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.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Um estudo sobre o erro ortográfico no 1º e 2º CEB – da identificação às estratégias de intervenção

Relatório de estágio apresentado à Escola Superior de Educação de Santarém do Instituto Politécnico de Santarém para obtenção do grau de Mestre em ensino do 1.º e do 2.º ciclo do ensino básico

Seleção de características para identificação de diferentes proporções de tipos de fibras musculares por meio da eletromiografia de superfície

Skeletal muscle consists of muscle fiber types that have different physiological and biochemical characteristics. Basically, the muscle fiber can be classified into type I and type II, presenting, among other features, contraction speed and sensitivity to fatigue different for each type of muscle fiber. These fibers coexist in the skeletal muscles and their relative proportions are modulated according to the muscle functionality and the stimulus that is submitted. To identify the different proportions of fiber types in the muscle composition, many studies use biopsy as standard procedure. As the surface electromyography (EMGs) allows to extract information about the recruitment of different motor units, this study is based on the assumption that it is possible to use the EMG to identify different proportions of fiber types in a muscle. The goal of this study was to identify the characteristics of the EMG signals which are able to distinguish, more precisely, different proportions of fiber types. Also was investigated the combination of characteristics using appropriate mathematical models. To achieve the proposed objective, simulated signals were developed with different proportions of motor units recruited and with different signal-to-noise ratios. Thirteen characteristics in function of time and the frequency were extracted from emulated signals. The results for each extracted feature of the signals were submitted to the clustering algorithm k-means to separate the different proportions of motor units recruited on the emulated signals. Mathematical techniques (confusion matrix and analysis of capability) were implemented to select the characteristics able to identify different proportions of muscle fiber types. As a result, the average frequency and median frequency were selected as able to distinguish, with more precision, the proportions of different muscle fiber types. Posteriorly, the features considered most able were analyzed in an associated way through principal component analysis. Were found two principal components of the signals emulated without noise (CP1 and CP2) and two principal components of the noisy signals (CP1 and CP2 ). The first principal components (CP1 and CP1 ) were identified as being able to distinguish different proportions of muscle fiber types. The selected characteristics (median frequency, mean frequency, CP1 and CP1 ) were used to analyze real EMGs signals, comparing sedentary people with physically active people who practice strength training (weight training). The results obtained with the different groups of volunteers show that the physically active people obtained higher values of mean frequency, median frequency and principal components compared with the sedentary people. Moreover, these values decreased with increasing power level for both groups, however, the decline was more accented for the group of physically active people. Based on these results, it is assumed that the volunteers of the physically active group have higher proportions of type II fibers than sedentary people. Finally, based on these results, we can conclude that the selected characteristics were able to distinguish different proportions of muscle fiber types, both for the emulated signals as to the real signals. These characteristics can be used in several studies, for example, to evaluate the progress of people with myopathy and neuromyopathy due to the physiotherapy, and also to analyze the development of athletes to improve their muscle capacity according to their sport. In both cases, the extraction of these characteristics from the surface electromyography signals provides a feedback to the physiotherapist and the coach physical, who can analyze the increase in the proportion of a given type of fiber, as desired in each case.

Valor de Mercado como Indicador de Competitividade: Identificação de Empresas Brasileiras com Alto Desempenho no Longo Prazo

O mercado de ações no Brasil tem se desenvolvido de forma consistente nos últimos anos, o IBOVESPA saiu de um patamar de 4000 pontos em janeiro de 1988 para mais de 50 mil pontos em 2012.O objetivo deste trabalho é identificar empresas nacionais que tenham alcançado retornos excedentes de mais de 200% em relação ao mercado e ao setor onde estão inseridas, por um período ininterrupto mínimo de quinze anos, após um período de valorização próxima da média do mercado.A pesquisa é descritiva, quantitativa, com delineamento documental.Foram identificadas sete empresas que, formando um portfólio, teriam apresentado um rendimento de 981% no período compreendido entre 1997 e 2012, um retorno excedente de 389% sobre o IBOVESPA nesses quinze anos. Concluiu-se que o valor de mercado utilizado como indicador de competitividade pode auxiliar na identificação de empresas que apresentam estabilidade na condução dos fundamentos da administração operacional e financeira.

Estudo da composição e das propriedades bioactivas de plantas produtoras de bagas

O consumo de frutas e vegetais tem sido associado à prevenção de várias doenças crónicas, nomeadamente doenças cardiovasculares, diabetes, cancro e outras que envolvam processos inflamatórios. As bagas destacam-se pelo seu elevado conteúdo em polifenóis, cujas propriedades antioxidantes contribuem para a manutenção da saúde humana. O presente trabalho teve como alvo de estudo as diferentes partes morfológicas (bagas e folhas) de espécies produtoras de bagas, nomeadamente Elaeagnus umbellata, a Rubus grandofolius, a Sambucus lancolata, a Vaccinium padifolium e a Vaccinium cylindraceum, tendo em vista a sua valorização como produtos alimentares e/ou nutracêuticos. A caracterização físico-química destas espécies permitiu determinar que o teor total de sólidos solúveis (TSS) das bagas varia de 4,4 a 16,5 °Brix. As bagas demonstraram ser a parte morfológica com teor de humidade mais elevado. A análise do perfil fenólico por HPLC-DAD-ESI/MSn, no modo negativo, dos extractos metanólicos mostrou que as folhas apresentam maior conteúdo de compostos fenólicos, comparativamente às bagas. Os ácidos hidroxicinâmicos (derivados dos ácidos cafeicos, cumárico e ferúlico), os ácidos cafeoilquínicos, bem como os flavonóis-O-glicosilados (derivados da quercetina e canferol) predominam nestas espécies. A análise pelo modo positivo permitiu a identificação de antocianinas glicosiladas (delfinidina, cianidina, petunidina, peonidina e malvidina) nas bagas e folhas jovens da espécie Vaccinium padifolium. Os ensaios in vitro de simulação da digestão gastrointestinal permitiram compreender a sua influência na actividade antioxidante dos extractos. Após a digestão, as folhas continuam a apresentar maior capacidade antioxidante do que as bagas. Adicionalmente, concluiu-se que as enzimas presentes neste processo têm menor influência do que o pH e a força iónica dos sucos digestivos. O estudo do efeito inibitório in vitro dos extractos sobre a actividade de enzimas responsáveis pelo metabolismo dos hidratos de carbono permitiu determinar que os viii Joana Pinto (2016) extractos foram mais eficientes na inibição da actividade da α-glucosidase do que na inibição da actividade da α-amilase.

Atuação das equipes da atenção primária na identificação precoce do câncer infanto-juvenil

Identificar o conhecimento de profissionais da atenção primária sobre a identificação precoce do câncer infanto-juvenil e descrever o desempenho das equipes de saúde antes da realização de treinamentos para identificação precoce do câncer infanto-juvenil. Método: Os dados foram obtidos por um questionário e grupo focal com 30 profissionais de uma Unidade de Saúde da Família, e analisados por temas geradores. Resultados: Os profissionais possuem conhecimentos sobre a identificação do câncer infanto-juvenil, e, demandam conhecer mais sobre os sinais e sintomas para identificação precoce, e a prestação de uma assistência sistematizada. Conclusão: Torna-se fundamental estimular a qualificação dos profissionais da atenção primária para a identificação precoce e o fortalecimento de uma rede de assistência que proporcione atendimento integral e a redução no retardo do diagnóstico de câncer infanto-juvenil

Auditoria e identificação de propostas de melhoria no SGA da Funfrap

O compromisso das organizações com as obrigações decretadas ao nível do ambiente, segurança e qualidade emancipa a implementação de Sistemas de Gestão e o processo de auditar. Neste trabalho, decorrente do estágio curricular no âmbito do Mestrado em Engenharia do Ambiente, está evidenciada a importância dos Sistemas de Gestão Ambiental e as razões preponderantes para que sejam auditados. O trabalho pretendeu auditar e avaliar a conformidade do sistema de gestão ambiental, de acordo com a NP EN ISO 14001:2004, de uma indústria de fundição de metais, Funfrap -. Fundição Portuguesa SA, a fim de identificar oportunidades de melhoria no desempenho ambiental da organização. No âmbito do estágio foram ainda realizadas outras atividades, incluindo campanhas de sensibilização, inventário de resíduos sólidos, ferramentas da metodologia World Class Manufacturing, formação sobre a separação de resíduos nos postos de trabalho, acompanhamento de um ensaio na estação de tratamento de águas residuais industriais e apoio para o desenvolvimento do relatório ambiental anual. Da auditoria interna identificaram-se e corrigiram-se nove não conformidades menores e cinco observações. Da auditoria externa foram identificadas apenas duas não conformidades, nenhuma delas relacionadas com as detetadas anteriormente. Além deste resultado foram sugeridas algumas medidas para alcançar um melhor desempenho ambiental da organização.

Escala de perceção dos alunos sobre a sua identificação escolar: construção e estudo psicométrico

O presente estudo descreve o processo de construção e estudo psicométrico de uma escala que mede a perceção dos alunos dos ensinos básico e secundário sobre o seu grau de identificação escolar. Os questionários foram aplicados a uma amostra de 1089 alunos dos 6º, 7º, 9º e 10º anos de escolaridade (mediana da idade=13 anos) em Portugal continental. Uma subamostra foi submetida a análise fatorial exploratória (n1=354), sendo uma segunda sub-amostra sujeita a uma análise fatorial confirmatória (n2=388). As análises revelaram uma estrutura tridimensional: (a) Valorização pessoal/intrínseca, α=.74; (b) Valorização no sentido prático/utilitarista, α=.74; e (c) Sentimentos de pertença e bem--estar, α=.78. As escalas revelam-se úteis e adequadas para avaliar as perceções dos alunos sobre a sua identificação escolar.

Conceções alternativas sobre o sistema respiratório humano: da sua identificação à sua mudança

Relatório de Estágio apresentado à Escola Superior de Educação de Lisboa para obtenção de grau de mestre em Ensino do 1.º e 2.º Ciclos do Ensino Básico

Identificação do potencial renovável para produção de electricidade na perspectiva da microgeração

Tese submetida à Universidade de Lisboa, Faculdade de Ciências e aprovada em provas públicas para a obtenção do Grau de Doutor em Energia e Ambiente (especialidade em Energia e Desenvolvimento Sustentável).

Identificação do transcrito CYP1A no peixe Phalloceros caudimaculatus e resposta para a exposição a beta-naftoflavona e amostras ambientais de sedimento

Contaminantes orgânicos, como os hidrocarbonetos policíclicos aromáticos (HPAs), podem atingir corpos da água e possuem potencial para causar efeitos tóxicos em organismos. A exposição aos HPAs causa indução nos níveis de citocromo P450 1A (CYP1A) em peixes, e portanto, é utilizado como um biomarcador de contaminação ambiental. O guarú Phalloceros caudimaculatus ocorre naturalmente em ambientes aquáticos dulcícolas e mixohalinos na América do Sul. O presente estudo identificou a sequência nucleotídica do transcrito CYP1A de P. caudimaculatus, que codifica uma proteína com 521 aminoácidos, e que apresenta 91% e 70% de identidade com CYP1A de killifish e paulistinha, respectivamente. A partir desta sequência foi possível realizar a avaliação dos níveis de mRNA de CYP1A deste peixe por RTq-PCR. Foi realizada uma caracterização de sua indução órgão- e tempo-dependente frente a exposição ao HPA beta-naftoflavona (BNF) e ao elutriato preparado a partir de sedimento de dois corpos da água possivelmente contaminados com HPAs. Foi constatado um aumento significativo nos níveis de mRNA de CYP1A em fígado, brânquia, intestino, cérebro, nadadeira anal de macho adultos e em alevinos na primeira hora de exposição a 1 µM de BNF, em relação ao grupo controle. O rim e as nadadeiras caudal e dorsal apresentaram indução de CYP1A após duas horas de exposição ao BNF. As maiores induções nos peixes dos grupos expostos ao BNF em relação ao controle foram de 176 no rim e 122 vezes no cérebro, observadas respectivamente após 8 e 48 horas de exposição. Os níveis de mRNA de CYP1A nos órgãos e tecidos de alevino, mantiveramse induzidos pela exposição ao BNF até o final das 96 horas de exposição. A exposição dos peixes ao elutriato produzido a partir dos sedimentos coletados em dois locais potencialmente contaminados causou indução do CYP1A no fígado de 22 e 122 vezes em relação ao controle. Os resultados demonstram que a indução de CYP1A em Phalloceros caudimaculatus ocorre em tempos curtos de exposição, além da variação de acordo com o tempo de exposição e com o órgão analisado. Além disso, foi demonstrado que tecidos externos também podem ser utilizados para tais análises e que o elutriato feito a partir de sedimento de locais que recebem descargas de contaminantes podem causar indução de CYP1A nos organismos.

Molecular markers for diabetic nephropathy

Type 2 diabetes is one of the most common metabolic disorders in the world. Globally, the prevalence of this disorder is predicted to increase, along with the risk of developing diabetic related complications. One of those complications is diabetic nephropathy, defined by a progressive increase in proteinuria and a gradual decline in renal function. Approximately 25% to 30% of type 2 diabetic individuals develop this complication. However, its underlying genetic mechanisms remain unclear. Thus, the aim of this study is to contribute to the discovery of the genetic mechanisms involved in the development and progression of diabetic nephropathy, through the identification of relevant genetic variants in Portuguese type 2 diabetic individuals. The exomes of 36 Portuguese type 2 diabetic individuals were sequenced on the Ion ProtonTM Sequencer. From those individuals, 19 did not present diabetic nephropathy, being included in the control group, while the 17 individuals that presented the diabetic complication formed the case group. A statistical analysis was then performed to identify candidate common genetic variants, as well as genes accumulating rare variants that could be associated with diabetic nephropathy. From the search for common variants in the study population, the statistically significant (p-value ≤ 0.05) variants rs1051303 and rs1131620 in the LTBP4 gene, rs660339 in UCP2, rs2589156 in RPTOR, rs2304483 in the SLC12A3 gene and rs10169718 present in ARPC2, were considered as the most biologically relevant to the pathogenesis of diabetic nephropathy. The variants rs1051303 and rs1131620, as well as the variants rs660339 and rs2589156 were associated with protective effects in the development of the complication, while rs2304483 and rs10169718 were considered risk variants, being present in individuals with diagnosed diabetic nephropathy. In the rare variants approach, the genes with statistical significance (p-value ≤ 0.05) found, the STAB1 gene, accumulating 9 rare variants, and the CUX1 gene, accumulating 2 rare variants, were identified as the most relevant. Both genes were considered protective, with the accumulated rare variants mainly present in the group without the renal complication. The present study provides an initial analysis of the genetic evidence associated with the development and progression of diabetic nephropathy, and the results obtained may contribute to a deeper understanding of the genetic mechanisms associated with this diabetic complication.