Avaliação dos efeitos tóxicos in vitro e in vivo do extrato hidroetanólico dos frutos de Genipa americana L. (Rubiaceae) em camundongos Swiss

The therapeutic use of medicinal plants has contributed since antiquity in a beneficial way for health. However, many species lacks of scientific evidence which provide basis for their use in therapeutic practice. In this context is the Genipa americana L. species (Rubiaceae), popularly known as jenipapo and used to treat syfilis, ulcer and hemorrhagic disturbs. It's also used against bruising, as tonic and as aphrodisiac. Due this species lacks toxicological studies, the aim of this study was to evaluate the toxicity in vivo (acute and sub-chronic toxicity) and in vitro (cytotoxicity) of the hydroethanolic extract from G. americana fruits. The hydroethanolic extract of G. americana fruits was prepared by maceration. A preliminary phytochemical analysis was performed to assess the presence of secondary metabolites in the extract. The cytotoxicity study of the extract (0.1, 1.0, 10, 100 and 1000 mg / 100 ul) were performed against normal cells (3T3) and tumor (786-0, HepG2 and B16), analyzed by the MTT assay. To evaluate the acute (single dose of 2000 mg / Kg) and subchronic (100, 500 and 1000 mg / kg for 30 days) toxicity Swiss mice of both sexes were used. At the end of the experiment, blood samples and organs were collected for analysis. Data between groups were compared by t test or ANOVA with Dunnett's post-test with 5% significance level. The phytochemical study of the extracts mainly indicated the presence of iridoids. Results for cytotoxicity tests showed up to 70% inhibition of B16 cell line at a dose of 1000 mg / 100 ul, and up to 29% inhibition of 786-0 at a dose of 10 ug / 100 ul. The extract did not cause death in 3T3 and HepG2 cells. During the in vivo assays, there were no animal deaths. Analysis of blood samples revealed that the animals submitted to the evaluation of acute toxicity had changes in AST and ALT, and that the animals evaluated for subchronic toxicity showed changes in the relative wet weight of the kidney and plasma urea concentration. No differences were observed between groups on histopathological evaluation of the collected organs. Despite the changes found in the in vivo toxicity tests, using the criteria described by the OECD Guidelines, it is suggested that the hydroethanolic extract of the fruits of the G. americana is classified as low toxicity. The cytotoxicity of the extract suggests that they have potential against melanoma cell lines (B16).

Testes para a avaliação da qualidade fisiológica de sementes de Moringa oleifera Lam

The evaluation of seed vigor is an important factor for detection of lots of high quality seeds, so that development of procedures to evaluate the physiological potential has been an important tool in quality control programs seeds. In this sense the study aimed to adapt the methodologies of accelerated aging, electrical conductivity and potassium leaching to evaluate Moringa oleifera seed vigor LAM.. Therefore, four lots of moringa seeds were subjected to the germination tests, seedling emergence, speed of emergence index, emergence first count, length and dry mass of seedlings and cold test for their physiological characterization, in addition to accelerated aging, electrical conductivity and potassium leaching. The experimental design was completely randomized with four replications of 50 seeds and the means compared by Tukey test at 5% probability. For accelerated aging the periods were studied aging 12, 24 and 72 hours at 40, 42 and 45°C. For the electrical conductivity test was used to a temperature of 25°C for periods of 4, 8, 12, 16 and 24 hours of immersion in 75 to 125 mL of distilled water, using 25 to 50 seeds, and for potassium leaching test samples were used 25 to 50 seeds, placed in plastic cups containing 70 and 100 mL of distilled water at 25°C for periods of 1, 2, 3, 4, 5 and 6 hours. From the results obtained, it can be inferred that the methods best fit for the accelerated aging test Moringa seeds were a temperature of 40°C for 12 to 72 hours, 42°C 72 hours 45°C 24 hours . In the electrical conductivity test Moringa seeds, the combination of 50 seeds in 75 mL distilled water for a period of immersion of 4 hours and 50 seeds in 125 mL of 4 hours were efficient for the differentiation of lots of Moringa seeds as to vigor and for potassium leaching test moringa seeds, the combination of 50 seeds in 100mL of distilled water allowed the separation of lots of four levels of vigor, at 2 hours of immersion, showing promise in evaluate the quality of moringa seeds.

Testes para a avaliação da qualidade fisiológica de sementes de Moringa oleifera Lam

The evaluation of seed vigor is an important factor for detection of lots of high quality seeds, so that development of procedures to evaluate the physiological potential has been an important tool in quality control programs seeds. In this sense the study aimed to adapt the methodologies of accelerated aging, electrical conductivity and potassium leaching to evaluate Moringa oleifera seed vigor LAM.. Therefore, four lots of moringa seeds were subjected to the germination tests, seedling emergence, speed of emergence index, emergence first count, length and dry mass of seedlings and cold test for their physiological characterization, in addition to accelerated aging, electrical conductivity and potassium leaching. The experimental design was completely randomized with four replications of 50 seeds and the means compared by Tukey test at 5% probability. For accelerated aging the periods were studied aging 12, 24 and 72 hours at 40, 42 and 45°C. For the electrical conductivity test was used to a temperature of 25°C for periods of 4, 8, 12, 16 and 24 hours of immersion in 75 to 125 mL of distilled water, using 25 to 50 seeds, and for potassium leaching test samples were used 25 to 50 seeds, placed in plastic cups containing 70 and 100 mL of distilled water at 25°C for periods of 1, 2, 3, 4, 5 and 6 hours. From the results obtained, it can be inferred that the methods best fit for the accelerated aging test Moringa seeds were a temperature of 40°C for 12 to 72 hours, 42°C 72 hours 45°C 24 hours . In the electrical conductivity test Moringa seeds, the combination of 50 seeds in 75 mL distilled water for a period of immersion of 4 hours and 50 seeds in 125 mL of 4 hours were efficient for the differentiation of lots of Moringa seeds as to vigor and for potassium leaching test moringa seeds, the combination of 50 seeds in 100mL of distilled water allowed the separation of lots of four levels of vigor, at 2 hours of immersion, showing promise in evaluate the quality of moringa seeds.

Desenvolvimento e validação de testes de fluxo lateral para a detecção de cultivares geneticamente modificados e de aflatoxinas em produtos agrícolas

The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I® e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 μg/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.

Desenvolvimento e validação de testes de fluxo lateral para a detecção de cultivares geneticamente modificados e de aflatoxinas em produtos agrícolas

The expansion of cultivated areas with genetically modified crops (GM) is a worldwide phenomenon, stimulating regulatory authorities to implement strict procedures to monitor and verify the presence of GM varieties in agricultural crops. With the constant growing of plant cultivating areas all over the world, consumption of aflatoxin-contaminated food also increased. Aflatoxins correspond to a class of highly toxic contaminants found in agricultural products that can have harmful effects on human and animal health. Therefore, the safety and quality evaluation of agricultural products are important issues for consumers. Lateral flow tests (strip tests) is a promising method for the detection both proteins expressed in GM crops and aflatoxins-contaminated food samples. The advantages of this technique include its simplicity, rapidity and cost-effective when compared to the conventional methods. In this study, two novel and sensitive strip tests assay were developed for the identification of: (i) Cry1Ac and Cry8Ka5 proteins expressed in GM cotton crops and; (ii) aflatoxins from agricultural products. The first strip test was developed using a sandwhich format, while the second one was developed using a competitive format. Gold colloidal nanoparticles were used as detector reagent when coated with monoclonal antibodies. An anti-species specific antibody was sprayed at the nitrocellulose membrane to be used as a control line. To validate the first strip test, GM (Bollgard I® e Planta 50- EMBRAPA) and non-GM cotton leaf (Cooker 312) were used. The results showed that the strip containing antibodies for the identification of Cry1Ac and Cry8Ka5 proteins was capable of correctly distinguishing between GM samples (positive result) and non-GM samples (negative result), in a high sensitivity manner. To validate the second strip test, artificially contaminated soybean with Aspergillus flavus (aflatoxin-producing fungus) was employed. Food samples, such as milk and soybean, were also evaluated for the presence of aflatoxins. The strip test was capable to distinguish between samples with and without aflatoxins samples, at a sensitivity concentration of 0,5 μg/Kg. Therefore, these results suggest that the strip tests developed in this study can be a potential tool as a rapid and cost-effective method for detection of insect resistant GM crops expressing Cry1Ac and Cry8Ka5 and aflatoxins from food samples.

Testes cosmológicos aplicados a modelos de energia escura

A significant observational effort has been directed to investigate the nature of the so-called dark energy. In this dissertation we derive constraints on dark energy models using three different observable: measurements of the Hubble rate H(z) (compiled by Meng et al. in 2015.); distance modulus of 580 Supernovae Type Ia (Union catalog Compilation 2.1, 2011); and the observations of baryon acoustic oscilations (BAO) and the cosmic microwave background (CMB) by using the so-called CMB/BAO of six peaks of BAO (a peak determined through the Survey 6dFGS data, two through the SDSS and three through WiggleZ). The statistical analysis used was the method of the χ2 minimum (marginalized or minimized over h whenever possible) to link the cosmological parameter: m, ω and δω0. These tests were applied in two parameterization of the parameter ω of the equation of state of dark energy, p = ωρ (here, p is the pressure and ρ is the component of energy density). In one, ω is considered constant and less than -1/3, known as XCDM model; in the other the parameter of state equantion varies with the redshift, where we the call model GS. This last model is based on arguments that arise from the theory of cosmological inflation. For comparison it was also made the analysis of model CDM. Comparison of cosmological models with different observations lead to different optimal settings. Thus, to classify the observational viability of different theoretical models we use two criteria information, the Bayesian information criterion (BIC) and the Akaike information criteria (AIC). The Fisher matrix tool was incorporated into our testing to provide us with the uncertainty of the parameters of each theoretical model. We found that the complementarity of tests is necessary inorder we do not have degenerate parametric spaces. Making the minimization process we found (68%), for the Model XCDM the best fit parameters are m = 0.28 ± 0, 012 and ωX = −1.01 ± 0, 052. While for Model GS the best settings are m = 0.28 ± 0, 011 and δω0 = 0.00 ± 0, 059. Performing a marginalization we found (68%), for the Model XCDM the best fit parameters are m = 0.28 ± 0, 012 and ωX = −1.01 ± 0, 052. While for Model GS the best settings are M = 0.28 ± 0, 011 and δω0 = 0.00 ± 0, 059.

Testes cosmológicos aplicados a modelos de energia escura

A significant observational effort has been directed to investigate the nature of the so-called dark energy. In this dissertation we derive constraints on dark energy models using three different observable: measurements of the Hubble rate H(z) (compiled by Meng et al. in 2015.); distance modulus of 580 Supernovae Type Ia (Union catalog Compilation 2.1, 2011); and the observations of baryon acoustic oscilations (BAO) and the cosmic microwave background (CMB) by using the so-called CMB/BAO of six peaks of BAO (a peak determined through the Survey 6dFGS data, two through the SDSS and three through WiggleZ). The statistical analysis used was the method of the χ2 minimum (marginalized or minimized over h whenever possible) to link the cosmological parameter: m, ω and δω0. These tests were applied in two parameterization of the parameter ω of the equation of state of dark energy, p = ωρ (here, p is the pressure and ρ is the component of energy density). In one, ω is considered constant and less than -1/3, known as XCDM model; in the other the parameter of state equantion varies with the redshift, where we the call model GS. This last model is based on arguments that arise from the theory of cosmological inflation. For comparison it was also made the analysis of model CDM. Comparison of cosmological models with different observations lead to different optimal settings. Thus, to classify the observational viability of different theoretical models we use two criteria information, the Bayesian information criterion (BIC) and the Akaike information criteria (AIC). The Fisher matrix tool was incorporated into our testing to provide us with the uncertainty of the parameters of each theoretical model. We found that the complementarity of tests is necessary inorder we do not have degenerate parametric spaces. Making the minimization process we found (68%), for the Model XCDM the best fit parameters are m = 0.28 ± 0, 012 and ωX = −1.01 ± 0, 052. While for Model GS the best settings are m = 0.28 ± 0, 011 and δω0 = 0.00 ± 0, 059. Performing a marginalization we found (68%), for the Model XCDM the best fit parameters are m = 0.28 ± 0, 012 and ωX = −1.01 ± 0, 052. While for Model GS the best settings are M = 0.28 ± 0, 011 and δω0 = 0.00 ± 0, 059.

Avaliação e correções de testes de hipóteses em modelos de sobrevivência com fração de cura

Survival models deals with the modelling of time to event data. In certain situations, a share of the population can no longer be subjected to the event occurrence. In this context, the cure fraction models emerged. Among the models that incorporate a fraction of cured one of the most known is the promotion time model. In the present study we discuss hypothesis testing in the promotion time model with Weibull distribution for the failure times of susceptible individuals. Hypothesis testing in this model may be performed based on likelihood ratio, gradient, score or Wald statistics. The critical values are obtained from asymptotic approximations, which may result in size distortions in nite sample sizes. This study proposes bootstrap corrections to the aforementioned tests and Bartlett bootstrap to the likelihood ratio statistic in Weibull promotion time model. Using Monte Carlo simulations we compared the nite sample performances of the proposed corrections in contrast with the usual tests. The numerical evidence favors the proposed corrected tests. At the end of the work an empirical application is presented.

Protótipo de testes para aprendizagem com classificadores múltiplos

Os classificadores múltiplos são processos que utilizam um conjunto de modelos, cada um deles obtido pela aplicação de um processo de aprendizagem para um problema dado. Combinam vários classificadores individuais, em que para cada um deles são utilizados dados de treino para gerar limites de decisão diferentes. As decisões produzidas pelos classificadores individuais contém erros, que são combinados pelos classificadores múltiplos de forma a reduzir o erro total. Estes têm vindo a ganhar uma crescente importância devido principalmente ao facto de permitirem obter um melhor desempenho quando comparado com o obtido por qualquer um dos modelos que o compõem, principalmente quando as correlações entre os erros cometidos pelos modelos de base são baixos. A investigação nesta área tem crescido, tornando-se uma área de investigação importante. No entanto, para que o desempenho seja melhor do que o desempenho obtido por cada classificador individualmente, é necessário que cada um deles produza uma decisão diferente originando uma diversidade de classificação. Esta diversidade pode ser obtida tanto pela utilização de diferentes conjuntos de dados para o treino individual de cada classificador, como também pela utilização de diferentes parâmetros de formação de diferentes classificadores. Apesar disso, a utilização de classificadores múltiplos para aplicações no mundo real pode apresentar-se como dispendiosa e morosa. Tem-se notado nos dias de hoje que o desenvolvimento web tem vindo a crescer exponencialmente, assim como o uso de bases de dados. Desta forma, combinando a forte utilização da linguagem R para cálculos estatísticos com a crescente utilização das tecnologias web, foi implementado um protótipo que facilitasse a utilização dos classificadores múltiplos, mais precisamente, foi desenvolvida uma aplicação web que permitisse o teste para aprendizagem com classificadores múltiplos, sendo utilizadas as tecnologias PHP, R e MySQL. Com esta aplicação pretende-se que seja possível testar algoritmos independentes do software em que estejam desenvolvidos, não sendo necessariamente escritos em R. Nesta Dissertação foi utilizada a expressão “classificadores múltiplos” por ser a mais comum, apesar de ser redutora e existirem outros termos mais genéricos como por exemplo modelos múltiplos e ensemble learning.

Estudo comparativo da estereopsia com três testes diferentes: TNO®, Fly Stereo Acuity Test® e StereoTAB® em estudantes do ensino superior

Estereopsia define-se como a perceção de profundidade baseada na disparidade retiniana. A estereopsia global depende do processamento de estímulos de pontos aleatórios e a estereopsia local depende da perceção de contornos. O objetivo deste estudo é correlacionar três testes de estereopsia: TNO®, StereoTAB® e Fly Stereo Acuity Test® e verificar a sensibilidade e correlação entre eles, tendo o TNO® como gold standard. Incluíram-se 49 estudantes da Escola Superior de Tecnologia da Saúde de Lisboa (ESTeSL) entre os 18 e 26 anos. As variáveis ponto próximo de convergência (ppc), vergências, sintomatologia e correção ótica foram correlacionadas com os três testes. Os valores médios (desvios-padrão) de estereopsia foram: TNO® = 87,04’’ ±84,09’’; FlyTest® = 38,18’’ ±34,59’’; StereoTAB® = 124,89’’ ±137,38’’. Coeficiente de determinação: TNO® e StereoTAB® com R2=0,6 e TNO® e FlyTest® com R2=0,2. O coeficiente de correlação de Pearson mostra uma correlação positiva de entre o TNO® e o StereoTAB® (r=0,784 com α=0,01). O coeficiente de associação de Phi mostrou uma relação positiva forte entre o TNO® e StereoTAB® (Φ=0,848 com α=0,01). Na curva ROC, o StereoTAB® possui uma área sob a curva maior que o FlyTest®, apresentando valor de sensibilidade de 92,3% para uma especificidade de 94,4%, tornando-o num teste sensível e com bom poder discriminativo.

Citotoxicidade da associação de agrotóxicos da rizicultura em hepatócitos de Zebrafish

No plantio do arroz parte de um corpo d’água (rio, lago, lagoa) é desviado para a irrigação da plantação, e, posteriormente, a água utilizada nas lavouras é devolvida ao rio/lago/lagoa de origem. Assim, seja por lixiviação ou por qualquer outro fator, a água entra em contato com os agrotóxicos que, anteriormente, foram utilizados na plantação, podendo causar danos à qualidade do recurso hídrico e à fauna lacustre, devido à exposição a estes poluentes. O presente trabalho teve por objetivo verificar a citotoxicidade de agrotóxicos (herbicida e inseticida), utilizados na rizicultura no estado do Rio Grande do Sul, em células hepáticas da linhagem ZF-L. A partir da análise de funcionalidade de três alvos celulares diferentes, integridade da membrana celular, estabilidade lisossomal e atividade mitocondrial frente à exposição ao Roundup Transorb® , ao Furadan 350 SC® e à associação destes produtos. Foi analisada ainda, a capacidade de defesa das células, expostas aos poluentes escolhidos, no que diz respeito à atividade de proteínas extrusoras de xenobióticos, assim como à expressão de tais proteínas. A partir dos resultados obtidos foi verificado efeito citotóxico de ambos os agrotóxicos, bem como a mistura destes para todos os alvos verificados, apresentando ainda efeito inibitório à atividade de extrusão de xenobióticos pelas glicoproteínas P (P-gps). Apenas quando expostas ao inseticida e à mistura as células apresentaram um aumento na expressão de glicoproteínas (P-gp). Verificou-se a existência de correlação negativa entre a citotoxicidade apresentada, principalmente na atividade mitocondrial e na integridade lisossomo e a atividade das P-gps. Em conclusão, percebeu-se que as concentrações abaixo do permitido pela legislação brasileira, para os princípios ativos dos agrotóxicos testados, mostraram-se tóxicas para todos os alvos de citotoxicidade testados neste estudo, com exceção da mitocôndria, sugerindo que esta toxicidade apresentada pode ser devido aos surfactantes presentes nas formulações comerciais.