Desenvolvimento e validação de uma técnica quantitativa de análise de imagem para avaliação do teste do cometa corado pela prata

The comet assay is a method of DNA damage analysis widely used to quantify oxidative damage, crosslinks of DNA, apoptosis and genotoxicity of chemicals substances as chemical, pharmaceuticals, agrochemicals products, among others. This technique is suitable to detect DNA strand breaks, alkali-labile sites and incomplete excision repair sites and is based on the migration of DNA fragments by microeletroforesis, DNA migrates for the anode forming a “tail”, and the formed image has the appearance of a comet. The slides can be stained with fluorescence or silver, having differences in the microscopy type used for the analysis and the possibility of storage of the slides, moreover, the first one is a stained-method with more difficulties of accomplishment. The image analysis can be performed by a visual way, however, there is a disadvantage as the subjectivity on the results, that can be minimized by an automated method of digital analysis. This process was studied in this report with the aim to perceive the validation of the digital analysis turning it a quantitative method with larger reproductibility, minimizing the variability and imprecision due to the subjective analysis. For this validation we selected 50 comets photographed in a standardized way and printed, afterwards, pictures were submitted to three experienced appraisers, who quantified them manually. Later, the images were processed by free software ImageJ 1.38x, printed and quantified manually by the same appraisers. The intraclass correlation was higher to comet measures after image processing. Following, an algorithm of automated digital analysis from the measures of the comet was developed; the values obtained were compared with those 12 estimated manually after the processing resulting high correlation among the measures. The use of image analysis systems increases ...(Complete abstract click electronic access below)

Avaliação dos efeitos dos inseticidas malation e fipronil sobre a viabilidade e regeneração de girardia tigrina (Platyhelminthe, tricladida)

In this study were carried out toxicity tests and the evaluation of sub lethal effects of the insecticides malathion and fipronil, over the test system of Girardia tigrina, using for these analyses a great variety of methods. For evaluation of the lethal concentrations of the products, adult planarians were exposed to several different concentrations. All the tests were carried out in duplicate, maintained on the shelter of the light, on a temperature 24-26ºC and they had the duration of 96h, with observations at each 24h. The results were statically valued by the tests U of Mann- Whitney, Spearman-Karber, ANOVA and Pearson correlation. The effects over the system-test, in the different tested concentrations, varied in accordance with the time of exposition. The EC50 given by the test of Spearman-Karber was of 12,5mg/L for malathion and 13,4mg/L for fipronil. The evaluation of sub lethal effects was composed by analyses of the potential of regeneration; histological, for tissue damage evaluation; d; and the “comet assay”, for DNA damage detection. The specimens split up and exposed to the insecticides demonstrated, between other effects, retardation in development of the regenerative blastema. The histological evaluation has shown that exposed planarians to lower concentrations of the insecticides cause more significant damage against the tissues and some specific cell types. Through the comet assay, significant damages were noticed in the DNA in all the treatments, when compared with the control group. Such damages may result in reproduction and immunity alterations, which reduce the competitive efficiency and, consequently, the survival chances of these organisms.

Estudo fotodinâmico da clorofila a, azul de metileno e rosa bengala como agente inseticida contra Aedes aegypti

Photodynamic therapy, term introduced by von Tapeiner in 1900, can be defined as the administration of a non toxic drug, i.e., a dye, known as photosensitizer (FS), which subsequently will be illuminated with light of specific wavelength. PDT is based on the interaction among FS, oxygen and light, which through photochemical reactions cause cell death. The FS molecules must have a high probability to form the singlet state after the excitation, which can induce chemical changes in the neighborhood in two ways, called reactions type I and type II. The type II reaction is based on the exchange of energy to molecular oxygen, exciting it to its state of higher energy (singlet), which is highly reactive. The proposed mechanisms for cell death are linked to damage to the DNA, mitochondria and to the cytoplasmic membrane. Several pre‐clinical and clinical trials have been carried out and the PDT is already used in many countries for treatment mainly against certain types of cancer. The therapy also has been gaining strength in antimicrobial control, since the microorganisms have appeared increasingly resistant to current antibiotics. Another attempt to use the PDT is for the inactivation of macro‐organisms, such as micro‐crustaceans and mosquitoes. To this end I tested whether the photosensitizers methylene blue, rose Bengal and the chlorophyll a has insecticidal activity against the yellow fever and dengue vector mosquito, Aedes aegypti. Since these diseases have no effective treatments, its control is linked to the vector control, which has shown resistance to chemical pesticides used. Based on this, this work shows its importance, because it is a new type of mosquito control since all the photosensitizers used are low cost, do not generate toxic products at the concentrations used and showed good results in mortality. The best photosensitizer was rose Bengal... (Complete abstract click electronic access below)

Marcadores de superfície de Células-tronco: estudo de reação cruzada inter-espécies

Mesenchymal stem cells (MSCs) are adult multipotent cells with fibroblastoid morphology and adherent to plastic. Furthermore, they can be obtained from different sources. Besides bone marrow, these cells are taken from umbilical cord blood, umbilical vein, saphenous vein, peripheral blood, arteries, liver and fetal pancreas, placenta, dental pulp and adipose tissue. MSCs derived from adipose tissue are important because of the abundant number of cells that can be obtained from this tissue, easy access and little discomfort to the patient. This study compared two techniques for obtaining MSCs from adipose tissue: mechanical dissociation (MD) and enzymatic digestion (ED). We also analyzed the inter-species cross-reactions using commercial monoclonal antibodies directed against surface antigens of stem cells from different species: mouse, horse, rabbit, monkey and human. We found that MD technique is favorable in relation to ED within 15 days of culture, and ED is more efficient in the first days of culture. The data also showed that MD causes less damage to cellular DNA. About inter-species cross-reactions, the monoclonal antibody A69 directed against stem cells from rabbits, which can be used in veterinary medicine, particularly in research involving horses

Lactobacillus acidophilus CRL 1014 e extrato aquoso de yacon na evolução do processo de carcinogênese química de cólon

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

Polimorfismos GSTM1, GSTT1 e GSTP1 da enzima Glutationa S-transferase como fatores moduladores do fenótipo na anemia falciforme

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito quimiopreventivo e modulador de HLA-G (Human Leukocyte Antigen - G) de Morelloflavona

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

Expressão proteica das enzimas metabolizadoras de drogas no carcinoma de células escamosas de boca em pacientes jovens

Pós-graduação em Biopatologia Bucal - ICT

Expressão diferencial de mRNA em células de cultivo infectadas ple herpesvírus bovino 5

Pós-graduação em Medicina Veterinária - FMVZ

Marcadores de ativação imune, inflamação crônica e estresse oxidativo em pessoas que vivem com o HIV/Aids: a busca do melhor prognóstico

Pós-graduação em Doenças Tropicais - FMB

Filmes de borracha natural com nanopartículas de prata e pontos quânticos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bioestimulação de célula-tronco mesenquimal de rato sob ação da luz contínua e pulsátil de 630 nm utilizando LED

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

Bioestimulação de célula-tronco mesenquimal de rato sob ação da luz contínua e pulsátil de 630 nm utilizando LED

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

Avaliação da genotoxicidade em mulheres com síndrome dos ovários policísticos: impacto da dieta

Introduction: Polycystic Ovary Syndrome (PCOS), present in 6-12% of women of reproductive age, the criterion of Rotterdam, is characterized by hyperandrogenism, insulin resistance (IR) and its inflammatory state, exacerbated by obesity and factors associated with the increase in damage DNA. Weight loss, combined with healthy eating, acts restoring the reproductive and metabolic functions in the SOP, though its influence in reducing DNA damage in PCOS are unknown. Aim: To investigate whether there are differences between DNA damage markers and factors of cardiometabolic risk in women with PCOS and control, and evaluate the effectiveness of nutritional intervention in DNA damage markers and cardiometabolic risk markers in overweight and obese women with PCOS. Methods: the study was conducted in two studies and the participants were aged between 18 and 35 years. In the first study, a prospective case-control, were included 27 women diagnosed with PCOS and 20 controls. In the second study, clinical trial of nutritional intervention with 12-week calorie-restricted diet 500Kcal / day. The genotoxicity, DNA damage (intensity tail, tail moment and tail length) was evaluated by the comet assay. Anthropometric data, dietary intake, hormonal, biochemical and inflammatory were evaluated in different studies. Results: there was no significant difference between the DNA damage marker tail intensity (p = 0.18), tail moment (p = 0.76) and tail length (p = 0.109) in PCOS when compared to the control group. Data after nutritional intervention in PCOS women with overweight and obesity showed a decrease in DNA damage markers: tail intensity (24.35 ± 5.86 - pre-diet vs. 17.15 ± 5.04 -Post-diet) and tail moment (20.47 ± 7.85 - pre-diet vs. 14.13 ± 6.29 -post-diet) (p <0.001). Reduction of weight (3.5%) and decreased cardiometabolic markers IR and hyperandrogenism. Conclusion: women with PCOS have a worse cardiometabolic risk profile compared to control however similar genotoxicity identified by DNA damage. Nutritional intervention reduced the genotoxicity of overweight and obese women with PCOS, and reduce the factors of cardiometabolic risk.

Caracterização do material particulado e avaliação do risco ocupacional e mecanismos moleculares associados à queima artesanal da castanha de caju

Brazil is among the largest cashew nut producers of the world. However, the roasting process is still carried out artisanally, especially in the Brazilian semiarid region. In face of this occupational problem, the aim of this study was to perform a physical-chemical characterization of the particulate matter (PM) emitted by the roasting of cashew nuts, as well as to determine the occupational risk and molecular mechanisms associated. The most evident PM characteristics were the prevalence of fine particles, typical biomass burning morphologies such as tar ball and the presence of the elements K, Cl, S, Ca and Fe. In addition, atmospheric modeling analyses suggest that these particles can reach neighboring regions of the emission source. Polycyclic aromatic hydrocarbons (PAHs) with carcinogenic potential, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[a]anthracene, benzo[b]fluoranthene, chrysene, benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene and benzo[j]fluoranthene were the most abundant PAHs found in the two air monitoring campaigns. Among the identified oxy-PAH the benzanthrone (7H-benz[d,e]anthracen-7-one) had the highest concentration and the evaluation of lifetime cancer risk showed an increase of 12 to 37 cases of cancer for every 10,000 exposed people. Chemical analysis of roasted cashew nuts identified the PAHs: phenanthrene, benzo[g,h,i]perylene, pyrene and benzo[a]pyrene, besides the 3-pentadecilfenol allergen (urushiol analogue) as prevalent. Occupational exposure to PAHs was confirmed by the increase of urinary 1-hydroxypyrene levels and genotoxic effects were evidenced by the increase on micronuclei and nuclear bud frequency in exfoliated buccal mucosa cells among the exposed workers. Other biomarkers of effects such as karyorrhexis, pyknotic, karyolytic, condensed chromatin and binucleated cells also have their frequencies increased when compared to an unexposed control group. The investigation of the molecular mechanisms associated with the PM organic extract showed cytotoxicity in human lung cell lines (A549) at concentrations ≥ 4 nM BaPeq. Using non-cytotoxic doses the extract was able to activate proteins involved in the DNA damage response pathway (Chk1 and p53). Moreover, the specific contribution of the four most representative PAHs in the cashew nut roasting sample showed that benzo[a]pyrene was the most efficient to activate Chk1 and p53. Finally, the organic extract was able to increase persistently the mRNA expression involved in the PAHs metabolism (CYP1A1 and CYP1B1), inflammatory response (IL-8 and TNF-α) and cell cycle arrest (CDKN1A) for DNA repair (DDB2). The high PM concentrations and its biological effects associated warn of the serious harmful effects of artisanal cashew nut roasting and urgent actions should be taken to the sustainable development of this activity.