoencapsulation promoted protection, guaranteed the absorption of carotenoids in the intestine, and bioconversion to retinol in the liver, without causing liver alterations. Thus, EPG presents itself as a candidate for future clinical studies, aiming to evaluate the beneficial effects of treating diseases that involve vitamin A deficiency, such as obesity.

Tuberculosis is a worldwide health problem caused by Mycobacterium tuberculosis and aggravated by the resistance mechanisms developed by this bacterium against the drugs used in its treatment. Thus, the development of drugs capable of eliminating multidrug-resistant strains, characterized by those that have resistance to the most important drugs - rifampicin and isoniazid - becomes crucial. Thereby, this work aims to perform a structural and energetic study of the kanglemycin and rifampicin interactions in complex with RNA Polymerase; also BDM72201, BDM72719, BDM76060 and BDM76150 with the transcriptional repressor EthR2, as a way to boost the action of ethionamide. Using crystallographic data from these systems, calculations of the interactions of each ligand with their respective receptors were performed within the density functional theory (DFT), and the binding energy of each molecule with the amino acid constituents of the binding site was obtained by applying the molecular fractionation with conjugate caps (MFCC). Thus, results showed that kanglemycin is able to bind to RNA polymerase in the same way as rifampicin, having -63.61 Kcal/mol and -65.68 Kcal/mol total energy, respectively, and sharing 79 residues at the binding site. In the presence of the S447L mutation, often associated with rifampicin resistance, kanglemycin maintains its strong interaction with its target, generating an overall energy of -64.00 Kcal/mol. BDM72201, BDM72719, BDM76060 and BDM76150 components share 68 residues of the same binding site. BDM72201 and BDM72719 attach to the site in a weaker way, showing energies of -19.56 Kcal/mol and -21.56 Kcal/mol, respectively; whereas BDM76060 (-65.88 Kcal/mol) and BDM76150 (-66.13 Kcal/mol) produce stronger binding and have greater potential to act as EthR2 inhibitors. Therefore, it was evidenced that kanglemycin has great potential to be inserted into clinical practice as a rifampicin substitute and the BDM76150 as an ethionamide booster.

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus which has emerged or reemerged in the last twenty years. CHIKV infection causes varied outcomes including asymptomatic infection, mild disease, or severe disease with debilitating arthritis, causing an increase in disability-adjusted life-years (DALY). The purpose of the current study was to discover immune pathways associated with different outcomes of CHIKV infection by transcriptional profiling of whole blood from CHIKV-infected individuals. During the 2016 CHIKV outbreak in Brazil, whole blood samples were collected from CHIKV-infected individuals diagnosed by RT-qPCR or neutralization assays, and from control, non-infected individuals. Among the collected samples, four were from patients in the acute phase, six from recovered or mild symptoms ones, and two were asymptomatic. From whole blood cells extracted RNA, we performed an RNASeq analysis and mapped the sequenced fragments to the human or the CHIKV genomes. Libraries had a mean of 96% of transcripts mapping to the human genome, and 3 from acute subjects contained sequences mapping to the CHIKV genome. There was a mean of 490, 1463 and 370 differentially expressed genes (DEGs) between Asymptomatic, Acute, and Non-Acute transcriptomes, respectively. DEGs from the Acute group were strongly associated with type I interferon response with upregulation of STAT1, STAT2 and a series of other interferon-stimulated genes (ISGs) such as RSDA2 and IRF7. The number of ISGs differentially expressed by the group of people with asymptomatic infection is reduced, which may be related to a weak response by type I interferon. Another highlight among the set of DEGs of asymptomatic people is the underexpression of the coding gene of the protein Granzima A (GZMA), one of the main promoters of inflammation in arthritis. The enrichment analyzes with Gene Ontology terms, using DEGs, showed that the group of people in the acute phase of the disease had high expression in genes related to the process of elimination of the virus, inflammatory response and control of cell replication. An Ingenuity Pathway Analysis functional enrichment analysis showed that GDEs in the asymptomatic group of people are involved in suppressing leukocyte activation and decreasing cell movement of monocytes and leukocytes. Our findings suggest that an inflammatory state is present during acute symptomatic CHIKV infection, while there is a decrease in these markers during asymptomatic infection.

Amburana cearensis (Fabaceae), popularly known as imburana or cumaru, its very used in traditional medicine, what awakens the search for evidence of its pharmacological properties. The aim of this study was to evaluate the antimicrobial and modulating activities of crude extract and fractions obtained from seeds of A. cearensis, as well as to characterize the plant material by UPLC-MS-QTOF. The aqueous extract obtained in 50 mM sodium phosphate buffer was sequentially fractionated into ammonium sulfate and subjected to characterization by the Acquity UPLC (Waters) system coupled to a Quadrupole/Flight Time (QtoF, Waters) system. The antimicrobial activity was verified by microdilution assay, while the possible interactions between the natural product and penicillin, norfloxacin, gentamicin and fluconazole were determined using a sub-inhibitory concentration. Samples at concentration of 128 µg/mL showed significant interaction with a possible synergistic effect when associated with antibiotics and antifungal against Escherichia coli, Sthaphylococcus aureus and Candida albicans. In the characterization it was possible to identify in the crude extract and fractions, several phenolic compounds, organic acids, and some polyphenols in positive ionization mode. The present study with crude extract and fractions of A. cearensis may be an alternative source of antimicrobial isolated compound.

The green macroalgae Codium isthmocladum is a rich source of bioactive sulfated polysaccharides (SP). Besides being found in abundance in the Rio Grande do Norte coast, the SP from this alga have been already described about its anticoagulant and antinociceptive potential. On other hand, there are not any registers about the activities of this alga front of bacterial strains. In this context, the present study has as focus the extraction and purification of SP’s from the green alga C. isthmocladum, well as characterize them chemical and structurally. About its applicability, the SP’s were evaluated about the antioxidant capacity in many different in vitro ways, well has been evaluated the potential of this compounds as possible antibacterial and antibiofilm agents of normal and multiresistant bacteria. After being collected, the seaweed was treated with the purpose of obtaining a polysaccharide-rich isolate (crude extract). From a subsequent fractionation of this extract, five ketonic fractions were obtained (Ci 0.3, Ci 0.5, Ci 0.7, Ci 0.9, Ci 1.2). All fractions have a low content of proteins and phenolic compounds (> 1%). Ci 0.7 is the fraction with higher yield and sulfate content. All polysaccharides showed antioxidant capacity, but only Ci 0.7 and Ci 0.9 were able to inhibit the growth of resistant Staphylococcus aureus (MRSA) and Escherechia coli in adjuvant with several antibiotics, mainly with gentamicin. Both fractions were subjected to ion exchange chromatography and tended to generate two chromatographic peaks at different molarities, eluted with 2 M and 3 M NaCl, respectively for Ci 0.9 and 1 M and 2 M NaCl for Ci 0.7. Based on the data obtained from the chemical and structural analyzes, it is possible to affirm that these are sulfated homogalactans. The homogalactans from Ci-0.7-1M and Ci-0.9-3M presented antibacterial capacity and antibiofilm also against resistant strains of S. aureus

Anxiety and depression are symptoms associated with ethanol withdrawal that lead individuals to relapse. In the tryptophan pathway, the enzyme indoleamine 2,3 dioxygenase (IDO) is responsible for converting tryptophan to kynurenine. Deregulation of this pathway is associated with psychiatric disorders, including anxiety and depression. The present study tested the hypothesis that the kynurenine pathway participates in modulating the behavioral effects caused by short- and long-term ethanol withdrawal. In experiment 1, Wistar rats received increasing ethanol concentrations for 21 days and were subjected to a battery of behavioral tests 3, 5, 10, 19, and 21 days after ethanol removal. In experiment 2, the animals were subjected to the same ethanol exposure protocol, euthanized 3 days (short term) or 21 days (long term) after ethanol removal. The brains were dissected to analyze the concentration of kynurenine (as a measure of IDO activity) in the prefrontal cortex, hippocampus, and striatum. In experiment 3, rats were subjected to behavioral tests on days 3, 19, and 21 after removing ethanol, and the brains were dissected for analysis of IDO activity in the areas mentioned above. Short-term ethanol withdrawal decreased the exploration of open arms in the elevated plus-maze. In contrast, in the forced swim test, rats subjected to long-term withdrawal showed a longer immobility time than control animals. The removal of ethanol did not alter the rats' locomotion or motor coordination (experiment 1). In experiment 2, the long-term withdrawal of ethanol increased the concentrations of kynurenine in the prefrontal cortex. In females, the administration of minocycline prevented the anxiogenic-like behavior promoted by the short-term ethanol withdrawal. This drug also prevented the effect of the long-term withdrawal on depressive-like behaviors. There was no locomotor alteration, neither in the concentrations of kynurenine in females (experiment 3). In conclusion, short-term ethanol withdrawal favored anxious behavior, while long-term withdrawal-induced depressive behavior. Prolonged ethanol withdrawal raised kynurenine levels, specifically in the prefrontal cortex in males, suggesting that the depressive responses observed after prolonged withdrawal may be related to increased IDO activity. In females, the administration of minocycline was able to reverse anxious and depressive behaviors caused by the withdrawal of ethanol, reinforcing that this pathway may be an option in the pharmacotherapy of ethanol withdrawal.

Introduction: Creatine is a supplement that has been the subject of research in several pathologies, with a wide possibility of clinical applications. Studies have shown the effect of creatine on increasing glucose uptake by elevating the expression of GLUT-4 receptors in metabolic disorders such as Type 2 Diabetes Mellitus, however, there are no studies that report the effect of creatine on the glucose metabolism of type Diabetes Mellitus 1. Thus, this work aims to evaluate the effect of creatine supplementation in diabetic rats induced by streptozotocin. Methods: Thirty-two Wistar rats were divided into 4 groups: (C) animals without diabetes and without creatine supplementation (n = 8), (CCr) animals without diabetes and with creatine supplementation (n = 8), (D) animals streptozotocin-induced diabetes mellitus (n = 8), (DCr) creatine-treated streptozotocin-induced diabetes mellitus (n = 8). Groups D and DCr received a single dose of streptozotocin (40 mg/kg i.p.). The following evaluations were performed: clinical, biochemical, as well as the analysis of redox state parameters in the renal tissue of the animals. Results: The groups D and DCr showed higher water and feed intake compared to groups C and CCr. In the weight variation in the experimental period, the diabetic groups showed significant weight loss in relation to the control groups. In the relative renal weight, the diabetic groups presented significant hypertrophy; in addition, the DCr group presented significantly greater renal hypertrophy than the D group. In fasting glycemia, the diabetic groups showed significant hyperglycemia in comparison with the control groups, however, the DCr group showed a significant improvement in relation to the group D. There was no statistical difference between the groups in serum creatinine and aspartate transaminase levels. In the quantification of urea and alanine transaminase, the diabetic groups showed a significant increase in comparison with the control groups, however, the DCr group showed a significant improvement in relation to the group D in both parameters. The enzymatic activity of catalase, superoxide dismutase and glutathione peroxidase were significantly lower in group D compared to control groups, however, the DCr group had restored activity like the control groups in both enzymes. In the quantification of the hydrogen peroxide content, the D group had significantly higher levels in comparison to the control groups, however, the DCr group had a level like the control groups in this parameter. We observed in the quantification of thiobarbituric acid reactive substances in the CCr group a significant increase in relation to the other experimental groups. In the quantification of carbonylated proteins, group D showed levels significantly lower in relation to the other groups, while the DCr group showed values statistically like the control groups. In the quantification of soluble thiols, there was no difference between the experimental groups. Conclusion: Creatine can improve metabolic profile in DM and restore redox state parameters in this condition. These results support creatine supplementation as a potential therapeutic agent.

Congenital generalized lipodystrophy (CGL) type 2 consists of a rare autosomal recessive syndrome characterized by almost complete lack of body fat at birth. LGC, genetically caused by loss of function mutations in the two alleles of the BSCL2 gene, was phenotypically characterized by hypertriglyceridemia, hyperglycemia, increased LDL-c, decreased HDL-c, and hypoadiponectinemia. These laboratory findings are important triggers for changes in redox and endoplasmic reticulum (ER) homeostasis. Therefore, our aim was to demonstrate if these intracellular mechanisms are part of this syndrome. Therefore, we collected the whole blood from people living in Northeastern Brazil with 0, 1 and 2 mutant alleles for the 325dupA mutation (rs786205071) in the BSCL2 gene. Through the RTqPCR technique, we evaluated the expression of genes responsible for triggering the antioxidant response, DNA repair and ER stress in leukocytes. Colorimetric tests were applied to quantify lipid peroxidation products and to evaluate the redox status of glutathione, as well as to access plasma energy metabolism panorama. Conventional PCR was also chosen to observe leukocyte mitochondrial DNA lesions. We also chose the immunoblot technique for the study of gene expression of chaperone proteins related to ER stress in leukocytes, as well as plasma adiponectin concentrations. Comparing to healthy group, subjects with the rs786205071 mutation in the 2 alleles showed increased transcription of NFE2L2, APEX1, OGG1 and αOGG1 in leukocytes, as well as the concentrations of malondialdehyde and the GSSG: GSH ratio in plasma. In addition, we also observed decreased in vitro polymerization rate of mitochondrial DNA and increased XBP1 splicing in leukocytes. In silico analyzes indicated a high probability of seipin rs786205071 belonging to the ER membrane, although the aforementioned mutation removes amyloidogenic regions from that protein. In addition, we observed a decrease in the transcription of BSCL2 in these leukocytes, associated to the absence of differences in the expression pattern of HSPA5 and P4HB chaperones. In parallel, these cells showed decreased DDIT3 transcripts, but without differences in the procaspase-3 cleavage pattern. Finally, we also observed a decrease in plasma concentrations of adiponectin and HDL-c. Together, results point to the presence of lipid lesions due to changes in redox homeostasis, associated with increased numbers of mitochondrial DNA damage and transcriptional recruitment of genes that participate of the antioxidant response and DNA repair. Although there is also evidence of ER stress, the rs786205071 mutation has been shown to be unlikely to directly generate this mechanism. In addition, the data suggest that there was no evidence of increased cell death and that decreases in adiponectin and HDL-c concentrations may act as potential triggers for changes in redox and ER homeostasis in CGL type 2.

Brazil is the world's largest producer of sugarcane. All parts of this cultivar are used as energy matrix for production not only of sugar, but also of biofuels and other products. Molecular studies with sugarcane develop slowly due to their genomic complexity; however, the discovery of new genes that may interfere in key processes of this organism is valuable for future contributions in the sugar- alcohol industry. This work aimed to characterize a sequence homologous to PHOSPHATIDYLINOSITOL-PHOSPHATIDYLCHOLINE TRANSFER PROTEIN SEC14 in sugarcane that has been identified in previous studies in subtractive libraries for the flowering process. The in silico characterization was performed by functional analysis of the gene, construction of protein-protein interaction networks, phylogeny, molecular dynamics of the three-dimensional model of the protein and co-expression analysis, in addition, an RT-qPCR expression assay was performed in plant tissues for late and early varieties, as well as in leaf tissues submitted to drought and oxidative stress, as well as expression analysis in different tissues of the plant. The results obtained using the bioinformatics tools show the existence of the CRAL-TRIO domain that is present in all homologous sequences essentially in the same position, revealing high conservation of the sequence SEC14 in plants. Interaction networks reveal that the protein homologous to SEC14 in A. thaliana can interact with the proteins of the PATTELIN family (PTL), SYNTAXIN (SYP), OXYSTEROL BINDING PROTEIN (ORP) , PHOSPHOLIPASE D (PLD), among others, whose main functions include transport of the lipid phosphatidylinositol, vesicle traffic, and can act on the action of phytormones such as abscisic acid, stress responses, development of floral organs and membrane integrity. The phylogenetic tree suggests that there are likely duplications of the SEC14 protein in plants. The three-dimensional model presents a probable hydrophobic pocket for lipid binding, described in other consolidated SEC14 models, indicating its structural conservation. Expression data reveal that the SEC14 gene was differentially expressed in the early juvenile phase variety and in the late mature variety; in addition, there was no significant difference in expression in leaf tissues of plants submitted to drought and oxidative stress, probably due to the juvenile stage of the analyzed organism. Finally, this study provides information for a better understanding of how the gene / protein of SEC14 acts on sugarcane. They reveal new knowledge about the three-dimensional structure of this protein in this culture; also brings new perspectives for molecular studies with plants of economic interest such as sugarcane.

Petroleum is one of the world's main source of energy, on Brazil pre-salt reservoir extraction has boosted oil production. However, such activity generates recurrent environmental contaminations, causing global problems. Microorganisms demonstrate the ability to degrade oil, so microbial metabolism can provide an opportunity to bioremediate contaminated environments and assist in reservoir management, contributing to oil quality or oil recovery. Identification of the microbiota in the reservoir provides essential data to understand the metabolism of oil degradation, but currently there are no analysis of the pre-salt microbiota. The objective of this work was to obtain and characterize consortia (indefinite and selected) capable of degrading petroleum. From a pre-salt oil sample three indefinite consortia and 26 isolates were obtained. Tests were conducted for consortia and isolates, such as: (i) oil degradation ability through the redox indicator test 2,6-Dichlorophenolindophenol (DCPIP) using 9 hydrocarbon sources (petroleum, soybean oil, olive oil, gasoline, diesel, kerosene, hexadecane, toluene and xylene) and glucose as a positive control; ii) production of biosurfactants; iii) cellular hydrophobicity; iv) biofilm production; v) enzymatic activity of hydroxylases; vi) presence of rhamnolipid and vii) growth curve. All isolates demonstrated the ability to degrade petroleum and some by-products by the DCPIP test, however only 46% used kerosene and 7% degraded xylene and toluene as carbon sources. The oil dispersion test showed that all isolates produce biosurfactants, yet only 84% of the bacteria possess the ability to emulsify kerosene. Lipase was produced by all isolates, however, only 4 strains produced esterase. The isolates were identified as belonging to the genera Bacillus (n= 9), Ochrobactrum (n = 11), Acinetobacter (n=2), Dermacoccus (n=1) e Staphylococcus (n = 2). The selected consortium was elaborated with eleven isolates that presented the best results in the functional tests. The estimation of degradation indicate that all consortia degraded 100% of oil, diesel, gasoline, soybean oil, olive oil and hexadecane, while degradation of kerosene, xylene and toluene was above 40%; and the emulsification index of the consortia was above 58%. The comparison of the results between the selected and the indefinite consortia shows similarities in the functional characteristics (growth curve, hydrocarbon degradation and biosurfactant production). It is concluded that the selected consortium presents the same potential of the native microbiota, being a good possibility for application of bioremediation; while indefinite consortia can provide data for reservoir management.

Chagas disease prow by the protozoan Trypanosoma cruzi affecting millions of people. The drugs were later that cytotoxic are not recognized in the chronic phase of the disease. In addition, the parasite has a genetic variable that causes different clinical manifestations and resistance to treatments. Potassium diethyl dithiocarbamate belongs to the class of dithiocarbamates which are highly versatile chemical compounds that allow interaction with metals and cause the increase of oxidative free radicals. T. cruzi strains were characterized as cultivated in the environment. This is an epimastigote and trypomastigote assay of T. and is evaluated by the resistance reduction technique and clear camera counting. It analyzed the mechanisms of parasitic death triggered by the compound against T. cruzi epimastigote within 24 hours. Mitochondrial damage due to DETC under T. cruzi epimastigote after 24 hours of treatment by membrane potential assay. They were also analyzed as morphological changes by scanning electron microscopy of the DETC action after 24 hours of treatment. Cellular cytotoxicity of DETC was also analyzed for 3T3 and RAW plants 24 hours after compound application and MTT assay analyzes. Researchers examined the inhibitory action of DETC on proteolytic proteins of T. cruzi extracts through zymography and analyzed by densitometry. To have that which dithiocarbamate has a high case in the development of T. cruzi carbonic one sought to evidence the anchorage of the DETC. From the antiparasitic assays was the first action antiparasitic activity under the strains variations and the concentration required to eliminate the amount of 0.235 µm to 0.790 µM up to 0.664 µM for epimastigote and 0.235 µM up to 0.790 For trypomastigote according to strain . DETC cellular cytotoxicity analysis showed moderate cytotoxicity against animal species. When analyzing the mechanisms of cell death, it was noted the need to determine the cell death processes by the disease anchorage, ie, evidencing the non-expression of phosphatidylserine. Through mitochondrial damage testing, it is possible to perform a strong movement of DETC in different strains inhibiting mitochondrial membrane potential, reaching 80% reduction. Through the analysis of scanning electron microscopy, we observed the discontinuity in the parasite's cell membranes, as well as the rupture and formation of pores. The zymography showed a reduction in the potential of proteolytic activity of parasite extracts that reached 36%. Finally, which is DETC, it has a high interaction capacity to inhibit carbonic α-anhydrase by analyzing the positioning of its anchorage in the protein structure. Therefore, the DETC is an excellent alternative for the alternative treatment of Chagas disease due to antiparasitic activity, not presenting high cytotoxicity and triggering actions on the specific parasite compartments. In addition, an α-Anhydrase was identified. as possible the DETC.

Chromium picolinate (PICCr) is a complex made up of three picolinic acid residues (in salt form) linked by coordination to a chromium III atom. PICCr supplementation by athletes and others has been increasing every year and without a determined dose. The hypothesis of this study is the safety of prescribing varying doses of chromium picolinate. After commercial acquisition of PICCr capsules from two different countries, England and Brazil, this work began by checking by Atomic Absorption Spectrometry, the masses contained in these capsules. We found a higher concentration of the mineral in the capsules of English origin, which was selected as a sample for the research. Four cell lines: murine macrophages (RAW-264.7), Chinese hamster ovary (CHO) cells, murine fibroblast (3T3) and human human hepatic adecarcinoma (HepG2) cells were exposed to varying concentrations of PICCr (5 to 20 µg /mL) CHO, 3T3 and HepG2 were found to have their ability to reduce (10 to 80%) decreased MTT (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazole). RAW 264.7 cells were not affected in this analysis. HepG2 cells when exposed to PICCr (20 to 50 µg / mL) had micronucleus numbers significantly similar to cells that were not exposed to PICCr. RAW 264.7 cells showed a decrease in nitric oxide release when they were exposed to bacterial wall liposaccharides (LPS). In in vivo assays, male Swiss mice were exposed to different doses of PICCr (1000 and 2000 µg / mL) for 15 days. After this period, it was found that the treated animals had lower weight gain and decreased plasma cholesterol, triglycerides and glucose levels. They did not present macroscopic or histological changes in the hepatic and renal tissues. Treated animals were also found to have 50% decrease in serum plasma iron levels. However, metallomics analyzes show that iron and hepatic tissue iron levels have not been affected, however, human studies are necessary to confirm these observations, and may boost future studies to better understand these effects and add economic and pharmacological value to the study. PICCr.

The drug development process have been improved by the add of 

other knowledge areas in the field of pharmaceutical sciences. 

Among these improvements, the introduction of the computational 

simulation was, with no doubt, one of the greatest landmarks for 

the field’s development. Nowadays, there are two main targets in 

drug development, the G protein-coupled receptors (GPCRs) and 

the immunotherapeutics. 5-HT1B receptors have been targeted for 

the treatment of migraine, since they act in the brain arteries 

contraction. By the other hand, class C GPCRs are potent 

neuromodulators, though small attention has been dedicated to 

them, such as the GABAB receptors. These receptor acts as potent 

inhibitors of the neuronal signal, leadind to impairments in the 

neurotransmitters release and closure of ionic channels. Despite its 

pharmacological relevance, only baclofen is approved by US-FDA as 

a molecule targeting GABAB, being used to treat neuropathic pain. 

For the class of immunotherapeutics, the most remarkable are the 

monoclonal antobodies (mAtb) which impair the binding between 

checkpoint proteins of T cells and their ligands in cancer cells. The 

main target for these mAtb is the programmed cell death protein 1 

(PD-1) and its ligands, PD-L1 and PD-L2. In this sense, this work 

propose a quantum biochemistry evaluation of the interactions 

made by compound thar act in 5-HT1B, PD-1 e GABAB receptors 

aiming to deepen the knowledge and describe the key points for the

 docking and stabilization of these compunds within the binding site. 

Thus, we take into account the crystallographic structure of the 

three receptors, which were fractionated in amino-acid residues by 

the molecular fractionation with conjugate caps (MFCC) scheme for 

posterior quantum (DFT) calculation. By our results, it was possible 

to predict the relevance of the amino-acids that compose the 

binding site for the three receptors, including the residues ASP129, 

ASP352, ASP123, GLU198, ASP204, PHE330, LEU126, PHE351, 

ILE130, VAL201, VAL200, THR355 e ARG114, that are part of the 

binding site for dihydroergotamine-5-HTR complex, the residues 

SER130, GLY151, SER153, HIS170, TYR250, TRP278, GLU349,

VAL201, SER152, SER154, GLN348, ARG168 e TRP65, which 

compose the GABAB’s binding site for GABA, baclofen, SCH50911 

and 2-hydroxysaclofen, as well as the lisine residue K131 from PD-1

 as the most relevant for the coupling of pembrolizumab, nivolumab 

and PD-L1. These residues shown the most intense attraction of 

repulsion energies, forming the key point for the ligand’s anchoring 

and stabilization. Through these data, it was possible to identify and 

describe the most important regions for ligands and receptors, as 

well as explain and differentiate, in a molecular and energetic level, 

the binding affinity experimentally found for these ligands.

The Northeast region is one of the largest shrimp producers in Brazil, with emphasis on the states of Rio Grande do Norte and Ceará. One of the biggest problems that the shrimp industry has faced in the last decades is the increase in the infectious diseases incidence, which cause great economic losses for the sector. Among these diseases, the most common are those caused by White Spot Syndrome Virus (WSSV), Penaeus stylirostris densovirus (PstDNV) and Infectious Myonecrosis Virus (IMNV). The genome study of these viruses is of fundamental importance to understand mechanisms essential to their survival and, thus, to develop effective tools for detection and control. In this context, the general objective of this work was to study the IMNV, WSSV and PstDNV genomes and to contribute with new information on the biology of these pathogens. Chapter 1 describes the sequences study of the Totiviridae family members. The amino acid alignment of ORF1 indicates that the artiviruses are the only members of the Totiviridae family that have the 2A-like cleavage sites and two proteins previously predicted for the IMNV. A possible cleavage site upstream to the capsid protein has also been identified in these genomes. Protein models revealed conserved structures for the two proteins, indicating that they are probably involved in the formation of viral protrusions and RNA packaging. Chapter 2 addresses the sequencing and analysis of the genome of a Brazilian WSSV isolate (WSSV-BR). The genome was sequenced using the Ion Torrent platform and assembled using an alternate pipeline in which a WSSV chimera genome was used as the reference sequence. The genome had a size of 292,912 bp, 184 possible ORFs and nine homologous regions (hrs). Identity and phylogenetic analyzes have shown that Brazilian WSSV is closer to isolates from Thailand and Mexico and that probably these three viruses share a common evolutionary origin. In addition, it has been noted that the WSSV evolutionary history may be related to recombination events and that these events, somehow, can be traced by analyzing the homologous regions of WSSV. Chapter 3 discusses about the sequencing and analysis of a possible new Brazilian variant of PstDNV (PstDNV-BR17), which does not cause symptoms in shrimps in field. The identity comparison and phylogenetic analysis showed that the new isolate was part of the PstDNV infectious lineage, which was unexpected. Secondary and tertiary structure analyzes of viral proteins also did not show differences among Brazilian isolates. However, results of the 5'UTR region analysisrevealed peculiarities that suggest that this region may be involved in the PstDNV virulence mechanisms, which would explain the difference observed in the field. All these results together contribute to increase the knowledge about the main viruses that affect the global shrimp farming.

Oxidative stress occurs in cells when there is an imbalance between the amount of antioxidants and oxidants in favor of the latter, which therefore can cause more than 100 diseases. In order to combat this situation, exogenous antioxidants can be used. For this reason, there is a search for different sources of antioxidant molecules, including semi-synthetic or synthetic compounds. Polysaccharides are natural molecules whose antioxidant capacity varies according to the polysaccharide type and can be modified with polysaccharide conjugation with different groups such as gallic acid (GA). Chitosan and dextran are two of the most abundant polysaccharide families in the nature and have several applications. However, they have a low antioxidant potential, which restricts their use. In this work, using a green conjugation method, it was possible to conjugate GA to chitosan and dextran and the conjugation was confirmed by the phenolic contend dosage, infrared and nuclear magnetic resonance spectrometries. It was possible to incorporate 10.2 ± 1.5 mg GA/chitosan g, which resulted in a 2-fold increase in total antioxidant capacity (TAC), 5-fold increase in reducting power, and the occurrence of a ferric chelation activity (about 60%) not observed with native chitosan. In addition, the conjugate was also able to prevent the exacerbated formation of calcium oxalate crystals in vitro, which was observed with the native chitosan. As regards to dextran, it is worth noting that this is the first report of GA conjugation to this polysaccharide, which in this case was 36.8 ± 1.4 mg GA/dextran g. Due to its novelty, the process of conjugation of dextran with GA was subjected to a factorial design in order to understand how the variables (amount of peroxide and amount of GA) influenced the synthesis of conjugated dextran; in its antioxidant activities; and to obtain an ideal conjugation condition of GA to dextran. It was verified that the amount of GA in the conjugation process is inversely proportional to the antioxidant activities, and that the ideal condition for production of a molecule with maximum antioxidant potential is that produced with 1.2M of hydrogen peroxide and a 1:1 ration of GA to dextran. The ideal molecule had CAT of 60 mg AA/dextran g, copper chelation of about 50%, and a reducing power close to 90%. In short, the GA green conjugation method was efficient for both chitosan and dextran and in both cases it was possible to increase the antioxidant potential of these polysaccharides, indicating a feasible use of these derivatives as substituents of their native molecules in various applications.

Some antioxidant compounds are able to reduce the levels of reactive oxygen species (ROS) and consequently reduce the deleterious effects of ROS in osteoblasts, thus avoiding an imbalance in bone tissue homeostasis, since damage to osteoblasts converges to bone fragility. Fucans and fucoidans are polysaccharides rich in sulfated L-fucose, they are found mainly in brown seaweed, this group of polysaccharides are attributed pharmacological activities as anticoagulant, anti-tumor and antioxidant. Considering its antioxidant character and vast abundance of macroalgae on the Brazilian coast, the present study aimed evaluate the antioxidant and osteogenic potential of rich fractions of fucoidan from the brown macroalgae Dictyota mertensii – a tropical algae easily found in the Brazil’s northeast coast, through the presence of these compounds in pre-osteoblastic and mesenchymal cells. Thought techniques of precipitation in increasing volumes of acetone, 6 fractions rich in fucoidan (FRFs) were obtained from the D. mertensii algae: F03, F0.5, F0.7, F1.0, F1.5 and F2.1, confirming the presence of fucoidans by physicochemical analyzes. Except for F0.3, which only had relevant activity in the test of total antioxidant capacity, the other fractions had antioxidant activity in vitro in most of the assays evaluated. Hydroxyl radical and superoxide ions scavenging activity were not detected activity higher than 50% in none of the FRFs at the concentrations studied. Similarly, when MCT3-E1 pre-osteoblastic cells were exposed to an oxidative stress by hydrogen peroxide (H2O2) and treated with FRFs, the MTT reduction test showed that, except F0.3, all others FRFs had protective effect, with emphasis on F0.7, F1.5 and F2.1. This protective effect was also observed in the evaluation of the activity of caspase-3 and caspase-9, again showing that cells damaged by H2O2 and treated with F0.7, F1.5 and F2.1 fractions had less apoptotic activity, being protective for the cells. The activity of the antioxidant enzyme superoxide dismutase (SOD) such as alkaline phosphatase (ALP) – a marker of osteoblastic activity, are also increased in the MC3T3 cells treated with the FRFs, when compared to the control even in the presence of H2O2, this indicates a osteoblast differentiation. To evaluate the osteogenic activity of FRFs, human mesenchymal cells (Msc) were treated with the FRFs and evaluated the viability of these cells by the MTT reduction assay. As noted, the FRFs studied are not toxic to Msc cells when applied at up to 0.1 mg / mL; moreover, through cytochemistry, it was observed that on days 14 and 21 Msc treated with the fractions F0.7 and F1.5 increased the synthesis of collagen and alkaline phosphatase, the first one intensely more at the beginning and the second later. These data indicate a possible induction of these mesenchymal cells to osteoblasts, which is confirmed by scanning electron microscopy (Mev), through which it is observed mineralization points in the cells treated with these fractions, confirmed by energy-dispersive X-ray spectroscopy (EDS). The results described here indicate that F0.7 and F1.5 fractions obtained from D. mertensii algae as well as antioxidants present an osteogenic potential.

Thrombin, the main coagulation protease, is capable of activating countless cellular responses through activation of the protease activating receptor 1 (PAR-1), including several signalling pathways in cancer. In melanoma, activation of PAR-1 triggers cellular responses that favour the cancer development, such as platelet aggregation, proliferation, migration, oncogene expression and metastasis, as well as acting directly on angiogenesis, one of the primordial events in tumour biology. Thus, since thrombin intrinsically enables tumour progression through PAR-1, biomolecules capable of inhibiting this protease become interesting models in cancer research. The present work describes the structural characteristics and in vitro and in vivo antitumour potentials of a chondroitin sulfate isolated from the Litopenaeus vannamei shrimp cephalothorax (sCS). The compound was obtained after proteolysis, acetone treatment and purification by ion-exchange chromatography and desalted using gel filtration. sCS presented a 12KDa average molecular mass and analysis by enzymatic degradation and NMR revealed that the compound shows the unique GlcA 3-O-sulfate residue, as well as GalNAc 4-O-sulfate, GalNAc 6-O-sulfate and GalNAc 4,6-di-O-sulfate residues. Similar to heparin, sCS was able to inhibit important activities for tumour progression. sCS reduced 66% of murine melanoma (B16F10) cell migration in the wound healing assay and 70% when analyzed by the transwell assay. Although the shrimp compound was unable to inhibit cell proliferation or to induce apoptosis and/or cell death after 24h incubation, it showed an important long-term antiproliferative effect, reducing the tumour cell clonogenicity by 75% at 100 µg/mL concentration (p <0.001), as well as inhibiting the anchorage-independent cell proliferation, an important indicator of tumour malignancy. These results may be inferred by the inhibitory effect presented by these GAGs on the in vitro gene expression of genes related to melanoma development, such as Cx-43, MAPK, RhoA, PAFR, NFKB1 and VEGFA gene. sCS compound also inhibited, in vitro, crucial molecules for tumour aggressiveness such as melanin and TNF-α by 52% and 75%, respectively. These results may explain the significant reduction in melanoma tumour growth (89% reduction) and the decrease of IL-1β, TNF-α and VEGF plasma levels (32%, 65% and 88% inhibition, respectively) in mice treated with 300 µg/kg/day of sCS. Additionally, sCS and heparin reduced in vivo mRNA levels of all evaluated genes, including PAR-1, as well as the immunohistochemical labelling for PAR-1 and VEGF in tumour and lung samples. The data shown here point to sCS as a potential biotechnological model for the development of new molecules which may be used in cancer therapy at the molecular level.

Considering the importance of the work using medicinal plants and also to prospect biomolecules, the aim of this work was to evaluate the antioxidant and antiproliferative activity of cambuí (Myrciaria tenella (DC.) O.Berg) and chia (Salvia hispanica L.) extracts. It was obtained extract using fresh leaves and a serial extraction approach using apolar to polar solvent. Then, it was obtain the following extracts: hexane extract (EHC and EHSh); chloroform (ECC and ECSh); ethanolic (EEC and EESh); methanolic (EMC and EMSh) and final aqueous (EAFC and EAFSh). Furthermore, it was also made an aqueous extract (EAC and EASh). These extracts were phytochemically characterized by measuring sugar, protein and total phenol. It was also analyzed by TLC and UHPLC. The antioxidant activity was measured using different in vitro assays (CAT, reducing power, iron and copper chelation, hydroxyl, superoxide and DPPH) and in vivo assays (ROS, SOD-3 expression and survival under stress conditions). Moreover, the toxicity from these extract were also analyzed using cell culture and the Caenorhabditis elegans model. In relation to cell lines it was measured the MTT reduction on murine fibroblasts (3T3), the human cervical cancer cells (HeLa) and the gastric adenocarcinoma (AGS). The tumour cell lines were also analyzed by flow citometry. The Caenorhabditis elegans model was evaluating the egg hatching rates and animal size. The results obtained here showed that the M. tenella polar extracts (EEC, EMC, EAFC) had phenolic compounds and it was identified the presence of rutin in EEC and probably at EMC. In M. tenella nonpolar extracts (EHC and ECC) it was identified the presence of terpenes as ursolic acid. The in vitro antioxidant assays showed that these extracts were able to scavening ROS and chelate metals. Furthermore, in vivo antioxidante assays using C. elegans model showed that EEC was able to reduced ROS content by up to 74% when compared to untreated control. For cell lines, it was observed that M. tenella extracts did not change the MTT reduction, on the other hand it was observed a reduction on cell viability up to 82.61% and 84.68% for HeLa and AGS, respectively. Flow cytometry data showed that the cell viability reduction for AGS was correlated to necrosis (80.7%) and late apoptosis (16.5%). The data obtained for chia (S. hispanica) showed a similar phytochemical profile, the terpenes presence in EHSh and ECSh and the presence of ursolic acid. The presence of phenolic compounds in all extracts was verified by UHPLC. In addition, it was observed that these extracts were able to donate electrons to the oxidized substrate and to chelate metals as iron and copper. In vivo assays showed that the C. elegans pre-treatment with EASh reduced the ROS levels to 29.76%. Thus, it was observed that S. hispanica extracts were able to reduce the cell viability from HeLa and AGS cell lines by up to 82.62% and 66.5% and for HeLa it was related to necrosis (19.6%), on the other hand for the AGS was associated to initial apopthosis (8.09%) and to late apoptosis (7.16%). Then, M. tenella and S. hispanica leaves may be considered as a potential source of biomolecules with antioxidant and antiproliferative activity.

The use of medicinal plants has been a common practice in folk medicine. This knowledge has been based on the experience that it has been passed though many generations. However, many times this knowledge do not have any scientific base. The molecules responsive for these activities are the secondary metabolic produce as a response to environmental conditions (biotic or abiotic stress). The Mata atlântica biome has a rich flora, which needs to be studied for its biological activities, and its potential to identify new biomolecules. In order to do this, six extracts (HE, CE, EE, ME, WEE e WE) were obtained from Coccoloba alnifolia leaves and then it was evaluated the antioxidant and the biological potential using different in vitro and in vivo systems. First, it was measured the sugar content, proteins and phenolic compounds. Based on these results, qualitative experiments were carried out to determine the secondary metabolites present in these extracts. It was detected the presence of phenols, flavonoids, saponins, triterpenic, and steroidal nuclei and unsaturated steroidal nuclei and vitexin and isovitexin. After that, it was evaluated the antioxidant potential by seven in vitro assay: Determination of Total Antioxidant Capacity (TAC), DPPH, Redution Power test, Metal Ion Chelation, Copper Chelation, Superoxide Ion Scavenging test and Hydroxyl Radical Scavenging Activity. These assays allowed us verify that the extracts presented antioxidant activity at extract concentrations: 100, 250 and 500 µg/mL. It was observed the ethanolic, methanolic and aqueous extracts had activity in TAC, reducing power, scavenging of the hydroxyl radical. The Pearson´s correlation showed that these results may be associated to the presence of phenolic compounds and sugars. It was also evaluated the antioxidant potential using in vivo models as Caenorhabditis elegans. This assay showed that the ethanolic extract increased its survival by approximately 30% in the presence of an oxidant stressor. Furthermore, the ethanolic extract was able to decrease the basal reactive species generated by the basal metabolism from C. elegans. Moreover, the wheat coleoptiles system showed that some extracts may have phytotoxic activity. The third in vivo model used in this work was cell culture. When it was used the 3T3 lineage it was observed that these extracts were not cytotoxic. However, it was not observed any antiproliferative activity in tumor cell lineages. In sumary, the data obtained here showed that using in vitro and in vivo assays that these extracts in special ethanolic and aqueous had an interesting antioxidant potential and it will be important to go further in this research.

Commiphora leptophloeos (Mart.) J. B. Gillett, folk knwon as “Imburana de espinho”, is a native plant from Caating. This plant is popular used for diferentepatologies. However, few scientific data is available related to this biological activities. In this work, it was obtained extracts from fresh leaves through a serial extraction using different solvents in growing polarity order it was obtained the following extracts: hexanic (EH), chloroformic (CE) ethanolic (EE), methanolic (EM), residual aqueous (EAR). Besides, it was done another extract called as water extract (EA) similar as the one produced in folk medicine. The phytochemical characterization was done by measuring the phenolic compounds, total proteins and sugars. It was also done a qualitative assay by thin-layer chromatography (TLC) and by ultra-high performance liquid chromatography (UHPLC). Furthermore, it was also analyzed in vitro antioxidant activity and in vitro cell viability by MTT assay. The results obtained with TLC showed the presence of phenolic compounds, terpenes, flavonoids, phenolic acids, tannins and saponins in special for the EE and EM. The Co-TLC analysis showed the presence of flavonoids luteolin and isoquercetina for the EE and EM. Moreover, UHPLC analysis identified rutin in these samples. The antioxidant assays showed that all extracts have the antioxidant capacity in vitro, in special for the EE and EM. Based on these results, it was analyzed the EE effect on the animal model Caenorhabditis elegans. This in vivo assay showed that the EE was able to reduce in 50% of the ROS intracellular levels from this animal model. Furthermore, the MTT assay showed that EE was not cytotoxicfor 3T3 cell lineage; on the other hand, for the B16-F10 cell lineage (tumor cell) it was observed a 45% reduction. Taken together, these results propose that C. leptophloeos has a potential source of bioactive compounds with antioxidant and antitumor potential.

The inflammatory response is a process of defense of the organism, however its exacerbated action generates an imbalance, making necessary the use of anti-inflammatory compounds. The search for new molecules that act on the inflammatory and antioxidant response has intensified. In this scenario, heparinoid, purified from shrimp has demonstrated ability to modulate various processes of the inflammatory response, an interest in evaluating the production of cytokines and the reactive metabolites has arise, since these molecules have their production increased during the process inflammatory. For this, we evaluated the leukocyte migration and the levels of cytokines in a model of peritonitis induced by LPS. Then, we investigated a compound cytotoxicity, nitric oxide production and intracellular reactive species in LPS-induced macrophages. Lastely, by performing the antioxidants of the testes, determining a chelation of ferrous ions, as well as an inhibition of reactive species, superoxide anion and hydroxyl radical. In addition, we evaluated lipid peroxidation and radical DPPH. The purification results related to heparinoid were extracted efficiently. The compound also reduced the leukocyte rate by 50%, to the injury site. In addition to reducing the levels of all cytokines tested, with emphasis on TNF-α, which decreased by 57%. Heparinoid maintained 90% cell viability, even at the highest concentration (100 μg/mL), reduced the production of nitric oxide, and intracellular reactive species, suggesting an antioxidant action. In the ferrous ion chelation test, heparinoid reduced the levels of this ion by 48% at 100 μg/mL. The compound also decreased the production of superoxide anion and hydroxyl radical. In lipid peroxidation, heparinoid inhibited about 60% of lipid oxidation at the lowest concentration tested (10 μg/mL). With respect to DPPH, heparinoid showed a reduction of approximately 70% in the lowest concentration. Therefore, we may suggest that heparinoid would be modulating the inflammatory response via cytokines and antioxidant mechanisms.

Histoplasmosis is a worldwide fungal disease that affect mainly immunocompromised patients. In Brazil, the mortality rate of histoplasmosis may achieve up to 72%, mainly due the troubles in the diagnosis of this disease. The fungal culture is the choose method to diagnosis histoplasmosis, although may take up to 8 weeks to conclusive results. Histopathology, however, is unable to identify the infectious agent in the sample, although it is indicative of infection. The immunoassays have sensitivity influenced by immune status of the patient and clinical manifestations, and often cross-reacting with some other fungi or bacteria. Molecular methods have been applied in diagnosis of histoplasmosis. However, these methods are not available in many laboratories and require well-trained experts. The objective of this work was to develop a LAMP assay (Loop-mediated Isothermal Amplification) to detect H. capsulatum in clinical samples. Due to its isothermal character, this reaction does not depend of thermocyclers, allowing to analyze the result in an alternative way to the agarose gel. The LAMP assay has been developed to detect ITS region in the genome of H. capsulatum, acquiring good sensitivity (limit of detection of 1 fg/µL) and being able to detect all tested genetic variants. In addition, the LAMP assay was able to directly detect DNA extraction-free health blood spiked with yeasts of Histoplasma.

Toxoplasmosis is a zoonotic worldwide disease caused by the protozoan Toxoplasma gondii(T. gondii), which can infect several warm-blooded animals, including humans. The most severe symptoms are usually observed in immunosupressed patients and could even be fatal. Due to the great biological diversity of non-clonal T. gondii isolates circulating in the southern hemisphere of the terrestrial globe, serious complications such as encephalitis, myocarditis and ocular damage can also be observed in immunocompetent individuals. Recently, a study revealed that T. gondii is also associated to the delineation of neurodegenerative diseases and a few types of cancers. Advances in immunoinformatics have been contributing with strategies to improve research on immunodominant epitopes contained in proteins from microorganisms, which can be recognized by host B and T cells. The study aims were to screen, analyze and select B and T cell epitopes contained in the Toxoplasma gondii AMA1, GRA7 and SAG1 proteins, as well as to construct chimeric peptides formed by the junction of B and T cell epitopes, and to analyze their immunogenic potential in silico and in vitro approaches. Four chimeric peptides were constructed, which showed binding affinities with murine and human MHC molecules in silico analyzes. A multi-antigenic epitopes chimera (TgAGS / BsT) was constructed from Toxoplasma gondii AMA1, GRA7 and SAG1 proteins by in silico analyzes, and expressed in recombinant system. The potential as diagnostic markers was confirmed by IgG recognition in serum-positive (n=10) for Toxoplasmosis by ELISA. Through antigenic stimuli in PBMC culture from immunocompetent serum-negative and serum-positive Toxoplasmosis persons, the expression of CD25 ⁺ in CD3 ⁺ cells was induced in the positive group of persons, with significance results (p<0.05), when compared to negative individuals. In addition, the production of IFN- and IL-2 cytokines were found in supernatant cultures stimulated by the chimeric peptides. These findings open perspectives for the development of vaccines and diagnostic reagents for Toxoplasmosis through chimeric products formed by B-cell epitopes and T.

Bothrops snakes are responsible for about 90% of snakebites in Latin America. Antivenom serum therapy, however, still has low efficacy against local effects, hard access in some regions, in addition to high cost and potential risk of adverse reactions. In this context, the main objective of this work is to contribute with complementary alternatives to antivenom therapy, with emphasis on the species Bothrops erythromelas, which is a snake of epidemiologic relevance in Northeastern Brazil, but that, until this moment, still lacks further studies and that is not included in the antigenic mixture for production of Brazilian bothropic antivenom. For this, two main work fronts were conducted: (1) characterization of the local and systemic toxic effects induced by this venom, in order to better understand its toxicity and thus to develop more effective strategies for its treatment; and (2) evaluation of the effectiveness of the plant species Jatropha gossypiifolia against the toxic effects of the venom under study, aiming at its use as raw material for future antiophidic herbal products, which may complement the efficacy of antivenom therapy. Through the experimental envenoming studies in mice, it was observed that B. erythromelas venom produced an intense local inflammatory picture, involving the direct participation of enzymatic components of the venom, as well as endogenous inflammatory mediators, which can be used as therapeutic targets for the treatment of local envenoming. In relation to systemic toxicity, venom produced in mice quite pronounced effects on hemostasis, in addition to systemic hemorrhage and a certain degree of renal and hepatic toxicity, which could be visualized by means of the alteration of several haematological, hemostatic and biochemical parameters. Through inhibition studies of these effects, it is observed that, in general, the tested antivenoms in fact have limited efficacy against the enzymatic activities in vitro and the local effects of the venom in vivo, despite the presence of immunological recognition, which indicates the presence of cross-reactivity with highly immunogenic but little toxicologically relevant components to the toxic action of the venom. Aqueous leaf extract of J. gossypiifolia, in turn, was able to significantly reduce the local and systemic effects of venom, which could be associated with its direct action on snake toxins, as well as an indirect action on the endogenous mediators. An herbal gel for use as a topical adjunct in the treatment of snake envenoming was developed and promising results were obtained when the association of this gel with antivenom was found to be able to significantly improve the efficacy of the treatment of local tissue damage induced by B. erythromelas. Phytochemical analyzes indicate that flavonoids are the major compounds of the plant species, and may be, at least partially, the main class of substance responsible for the activities presented. In conclusion, the results obtained to date demonstrate the potential of the plant species J. gossypiifolia as an adjuvant in the treatment of bothropic envenoming, and this study could an initial kick-off for the future development of herbal antiophidic products with genuinely Brazilian raw material for complementation of the current antivenom therapy in the treatment of bothropic accidents.

Sugarcane is one of the most important producing monocultures in Brazil as a source of first generation renewable biofuel, as well as sugar and other derivatives. The edaphoclimatic conditions of the Northeast region may disfavor their cultivation, affecting the plants to bloom early. This has a negative impact and may lead to a reduction in the production of alcohol and sugar. Molecular studies using genes involved in the flowering pathway is of extreme economic importance. In this work, the main focus it the role of the ScCAM sequence (Calmodulin sequence in sugarcane). This sequence was previously identified related to flowering process. In order to understand the possible association of this sequence to flowering process in sugarcane different approaches were used here. It was used bioinformatics, protein-protein interaction (two-hybrid assays), analysis of transgenic plants Nicotiana tabacum with the super-expression cassette in antisense orientation and histological analysis from apical meristem from sugarcane from production field. First, bioinformatics analyzes allowed us to verify the conservation of the nucleotide sequence by comparing the ScCAM sequence with the sequences of AtCAM (Arabidospis thaliana), which presented 99% identity. A phylogenetic tree was done using the coding nucleotide sequences (CDS). It was observed a high conservation of calmodulin in plants. Based on these results, a 3D modeling from the ScCAM sequence was done. This model was overlapped to the AtCAM 3D model for the 4AQR crystal. This overlapped showed the EF-hands motifs and the entire protein structure. This data allowed us to propose that ScCAM may be a functional protein. After that, an interaction network was done using the AtCAM7 sequence using Cytoscape. The Clusters obtained were enriched and it showed proteins that may be interacting as flowering induction as well as it had a role in the oxidation/reduction homeostasis. The two hybrids assays identified some clones and tow of these clones had homology to A. thaliana ribosomal proteins L31-1 and L19-1 (Uniprot access: Q9SLL7). These results suggest that the ScCAM protein may interact with ribosomal proteins. The transgenic plants having the overexpression cassete in antisense (ScCAM-AS) orientation showed an increase in the germinated potential of the transgenic seeds ScCAM-AS, an increase of inflorescence branches number and fruit. Furthermore, it was observed an increase of root hair when compared to transgenic plants having the empty vector (control) or wild-type plants.

For leaves, it was not observed any changes at morphometric parameters. The only modification that was observed for these plants were the height, inflorescence and fruits. In parallel, it was also analyzed the shoot apical meristem from sugarcane plant at production field using early and late-flowering cultivar. This analysis showed that the flowering induction may be happen 30-60 days before for early flowering cultivar. Similar results were observed for the transgenic plants. In this way, the results obtained here reinforce the hypothesis that the CAM sequence may be associated to the flowering process as well as stress signaling pathway by modification from physiological processes, then it may be remodeling apical and root meristems. Moreover, changing flowering time and protein interaction network. In addition, the results obtained with the two-hybrid system suggest that ScCAM may act together with the associated ribosomal 60S protein in the participation of the formation of ribosomal subunits by activating or inhibiting certain genes.

The productivity of any cultivar is directly related to the stability of its genome. Then, if this genome suffers any damage or alteration in its sequence it can have consequences that can directly affect its development/growth. The response to DNA damage occurs through the repair that is achieved through different pathways, one of which is the base excision pathway (BER), whose studies in plants are based on Arabidopsis thaliana and rice. One of the obstacles in advancing research would be the complexity of the plant genome present in some cultivars of agroeconomic importance - such as sugarcane – because of this, many trials and studies use diploid model organisms. This work proposes to fill a gap in the knowledge of the BER in plants, especially for sugarcane. In a first work approach, a sequence homologous to the AP endonuclease of Arabidosis thaliana (AtARP) called the ScARP1 was used. This was the subject of an enzymatic characterization assay. The second approach was to identify and compare the different proteins associated to BER pathway in sugarcane and plants and evaluated its conservation, duplication. In order to make the first approach, the ScARP1 protein was cloned, expressed and purified. With th e protein purified different assays were done to evaluate its enzymatic efficiency (considering temperature, enzymatic cofactors and salt concentration), as well as the substrates that would be recognized by this enzyme. It was observed that ScARP1 only had AP endonuclease activity since it did not recognize other substrates with lesions. In addition, the existence of enzymatic complementation of ScARP1 in protein extracts from the Arabidopsis arp^-/- mutant was also examined. Partial complementation was observed. For the second approach of this work, it was considered the previous results, where a duplication in sugarcane was verified for the sequence AP endonuclease (ScARP1 and ScARP3), furthermore the recent work from the BER pathway in plants. Based on these data, a search was done on the sugarcane databases (SUCEST-FUN) for BER homology sequences in plants. The results obtained with this search were used for phylogenetic analyzes (via TaxOnTree) and Bayesian inferences (via BEAST v2.4.8) to verifying the occurrence of duplications on the BER components.The sequences found were characterized as the presence of conserved domains, besides some of them were modeled, creating hypothetical models. Some of the putative sugarcane proteins identified differ in their structure with the reference proteins of A. thalina, in addition to duplications were observed in some plant families except for others, indicating possible differences in the BER pathway mechanism. The results obtained with this work provided new information regarding characterization as well as the enzymatic characterization of the components of the BER pathway, increasing knowledge about this pathway in plant organisms.

Scorpions of the genus Tityus belong to the Buthidae family and are responsible for most of the reported envenomation accidents in Brazil. Envenomation is often characterized by local symptoms as pain, paresthesia, edema and erythema. Scorpion venoms are complex mixtures containing many toxic peptides and proteins. The study of toxic composition of scorpion venom is especially important for the development of effective treatments for cases of scorpionism. Additionally, toxins are molecules that may to have potential biotechnological applications. In this study, we present the proteomic and functional characterization of the Brazilian scorpions venoms Tityus stigmurus, Tityus neglectus and Tityus pusillus, species widely distributed in the northeastern of the Brazil. T. stigmurus is recognized as the main responsible for medical poisoning in the Northeast region. Although studies have been reported for this species, a large number of components of the venom remain unknown. Tityus neglectus and Tityus pusillus do not present study of chemical or biological characterization of the venom. The proteomic characterization of venoms were performed by bottom-up proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS) performed in a LTQ (Linear Trap Quadrupole) Orbitrap Velos. Peptide spectrum matching (PSM) search was performed using the PatternLab platform, and de novo sequencing using PEAKS Studio 8.0. The databases containing all the protein entries for order Scorpiones from UniProtKB (UniPro Knowledgebase) and NCBI (National Center for Biotechnology Information), and protein amounts was estimated using the normalized spectral abundance factor (NSAF). For the functional characterization, enzymatic assays as phospholipase A₂, hyaluronidase and fibrinogenolytic, PT and aPTT tests, antimicrobial activity, cell viability assay, nitric oxide (NO) production, lethality determination and evaluation of effects of envenomation were performed. The proteome analyzes for T. stigmurus revealed the presence of 2 new families of toxins, serine proteases and phospholipases A₂, described for the first time in this venom. The ions channels modulators peptides account for approximately 70% of the peptides identified, outlining the importance of neurotoxins. In the venoms of T. neglectus and T. pusillus, families of proteins such as NaTx, KTx, CRISPs, AMPs, hypotensins, serine protease inhibitors, serine proteases, metalloproteases, phospholipases A₂ and hyaluronidases were identified. The three venoms presented hyaluronidase and fibrinogenolytic activity with the prominent participation of metalloprotease enzymes. The antimicrobial activity was not verified, and the action of phospholipases was verified only in T. neglectus venom. Cytotoxic activity was verified in Hek cells for T. stigmurus and T. neglectus venoms. T. neglectus also demonstrated cytotoxicity for RAW 264.7 and 3T3, as well as modulating effect of nitric oxide production. The T. stigmurus venom showed high toxicity to BALB/c mice (LD₅₀ = 0.575 mg/kg) and the effects of poisoning were described. The biological tests associated with the technology of proteomic analysis contributed to a better elucidation of composition and characterization of T. stigmurus venom. The approach presented in this study comprised the most complete description of the molecular repertoire for T. stigmurus scorpion, which together with T. neglectus and T. pusillus constitute sources of molecules with potential pharmacological and/or biotechnological application.

Cryptococcosis, caused by the fungal species Cryptococcus neoformans or Cryptococcus gattii, is one of the most important systemic and/or opportunistic diseases in the world. Each species has four genotypes, usually accessed by PCR-RFLP of the URA5 gene, which present differences in their ecology, epidemiology, geographical distribution and antifungal susceptibility. Easier accessible molecular markers are attractive for rapid recognition of genotypes or relevant characteristics such as virulence and antifungal susceptibility. In this way, group I autocatalytic introns in the mitochondrial LSU rRNA were evaluated as potential molecular marker for the genotypes of C. neoformans and C. gatti, as well as their relationship to antifungal susceptibility. Seventy-seven Brazilian isolates were used, most of the genotype VNI (39 strains) followed by 20 VGII, 5 VNIV, 4 VNII, 3 VNIII, 2 VGI, 2 VGIII and 2 VGIV. The introns Cne.mL2449 and Cne.mL2504 were amplified in a single PCR with complementary primers to the flanking region of the introns LSU rRNA gene. PCR products showed a significant polymorphism between C. neoformans and C. gattii genotypes. Sequencing of the PCR products indicated that some strains had none, one, two, three or four introns followed. This new two introns, not previously described in the mitochondrial genome of Cryptococcus, were named Cne.mL2439 and Cne.mL2584 in C. neoformans and Cga.mL2439 and Cga.mL2584 in C. gattii. Cne.mL2439/Cga.mL2439 introns were classified as belonging to IB2, whereas Cne.mL2584/Cga.mL2584, as belonging IA1 subclass. Interestingly, genotypes with some intronless strains, VNI, VGII, VGI and VNIV, are those known to be more virulent and less susceptible to antifungal agents. Here, we observed that those intronless isolates had significant higher MICs values for 5-flucytosine. The findings suggest that these elements can be used as potential molecular markers for antifungal resistance. Finally, phylogenetic analyzes suggested high sequence similarity between the introns Cne.mL2449, Cne.mL2504, Cne.mL2439/Cga.mL2439 and Cne.mL2584/Cga.mL2584 with other mitochondrial introns present in the genes COX1, COX2, COX3, NAD5, ATP9, COB, LSU of fungi supporting the “introns early” hypothesis, as well as its dispersion to heterologous sites by reverse splicing.

Silver nanoparticles (AgNPs) have several biomedical applications, among them the antiproliferative potential. Numerous types of synthesis of silver nanoparticles are found in the literature, however, a type of synthesis less aggressive and more economically viable, a green synthesisl, has gained value among the others. In this synthesis process, it is necessary to use a reducing agent and stabilizer which are non-toxic to animal cells and to the environment. In this context, an association of biomolecules of marine origin with biological activities already described, such as the polysaccharide acids of algae, to the AgNPs gain importance. Many acidic polysaccharides (PA) extracted from marine algae are distinguished by their different activities, such as the algae Spatoglossum schröederi. These fucans have antioxidant, antiangiogenic, antitumor activity, among others. Due to the few reports of AgNPs synthesis involving algae polysaccharides, the objective of this work was to synthesize AgNPs with a rich fraction of alginic acid and fucan A from the S. schröederi algae by a green synthesis process and to test the AgNPs obtained on the lineage of melanoma B16F10. The PA of the S. schröederi algae was extracted by a proteolytic digestion process and fractionated with acetone. The higher yield fraction rich in alginic acid and fucan A,   F0.5 v, was used for synthesis of the AgNPs. The characterization of the AgNPs obtained contemplated an analysis of UV light absorption, chemical composition, average diameter, polydispersity, zeta potential, infrared spectroscopy (FTIR), dispersive energy spectroscopy (EDS), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The analyzes by UV-visible spectroscopy in the range between 400 and 440 nm confirmed the formation of AgNPs, they presented 196 ± 13 nm diameter, polydispersion values below 0.4 and negative zeta potential. The chemical dosages, FTIR and EDS revealed that most AgNPs are composed of acidic polysaccharides. The images obtained by SEM and AFM indicated a spherical format for AgNPs. The antiproliferative activity was evaluated by the MTT method against B16F10 cell lines and 3T3 fibroblasts, and by flow cytometry, its action on death and cell cycle was investigated. The AgNPs affected the ability to reduce MTT from the 3T3 and B16F10 lineages, but these were evidenced for a B16F10, the AgNPs (0.5 mg/mL) were able to reduce by around 50%, while a silver ion and F0.5 v at any of the concentrations influenced the reduction of MTT. Flow cytometry analyzes indicated a high labeling rate for annexin V and propidium iodide for the B16F10 cells exposed to AgNPs, in addition to an increase in the percentage of cells in SubG1 in detriment to the other phases. Additional studies are needed to fully elucidate the mechanism of antiproliferative action of AgNPs and to discover other properties of the fraction that are enhanced by the synthesis of nanoparticles. However, it can be concluded that it is possible to synthesize AgNPs with acidic polysaccharides from S. schröederi using a green method and that these AgNPs showed more pronounced antiproliferative activity than their precursors.

Sugarcane is monocot that has a huge economic importance as it is used for ethanol and sugar production. This plant may grow in tropical and subtropical regions. However, the abiotic conditions as drought, salinity, high temperatures may reduced its productivity. Considering this, the aim of this work was to study the effect of changes of gravity forces such as hypergravity and microgravity on sugarcane transcriptome. In order to make this, sugarcane plants were submitted to a VSB-30 flight with 06 min. of microgravity. When these plants were recovered leaves and roots were separated, RNA was extracted and sequenced using Illumina plataform. The sequence received was analyzed using different bioinformatics tools.The transcriptome analysis from leaves and roots allowed us to identify 1387 constitutive genes and tissue specific genes for vertical root (454 genes), horizontal roots (478 genes), vertical leaves (147 genes) and leaves horizontal (49 genes). The constitutive genes were analyzed using the DESeq, which identified 154 genes that were specific. On the other hand, the tissue specific genes were evaluated by AgriGO tool and it was observed using some bioinformatics tolls that sugarcane plants in vertical orientation had probably differential expression for genes associated to nitrogen metabolism. It was also analyzed the possible interactomic network and it was observed that the root tissue had possible a complex response involving different proteomic interaction. Interactoma tool showed the presence of 12 clusters for vertical roots and 13 cluster for horizontal orientation in roots. These networks identified suggested a possible relation to nitrogen metabolism and amino acid biosynthesis. This work allowed us to identifiy what may be affected in the transcriptome during the VSB-30 flight and the data presented here suggested possible gene/protein network that may be changed due to hypergravity and microgravity.

The bacterial is metabolically diverse and represents a huge source of molecules with new biotechnological applications. Although this kingdom it is very rich, microbiological techniques of cultivation and identification are limited to access and identify this favorable source. Soon metagenomics stands out as an innovative tool to study bacterial communities independently of cultivation, and in addition, to extract new proteins and organisms with biotechnological application. The present study aimed to identify, both taxonomically and functionally, microorganisms residing in the bacterial community of petroleum wells involved in the degradation of oil and the production of surfactant molecules. To do so, samples of production water and well rocks are subjected to DNA extraction, as well as the preparation of cultures subjected to selection of petroleum degrading organisms and surfactant producers. Subsequently, all samples were sequenced through new generation sequencing platforms. The bioinformatics results show that the communities living in the rocks and in the production water were predominantly belonging to the Halomonas and Marinobacter genera, respectively. While the results of the consortia classification show a predominance of degrading and biosurfactant producers of genera Microbacterium, Ochrobactrum, Devosia and Paenibacillus in the R2 consortium, and Sphingobacterium, Bacillus and Lysinibacillus in the R1 consortium. Functional tests show that such bacteria are producers of biosurfactants to stabilize an emulsion as well as reduce interfacial tension between oil and water. Using homology comparisons with a protein custom database, it was possible to identify that such surfactant molecules were synthesized in Lichenysin, Plipastatin and Pultisolvin operons. This data refers to the degradation tests by means of the oxy-redox indicator, showing that there was predominance of degradation routes of linear as well as polycycloaromatic alkanes. These results indicate that isolated reservoir consortia have potential to increase the fluidity of the oil by degrading it, as well as the production of surfactants that significantly reduce the interfacial tension between water and oil, therefore showing potential for biotechnological application.

Human mesenchymal stem cells (hMSC) differentiation into osteoblast follows a specific gene expression program, committing itself initially under Wnt and BMPs pathways influence then differentiating into osteoblasts. However, specifically activated pathways during the first three days of differentiation are still poorly studied. Considering next generation sequencing technologies efficiency, and the lack of characterization in early hMSC differentiation process, in this work we used Illumina RNA-Seq to investigate the changes in these cells transcriptome. We used ex-vivo cultures of two human umbilical cord veins. Data from the complete transcriptome were analyzed in Transcriptogramer for the production of an ordered list of functionally associated genes, obtained by the mean of the gene expression taken on neighboring genes in this list, thus facilitating their biological interpretation. To study gene ontology and gene expression profile design during osteogenesis, the metabolic processes and molecular functions significantly altered during the course of the differentiation process were analyzed in several tools (REVIGO, GOrila, PANTHER, LNCipedia and NONCODE) and properly described. During hMSC differentiation, an increase in expression of osteoblastic phenotype genes was observed as soon as the first day of differentiation started. Noncoding RNAs were also identified, depending on the evolution of the differentiation process, as well as genes involved in the formation of membrane rafts, on the third day of differentiation. During the third day of induction, cell differentiation regulation genes and other important genes on biological processes that precede differentiation, such as cell adhesion, signaling and external chemical factors response, have already increased expression. The study of gene expression during these first three days also revealed the decrease in the expression of groups of genes crucial to basal metabolism maintenance, RNA degradation and cytoskeleton organization, thus indicating that the cellular changes that lead the cell into differentiation may originate during the first three days of osteogenic treatment.

Leprosy is a chronic, slowly evolving infectious disease with a broad spectrum of clinical manifestations caused by Mycobacterium leprae. The state of Rio Grande do Norte has a low detection coefficient for new cases, but some localities have focal areas with high detection rates, such as the city of Mossoró, which had a detection rate of 39.73 per 100,000 habitants in 2012. After exposure to the bacillus, about 10% of people develop disease, with a spectrum of presentations, ranging from the tuberculoid pole (tuberculoid-tuberculoid, borderline-tuberculoid), the borderline (borderline-borderline), to the lepromatous pole (lepromatous-lepromatous). The World Health Organization also classifies the disease according to the number of lesions, for therapeutic purposes, so cases with up to five lesions are classified as paucibacillary (BP), and cases with more than five lesions, multibacillary (MB). Approximately one third of people with leprosy develop immunopathological reactions, classified as type I, type II and neuritis. The post-infection evolution with M. leprae is influenced by environmental factors and by the genetic repertoire of the host. Therefore, in order to contribute to the knowledge of the association of genetic factors with susceptibility to leprosy, the objective of the present study was to analyze single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy in the newborn. The study design was casecontrol with recruitment of cases of leprosy and contacts. The participants were phenotypically characterized according to exposure to M. leprae, considering the presence of anti-LID-NDO antibody and clinical presentation. The amount of antibodies varied according to the operational classification of the leprosy case and the type of reaction, being higher in MB cases and type II reaction. All participants were also genotyped using Immunochip. For analysis of the genotyping data were considered: leprosy vs contacts, reaction vs non-reaction, and anti-LIDNDO antibody rate. A total of 55 SNPs showed association with leprosy and antibody levels. In the leprosy vs contacts group, 10 SNPs showed association, 3 related to the immune response (FASLG, TNFS18, EBF1 and ICOSLG), as well as one SNP close to VDR, whose protein is related to immunomodulation associated with vitamin D3 in monocytes, macrophages and activated lymphocytes. In the reaction vs. non-reaction group, the association of 30 SNPs, 20 close to genes known as susceptibility to psoriasis, and one SNP close to the UBD gene were observed. In the continuous LID-NDO antibody rate results, the association with 15 SNPs, one related to the THEMIS gene, which encodes a regulatory protein in T-cell selection was seen. Among the 55 SNPs associated with one of the phenotypes, 4 are found in coding regions. The results indicate that Immunochip is a tool to identify genes susceptible to leprosy development and its reactions. Together the data suggest that susceptibility to leprosy and the development of leprosy reactions are linked to the cellular and humoral immune response of the host and could potentially be modulated.

Corn cob is an agricultural by-product that annually produces a huge amount of waste estimated at thousands of tons, and it is a source of xylan, a bioactive polysaccharide. In this article, silver nanoparticles containing xylan (nanoxylan) were synthesized using an environmentally friendly synthesis method. So for such, we extracted the xylan from corn cob using ultrasound. Proteins or phenolic compounds did not contaminate this xylan. In addition, HPLC analysis showed that it contains xylose: glucose: arabinose: galactose: mannose: glucuronic acid in a molar percentage ratio of 50: 21: 14: 9.0: 2.5: 2.5, respectively. The formation of nanoxylan was analyzed by UV–vis spectroscopy at kmax = 469 nm. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of silver and xylan in nanoxylan. Dynamic Light Dispersion (DLS) and Atomic Force Microscopy (AFM) showed size of ± 102.0 nm and spherical shaped nanoparticles, respectively. DLS also showed nanoxylans were stable for 12 months. Coupled plasma optical emission spectrometry (ICP OES) showed nanoxylan contain 19% of silver. Nanoxylan reduced viability of the promastigotes of Leishmania amazonensis (L. amazonensis) (IC50 25 μg/mL), while xylan was not effective. In addition, nanoxylan showed antifungal activity on Candida albicans (MIC = 7.5 μg/mL) Candida parapsilosis (MIC = 7.5 μg/mL) and Cryptococcus neorformans (MIC = 7.5 μg/mL). The data obtained here lead us to the conclusion that it is possible to synthesize silver nanoparticles with xylan and that these nanoxylans showed an antileishimanial and antifungal activities superior to the polysaccharide used for its synthesis, and that this can be used as a promising antiparasitic agent against these microorganisms.

Tityus stigmurus it’s the predominant scorpion specie in Northeast region from Brazil, it’s considered one of the main cause of scorpion accidents in this region. In T. stigmurus venom, which is a complex mixture of high and low molecular mass molecules, was identified an antimicrobial peptide denominated Stigmurin. Antimicrobial peptides are small molecules considered the first line of defense against microorganisms, they show broad spectrum action. Many authors have proved that these peptides can also be effective on cancerous cells. Mutation in these molecules sequence has been held aiming the increase in the activity maintaining its low toxicity. Therefore, it is proposed the characterization of the structure in silico and by circular dichroism, as well the antimicrobial, antiparasitic, anti-proliferative and hemolytic activities of two analog peptides from Stigmurin, denominated StigA3 and StigA4. We performed an in silico analysis for these peptides, where we were able to observe α-helix structure, which was confirmed by circular dichroism. We were also able to find that the analog peptides net charge and hydrophobic moment were higher than Sitgmurin’s, which can explain the increase in antimicrobial and antiparasitic activity. The peptides StigA3 e StigA4 showed activity on cancerous cells similiar to the native peptide, except when tested on a normal cell we were able to find that they’re less toxic. Therefore, these results indicate a potential biotechnological application for the analogs peptides, even as prototype to new therapeutic agents.

Caatinga is an exclusive brazilian biome, marked by semi-arid climate, hot, lack of rain, high evaporation, strong insolation all year round, shallow and Stony soils. This biome stands out for the high endemism index and scientific unfamiliarity. Plants show evolution of intrinsic mechanisms of external environmental signals perception and their respectives metabolic responses in order to survive in this environment with peculiar edaphoclimatic conditions. These responses may modify the secondary metabolites profile, increasing or decreasing the amount and even modifying their profile. This can be interesting in the metabolites used for medicinal purposes. Erythroxylum pungens, which has unexplored potential, is outstanding in Caatinga biome as bioactive alkaloids source. Thus, the objective of this work was to investigate total proteome and metabolic fingerprint of E. pungens besides tropane alkaloids bioprospection with cytotoxic potential evaluation. It was possible to identify, by mass spectrometry, seven known tropane alkaloids; 3-(2-methylbutyryloxy)tropan-6, 7-diol as well as 3-(2-methylbutyryloxy)tropan-6, 7-diol were isolated and characterized by 1D and 2D NMR for the first time. N,N-Dimethyl-1-H-indol-3-ethanamine was isolated and characterized from roots. In addition, the assay against five cell lines indicated a selective potential of 3-(2-methylbutyryloxy)tropan-6, 7-diol against prostate tumor cells. This result was reinforced by docking studies. Iit was possible to identify 1746 proteins in the leaves, 1779 in the stem and 1026 in the root of E. pungens. Through the recovery of the GO terms, it was possible to evaluate processes in which these proteins are involved, verifying the same process profile for three organs of E. pungens, with emphasis on biotic, abiotic, chemical stress responses, as well as responses to oxidative stress. Were observed significant proteins levels involved in photosynthesis of E. pungens leaves, photorespiration, as well as many proteins related to stress responses: proteins from 14-3-3 family, peroxidases, catalases, superoxide dismutase and shock proteins, which can also act on protein turnover. Five proteins homologous to tropinone reductase were still identified, and the study of alkaloid production in all organs suggested that even under chronic stress conditions, E. pungens maintains the production of these special metabolites. Finally, in E. pungens in water stress by irrigation suspension in greenhouse, it was observed that this species invest in the increase of proteins related to the responses to water stress, it significantly increases free proline content in cytosol which act as an important osmoprotector. Furthermore, the plant maintains alkaloids production that appear to be more related to development stage of the plant than stress condition in which it is inserted. The results of this work seek to contribute to knowledge of E. pungens phytochemistry and physiology, as well as to promote the species and consequently conservation of Caatinga biome.

Chromobcterium violaceum (C.violaceum) is a Gram-negative bacteria, which has been found at tropical and subtropical regions. Some proteomic studies performed with this bacterium have demonstrated its ability to adapt to environmental challenges such as high iron concentration and oxidative stress exposure. However, no study was made with this specie submitted to simulated microgravity (SMG). SMG refers to conditions in which the gravity is artificially reduced to less than 1xg. Life on Earth has evolved at 1xg and study SMG is important to understand the overall changes that organisms might face in space travels. Therefore, the aim of this study was to characterize the response of C. violaceum, as free-living model organism, cultured at MGS, using proteomics techniques in order to understand how this bacterium response to this stress. The SMG was achieved by rotating the vessel around the horizontal axis perpendicular to the gravitational vector in Rotating Cell Culture Systems (RCCS4). SMG was conducted at a speed of 40 rpm for a period of 12 hours to obtain the growth curve every 2 hours. Total protein extraction was made in two times: 5 and 12 hours, corresponding to early (MG5) and late (MG12) exponential phase, respectively. After trypsinization, samples were analyzed with Q-TOF mass spectrometer. In our results, we detected 155 proteins during MG5, from which 18 proteins were upregulated, 19 down-regulated and 17 proteins were exclusive when compared to GN5. In MG12 were identified 173 proteins, from which 17 were upregulated, 22 down-regulated and 28 exclusive when compared to control. When comparing the amount of proteins identified in both early (5 hours) and late (12 hours) exponential phase, we detected 212 proteins during MG5 and 192 during MG12 from which 144 of them are common to both phases. We also observed a decrease of C. violaceum growth at MGS when compared to bacterial cultures submitted to normal gravity. Proteins correlated with transcriptional processes and release of energy through aerobic pathways were down-regulated, while proteins involved with the inhibition of transcription, anaerobic pathway and cell survival had their expression increased, indicating a decrease in metabolism and proliferation of C. violaceum. MGS can cause alterations in respiratory metabolism, decreasing of transcription and translation rates and, consequently, C. violaceum proliferation. These data can be understood as a strategy for maintenance and survival at reduced gravity environment.

Glucanas são polissacarídeos com diversas atividades farmacológicas e biológicas descritas. Contudo, há pouco relatos sobre as atividades das dextranas com figuração do tipo α (alfa). Nesse contexto, dextranas (α-D-glucanas) extraídas da bactéria Leuconostoc mesenteroides, com massas moleculares de 10 (D10), 40 (D40) e 147 (D147) kDa e seus derivados fosforilados P10, P40 e P147 foram avaliados quanto ao seu potencial antioxidante, anticoagulante e imunomodulador pela primeira vez, com o intuito de elucidar compostos com atividades potentes e pouco tóxicas. Análises de espectroscopia de infravermelho, composição monossacarídica e dosagens químicas comprovaram que estas dextranas são o mesmo polissacarídeo, mas com massas moleculares diferentes, além de confirmar o sucesso da fosforilação. Nenhuma das dextranas tem atividade anticoagulante. No teste do poder redutor verificou-se que D147 e P147 foram duas vezes mais potentes que as outras dextranas. Por outro lado, as seis amostras tiveram atividade semelhante (50%) em sequestrar o radical OH. Frente ao sequestro do íon superóxido, apenas D10 teve uma atividade pronunciada (50%). D40 foi a única dextrana nativa com ação imunomodulatória, pois estimulou em dobro a proliferação de macrófagos murínicos (RAW 264.7) e dobrou a liberação de NO por essas células, tanto na ausência como na presença de LPS. Além disso D40 teve maior atividade sequestradora (50%) de peróxido de hidrogênio, o que fez com que ela também fosse a dextrana mais potente em inibir peroxidação lipídica (70%). Já P147 apresentou maior atividade quelante de íons ferro e cobre (~85%), além de aumentar o triplo a liberação de NO na ausência e na presença de LPS. P10 provou ser o composto mais efetivo sobre a proliferação de macrófagos. Os dados indicam que dextranas com massa aproximada de 40 kDa são as ideais para serem utilizadas como antioxidantes e imunomoduladores, podendo ter suas atividades potencializadas com o processo de fosforilação. Contudo, estudos futuros com D40 e outras dextranas de massa semelhante confirmarão esta hipótese.

Os polissacarídeos de algas marrons são compostos heterogêneos com importantes propriedades bioativas. A alga marrom Lobophora variegata sintetiza diferentes grupos de fucanas, polissacarídeos sulfatados com várias propriedades biológicas. Estes polissacarídeos foram obtidos por prévia delipidação das algas, proteólise e fracionamento em diferentes volumes de acetona. O presente estudo teve como objetivo, extrair e fracionar o polissacarídeo sulfatado com acetona 0,8v. A fração foi escolhida por exibir uma alta proporção de açúcares totais/sulfato (1.5) e foi denominada de LV. A fucana LV foi avaliada quanto a sua possível ação no processo inflamatório em ratos, ação anti-proliferativa contra células de carcinoma de cólon humano (HT-29) e ação imunomodulatória em macrófagos murinos (RAW 264.7). A ação de LV sobre a reação de hipersensibilidade por contato, mediadas pelo uso de óleo de cróton e oxazolona, foi confirmada pela redução significativa do edema inflamatório nas diferentes doses testadas. Os grupos de animais tratados com LV apresentaram uma diminuição significativa no recrutamento de células leucocitárias ao local da inflamação e redução do dano tecidual, comprovados pela baixa atividade da enzima mieloperoxidase. Essa fração também teve sua ação avaliada sobre células do carcinoma de cólon humano HT-29 e células de macrófagos RAW 264.7. Esta fucana mostrou ação citotóxica em altas concentrações (100 e 200 µg/100 µL) possivelmente pela indução de apoptose na linhagem celular HT-29. LV demonstrou também possuir efeito citostático nas fases S e G2/M e acumulo de células na fase G1. Esta fucana teve ação antioxidante sobre H₂O₂ em culturas em células HT-29. Em células RAW 264.7 foi visto ação proliferativa da LV e ação imunomoduladora acentuada com e sem lipopolissacarídeo (LPS). Em um estudo comparativo de fucanas de duas espécies de algas marrons, estruturalmente diferentes, L. variegata (LV) e F. vesiculosus (FV), pode ser visualizado efeito proliferativo sem dose dependência para LV enquanto que para FV foi observado um efeito anti-proliferativo dose-dependente em células RAW 264.7. As fucanas LV e FV produziram aumento nos níveis de óxido nítrico nas concentrações testadas. Foram também observados efeitos imunomoduladores expressivos para as citocinas IL-6 e TNF-α nas células tratadas apenas com LV, como nas células co-estimuladas com LPS. Os dados sugerem que LV ação anti-proliferativa, anti-inflamatória e imunomoduladora. Esta última, por vias distintas de sinalização.

Reserve mobilization, metabolite partitioning, and reserve-degrading enzyme activities were studied in sunflower seedlings cultivated in vitro under 12-h photoperiod or in the absence of light to investigate the involvement of source-sink relation and carbon starvation in the mechanisms that regulate reserve mobilization during the exposure to continuous darkness. At the first 24 h of treatment (acclimation phase), the absence of light did not affect growth, but restricted carbon and nitrogen utilization, indicated by sugar and amino acid accumulation in the different seedling parts. After 5 days of treatment (survival phase), extended exposure to darkness limited growth and retarded storage lipid and protein mobilization due to carbon starvation, evidenced by the depletion of carbohydrates in cotyledons and hypocotyl, as well as the consume of amino acids in hypocotyl and roots. Alterations in the source-sink relation may have been a response to extended darkness, instead of a mechanism utilized to regulate reserve mobilization, as these alterations cannot be associated with negative feedback mediated by metabolite accumulation. Storage lipid degradation depended, at least in part, on mechanisms that co-ordinately regulate the activities of lipases and isocitrate lyase, but the activities of acid proteases and amylases could not be directly related to the regulation of storage protein and starch hydrolysis, respectively. Taking these results together, it is possible to suggest that reserve mobilization in sunflower seedlings cultivated in the dark may be regulated by mechanisms that perceive the absence of light and predict carbon starvation, adjusting reserve utilization according to future energy demands to allow, at least in short term, seedling survival.

During the post-germinative development, drought and toxic salts concentrations can affect physiological and metabolic processes of cotyledonary tissues, such as growth, enzyme activity and mobilization of lipid reserves, besides the generation of a cellular redox imbalance. However, studies generally report metabolic changes throughout the development under stresses applied only at the time of sowing. Therefore, this study aimed to evaluate the phenological and physiological changes caused by drought and salt stresses applied at the same time during sowing or during the seed-to-seedling establishment. In addition, this study characterizes the activity of APX, CAT and SOD enzymes and APX, CAT, ICL and MLS mRNA expression of safflower seedlings subjected to stress only during the seed-to-seedling transition. We found that NaCl and PEG decreased the germination rate, root length, germination speed index, cotyledons water content and humidity. However, in both treatments was observed lower levels of lipid peroxidation, SOD and CAT activity and CAT expression. The activity and expression of APX remained high during treatments, as well as MLS expression. From this, we conclude that NaCl and PEG treatments affected the phenological and biochemical aspects evaluated during seed-to-seedling transition and that APX system and H2O2 content may be linked to the maintenance of photosynthetic establishment.

In recent discussions on environmental issues, air pollution has been considered an important environmental risk factor, and, consequently, a burden to human health. Several poluents are released daily by natural or human activities, causing the air to be improper and harmful to the welfare of humans and ecosystems. In the Amazon region, deforestation and forest fires have been causing damage to the exposed population. Studies already demonstrated that airborne particles can lead to serious cardiorespiratory effects, including DNA damage. Therefore, the aim of this study was to evaluate oxidative stress, mitochondrial integrity and cell death caused by organic chemical compounds from particulate matter smaller  than 10 μm (PM₁₀) originated from biomass burning of the Amazon forest, as well as the effects of retene, a biomass burning marker, in human lung epithelial cells (A549). It was evaluated reactive oxygen species (ROS) generation  (DCF and MitoSOX) and autophagy process by expression and distribution of LC3 in A549 cells exposed to 200 μg/mL and 400 μg/mL for 24 h and 72 h. Likewise, it was examined the effects of retene on oxidative stress on the concentrations of 3,3 ng/mL, 10 ng/mL and 30 ng/mL. Also, mitochondrial function and cell death was observed with TMRM and Mitotracker dyes and annexin and propidium iodide markers, respectifully. Regarding the extracted organic particulate matter, this led to the increased production of reactive oxygen species and intracellular mitochondrial superoxide. Additionally, PM₁₀ exposure triggered the formation of autophagosomes, suggesting increased autophagy. In the biological analysis with retene, the data showed that this compound led to an increase in reactive oxygen species and mitochondrial superoxide, hyperpolarization of the mitochondrial membrane, as well as increased mitochondrial content at all times. However, the retene was only able to induce cell death in the greatest concentration used and over a 72-hour period. From these results, it is important to emphasize the reduction of emissions by biomass burning, searching for new control policies. In addition, the toxicity of the retene raises an alert about the inclusion of this compound in the risk assessment of PAHs, mainly because it is a biomass burning marker.

Polissacarídeos de algas podem ter sua síntese, sua estrutura e propriedades farmacológicas modificadas devido a alterações de fatores ambientais. Porém, poucas algas já foram analisadas com essa ótica. A alga verde C. cupressoides var. flabellata Børgesen é uma alga abundante na costa do Rio Grande do Norte e já foi demostrando que esta alga coletada na mesma época, mas em praias com grau de salinidade diferentes, sintetizava polissacarídeos sulfatados (PS) com propriedades diferentes, inclusive atividade anticoagulante. Por isso, o objetivo do presente trabalho foi avaliar a influência do período de coleta e de parâmetros ambientais na composição química e atividade anticoagulante de PS obtidos de algas verdes do litoral potiguar, inclusive da C. cupressoides, bem como obter, caracterizar e avaliar o potencial anticoagulante de pelo menos um PS da alga C. cupressoides. Inicialmente, foram obtidos extratos ricos em polissacarídeos sulfatados (ERPS), por proteólise seguido por precipitação com metanol, da alga C. cupressoides coletada mensalmente, durante um ano na praia de Búzios, Nísia Floresta/RN. Observou-se que havia variações no rendimento da extração, composição química e atividade anticoagulante dos ERPS da C. cupressoides de acordo com o mês de coleta, sendo o mês de março aquele em que se obteve ERPS com maior potencial anticoagulante, vale salientar que esta atividade foi maior que a do Clexane®, uma heparina de baixo peso molecular comercial. Ao se analisar a influência de fatores ambientais do local de coleta no rendimento, composição química e atividade anticoagulante observou-se que existe uma correlação positiva significativa (p < 0,05) entre o rendimento da extração dos ERPS e a salinidade da água do mar e a insolação; para a quantidade de sulfato observou-se uma correlação negativa significativa (p < 0,05) com a salinidade da água do mar. Já a quantidade de açúcares totais teve uma correlação negativa significativa (p < 0,05) com: sólidos totais, sódio, cloreto e insolação. Como o mês de março foi o mês com ERPS com maior potencial anticoagulante, resolveu-se purificar, caracterizar e avaliar o potencial anticoagulante de PS extraídos neste mês. Após proteólise e fracionamento com volumes crescentes de acetona obteve-se quatro frações polissacarídicas da C. cupressoides (CCB-0.3, CCB-0.5, CCB-1.0 e CCB-2.0), como a CCB-0.5 apresentou maior atividade anticoagulante, esta foi submetida a cromatografia de troca-iônica e eluída em duas novas frações (FI e FII), após eletroforese em gel de agarose, coloração da lâmina com azul de toluidina e descoloração, observou-se o aparecimento de uma única banda em ambas as subfrações, o que indica a presença de uma única população de PS, o que permite inferir que estes PS foram purificados. Análises por cromatografia líquida de alta eficiência (HPLC) indicam que as subfrações FI e FII tratam-se de glucogalactanas. Estas glucogalactnas sulfatadas exibiram atividade anticoagulante pela via intrínseca (teste de aPTT), pela via extrínseca (teste PT) e pela via comum (teste TT) da cascata de coagulação. Um resultado interessante foi que a atividade no teste de aPTT das glucogalactanas sulfatadas foi similiar a atividade do Clexane®. Além disso, estes PS foram capazes de inibir parcialmente a trombina. Isto é indicativo que os PS da C. cupressoides podem estar agindo em diversas proteases da cascata de coagulação. Porém, mais estudos são necessários para explicar detalhadamente quais os alvos de ação destes polímeros. Por fim, analisou-se a influência do período de coleta e de fatores ambientais em ERPS de outras algas verdes do litoral do RN (Caulerpa prolifera, Caulerpa racemosa var. occidentalis, Caulerpa sertularioides e Codium isthmocladum) coletadas também mensalmente, durante um ano na praia de Búzios, Nísia Floresta/RN; e observou-se que, da mesma forma da C. cupressoides, houve variações no rendimento, composição química e atividade anticoagulante para os ERPS algas verdes C. prolifera, C. racemosa, C. sertularioides e C. isthmocladum. No entanto, um fato interessante é que cada alga responde de forma diferente as condições ambientais do local de coleta. Estes dados indicam que dependendo do período do ano que a alga é coletada, os PS extraídos destas espécies de algas podem ter suas estruturas químicas afetadas e, consequentemente, suas atividades biológicas poderão ser distintas. No entanto, como descrito acima, cada espécie de alga responde de forma diferente as mudanças ambientais do local de coleta. Estes tipos de estudos levam ao esclarecimento de quais seriam as melhores condições para se obter o PS com as características estruturais e biológicas de interesse, o que é fundamental para o uso destes polímeros na indústria.

A resistência aos antimaláricos disponíveis no mercado leva à necessidade do desenvolvimento de novos compostos com novos alvos farmacológicos. Os derivados de naftoquinonas são descritos como compostos líderes promissores para o desenvolvimento de fármacos antimaláricos. Em vista disso, nós avaliamos a atividade antiplasmodial in vitro de três derivados de hidroxinaftoquinonas contra o estágio intraeritrocítico assexuado de Plasmodium falciparum, assim como parâmetros toxicológicos in vitro e in vivo e investigamos um provável mecanismo de ação relacionado à via dos isoprenóides através de marcações metabólicas de precursores da via com trítio radioativo, complementado com estudos de docking com um template da octaprenil pirofosfato sintase. Os derivados de hidroxinaftoquinonas analisados tiveram boa atividade antiplasmodial, com IC₅₀ menor que 20 μM para a cepa 3D7 e menor que 50 μM para a cepa Dd2. A janela terapêutica é segura, com índice de seletividade variando entre 36,7 e 143,0. Os compostos não causaram hemólise nas doses testadas (10 e 50 vezes maiores que as respectivas IC₅₀), e não desencadearam sinais de toxicidade no teste de toxicidade aguda in vivo apesar de o composto 4a ter promovido esteatose hepática e hemorragia no tecido renal. Considerando um provável mecanismo de ação, os derivados de hidroxinaftoquinonas parecem inibir a síntese dos precursores isoprênicos, principalmente a menaquinona e o tocoferol e os estudos de docking revelaram nove possíveis interações com alta energia em quatro sítios de ligação diferentes com um template da octaprenil pirofosfato sintase. Em nossos resultados, o composto 4c foi o mais promissor, visto que possuiu o menor IC₅₀ no teste antiplasmodial in vitro, menor citotoxicidade in vitro e toxicidade aguda in vivo, além de ter inibido os três produtos da via dos isoprenóides testados, podendo ser considerado um candidato padrão para o processo de “hit-to-lead.

Acidentes com animais peçonhentos representam um sério problema de saúde pública em diversos países do mundo, destacando-se os acidentes ofídicos e escorpiônicos. No Brasil, as serpentes do gênero Bothrops são as de maior relevância clínica, evidenciando-se as espécies Bothrops erythromelas e Bothrops jararaca. Com relação aos escorpiões, os pertencentes ao gênero Tityus incluem os de maior importância médica, sendo o escorpião Tityus serrulatus o principal responsável pelos casos de maior gravidade. O tratamento para o envenenamento consiste em administração de soros antiofídico e antiescorpiônico. Vacinas que utilizam antígenos puros e vias de administração alternativa requerem o uso de adjuvantes potentes e um sistema de entrega de antígeno eficaz. Nanossistemas vêm sendo investigados como sistemas de entrega de macromoléculas terapêuticas. A quitosana, devido as suas propriedades, tem sido extensivamente investigada na formulação de nanocarreadores, particularmente de genes e proteínas. Este estudo teve como objetivo a obtenção de nanopartículas de quitosana (CNP) com base na gelificação iônica para a entrega de proteínas/peptídeos terapêuticos utilizados na imunoterapia e avaliação do potencial imunoadjuvante dessas nanopartículas na produção de soros antivenenos. CNP foram obtidas por gelificação iónica, com tamanho médio de 200 nm, caracterizadas físico-quimicamente e o perfil de liberação avaliado, demonstrando se tratar de um sistema de liberação modificado. A estabilidade dos sistemas foi avaliada por 7 semanas, observando-se uma maior estabilidade dos sistemas associados aos venenos. Animais experimentais foram imunizados durante 6 semanas com 100 µL de veneno das serpentes através injeções subcutâneas, em diferentes concentrações (5,0 ou 10,0%), encapsuladas em CNP ou associados ao hidróxido de alumínio (HA). Os resultados demonstram que os títulos de anticorpos obtidos para os animais vacinados com os nanossistemas foram equivalentes ou maiores aos obtidos para os animais vacinados com o HA, com a vantagem dos nanossistemas serem menos inflamatórios que o HA, exigindo uma menor quantidade de antígeno a ser administrada, por se tratar de um sistema de libertação modificada. Esse trabalho revela a obtenção, caracterização físico-química e avaliação da produção de anticorpos de um novo nanossistema, a base de venenos de animais peçonhentos e nanopartículas de quitosana, com potencial aplicação na soroterapia.

Dengue is considered the most important human arboviruses, and is a serious public health problem. This disease is endemic in tropical and subtropical regions, reaching more than 100 countries, which means the half of world population, and those epidemies have been appellant. It is estimated that Dengue virus could cause 390 millions of infections each year, and is an important cause of morbidity. Although this scenario, actually doesn’t exist a dengue antivirus still. Sorts of studies have showed antivirus activity of seaweed sulfated polysaccharides against enveloped viruses, which action seems to be associated to initials steps of infection process. In this study, was evaluated the potential antiviral activity of crude extracts rich in sulfated polysaccharides obtained from the brown algae Dyctiota menstrualis (EBDMens) and Dyctiota mertensii (EBDM), and green algae Codium isthmocladium (EBCl) and Caulerpa sertularioides (EBCS), tested against Dengue virus 2 (DENV-2) in Vero cell line, by using different methodological strategies (simultaneous treatment, post-infection treatment, cell pre-treatment, virus pre-treatment, adsorption, post-adsorption and penetration). The extracts cytotoxicity was evaluated by MTT reduction assay. The study of antiviral activity was determinated by quantifying viral RNA load using RT-qPCR, after 120 hours of infection. None extract has showed toxicity against Vero Cells in MTT reduction assay with 100 µg/mL concentration. All extracts have shown antiviral activity when added during the first 90 minutes of infection, and a significant reduction of viral RNA number of copies after 120 hours. The EBDMens and EBDM extracts were more efficient to cell and virus pre-treatment assays, and the first has showed higher antiviral activity than EBDM during viral adsorption. The EBCI and EBCS extracts have presented antiviral activity very similar in cell pre-treatment assay. EBCI was more efficient in the reduction of DENV-2 adsorption in Vero cell. However, EBCS has showed more efficient in cell pre-treatment and penetration assays. Between the extracts evaluated, EBCS seems to be more effective against DENV-2. These results have showed potential action of extracts rich in sulfated polysaccharides with antivirus activity, suggesting that they act in the initials steps of infection process of DENV-2.

Seaweeds are important sources of neutral and negative polysaccharides with important industrial and pharmacological potential. The brown macroalgae Lobophora variegata is known for presenting sulfated polysaccharides (heterofucans) and neutral (laminarins). In this work, it is shown getting 6 polysaccharide fractions (F0.3, F0.5, F0.8, F1, F1.5 and F2). From the data of physicochemical characterization of fractions, chose to F1 for purification of its polysaccharide components. Liquid chromatography of molecular exclusion analysis carried observation that F1 shows polysaccharide of low (~ 5 kDa) to high (> 100 kDa) molecular mass. These components were separated according to their molecular mass, using separation by molecular size devices. Thus, there was obtained subfractions with an average polysaccharide size of 5, 20, 40, 75 and above 100 kDa, named F1-5, F1-20, F1-40. F1-75 and F1>100 kDa, respectively. These subfractions were analyzed physico-chemically by agarose gel electrophoresis, high-performance liquid chromatography, as by analysis of their antioxidant, immunomodulatory and cytotoxic / antiproliferative properties. As a result, we detected a total sugar content above 55% in all subfractions, since proteins and phenolic compounds were observed. The fractions F1-5, F1-20 and F1-40 showed only glucose (glucans), as F1-75 and F1> 100 exhibit glucose, galactose and fucose (heterofucans). All subfractions showed chelating activity of metals, but only F1-75 and F1>100 were more effective in sequestering radicals. The production / release of nitric oxide (NO) by RAW cells was increased only in the presence F1-5 (10 µM, p <0.05), F1-75 (10 µM, p<0.001) and, especially, F1>100 (10 µM, p <0.001) whose presence has increased by about 12 times the amount of NO. However, when these cells were activated with LPS only F1-20 and F1>100 (10 µM) (p<0.001) yielded production / release of NO. The other fractions did not interfere in the LPS action. Only F1-5 and F1>100 stimulated the production / release of cytokines. Only F1>100 (10 µM) was cytotoxic to line 3T3 (fibroblast) (p<0.01). None of subfractions showed cytotoxicity effect to cell line RAW 264.7 (macrophages) or tumor cell line Hep-G2 (hepatocellular carcinoma). On the other hand, only F1-40 did not showed cytotoxic activity (p>0.05) against 786-0 renal carcinoma cells. The highest cytotoxicity values were obtained with the subfraction F1-20, in the case against B16F10 cells (melanoma). Flow cytometry data indicate that this subfraction promotes stopping of cells in the G1 phase of the cell cycle (10 µM, p<0.01). Nuclear magnetic resonance analysis led to the proposal that F1-20 is a β-glucan formed by β-(1à3) glucosides with branches, comprising a glucose residue at position 6. The ratio is nine glucose residues 1,3 connected to each branching point. The data point F1-20, being an antioxidant, immunomodulating and cytotoxic as a compound pluripotent whose potential should be further investigated.

Chromobacterium violaceum is a ß-proteobacteria commonly found around tropical and subtropical regions throughout the globe. It produces many metabolites with biotechnological properties such as antitumoral peptides, antibiotics and polymers that have potential to replace the oil-based ones. Although it has been extensively studied over the past 40 years, there are many aspects of C. violaceum that remains unclear until today. We have conducted a biochemical study on the homologous recombination (HR) machinery of C. violaceum, mainly in RecA and its paralog, RadA/Sms. We performed in vitro assays from initial and late steps of HR such as D-loop formation and branch migration, respectively, with their corresponding molecular actors and how RadA/Sms influenced each one. We observed cvRadA/Sms influences negatively D-loop formation promoted by cvRecA and through pull-down assay we have observed an interaction between these two proteins. We also observed the DNA-binding preference of cvRadA/Sms and cvRecA and observed that this protein binds preferentially to dsDNA instead ssDNA, unlike cvRecA. No involvement of cvRadA/Sms on branch migration reactions was detected. In this work, we also described, for the first time, the isolation, sequencing and annotation of a new plasmid from C. violaceum, which we named ChVi1 and has 44,236 base pairs, 39 predicted open reading frames (ORFs) and, possibly, two origins of replication. Most of the ORFs codes for hypothetical and structural bacteriophage proteins. By using restriction digestion and Next-generation sequencing (NGS) we also looked for the presence of a similar plasmid in other seven C. violaceum strains isolated from amazon region. Our analysis suggest the presence of a plasmid similar to ChVi1 in two of these strains. The present work describes for the first time a biochemical characterization of RadA/Sms and RecA from C. violaceum which have different roles in HR. Moreover, the discovery of ChVi1 opens a path to further explore C. violaceum’s biology.

Cancer is a term used to represent a set of more than 200 diseases, including malignant tumors of different localizations. There are several mechanisms that contribute to carcinogenesis: sustained proliferative signal, deregulation of cellular energy, evasion of apoptosis, angiogenesis induction, unlimited replication, among others. Among the main types of cancer, skin cancer stands out: arises in melanocytes and is the most common in Brazil, accounting for 30% of all malignant tumors registered in the country. Melanomas, at an early stage, can be treated with surgery, but the most advanced cancers require other treatments. In this work a Kunitz-type trypsin inhibitor was purified from Mimosa regnellii Benth (ITJ) legume seeds, partially characterized and evaluated for its toxicity against tumor cell lines, specifically acting in cell line B16-F10, with an IC₅₀ of 0.65 uM, showing no toxicity compared to non-transformed cell lines. Its ability to induce cell death by apoptotic pathway in mouse B16-F10 melanoma cells was evaluated by flow cytometry with Annexin V-FITC / PI markers. In addition, the inhibitor was also evaluated for its ability to: change the mitochondrial membrane potential, visualized by flow cytometry experiments using Rhodamine123 probe and confocal microscopy with Mitotracker Red marker; Reactive oxygen and nitrogen species release through specific probes visualized by microscopy techniques; Liberation of cytosolic calcium, an event that influences apoptosis activation; Inhibit angiogenic activity on rabbit endothelial cells through experiments of inhibition of new vessel formation in Matrigel; Analysis of VEGF expression by western blotting techniques and reduction of IL-6 expression analyzed by confocal microscopy; Inhibition of cell migration process by induction of wound assay, analyzed by microscopy and, finally, cell morphologic changes of B16-F10, analyzed by incubation with specific antibodies of extracellular matrix components and intermediate filaments of melanoma cells, conducted in fluorescence microscopy. All these combined results favor the construction of a possible ITJ action mechanism in the induction of cell death by apoptosis in B16-F10 cells, where the inhibitor would act primarily by altering the mitochondrial function, wherein the trigger initiation of the apoptotic process is the loss of mitochondrial membrane potential, release of cytosolic calcium and ROS, activating a death pathway dependent on caspases. These results suggest that ITJ has the potential to be used as a drug adjuvant treatment for melanomas, due to their specificity and low-dose when compared to other bioactive molecules.

Background Dog is the main domestic reservoir of Leishmania infantum and shows a wide spectrum of clinical manifestations, varying from mild weight loss to severe hepatosplenomegaly and body spread lesions, classical signs of canine visceral leishmaniasis (CVL). The goal of this study was to evaluate the gene expression profile of spleen and peripheral blood cells from Leishmania-infected dogs and investigate microstructural alterations in spleen, liver, gut and skin in order to determine possible molecular pathways and histopathological process underlying the mechanisms related to disease progression and resistance. Methodology: 21 animals were recruited to this study at Zoonotic Control Center in Natal-RN and a sub group was selected for transcriptomic studies. Spleen fragments were obtained from 5 mongrel dogs, one of them showing negative serology for SLA, two animals were classified as asymptomatic and other two as symptomatic. In addition, spleen, liver, gut and blood parasitism were estimated by qPCR. Total RNA was extracted and cDNA libraries were constructed and sequenced in an Illumina platform. The reads obtained were filtered and aligned against Canis familiaris genome using Bowtie. Differential gene expression was analyzed using the Cufflinks workflow, functional annotation and molecular features for each gene was obtained from Gene Ontology and KEGG database using STRINGdb. A fragment of liver, gut, skin and another spleen fragment were collected, processed and stained by HE and immunofluorescence for histopathological analysis. Results: We observed a marked difference in gene expression profile of spleen and peripheral blood samples from symptomatic dogs compared to asymptomatic ones. Approximately, 756 genes were upregulated in the spleens of symptomatics animals, as CTLA4, CCL2 and IL27, furthermore, about 1,190 genes were downregulated in the same group of animals, as BMP6, C1QB and C1QC. After an enrichment analysis for molecular pathways associated to visceral leishmaniasis pathogenesis, we found that NK cells activation, toll-like and NOD-like receptors pathways is highly expressed in the spleen of symptomatic ones, while GPI-anchors synthesis and immuneprotesome activation related genes is downregulated in these dogs. Additionally, we observed that in symptomatic animals, the cytoarchitecture of spleen is altered, where white and red pulp delimitations is partially lost. We still observed an intense inflammatory infiltrate in the liver, gut and skin of these animals. Confirmig the RNA-Seq findings, we observed by immunofluorescence CTLA4, CCR7 and NLRP3 is the spleen of symptomatic dogs. Conclusions: Taken together, these results suggests that misregulation of mechanisms classically described as acting in innate immunity is involved in CVL pathogenesis and an efficient antigen presentation maybe decisive for resistance to clinical signs development and tissue integrity during L. infantum infection in dogs.

Cana-de-açúcar é um cultivar muito importante economicamente por  apresentar diversos produtos finais como açúcar e bioetanol. A cana-de- açúcar é uma planta monocotiledônea cultivada em regiões tropicais e  subtropicais, seu genoma é octaploíde e devido a isto há uma  variabilidade genética nos diferentes cultivares. Um dos problemas  encontrado é o florescimento precoce para alguns cultivares, que pode  promover uma perda em até 60% na produção do suco, consequentemente na produção de açúcar ou bioetanol. Apesar de sua importância econômica, muito pouco é conhecido molecularmente sobre o processo de floração em cana-de-açúcar. Desta forma, neste trabalho nosso objetivo foi de caracterizar um gene homólogo a proteína HINT1, que foi identificado em bibliotecas subtrativas para florescimento, utilizando diferentes ferramentas moleculares. Os resultados obtidos com  os ensaios de dois-híbridos encontrou uma interação proteica com a proteína homologa à SUBTILISIN-like. Variações em sua estrutura gênica e na região promotora foram encontradas em cópias de cromossomos  artificiais bacterianos, além de duas proteínas distintas. Análises  filogenéticas mostraram que as sequências encontradas em  monocotiledôneas estão agrupadas. Outra análise realizada utilizando plantas contendo cassetes de superexpressão do gene HINT no sentido senso levou à baixa estatura vegetal, formação de órgãos fusionados e  indiferenciação meristemática, impedindo a formação de órgãos reprodutivos e consequente perda da produtividade. Já a superexpressão  do gene HINT no sentido antissenso gera plantas com apenas com  estatura menor que o controle, mas sem aparentes anormalidades nos  tecidos aéreos. Isto indica que HINT de cana de açúcar está relacionado ao desenvolvimento vegetal, havendo várias possibilidades de interações para a regulação do processo de indução floral.

O termo vitamina E refere-se a um grupo de oito compostos moleculares que diferem em estrutura e biodisponibilidade, sendo o RRR-alfa-tocoferol a forma mais ativa biologicamente. A composição de vitamina E no leite materno sofre variações ao longo da lactação, sendo o leite colostro mais rico neste micronutriente quando comparado ao leite de transição e maduro. Os recém-nascidos, especialmente os prematuros são mais susceptíveis a deficiência de vitamina E e para prevenir os danos causados por esta deficiência tem sido proposta a suplementação do neonato com este micronutriente, porém não existe consenso para realização desta intervenção. Assim, a suplementação materna com RRR-alfa-tocoferol no pós-parto pode ser uma boa alternativa para manter os níveis de alfa-tocoferol elevado por um período de tempo mais prolongado e, consequentemente, fornecer ao recém-nascido prematuro quantidades adequadas de vitamina E. Esse estudo objetivou avaliar o efeito da suplementação com 400 UI de acetato de RRR-alfa-tocoferol em mulheres com partos prematuros sobre a concentração de alfa-tocoferol no leite materno colostro, transição e maduro. Participaram do estudo 89 puérperas adultas saudáveis, que foram distribuídas no grupo controle (n = 51) e grupo suplementado (n = 38). Foram coletadas amostras de sangue e leite colostro logo após o parto (leite 0h), leite colostro 24 horas após a primeira coleta (leite 24h), leite de transição 7 dias após o parto (leite 7d) e leite maduro 30 dias após o parto (leite 30d). A suplementação no grupo suplementado foi realizada após a coleta de sangue e leite 0h. As análises de alfa-tocoferol foram feitas por cromatografia líquida de alta eficiência. Valores séricos de alfa-tocoferol menores que 516 μg/dL foram considerados indicativos de deficiência. A concentração média de alfa-tocoferol no soro das parturientes do grupo controle foi 1159,8 ± 292,4 μg/dL e no grupo suplementado foi 1128,3 ± 407,2 μg/dL, (p = 0,281). Todas as puérperas apresentaram estado nutricional em vitamina E adequado. Em ambos os grupos, foi possível observar que a concentração de vitamina E no leite colostro foi maior em relação ao leite de transição e maduro. No grupo suplementado a concentração de alfa-tocoferol aumentou em 60% no colostro 24 horas após a suplementação, passando de 1339,3 ± 414,2 μg/dL (leite 0h) para 2234,7 ± 997,3 μg/dL (leite 24h). Enquanto que o grupo controle os valores no colostro 0h e colostro 24h foram semelhantes 1512,0 ± 693,2 μg/dL e 1357,2 ± 735 μg/dL, respectivamente (p = 0,681). No leite de transição do grupo controle o valor de alfa-tocoferol foi 875,3 ± 292,4 μg/dL e no grupo suplementado 1352,8 ± 542,3 μg/dL, com aumento de 35% no grupo suplementado em relação ao controle (p < 0,001). No leite maduro foi encontrado 426,6 ± 187,5 μg/dL de alfa-tocoferol para o grupo controle e 416,4 ± 214,2 μg/dL para o grupo suplementado (p = 0,853). Apenas o leite 24h do grupo suplementado atendeu o requerimento nutricional de alfa-tocoferol (4 mg/dia) do recém-nascido. Não foram encontradas associações entre o alfa-tocoferol do soro e leite 0h, assim como entre o leite materno em suas distintas fases, tanto no grupo controle, quanto no grupo suplementado e tais resultados evidenciam que o transporte deste micronutriente para o leite ocorre de maneira controlada e limitada. Dessa forma, a suplementação materna com vitamina E eleva a concentração de alfa-tocoferol no leite colostro e de transição e não influencia a concentração no leite maduro. Apenas o aumento no leite colostro foi suficiente para atingir o requerimento nutricional do recém-nascido prematuro.

Sementes de plantas são reservatórios de moléculas com grande potencial para elaboração de bioprodutos e por este motivo uma especial atenção tem sido direcionada na busca de proteínas bioativas com ação antimetabólica e propriedades farmacológicas. Uma lectina das sementes de Canavalia maritima (CML) foi purificada em dois passos, pelo fracionamento com sulfato de amônio seguido pela cromatografia de afinidade em coluna Sephadex G-50. CML foi analisada por SDS-PAGE e parte de sua sequência de aminoácidos determinada por espectrometria de massas. Os 20 resíduos resultantes foram submetidos a uma procura local em bancos de dados e alinhados (BLASTp), resultando em 100% de identidade com a ConM. ConM foi testada contra eritrócitos do sangue periférico humano e foi constatado a ausência de toxicidade quando incubados com até 1000 µg/mL da proteína. Já contra células mononucleares, a lectina não foi significativamente tóxica nas concentrações de 1, 2,5 e 5 µg/mL, mas o foi a partir da concentração 7 µg/mL. Outros ensaios de citotoxicidade revelaram que a ConM não é tóxica para a linhagem RAEC quando testada com o dobro da maior concentração proteica que diminuiu a viabilidade das linhagens tumorais A549, 786-0, HT29 e HeLa no intervalo entre 24 e 72 horas, com as concentrações variando de 1 a 10 µg/ml. ConM foi capaz de aglutinar as formas promastigotas de Leishmania spp na concentração de 6,25 µg/ml. Quanto a ação contra Candida spp, nenhuma das concentrações testadas (1 a 500 µg/ml) foi capaz de interferir no crescimento. Quando avaliada a atividade bacteriostática, a ConM nas concentrações variando de 0,39 a 400 µg/ml não inibiu o crescimento de Escherichia coli, já para Staphylococcus aureusa concentração de 25 µg/ml se mostrou ativa, reduzindo o crescimento em 75 %. As lectinas de plantas são proteínas com atividades biológicas relacionadas a defesa, mas que podem ser utilizadas, heterologamente, como interferentes no metabolismo de outros organismos. O reconhecimento celular específico relacionado a estas proteínas as tornam alvos na busca e exploração de suas potencialidades biotecnológicas. Logo, se torna lógico pensar que estratégias para desenvolver novas moléculas e métodos de diagnósticos e tratamento contra células tumorais e patógenos são promissoras e devem ser desenvolvidas.

As vitaminas A e E são nutrientes que possuem natureza lipofílica e atuam em vários processos biológicos importantes, como a imunidade, reprodução, crescimento e desenvolvimento. Estas vitaminas são essenciais na fase inicial da vida e devem ser transferidas adequadamente da mãe para o filho durante a gestação e a lactação. A suplementação materna com vitamina A é uma das estratégias de controle de sua deficiência no grupo materno-infantil. Entretanto, estudos com animais evidenciaram que a suplementação com altas doses de vitamina A reduziu os níveis de alfa-tocoferol (vitamina E) no soro e no leite. Assim, o objetivo deste trabalho foi avaliar a influência da suplementação materna com vitamina A sobre a concentração de retinol e alfa-tocoferol nos leites colostro e maduro de lactantes. Puérperas a termo e saudáveis foram aleatoriamente distribuídas nos grupos controle (n = 44) e suplementado (n = 44). Amostras de sangue e leite colostro foram coletadas no pós-parto imediato e uma amostra de leite maduro foi coletada após 30 dias. O grupo suplementado recebeu uma suplementação com palmitato de retinila (200.000 UI), sob a forma líquida, imediatamente após a primeira coleta de colostro. O retinol e o alfa-tocoferol das amostras biológicas foram analisados por Cromatografia Líquida de Alta Eficiência (CLAE). Os pontos de corte séricos para deficiência foram 20 µg/dL para a vitamina A e 516 µg/dL para a vitamina E. As concentrações médias de retinol e alfa-tocoferol no soro das lactantes foram 46,4 ± 15,9 µg/ dL e 1.023,6 ± 380,4 µg/ dL, respectivamente, sendo consideradas adequadas. No grupo suplementado, verificou-se um aumento significativo dos níveis de retinol no leite colostro, 24 horas após intervenção (p<0,001). Entretanto, não foi observada diferença estatística entre a concentração de retinol no leite maduro dos grupos avaliados (p>0,05). Além disso, após a suplementação materna com vitamina A, houve uma redução significativa na concentração de alfa-tocoferol no leite colostro (p<0,05), que correspondeu a um declínio de 16,4% dos níveis de vitamina E, corroborando com resultados obtidos em estudos com animais. Por outro lado, a administração do suplemento não influenciou os níveis de alfa-tocoferol no leite maduro (p>0,05). Diante disso, conclui-se que a suplementação materna com altas doses de vitamina A aumentou os níveis desse micronutriente no leite colostro, porém, reduziu a biodisponibilidade do alfa-tocoferol nessa secreção, o que pode trazer prejuízos à saúde do neonato, que possui reservas limitadas de vitamina E ao nascimento.

Os produtos naturais derivados de plantas servem de matéria-prima para a síntese de fitoterápicos, sendo amplamente utilizados por várias populações em todo o mundo. Dentre estes compostos, destacam-se as saponinas e seus metabólitos. Seu intenso uso é decorrente de suas propriedades bioativas, somado a sua similaridade estrutural a hormônios. As saponinas derivam-se em sapogeninas, como, por exemplo, a Diosgenina e a Hecogenina. Além de serem utilizadas na indústria farmacêutica para a produção de hormônios e anti-inflamatórios esteroidais, estas duas sapogeninas também são empregadas como fitoterápicos, estando presentes também em vegetais que compõem a dieta de muitas populações. Apesar da vasta utilização destes compostos, existe uma falta de informações sobre a toxicidade genética, o que torna o consumo destas sapogeninas um risco para as populações. Diante deste contexto, o presente trabalho teve como objetivo investigar as potencialidades tóxicas, em nível celular e de DNA, da Diosgenina e da Hecogenina. Ensaios em células HepG2 para a avaliação da citotoxicidade, genotoxicidade, e mutagenicidade foram realizados. Considerando os resultados obtidos com a Diosgenina, esta sapogenina mostrou efeito citotóxico em concentrações acima de 30 μM, assim como induziu significativamente incrementos na frequência de danos no DNA e de micronúcleos. Por outro lado, a Hecogenina somente se revelou citotóxica acima de 150 μM, porém em concentrações abaixo deste valor, foi capaz de induzir danos ao material genético sem promover um aumento na frequência de micronúcleos. A análise do conjunto destes dados sugere que o consumo de alimentos, fitoterápicos ou de medicamentos que contenham a Diosgenina na sua composição apresenta um risco maior de toxicidade, quando comparados com as formulações a base de Hecogenina, uma vez que a Diosgenina apresenta um potencial mutagênico demonstrado por meio de um aumento significativo na frequência de micronúcleos que são biomarcadores de instabilidade genômica.

A produção de nanoparticulas de prata com extratos naturais tem sido apontada como uma excelente alternativa para potencializar ou fornecer novas aplicabilidades a extratos. Extratos de polissacarídeos sulfatados de algas (ASP) apresentam propriedades importantes nas áreas da nutrição e farmacologia, porém há poucos relatos da produção de nanopartículas de prata com extratos ricos em polissacarídeos sulfatados (SPN). Assim, neste trabalho sintetizou-se SPN de algas encontradas no Brasil: Spatoglossum schröederi, Dictyopteris justii, Sargassum filipendula  e Dictyota mertensii. A obtenção dos extratos ricos em polissacarídeos ocorreu por proteólise seguida por precipitação com metanol. A síntese das diferentes nanopartículas ocorreu com a adição de soluções de prata 1 mM em soluções dos diferentes polissacarídeos e mantidos em repouso. Posteriormente as amostras foram centrifugadas e liofilizadas. A formação SPN foi confirmada por espectroscopia UV/visível, microscopia eletrônica de varredura e microscopia de força atômica. O tamanho das SPN foi de 108 ± 2 nm; 82 ± 1nm; 288 ± 52 nm; 104 ± 2 nm para S. schröederi; D. justii; S. filipendula; D. mertensii, respectivamente e se manteve estável por catorze meses. Os potenciais zeta e as formas das SPN foram negativas e arredondadas, respectivamente, e resultados de diversos testes in vitro mostraram que as SPN potencializam as atividades antioxidantes de ASP. As SPN também foram biocompatíveis com células normais 3T3 (fibroblastos murinicos). Por outro lado, SPN de S schröederi e de D. mertensii tiveram atividade citotóxica (~60%) frente as células de melanoma murínico (B16F10) e acredita-se que a maior atividade citotóxica destas SPN ocorram devido aos seus pequenos tamanhos. SPN também tiveram grande capacidade antibacteriana. Nanopartículas de D. justii e S. filipendula tiveram os melhores resultados, sendo necessário somente 50 µg/mL para a morte da bactéria E. coli e 100 µg/mL para a morte de S. aureus. Hipotetíza-se que esta atividade ocorra por liberação de prata pelas SPN. SPN também foram capazes de induzir a produção de óxido nítrico e citocinas com perfil semelhante a os seus respectivos ASP, com exceção para SPN de S. schröederi. Em geral os resultados revelaram que a síntese de SPN a partir das ASP potencializa efeitos antioxidantes, citotóxico e antibacteriano, além de apresentar o mesmo efeito imunomodulador de seus respectivos ASP. Os dados obtidos levam a propor que a síntese de SPN constituiu-se como um possível mecanismo potencializador de atividades biológicas.

Alginate is a linear polymer compound of D-mannuronic acid(M) linked (1 → 4) at its epimer in C-5 of L-guluronic acid (G). An alginate was isolated and partially purified from brown seaweed D. delicatula (DYN). DYN was chemically sufated (DYS). DYS showed 22% of sulfation and the rate of total sugars was around 66 and 59% for DYN and DYS respectively. DYN DYS and showed a ratio M G of 0.86 and 1.1, respectively. IR signals at 1221cm-1 confirmed the sulfation of DYS. DYN presented anticoagulant activity about intrinsic coagulation pathway, while DYS did not alter the clotting times for aPTT and PT. DYN and DYS also showed antioxidant properties especially ferric chelation where DYN reached about 97% and the OH sequestration where both averaged 85%. In 3T3 cells, DYN indicated proliferation in 24h and 48h of incubation and no cytotoxicity. For tumor lines HeLa and b16, and DYN and DYS decreased significantly the cell viability, especially after 48 hours (about 71% of inhibition). Given the results obtained in this study, it is clear that the sulfation is not decisive for the activity of D. delicatula alginate. In general, DYS and DYN shown as potential compounds to be used pharmacologically, and also in the food industry for revealing be great antioxidant.

A pré-eclâmpsia é uma doença que afeta 3-8% das mulheres grávidas. Os fatores de risco para essa doença não são completamente compreendidos, mas incluem desregulação da resposta imune oriundos de defeitos na placentação, fatores ambientais e genéticos. O presente estudo teve como objetivo investigar associação variação na quantidade de receptores de citocinas pró-inflamatórias (IL-1R, IL-6R e TNF-αR) estariam envolvidos com a pré-eclâmpsia. Receptores de citocinas (IL-1R2, TNF-αR1 e IL-6R) foram avaliados em células mononucleares das grávidas normotensas (controle n=11) e grávidas com pré-eclâmpsia (PE, n=24). Mulheres com pré-eclampsia tinham peso mais elevado no início da gravidez (p=0.0171). Foi observado uma diminuição de monócitos clássicos, mas não de monócitos intermediários e não-clássicos na pré-eclâmpsia. A frequência dos receptores de citocinas proinflamatórias IL-1R2, TNF-αR IL-6R aderidos a membrana das subpopulações de monócitos (clássicos, intermediários e não clássicos) e linfócitos (CD3+CD4+ e CD3+CD8+) estavam diminuídas em pacientes com pré-eclâmpsia, quando comparados com grávidas normais. A redução na quantidade de receptores de citocinas IL-1R2, TNF-αR1 e IL-6R em monóciots e linfócitos pode ser um fator mantenedor do estado inflamatório na pré-eclampsia.

Envenenamentos causados por animais peçonhentos são um grave problema de saúde pública, enquadrando-se nesse cenário, principalmente, os escorpiões e as serpentes. Tityus serrulatus é considerado o escorpião mais venenoso da America do Sul, devido à alta toxicidade do seu veneno, responsável por causar acidentes graves, principalmente em crianças. A espécie Bothrops jararaca é uma serpente que possui no seu veneno uma mistura complexa de enzimas, peptídeos e outras moléculas. As toxinas do veneno de B. jararaca induzem respostas inflamatórias locais e sistêmicas. O tratamento de escolha para os casos graves de envenenamento é a administração intravenosa do antiveneno especifico. Porém, nem sempre esse tratamento está acessível para os moradores de zonas rurais, que fazem uso de extratos de plantas medicinais. Nesse contexto, extratos aquosos, frações e compostos isolados de Aspidosperma pyrifolium (pereiro) e Ipomoea asarifolia (salsa), usadas na medicina popular, foram objeto de estudo deste trabalho para avaliar a atividade anti-inflamatória nos modelos de peritonite induzida por carragenina e peritonite induzida pelo veneno de T. serrulatus (VTs), e nos no modelo de edema local e infiltrado inflamatório induzido pelo veneno de B. jararaca, administrados pela via intravenosa. Os resultados dos ensaios de citotoxicidade utilizando o ensaio MTT demonstraram que os extratos aquosos das espécies vegetais apresentaram baixa toxicidade para células provenientes de fibroblasto de embrião de camundongo (3T3). A análise química dos extratos por Cromatografia Líquida de Alta Eficiência revelou a presença do flavonóide rutina, na A. pyrifolium, e rutina, ácido clorogênico e ácido caféico, na I. asarifolia. Quanto à avaliação farmacológica, os resultados demonstraram que o pré-tratamento com extratos aquosos e frações reduziram a migração de leucócitos totais para a cavidade abdonimal no modelo de peritonite causada por carragenina e no modelo de peritonite induzida por veneno de T. serrulatus. E ainda, esses grupos apresentaram atividade antiedematogênica, no modelo de edema local causado pelo veneno de B. jararaca, e reduziram o infiltrado inflamatório para o músculo. Os soros (anti-aracnídico e anti-botrópico) específicos para cada veneno atuaram inibindo a ação inflamatória dos venenos e foram utilizados como controles. Os compostos identificados nos extratos também foram testados e, assim como os extratos das plantas, exibiram efeitos anti-inflamatórios significativos, nas doses testadas. Dessa forma, esses resultados dão evidências, às plantas estudadas, de potencial atividade anti-inflamatória. Esse é o primeiro estudo que avaliou os possíveis efeitos biológicos de A. pyrifolium e I. asarifolia, mostrando o potencial biológico que essas espécies possuem.

A leishmaniose visceral é uma doença endêmica em muitos países, incluindo o Brasil. O protozoário Leishmania infantum é o agente etiológico da LV, sendo este patógeno transmitido pela picada das fêmeas dos flebotomíneos, durante o repasto sanguíneo. A maior parte dos indivíduos quando expostos ao parasita não desenvolvem a doença, pois apresentam um predomínio da resposta celular Th1. Aqueles que desenvolvem, apresentam sinais como febre, perda de peso, hepatoesplenomegalia e um comprometimento da resposta imune celular, específica a antígenos de Leishmania. Nós avaliamos se essa anergia observada durante a doença ativa poderia estar associada com alterações nas moléculas coestimulatórias de linfócitos T ou em seus ligantes em monócitos CD14+. Encontramos que durante a leishmaniose visceral ativa há aumento na porcentagem de CTLA-4 em linfócitos T CD4+ (p=0,001) e ICOS em linfócitos T CD4+ e CD8+ (p=0,002 para CD4+ e p=0,003 para CD8+) após estímulo por antígeno solúvel de Leishmania (SLA), e que há maior porcentagem dessas moléculas ex vivo, quando comparados indivíduos sintomáticos aos recuperados (p=0,04 para CTLA-4 em CD4+, e p=0,001 para ICOS em CD4+ e p=0,026 para CD8+). Além disso, encontramos uma maior expressão dos genes CTLA-4, OX-40 e ICOS, durante a LV ativa. As moléculas CD40, CD80, CD86, HLA-DR e ICOSL aparentemente não sofrem alteração durante a doença. Há produção de IFN-γ, após estímulo por SLA, em indivíduos sintomáticos; no entanto, há diminuição na razão entre IFN-γ/IL-10, sendo observada um aumento desta após a cura. Podemos sugerir que há o comprometimento de algumas vias de moléculas coestimulatórias durante a leishmaniose visceral, e que isto pode ser usado pelo parasita para garantir sua sobrevivência e proliferação.

Marine sponges are presented as a rich source of new bioactive compounds of great biotechnological interest. Here in this research we report the purification and characterization of a lectin from Aplysina fulva species, called AFL, that presents immunomodulatory activity, and it was also evaluated whether it possesses cytotoxic effects on cultured cells (normal, tumor and Leishmania). Total proteins were extracted from the sponge with Tris-HCl buffer 20 mM, pH 7.5, and fractionated by precipitation with acetone. The fraction obtained with 1 volume of acetone had greater hemagglutinating activity and was subjected to a column chromatography on guar gum matrix with 20 mM sodium tetraborate buffer, pH 7.5. It was then applied to a Superdex 75 column (AKTA-FPLC) containing 20 mM Tris-HCl buffer, pH 7.5. The purity of the lectin was attested in a HILIC (HPLC) column. The sequence of 15 residues of N-terminal amino acid of the lectin was determined by Edman degradation and showed no similarity to any other protein of the known databases. The molecular mass was set at 62 kDa by gel filtration on Superdex 75. Analysis after treatment with reducing agents enabled to deduce that the AFL is a homotetrameryc protein. Their hemagglutinating activity was reduced when the lectin was exposed in an acidic medium (pH <4) or when subjected to temperatures above 60 ° C. AFL showed specificity for partial lactose and D-glucose and D-galactose. The purified lectin LFA showed no cytotoxic effect on cancer cell lines (PANC, HeLa, HT-29, Bf16F10, A549) and fibroblast (MRC5). AFL was able to agglutinate living promastigotes of Leishmania amazonensis and Leishmania braziliensis, and had their activity reversed by the addition of lactose to the test. AFL did not decrease significantly the viability of Leishmania amazonensis. The AFL lectin showed mitogenic activity at concentrations from 40 ug / ml for mononuclear peripheral blood cells (lymphocytes and monocytes). The AFL lectin was able of induce the macrophage murine lineage (RAW264.7) release of TNF-α, but not IL-6 at a concentration of 10.0 mg / mL. When LPA was incubated with spleen cells from BALB / c mice was able to stimulate the production of IFN-γ and to promote the differentiation of T cells to a cellular Th1 immune response. The ability to modulate the immune response of the Th1-type lectins has never been reported in marine sponges. All results confirm the high potential that the lectin AFL, that appears as a new molecule capable of modulating the immune system and can be used as an adjuvant for vaccines against microorganisms, including the gender Leishmania.

O presente estudo teve como objetivo extrair, caracterizar e realizar uma análise prospectiva de atividades farmacológicas de polissacarídeos sulfatados da macroalga verde Caulerpa prolifera. Sete frações (CP-0.3/CP-0.5/CP-0.7/CP-0.9/CP-1.1/CP-1.5/CP-2.0) foram obtidas de C. prolifera por proteólise alcalina seguida de precipitação sequencial em acetona. As análises físico-químicas indicaram que C. prolifera sintetiza diferentes populações de heteropolissacarídeos sulfatados. Na análise da atividade anticoagulante, todas as frações, exceto CP-0.3, apresentaram influência sobre a via intrínseca da coagulação. Todas as frações apresentaram atividade antioxidante em 6 ensaios diferentes, sendo mais pronunciadas no teste de sequestro de peróxido de hidrogênio, com destaque para CP-0.3, CP-0.7 e CP-0.9 (quando se obteve até 61% de sequestro), no teste de quelação férrica (com destaque para CP-0.9, com 56% de quelação) e no teste de quelação cúprica (com destaque para CP-2.0, com quelação de 78%). Com relação a ação imunomoduladora, a presença de CP-0.3, CP-0.7 e CP-0.9 promoveu aumento da produção de óxido nítrico (ON) em macrófagos em até 48, 142 e 163 vezes, respectivamente. De maneira oposta, a síntese de ON caiu em 73% quando macrófagos ativados por LPS foram incubados concomitantemente com CP-2. A atividade anti-adipogênica das frações também foi avaliada e o destaque ficou com CP-1.5, na presença desta fração (0,2 mg/mL) a diferenciação de pré-adipócitos (3T3-L1) em adipócitos foi reduzida em 60%. Vale salientar, que a viabilidade das células 3T3-L1 não foi afetada pela presença de CP-1.5. O que não ocorreu com células de câncer cervical humano (HeLa) e de adenocarcinoma renal humano (786-0), já que a viabilidade dessas células caiu quando elas foram expostas as frações, o destaque foi para a redução da viabilidade em torno de 55% quando as células HeLa foram incubadas com CP-0.3, CP-0.5 e CP-0.9. Já com 786-0, o melhor resultado encontrado foi com o uso da fração CP-1.5, quando se obteve uma redução de ~75% da viabilidade celular. Não se identificou atividade leishmanicida ou microbicida contra a Klebsiella pneumoniae Carbapenemase (KPC) com ao se testar as frações contra estes organismos. No entanto, a proliferação de Staphylococcus epidermidis foi diminuída em 30% na presença de CP-1.5. Outro resultado relevante, foi a observação de que a formação de cristais de oxalato cálcio na presença das frações era alterada. Destaca-se CP-0.3, CP-0.5 e CP-1.1, já que na presença dessas frações identificou-se apenas a formação de cristais dihidratados e de tamanho médio 80% menor do que aquele verificado no grupo controle. Dados oriundos da avaliação do potencial zeta dos cristais formados na presença das frações e da avaliação de imagens de microscopia confocal de cristais formados na presença das frações marcadas fluorescentemente levaram a hipótese de que os polissacarídeos presentes nas frações interagem com o cálcio da rede cristalina e isso afeta o crescimento e a morfologia dos cristais formados. Os resultados aqui descritos levam a sugestão de os principais agentes responsáveis pelas atividades observadas com as frações seriam os polissacarídeos sulfatados, portanto passos posteriores de purificação desses polímeros, bem como a investigação de seus mecanismos de ação devem ser investigados. Por fim, os dados aqui obtidos levam a observação de que as frações ricas em polissacarídeos obtidas da alga C. prolifera apresentam um potencial multiterapêutico por serem anticoagulantes, antioxidantes, imunomoduladores, citotóxicas contra células tumorais, anti-adipogenicos, antibacterianas e alterarem a formação de cristais de oxalato de cálcio.

A expansão agrícola utilizando cultivares geneticamente modificados (GM) é fenômeno mundial. Esse fato tem impulsionado a implementação de legislações regulatórias para monitorar à presença de variedades GM em culturas agrícolas. Outra importante demanda mundial está relacionada ao consumo de alimentos contaminados por aflatoxinas. Essas moléculas constituem um classe de compostos extremamente tóxicos que causam efeitos danosos para a saúde humana e animal. Por isso, a segurança e qualidade dos produtos agrícolas são essenciais para os consumidores. Os testes de fluxo lateral são métodos alternativos promissores que podem ser utilizados tanto para a detecção de proteínas transgênicas expressas em culturas GM quanto para a detecção de compostos tóxicos em alimentos. Essa técnica apresenta vantagens adicionais quando comparado aos métodos convencionais, como: simplicidade, rapidez e baixo custo. Nesse estudo foram desenvolvidos dois testes de fluxo lateral, um para a identificação das proteínas Cry1Ac e Cry8 Ka5 expressas em cultivares de algodão GM e o outro, para a detecção de aflatoxinas em produtos agrícolas. O teste, para a detecção dos cultivares transgênicos, foi desenvolvido no formato sandwhich. Esse teste foi desenvolvido utilizando os anticorpos monoclonais 1B1 e 5H4, produzidos contra a proteína Cry1Ac, mas que apresentaram reação cruzada para a proteína Cry8Ka5. O monoclonal anti-Cry1B1 foi conjugado com nanopartículas de ouro coloidal (40 nm) e utilizados como reagente de detecção. O monoclonal 5H4 foi adsorvido na membrana de nitrocelulose, na região denominada de linha teste e utilizado como reagente de captura do teste. Na linha controle, foi adsorvido o anticorpo anti-mouse IgG. Esses testes foram validados utilizando amostras de folhas de plantas de algodão GM ( Bollgard I ® e Planta 50- produzida em nosso laboratório) e folhas provenientes de cultivares não GM ( Cooker 312). Os resultados demonstraram que esse teste foi capaz de distinguir eficientemente amostras GM de não GM. Além disso, também apresentou elevada sensibilidade, sendo capaz de detectar 0,06 μg das proteínas respectivas nos cultivares transgênicos. O teste para a detecção de aflatoxinas foi desenvolvido no formato competitivo. O anticorpo α-AFLA 3B6, produzido contra AFB1, apresentou reatividade cruzada contra as aflatoxinas AFB2, AFG1, AFG2 e AFM1 e por isso, foi utilizado para o desenvolvimento das fitas-testes. Nesse teste o anticorpo 3B6 foi conjugado com ouro coloidal (40 nm) e utilizado como reagente de detecção. O antígeno AFB1 foi adsorvido na linha teste e utilizado como reagente de captura e o anti-mouse IgG foi imobilizado na linha controle do teste. Para a validação, grãos de soja contaminados com o fungo Aspergillus flavus, foram utilizados. Esses testes também foram avaliados quanto à habilidade de detecção de aflatoxinas em amostras alimentares, incluindo leite e proteína texturizada de soja. Os resultados demonstraram que a fita eficientemente identificou amostras contendo aflatoxinas. Além disso, apresentou sensibilidade de 0,5 ng/mL ou 0,5 μg/Kg. Os resultados obtidos sugerem que as fitas testes desenvolvidas podem ser utilizadas como método rápido e de baixo custo para o screening de cultivares GM, expressando as proteínas Cry1Ac e Cry8Ka5, quanto para a detecção de aflatoxinas em amostras alimentares.

Complicações trombóticas são a segunda causa de morte em pacientes com câncer e a terapia anticoagulante com heparina tem sido amplamente indicada. Além de prevenir a ocorrência de tromboembolismo, a heparina também é capaz de inibir passos importantes da progressão tumoral, contribuindo para a redução da agressividade do tumor. Entretanto, seu aproveitamento clínico é limitado especialmente pela ocorrência de hemorragias, o que tem estimulado a pesquisa por análogos de heparina (heparinoides) com efeitos colaterais reduzidos. O presente trabalho descreve as características estruturais e o potencial anticoagulante e antitumoral de um heparinoide com baixo risco hemorrágico isolado do cefalotórax do camarão Litopenaeus vannamei. A molécula foi obtida após proteólise, tratamento com acetona e purificação por cromatografias de troca-iônica e gel filtração. O heparinoide do camarão apresentou uma massa molecular média de ~ 15 kDa e as análises por despolimerização enzimática e RMN revelaram que o composto compartilha características estruturais com a heparina, como o alto grau de N- e 6-O-sulfatação, além do baixo grau de N-acetilação, e também com o heparam sulfato (HS), como o alto conteúdo de ácido glucurônico. Além de inibir a formação de coágulo pelo ensaio de aPTT, o heparinoide foi capaz de promover a estabilização térmica da antitrombina, o que pode justificar suas atividades anti-Xa e anti-IIa. Semelhante à heparina de mamíferos, o composto de L. vannamei foi capaz de induzir a síntese de HS antitrombótico por células endoteliais (ECs) em todas as concentrações testadas. Quando avaliado com relação a seu efeito sobre as etapas da progressão tumoral, foi demonstrado que o composto isolado do camarão reduz mais de 80% da formação de estruturas tubulares em matrigel, e 86% da migração de células de melanoma murino (B16F10) por uma membrana permeável. Embora tenha sido incapaz de inibir a proliferação dessas células durante um período de 24 h, a molécula em estudo apresentou um importante efeito antiproliferativo em longo prazo, reduzindo cerca de 80% da clonogenicidade das células tumorais nas concentrações de 50 e 100 µg/mL (p<0,001), bem como a proliferação celular independente de ancoragem, um importante indicativo da malignidade do tumor. Esses resultados podem explicar a significativa redução do crescimento tumoral em camundongos C57BL/6 tratados com heparinoide nas doses de 50 e 100 µg/Kg/dia (82,2 e 97% de redução, respectivamente). Os resultados obtidos aqui demonstram que o heparinoide de L. vannamei pode ser um modelo promissor para o desenvolvimento de novos agentes capazes de auxiliar no tratamento oncológico, uma vez que o mesmo pode ser efetivo não apenas em reduzir a progressão do tumor, com também em prevenir as complicações trombóticas que ocorrem no paciente com câncer. Além disso, o heparinoide ainda oferece a vantagem de ser obtido a partir de uma fonte de baixo custo e possuir uma atividade hemorrágica negligente.

O alginato foi extraído da alga Lobophora variegata e parte foi sulfatado para fins comparativos. O extrato nativo apresentou 42% de açúcar total, 65% de ácido urônico, 0,36% de proteína e 0% de sulfato, enquanto a sulfatada apresentou 39%, 60%, 0,36% e 27,92% respectivamente. A presença do grupo sulfato pode ser verificada através da metacromasia com o corante azul de toluidina no sistema de eletroforese e vibração característica em 1262,34 cm^-1 na espectroscopia de infravermelho, atribuído a ligações S=O. Observou-se a formação de filmes e esferas de alginato nativo, onde a solução mais concentrada 6%, resultou em um filme mais espesso e consistente. O alginato nativo apresentou atividade proliferativa nas concentrações (25 e 50µg), (50 µg) e (100 µg) em linhagem celular 3T3 em 24h, 48h e 72h, respectivamente, já o sulfatado em (100 µg) em 24h. Apresentou também atividade antiproliferativa ou citotóxica em células da linhagem HeLa, com  (25 e 100 µg), (25 e 100 µg) e (25, 50 e 100 µg), para o nativo,  já para a sulfatada nas concentrações (100 µg) em 24h, (25, 50 e 100 µg) em 48h, e (50 e 100 µg) 72h. Quanto a atividade antioxidante, os alginatos sulfatados apresentam melhor varredura do radical hidroxila (0,3, 0,62, 1,25, 2,5 e em 5 µg /mL) e atividade antioxidante total 29,2 equivalente ao ácido ascórbico  enquanto o nativo apresentou 7,8mg/g equivalente de ácido ascórbico. Em relação ao poder redutor o extrato nativo obteve variação da absorbância entre 0,672 a 0,821nm enquanto a sulfatada oscilou entre 0,475 e 1,31nm de inibição relativa ao ácido ascórbico que a 0,25mg/mL teve alta ação redutora  com absorbância de 1,505 mostrando um baixo poder redutor de ambas as amostras frente a esse antioxidante sintético. Na quelação do íon ferro o AN apresentou efeito dose dependente promovendo um efeito de 99,5% atingindo um platô a partir de 0,31 mg/mL equiparado-se ao EDTA, já o AS mostrou uma baixa atividade quelante com 6,5% a uma concentração de 5 mg/mL

A úlcera gástrica é uma patologia inflamatória na qual ocorre um desequilíbrio entre os fatores protetores e agressores da mucosa, nos quais a elastase de neutrófilo humano (ENH) está inserida e cuja hiperatividade corrobora no processo inflamatório. Os inibidores de tripsina e quimotripsina da espécie Erythrina velutina têm ação anti-inflamatória atestada. Logo, esse estudo tem por objetivo avaliar o efeito gastroprotetor por ação inibitória sobre ENH de isolados proteicos com atividades antitríptica e antiquimotríptica de sementes de E. velutina em modelo experimental de úlcera gástrica. Para tal, os isolados proteicos foram obtidos por extração, precipitação com sulfato de amônio e cromatografia em coluna de afinidade com matriz sepharose, derivatizada com as respectivas enzimas imobilizadas inibidas por cada isolado. Foi determinado para o isolado proteico com atividade antitríptica a IC₅₀, a Ki e a estabilidade em diferentes pHs e temperaturas para tripsina e ENH. Dois grupos foram receberam o isolado proteico com atividade antitríptica em duas concentrações (0,2 e 0,4 mg/kg), um terceiro grupo com isolado proteico com atividade antiquimotríptica (0,035 mg/kg), como controle negativo, um quarto grupo que recebeu cloridrato de Ranitidina (50 mg/kg) e no controle positivo, um último grupo que recebeu solução salina 0,9%. Após cinco dias de administração, foi induzida nos cinco grupos de animais (ratas wistar, pesando entre 180 – 220 g com 8 semanas), a úlcera gástrica com etanol 99%. Como resultado, o isolado proteico com atividade antitríptica, apresentou alta afinidade e atividade de inibição sobre ENH com valores de Ki e uma IC₅₀ de 0,4µg/ml e 1,0µg/ml, além de estabilidade e em uma faixa de temperatura de 37 a 100°C e de pHs de 2 a 14. Ambos os isolados proteicos foram eficazes em proteger danos contra à mucosa gástrica, principalmente as lesões hemorrágicas. Além disso, não apresentaram efeitos tóxicos aos animais. Dessa forma, os inibidores de serinoproteases com atividade antitríptica e antiquimotríptica se mostraram eficientes gastroprotetores, despontando como uma terapêutica alternativa na prevenção da úlcera gástrica.

Proteínas do reparo por excisão de bases (BER) têm sido associadas a funções além do reparo e DNA. A apurínica/apirimidinica endonuclease 1 (APE1) é uma proteína multifuncional envolvida em diversas atividades celulares como ativação redox de fatores de transcrição, processamento de RNA e reparo de DNA. Alguns trabalhos têm descrito a ação da proteína 8-oxoguanina (OGG1) na correção de lesões oxidadas no promotor como passo para a transcrição de citocinas pro-inflamatórias. Apesar de ser notadamente importante na ativação redox de fatores de transcrição, como o fator nuclear κB (NF- κB) e AP-1, a atividade de reparo de APE1 ainda não foi associada à resposta inflamatória. Neste trabalho, foram utilizadas análises bioinformáticas e abordagens experimentais para investigar a relação entre a inibição do reparo de sítios abásicos no DNA pela MX, molécula sintética inibidora indireta da atividade de reparo de APE1, e a modulação de resposta inflamatória. Os resultados demonstraram que o tratamento de monócitos com lipopolissacarídeo (LPS) e MX reduziu a expressão de citocinas, quimiocinas e receptores toll-like, e regulou negativamente processos biológicos da imunidade, como ativação de macrófagos, e as vias ativadas pelo (NF-κB), fator de necrose tumoral (TNF-α) e interferon, sem induzir morte celular. A análise transcriptômica sugere que o tratamento LPS/MX induz disfunções mitocondriais, estresse de retículo endoplasmático e ativação de vias de autofagia, provavelmente ativadas pelo comprometimento da energética celular e/ou pelo acúmulo de danos ao DNA, nuclear e mitocondrial. Adicionalmente, propõe-se que a atividade de reparo de APE1 é requerida para a transcrição de genes inflamatórios pela interação com sítios abásicos no promotores específicos e recrutamento de complexos transcricionais durante a sinalização inflamatória. Este trabalho apresenta uma nova perspectiva acerca das interações entre a atividade do BER e a modulação de resposta inflamatória, e sugere uma nova atividade para a proteína APE1 como modular da resposta imune de maneira redox-independente.

Muitas espécies se especializaram em viver nos mais variados ambientes existentes demonstrando a notável capacidade de adaptação do mundo microbiano as mais diversas condições físico-químicas. Ambientes expostos à radiação natural e a metais são escassos ao redor do mundo, apresentando uma microbiota ainda desconhecida. Com um número total estimado entre 4 e 6 x 1030 microrganismos na terra, estes constituem um enorme pool biológico e genético a ser explorado. Abordagens metagenômicas, independentes de cultivo, proporcionam uma nova forma de acesso ao potencial genômico de amostras ambientais tornando-se uma importante ferramenta para elucidação de funções ecológicas, bem como para identificação de novas espécies e biomoléculas. Neste trabalho, o material genético ambiental de amostras de solo e água do Açude Boqueirão de Parelhas-RN, sob influência de radiação natural e da presença de metais, foi extraído, pirosequenciado, e as sequências geradas foram analisadas através de programas de bioinformática (MG-RAST e STAMP). Perfis taxonômicos comparativos de ambas as amostras mostraram alta abundância do Domínio Bacteria, seguida por uma pequena parcela atribuída aos Domínios Eucaryota, Archaea e Vírus. Proteobacteria, Actinobacteria e Bacterioidetes foram os filos que mostraram maior dominância em ambas as amostras. Importantes gêneros e espécies associados à resistência aos agentes estressores encontrados na região foram observados. Sequências relacionadas à replicação e reparo do DNA, ao estresse oxidativo e estresse pelo calor e a resistência a compostos tóxicos foram observadas, mostrando uma importante relação entre a microbiota e seu perfil metabólico, influenciada pelas variáveis ambientais regionais. Os resultados encontrados neste estudo adicionam valiosos e inéditos dados sobre a composição de comunidades microbianas nestas regiões.

Polissacarídeos sulfatados (PS) da alga vermelha comestível Gracilaria birdiae foram obtidos através de cinco diferentes condições de extração (GB1: água; GB1p: água/proteólise; GB1s: água/sonicação; GB1sp: água/sonicação/proteólise; GB2s: NaOH/sonicação; GB2sp: NaOH/sonicação/proteólise). O rendimento em massa seca (g) das extrações aumentou na seguinte ordem: GB2sp>GB1sp>GB1p>GB2s>GB1s>GB1. A quantidade de PS extraídas por cada condição, em contrapartida, aumentou em sequencia diferente: GB2sp>GB1p>GB1>GB1sp>GB1s>GB2s. Espectroscopia de infravermelho e eletroforese em gel de poliacrilamida em agarose demonstram que todas as condições extraíram o mesmo polissacarídeo sulfatado. Além disso, com base nos dados referentes à composição monossacarídica de cada condição de extração pode-se pressupor que o uso de sonicação fez com que se obtivesse outros polissacarídeos além dos PS. No teste de tempo de protrombina (PT), o qual avalia a via extrínseca da cascata de coagulação, nenhumas das amostras apresentaram atividade anticoagulante até uma concentração máxima de 0,1 mg/mL. Por outro lado, no teste de tempo de tromboplastina parcial ativada (aPTT), cuja via da cascata de coagulação avaliada é a intrínseca, todas os extratos de PS tiveram atividade positiva, com exceção da condição GB2sp. A atividade anticoagulante no teste de aPTT diminuiu na seguinte sequencia: GB1sp>GB2sp>GB1p>GB1>GB1s>GB2s. O teste de capacidade antioxidante total (CAT) dos polissacarídeos sulfatados também sofreu influência das variações de acordo com condição de extração, visto que com Gb2s e Gb1 foram obtidos os menores valores de atividade quando comparado às demais condições de extração. No presente trabalho, conclui-se que as condições de extração de PS influenciam nas atividades biológicas e na composição química dos PS obtidos e que portanto, o uso de uma condição em especial pode favorecer a obtenção de PS com uma atividade que se deseja em detrimento de outras. De acordo com os dados obtidos pode-se afirmar que NaOH/sonicação/proteólise foi a melhor condição para obter PS com atividade anticoagulante e antioxidante em Gracilaria birdiae.

Compostos derivados de fungos tem sido alvo de muitos estudos a fim de ampliar o conhecimento acerca de seu potencial bioativo. Polissacarídeos de Caripia montagnei já foram descritos por possuírem propriedades anti-inflamatória e antioxidante. Neste estudo, as glucanas extraídas do fungo Caripia montagnei foram caracterizadas quimicamente e seus efeitos sobre as lesões intestinais foram avaliados em diferentes intervalos de tratamento no modelo de colite induzida por ácido 2,4,6 - trinitrobenzenossulfónico (TNBS), verificou-se ainda sua ação sobre células do carcinoma de cólon humano, HT-29. Na análise realizada no composto obtido de C. montagnei foi verificado que este é formado principalmente, por carboidratos (96%) apresentando um baixo teor de compostos fenólicos (1,5%) e baixa contaminação protéica (2,5%). As análises por espectroscopia de infra vermelho (FT-IR) e ressonância magnética nuclear (RMN) mostraram que os polissacarídeos desta espécie de fungo são   α e β -glucanas. O dano colônico foi avaliado por análises macroscópicas, histológicas, bioquímicas e imunológicas. Os resultados mostraram a redução das lesões no cólon em todos os grupos tratados com as glucanas (GCM). GCM reduziram significativamente os níveis de IL-6 (50 e 75 mg/Kg, p < 0,05), uma importante citocina inflamatória. As análises bioquímicas mostraram que essas glucanas atuaram na redução dos níveis de fosfatase alcalina (75 mg/Kg, p < 0,01), óxido nítrico (p < 0,001) e mieloperoxidase (p < 0,001). Estes resultados foram confirmados pela redução da infiltração celular observado microscopicamente. O aumento da atividade da catalase, sugere um efeito protetor de GCM no tecido do cólon, o que confirma o seu potencial anti-inflamatório. GCM mostraram atividade citostática sobre as células HT-29, causando acúmulo de células na fase G1 e impedindo, assim, a progressão do ciclo celular. As glucanas deste estudo também mostraram habilidade em modular a adesão de células HT-29 ao Matrigel® e reduzir o estresse oxidativo nessas células. A atividade antiproliferativa contra células HT-29 exibida por GCM (50 e 100 µg/ 100 µL, p < 0,001) pode ser atribuída à sua ação citostática ou indução da apoptose por essas glucanas.

A dengue é a mais importante arbovirose da atualidade, sendo transmitida pela picada do mosquito Aedes aegypti. A ausência de uma vacina profilática torna o controle da dengue pautado principalmente no controle do inseto vetor.  Todavia os crescentes relatos de resistência e os danos ambientais causados pelos inseticidas químicos têm tornado urgente à busca por alternativas seguras. Foram preparados 21 extratos brutos (EB) de sementes de plantas da Caatinga. Como extrator foi utilizado o fosfato de sódio 50mM pH 8.0. Os extratos foram utilizados em bioensaios Aedes aegypti, ensaios de toxicidade  e submetidos a uma caracterização parcial para identificar a presença de moléculas bioativas. As análises inseticidas foram realizadas por probit e as diferenças entre os grupos foram constatada por Student’s t-test e ANOVA. Todos os extratos apresentaram atividade larvicida para L1 e L4 e, com exceção do extrato de G. americana, levaram ao óbito 100% das larvas (48h). Os EB de M. urundeuva, P. viridiflora, E. velutina, A. cearenses  e E. contortisiliquum  apresentaram os melhores resultados larvicidas. Do mesmo modo, todos os EB foram repelentes para a postura das fêmeas grávidas. Os EB de D. grandiflora, E. contortisiliquum, A. cearenses, C. ferrea e C. retusa  foram capazes de atrair as fêmeas para a posturas quando em baixas concentrações. Na concentração atrativa o EB de E. contortisiliquum foi capaz de levar a óbito 52% das larvas L1 em 48 h, enquanto que o EB de A. cearenses resultou na morte de 100% dessas larvas em 24h. Os extratos de A. cearenses, P. viridiflora, E. velutina, M. urundeuva e S. brasiliensis tiveram atividade pupicida, enquanto que os extratos de P. viridiflora, E. velutina, E. contortisiliquum, A. cearenses, A. colubrina, D. grandiflora, B. cheilantha,  S. spectabilis, C. pyramidalis, M. regnelli e  G. americana tiveram ação adulticidas. Todos os extratos apresentaram toxicidade para C. dubia e os EB de E. velutina e E. contortisiliquum não interferiram na viabilidade dos fibroblastos. Considerando todos os ensaios biológicos os extratos de A. cearenses, P. viridiflora, E. contortisiliquum, S. brasiliensis, E. velutina e M. urundeuva  foram considerados os mais promissores. Em todos os extratos foram identificadas pelo menos duas proteínas com potencial. O EB de E. contortisiliquum foi selecionado para ser fracionando, já que não apresentou atividade pupicida, indicando que seu mecanismo de ação larvicida e adulticida resultou da ingestão de compostos ativos pelos insetos. Além disso, esse extrato apresentou o maior teor de proteína (22 mg/mL) e a maior atividade inibitória para as tripsinas das larvas de A. aegypti. Como observado para o EB, as frações proteicas de E. contortisiliquum,  também apresentaram atividade larvicida, mas não foram pupicida, com destaque para F2 que apresentou maio atividade larvicida e menor toxicidade ambiental do que o EB de origem. A redução na atividade proteolítica das larvas alimentadas com o EB e as frações de E. contortisiliquum sugeriu que  inibidores de tripsina seriam os responsável pela atividade larvicida. Embora o aumento na purificação de um inibidor de tripsina de E. contortisiliquum (ITEc) tenha resultado na perda da atividade larvicida,  foi constatado que sua  ausência reduziu a eficácia das frações, indicando que o ITEc contribui, mas não é suficiente para a atividade larvicida do EB de E. contortisiliquum. Também não foi verificada atividade larvicida e adulticida na fração rica em vicilina, e nem evidências da contribuição dessa molécula para a atividade inseticida desse extrato. Os resultados mostram o potencial dos extratos salinos de sementes de planta da Caatinga e, em especial do EB e da F2 de E. contortisiliquum, no controle da população de A. aegypti e indicam que a eficácia desses extratos deve resultar da ação conjunta de diferentes compostos ativos, inclusive de natureza proteica, que atuando em diferentes mecanismos são capazes de comprometer a viabilidade dos insetos e  retardar o surgimento de resistências.

A Amazônia representa mais da metade das florestas tropicais remanescentes no planeta e compreende a maior biodiversidade do mundo, correspondendo aproximadamente a 60% do território brasileiro. Entretanto, o desmatamento e as queimadas que ocorrem na região têm causado sérios prejuízos para a população que está sendo exposta. Diante desta situação, o objetivo do presente estudo foi avaliar os compostos químicos assim como os efeitos celulares e moleculares após a exposição ao material orgânico extraído do material particulado menor que 10 µm (MP10) na região Amazônica. Com relação à composição química, a análise dos n-alcanos mostrou um predomínio da influência antrópica no período de queimadas na região. Além disso, observou-se um predomínio dos monossacarídeos marcadores da queima de biomassa. Também foram identificados os Hidrocarbonetos Policíclicos Aromáticos (HPA) e os seus derivados nas amostras coletadas na Amazônia. Os dados das concentrações dos HPA permitiram calcular o BaP-equivalente e observou-se que o dibenzo(a)antraceno contribui com 83% para o potencial risco carcinogênico. Já para o potencial risco mutagênico, o benzo(a)pireno é o HPA que apresenta uma maior contribuição nesta análise. Pode-se destacar que o reteno foi o HPA mais abundante. Este composto foi considerado genotóxico, além de causar morte por necrose nas células estudadas.  Nas análises biológicas, os dados mostraram que o MP10 orgânico é capaz de causar alterações genéticas tanto em células vegetais como em células do pulmão humano. Estes danos levaram a uma parada na fase G1 no ciclo das células expostas, aumentando a expressão das proteínas p53 e p21. Além disso, o MP causou morte celular por apoptose, aumentando a  marcação da histona -H2AX. Com resultados bem evidentes, o MP inalável também causou morte por necrose nas células do pulmão humano. Diante destes resultados, é importante enfatizar a importância da redução e um melhor controle da queima de biomassa na região Amazônica. Afinal, como descrito recentemente pela Organização Mundial de Saúde, pode-se afirmar que a redução da poluição do ar poderá salvar milhões de vidas.

A leishmaniose visceral (LV) é endêmica no Brasil, sendo a região nordeste a que apresenta maior incidência, apesar de nos últimos 30 anos ter aumentado o número de casos em outras regiões do país. A LV na América Latina é resultante da infecção por Leishmania infantum. No entanto, nem todas as pessoas infectadas desenvolvem doença; na verdade, a maioria apresenta resolução espontânea da infecção sem sintomas característicos de LV. Tradicionalmente a avaliação do perfil imunológico tem sido realizada utilizando a reestimulação de células mononucleares de sangue periférico em cultura. Esses estudos revelaram que pacientes com LV apresentavam inibição tanto da proliferação linfocitária quanto da resposta pró-inflamatória anti-Leishmania. O presente trabalho teve por objetivo avaliar a resposta imune na LV sintomática, na cura pós-tratamento e assintomática. Para isso, analisamos características imunofenotípicas relacionadas à ativação, Treg e memória de linfócitos, por citometria de fluxo, assim como a produção de citocinas ex vivo ou em cultura de sangue total. Para os voluntários com LV, foi realizado um estudo longitudinal com reavaliação imunológicas aos 4 e 14 meses após a cura clínica. O grupo controle incluiu pessoas provenientes de região endêmica, sendo dividido em 2 grupos: Controle Positivo, formado por pessoas que apresentaram sorologia e∕ou PCR anti-Leishmania positivos e Controle Negativo formado por pessoas com sorologia e PCR anti-Leishmania negativos. Durante a LV os linfócitos CD4 apresentam um maior perfil de ativação e memória, além de serem maiores produtores de citocinas em cultura, quando comparado aos linfócitos CD8, contudo essa ativação não é Leishmania específica, visto que ocorreu tanto em ausência (CD4⁺CD25⁺:10,60%, CD8⁺CD25⁺:5,36%; p<0,0001) quanto em presença de antígenos solúveis de Leishmania (SLA), (CD4⁺CD25⁺: 10,20%, CD8⁺CD25⁺:3,28%; p=0,0003). Há ativação de linfócitos durante a LV (CD4⁺CD69⁺: 4,9%), quando comparado aos grupos Controle Positivo (CD4⁺CD69⁺: 1,96% p=0,0045) e Negativo (CD4⁺CD69⁺: 1,35% p=0,0062), mas esta ativação também não é Leishmania específica. O perfil de ativação linfocitária permanece elevado mesmo 14 meses após o fim do tratamento, porém após a cura a ativação é Leishmania específica (CD4⁺CD25⁺ em ausência de SLA: 8,44%, presença de SLA: 10,70% p=0,0279). Linfócitos CD8⁺CD25⁺ foram capazes de produzir IFN-γ em presença de antígeno tanto em Controles Positivos (ausência de SLA: 5,17%, presença de SLA: 9,52% p=0,0391) como em LV curados (Curado 4 meses: ausência de SLA: 3,90%, presença SLA: 10,70% p=0,0098). Células presentes no sangue total de pessoas com LV ativa são capazes de produzir IFN-γ em resposta ao SLA (IFN-γ em ausência de SLA: 3,61 pg∕mL e em presença de SLA: 44,26 pg∕mL; p=0,0020), assim como LV recuperado (IFN-γ em ausência de SLA: 2,29 pg∕mL e presença de SLA: 139,80 pg∕mL; p=0,0005). Contudo o elevado nível de IL-10 parece estar inibindo a atividade pro-inflamatória de IFN-γ e TNF-α em pacientes na fase sintomática. Contrariamente as demais citocinas pro-inflamatórias, a cultura de sangue total do grupo LV ativa não apresentou produção de IL-2 Leishmania específica (em ausência de SLA: 2,42 pg∕mL e em presença de SLA: 2,56 pg∕mL). Com base nesses dados nós concluímos que a restauração da ativação de linfócitos e a diminuição da produção de IL-10, Leishmania específica, estão relacionados à um perfil imunológico protetor.

Glioblastoma multiforme (GBM) é a forma mais frequente e maligna dentre os tumores cerebrais em adultos e tem sido considerado o tipo de tumor mais agressivo e letal. A radiação ionizante (RI) é considerada o tratamento mais eficaz contra o GBM. No entanto, o GBM é altamente resistente à RI e apresenta altas taxas de recorrência. Células-tronco tumorais, um subconjunto de células de um tumor com capacidade de auto-renovação e resistência inerente à terapia são responsáveis, em parte, pela recorrência do GBM. A manutenção do estado indiferenciado das células-tronco é a função principal da Musashi1 (MSI1), uma proteína de ligação a RNA altamente expressa em numerosos tipos de cânceres, incluindo os gliomas. Para avaliar o possível envolvimento de MSI1 em resposta à radioresistência, linhagens de células de GBM foram expostas a raios-X. Os níveis de expressão de proteína e de mRNA de MSI1 foram determinados por Western Blot (WB) e por qRT-PCR, respectivamente. Utilizando ensaio clonogênico investigamos o efeito do silenciamento de Msi1 (Msi1-KD) na sobrevivência celular e os níveis de danos no DNA após tratamento foram avaliados por meio da expressão de gama-H2AX e ensaio cometa. A comparação do transcriptoma foi realizada através de análise de dados do RNASeq por ferramentas de bioinformática. De acordo com os resultados obtidos, a RI foi capaz de induzir a expressão de MSI1. Além disso, o silenciamento de MSI1 foi capaz de promover uma diminuição na sobrevivência celular e um aumento dos níveis de danos no DNA em relação ao controle. A análise do transcriptoma indicou que MSI1 regula a expressão de genes envolvidos em diversas vias relacionadas à tumorigênese e radioresistência. Ao integrar vários conjuntos de dados, verificou-se que os genes altamente expressos em câncer e que são alvos para terapias contra o GBM também apresentaram um padrão diferente de expressão entre as células silenciadas e as controle. Em conclusão, os resultados sugerem que MSI1 têm um papel chave na resposta à radioresistência e levantam a possibilidade de MSI1 ser utilizada como um novo alvo para terapias contra o GBM.

Leishmania infantum e Trypanosoma cruzi são tripanossomatídeos de importância médica e são, respectivamente, os agentes etiológicos da leishmaniose visceral (LV) e da doença de Chagas (DC) no Brasil. Pessoas infectadas por L. infantum ou por T. cruzi podem evoluir de forma assintomática, possibilitando a transmissão destes patógenos através de transfusão sanguínea e/ou órgãos. A avaliação da infecção por  T. cruzi está contemplada entre os exames realizados para triagem de doadores no Brasil, no entanto, não há disponibilidade de exames para Leishmania. Os testes sorológicos para T. cruzi são muito sensíveis, mas não específicos, podendo apresentar reações cruzadas com outros microorganismos. Desta forma, o objetivo desse estudo foi determinar a prevalência da infecção por Leishmania em doadores de sangue e avaliar se os teste sorológicos para T. cruzi detectam L. infantum. Dentre as 300 amostras de sangue de doadores, descartadas em 2011, 61 eram T. cruzi positivas, 203 eram de doadores com outras infecções e 36 eram oriundas de bolsas com baixo volume de sangue, mas sem infecção. Foram também avaliadas 144 amostras de doadores sem infecções e apto a doarem sangue, totalizando 444 voluntários. DNA foi extraído do sangue de todas as amostras para realizar PCR quantitativa (qPCR) a fim de detectar DNA de Leishmania. O creme leucocitário obtido de todas as amostras foi cultivado em meio Schneider e NNN suplementado. Todas as amostras foram avaliadas quanto a presença de anticorpo anti-Leishmania. Os resultados sorológicos apontam um percentual de 22% de infecção por Leishmania nas amostras de sangue obtidas das bolsas descartadas. Um total de 60% das amostras positivas no ELISA para T. cruzi foram negativas na RIFI, teste usado como confirmatório, ou seja, 60% falso positivos para Chagas. Dentre essas amostras falso positivas para Chagas, 72% foram positivas no ELISA para Leishmania caracterizando a ocorrência de reação cruzada entre os ensaios sorológicos. Do total de 300 culturas realizadas, 18 cresceram parasitas que foram tipados por isoenzimas e qPCR específica, sendo encontrada a espécie Leishmania infantum nas culturas. Dentre as 18 culturas, 4 foram provenientes de bolsas expurgadas por baixo volume e negativas em todas as sorologias do banco de sangue, demonstrando assim que há um risco real de transmissão de Leishmania por via transfusional. Conclui-se que em área endêmica para leishmanioses no Brasil, diagnósticos sorológicos realizados para detectar infecção por T. cruzi em doadores de sangue podem identificar a infecção por L. infantum e, apesar de ocasionar resultados falso positivos para Chagas, essa reatividade cruzada reduz o risco de infecção por Leishmania via transfusão sanguínea, visto que não são aplicados testes específicos detecção desse parasita. Desde modo, persiste a necessidade de se discutir a implementação de um teste de triagem sorológica específico para Leishmania em países endêmicos como o Brasil.

A cana-de-açúcar é uma das principais culturas brasileiras e importante, principalmente, pela produção de açúcar e biocombustível. Por isso, manter a qualidade das cultivares desta espécie tornou-se alvo das pesquisas envolvendo genética e bioquímica moleculares. Um dos objetivos destas pesquisas é descobrir informações úteis sobre o material genético que as cultivares da cana-de-açúcar possuem, para utilizá-las como ferramentas no melhoramento contra intempéries que afetam sua produção, muitas vezes, de forma drástica. O foco deste trabalho é a via de reparo de DNA conhecida por Reparo por Excisão de Base, mais precisamente, a enzima DNA-glicosilase MUTM (formamidopirimidina-DNA-glicosilase), a qual reconhece e repara guaninas oxidadas no DNA. A caracterização dos genes MUTM da cana-de-açúcar foi realizada a partir das análises de quatro BACs (Bacterial Artificial Chromosome) de uma biblioteca genômica da cultivar R570. Os resultados obtidos dos alinhamentos mostraram a presença marcante de elementos de transposição. Além disso, foi verificado que os genes MUTM foram altamente similares aos de Sorghum bicolor, tanto em sequências nucleotídicas e peptídicas, como na estrutura gênica. Foi analisado também que as regiões promotoras de genes MUTM em algumas gramíneas apresentam vários elementos reguladores de expressão, associados com o estresse oxidativo, indicando uma regulação por estresse oxidativo.

A utilização de plantas medicinais para a cura e tratamento de diversas afecções é uma prática comum no mundo e também no Brasil. Em várias regiões do Nordeste brasileiro o cacto Cereus jamacaru, mais conhecido como mandacaru, é utilizado popularmente no tratamento de várias doenças, incluindo aquelas relacionadas com problemas cardíacos, respiratórios, úlceras gástricas, escorbuto, e problemas renais. Contudo, há uma escassez na literatura científica que comprove cientificamente a aplicação popular do mandacaru. Assim como outras plantas, Cereus jamacaru sintetiza várias moléculas potencialmente bioativas, como polissacarídeos. Neste trabalho, três extratos aquosos ricos em polissacarídeos, MCA80, MPM e MCP60, foram obtidos dessa planta e analisados quimicamente, bem como, foi avaliado os seus potencias como agentes antioxidantes e citotóxicos. Os dados mostraram que todos os extratos são constituídos principalmente de polissacarídeos (89,42 a 95,76%), porém foram detectados contaminantes proteicos (>2 %) e fenólicos (3 a 8,87%). Todos os extratos são ricos em galactose, glicose e manose. Além disso, de MCA80 e MCP60 apresentam ácido glucurônico em sua composição. Os extratos apresentaram capacidade antioxidante total variando de 55,21 a 68,13 equivalentes de ácido ascórbico (EAA). Porém não apresentaram atividade sequestradora de íon superóxido. Por outro lado, eles apresentaram atividade redutora e quelação cúprica de forma dose-dependente. Nenhum dos extratos inibiu a redução do MTT na presença das células tumorais de próstata (PC-3). Por outro lado, as células HEK, HeLa e 786 tiveram seu poder redutor de MTT inibido em diferentes níveis na presença dos extratos. MCP60 foi o extrato mais efetivo, impedindo a redução do MTT em cerca de 80% na presença das células 786. Ensaios de fragmentação nuclear mostraram que esse extrato induz morte celular. Os dados indicaram que mandacaru sintetiza polissacarídeos bioativos com potencial como agentes antioxidantes e antitumorais. Em estudos futuros pretende-se purificar esses polissacarídeos e caracterizar seus mecanismos de ação antioxidante e antitumoral.

Os polissacarídeos sulfatados compõem um grupo complexo de macromoléculas com uma gama de importantes atividades biológicas, incluindo atividade antiviral, anticoagulante, antiproliferativa, antiherpética, antitumoral, anti-inflamatória e antioxidante. Esses polímeros aniônicos são amplamente distribuídos em tecidos de vertebrados, invertebrados e algas. As algas marinhas são as fontes mais abundantes de polissacarídeos sulfatados na natureza. Os polissacarídeos sulfatados de algas verdes são homo ou heteropolissacarídeos compostos por galactose, glicose, arabinose e/ou ácido glucurônico. Para estes polímeros são descritas atividades como anticoagulante, anti-inflamatória, antiviral, antiangiogênica, antitumoral. Porém, ainda há poucos estudos de elucidação e avaliação de atividades biológicas/farmacológicas de polissacarídeos sulfatados obtidos de algas verdes, por exemplo, há somente um trabalho relatando a atividade antinociceptiva de polissacarídeos sulfatados destas algas. Portanto este trabalho teve como objetivo obter polissacarídeos sulfatados da alga verde Codium isthmocladum e avalia-los como possíveis agentes antinociceptivos. Assim, no presente estudo, o extrato total de polissacarídeos da alga verde C. isthmocladumfoi obtido através de digestão proteolítica, seguida de fracionamento resultando em cinco frações (F0.3; F0.5; F0.7; F0.9 e F1.2) por precipitação sequencial com acetona. Com o teste de contrações abdominais observou-se que a fração F0.9 foi o mais potente antinociceptivo. F0.9 é composta principalmente por uma heterogalactana sulfatada. Ensaios mais específicos mostraram que o seu efeito é dose e tempo dependente, chegando ao máximo com 90 após a administração (10 mg/kg de animal). F0.9 se associa a receptores TRPV1 e TRPA1 e inibe sensação dolorosa em animais. Além disso, F0.9 inibe a migração de linfócitos em ensaios de peritonite induzida. Por outro lado estimula a liberação de NO e TNF-α. Esses resultados sugerem que F0.9 tem potencial para ser usada como fonte de galactana sulfatada antinociceptiva e anti-inflamatória.

As sementes oleaginosas armazenam óleos, um recurso natural de alto valor econômico devido a sua aplicabilidade como substituto aos derivados do petróleo. Porém, o tempo de armazenamento pode inviabilizar a semente provocando a redução da qualidade do óleo e perda da viabilidade da semente. Por este motivo, esforços científicos têm sido realizados para proporcionar um maior tempo de armazenamento e incrementar taxas de germinação e estabelecimento destas plantas com alto valor econômico. O estabelecimento da plântula depende da capacidade do vegetal transpassar a etapa de transição funcional, caracterizada pela mudança do estado heterotrófico para o autotrófico. O consumo das reservas de óleo depende das vias de β-oxidação e do ciclo do glioxilato. No entanto, o processo de transição envolve a aclimatação do vegetal à condição de organismo fotossintetizante, que em geral conta com a participação fundamental do sistema antioxidante do vegetal devido à produção de espécies reativas de oxigênio em várias reações do metabolismo primário e secundário. Neste estudo, a Catalase foi inibida durante a transição funcional de girassol e cártamo para analisar os efeitos provocados nos sistemas antioxidantes de APX e da SOD e verificar as modificações no padrão de expressão das enzimas marcadoras do ciclo do glioxilato, ICL e MLS. Constatou-se que diante da inibição da Catalase o sistema antioxidante da APX e da SOD permitem o estabelecimento da plântula. Verificou-se também que as duas oleaginoasas parecem acelerar a mobilização de reservas e o estabelecimento fotossintético quando a Catalase tem a atividade drasticamente reduzida.

A vitamina E consiste em oito formas homólogas, denominadas, alfa, beta, gama, delta tocoferóis e tocotrienóis. Biologicamente, o alfa-tocoferol (α-TOH) é o mais importante. Comercialmente são encontradas duas formas de α-TOH, uma natural (RRR-alfa-tocoferol) e uma sintética (all-rac-alfa-tocoferol). A forma sintética é obtida através da reação da trimetilhidroquinona com o isofitol sintético resultando em uma mistura de oito isômeros ópticos, metade na forma 2R e outra metade na 2S. Ambas as formas são absorvidas no intestino, entretanto no fígado ocorre uma discriminação em favor das formas 2R, devido à proteína de transferência de α-TOH, apresentar maior afinidade a estes estereoisômeros. Para recém-nascidos em aleitamento materno exclusivo, o leite materno é a fonte alimentar para manutenção dos níveis séricos de α-TOH. Os neonatos são considerados grupo de risco para deficiência de vitamina E, principalmente os pré-maturos. Sinais clínicos como trombocitose, anemia hemolítica, fibroplasia retrolental, hemorragia intraventricular, displasia bronco pulmonar e degeneração espinocerebelar podem ser encontrados caso ocorra uma baixa ingestão de α-TOH. Sendo assim, a suplementação materna nos pós-parto com α-TOH pode ser uma forma eficiente de aumentar os níveis da vitamina no leite materno e consequentemente o fornecimento do micronutriente para o recém-nascido. Entretanto, a maioria dos estudos com suplementação de vitamina E foram realizados em animais e pouco se sabe sobre o efeito da suplementação materna em humanos, bem como, sobre sua eficiência para aumentar os níveis de α-TOH no leite humano, em função da forma natural ou sintética. Participaram do estudo 109 mulheres, distribuídas em três grupos: controle sem suplementação (GC) (n=36), suplementadas com a cápsula natural (G_NAT) (n=40) e com a cápsula sintética (G_SINT) (n=33). Foram coletadas amostras de sangue para determinação do estado nutricional materno, e de colostro no contato inicial e após 24 horas pós-suplementação. As análises foram realizadas por Cromatografia Líquida de Alta Eficiência. Valores de α-TOH no soro inferiores 499,6 µg/dL foram considerados como deficientes. Foram utilizados o teste de Kruskal Wallis e teste de Tukey para confirmar o aumento de alfa-tocoferol no leite e a eficiência das cápsulas administradas. O consumo diário de α-TOH foi baseado na ingestão diária de 500 mL de colostro pelo recém-nascido e comparada com o requerimento nutricional para crianças de 0 à 6 meses de idade, 4 mg/dia. As parturientes apresentaram concentração média de α-TOH no soro de 1016 ± 52, 1236 ± 51 e 1083 ± 61 µg/dL,respectivamente nos grupos GC, G_Nat e G_SINT. Não foram encontradas mulheres com deficiência. O GC não apresentou variação nas concentrações de α-TOH no colostro. Já mullheres suplementadas com as formas natural e sintética aumentaram as concentrações de α-TOHno colostro em 57,6% e 39% respectivamente. Ao comparar os grupos suplementados foi observado uma diferença significativa (p=0,04), sendo a cápsula natural mais eficiente que a sintética em aproximadamente 49,6%. Individualmente 21,1% das mulheres forneceram valores inferiores as 4mg/dia de α-TOH, após a suplementação este índice declinou para 4,1%. Sendo assim, a suplementação materna no pós-parto se mostrou uma forma eficiente para aumentar os níveis de alfa-tocoferol no colostro, com a forma natural com maior eficiência.

Sementes de leguminosas são alimentos que compõem a base das dietas de diversas culturas ao redor do mundo, tendo uma importante contribuição nas necessidades diárias de nutrientes dos seres humanos. As globulinas 7S (vicilinas) são proteínas de reserva encontradas em sementes de leguminosas, e podem apresentam uma função adicional de defesa constitutiva do embrião contra pragas e patógenos. Neste trabalho uma vicilina de Anadenanthera macrocarpa - AmV (Angico-vermelho), foi purificada e caracterizada parcialmente. Seu efeito sobre o desenvolvimento, sobrevivência larval e emergência dos adultos de Callosobruchus maculatus foram avaliados pela determinação das LD₅₀, WD₅₀ e ED₅₀ em sistema de bioensaio. A purificação da vicilina foi iniciada por cromatografia de afinidade à quitina e posteriormente em cromatografia de gel filtração no sistema de FPLC, seguida por cromatografia de fase reversa em sistema HPLC. Por SDS-PAGE, AmV dissociou-se em quatro subunidades principais com aproximadamente 73, 70, 43 e 41 kDa, e quando submetida à eletroforese em condições nativas apresentou uma banda única de característica eletroforética ácida. Nos bioensaios, a WD₅₀ e a LD₅₀ para as larvas foram de 0,32% e 0,33% (p/p) respectivamente, já para os adultos a ED₅₀ foi de 0,096%. O provável mecanismo de ação foi avaliado por ensaios de digestibilidade da AmV in vitro, sendo observado o envolvimento de dois fragmentos de vicilinas imunorreativos contra anticorpo policlonal Anti-vicilinas de Erythrina velutina (Anti-EvV), de 22 e 13 kDa ligantes à quitina. A AmV na sua forma nativa foi reconhecida pelo anticorpo anti-EvV, indicando que existe uma provavel região conservada nas vicilinas, que pode corresponder à domínios de ligação à quitina. Estes resultados apontam para uma nova vicilina que pode vir a ser utilizada como um possível bioinseticida de origem proteica, de maneira a controlar o inseto praga C. maculatus, bem como corroborar com achados da literatura que demonstram em vicilinas de diferentes espécies a existência de regiões conservadas ligantes à quitina ainda não caracterizados.