Made available in DSpace on 2016-08-10T10:38:45Z (GMT). No. of bitstreams: 1 LUCIANA MAIA ESCHER DOS SANTOS.pdf: 3201320 bytes, checksum: f5c8c078f60bbba17912e8c39742ed0a (MD5) Previous issue date: 2013-06-24 / Molecular Biology shown to be an effective tool in forensic laboratories for the ability to identify an individual from minute amounts of biological samples such as blood, bones, semen, hair, teeth, nails, spittle, urine and other biological fluids recovered from the crime scene. One of the main biological evidence found at a crime scene are traces of bloodstains. This study aimed to analyze the molecular profiles of biological samples exposed to the chemiluminescent reagent luminol based on different storage times (48 hours and 30 days). With the measurement of all samples can be inferred that in the first 48 hours of storage, there was obtained DNA and varying concentrations after application of the chemiluminescent reagent. The samples were amplified by PCR using a multiplex system AmpFlSTR ® Select NGM and capillary electrophoresis. Molecular profiles were obtained complete and incomplete denoting specificity as the quality and quantity of the sample analyzed. The selection of molecular markers mini-STRs greatly contributed to the success of these profiles, allowing the study and analysis of degraded material. Our data suggest that the degradation of the DNA molecule exposed to the chemiluminescent reagent was higher in the samples compared to those containing diluted whole blood impregnation. Therefore, analyzing the bloodstains samples exposed to luminol in shorter storage provide molecular profiles compatible to clash samples. / A Biologia Molecular mostrou-se uma ferramenta efetiva nos laboratórios forenses pela capacidade de identificar um indivíduo a partir de quantidades ínfimas de amostras biológicas como sangue, ossos, sêmen, cabelo, dentes, unhas, saliva, urina, entre outros fluidos biológicos recuperados no local do crime. Uma das principais evidências biológicas encontradas em local de crime são vestígios de substâncias hematóides. Esta pesquisa visou a análise de perfis moleculares de amostras biológicas expostas ao reagente quimioluminescente a base de luminol em diferentes tempos de armazenamento (48 horas e 30 dias). Com a quantificação de todas as amostras pode-se inferir que nas primeiras 48 horas de armazenamento, obtiveram-se concentrações variáveis de DNA e após a aplicação do reagente quimioluminescente. As amostras foram amplificadas por PCR utilizando um sistema multiplex AmpFlSTR® NGM SElect e eletroforese capilar. Foram obtidos perfis moleculares completos e incompletos denotando inespecificidade conforme a qualidade e quantidade da amostra analisada. A seleção dos 17 marcadores moleculares mini-STRs em muito contribuiu para o sucesso desses perfis, permitindo estudo e análise de material degradado. Os dados indicam que a degradação da molécula de DNA exposta ao reagente quimioluminescente foi maior nas amostras diluídas em relação àquelas contendo impregnação com sangue total. A análise estatística das amostras com e sem exposição ao reagente quimioluminescente comparado ao grupo controle e em diferentes concentrações, mostrou não haver diferença estatísticamente significativa entre os valores quantificados obtidos. Ainda assim, analisar as amostras hematóides expostas ao luminol em menor tempo de armazenamento proporcionarão perfis moleculares compatíveis ao confronto de amostras.

Heterosis is defined as the superiority in performance of heterozygous genotypes compared to their corresponding genetically different homozygous parents. This phenomenon is already known since the beginning of the last century and it has been widely used in plant breeding, but the underlying genetic and molecular mechanisms are not well understood. In this work, a systems biological approach based on molecular network structures is proposed to contribute to the understanding of heterosis. Hybrids are likely to contain additional regulatory possibilities compared to their homozygous parents and, therefore, they may be able to correctly respond to a higher number of environmental challenges, which leads to a higher adaptability and, thus, the heterosis phenomenon. In the network hypothesis for heterosis, presented in this work, more regulatory interactions are expected in the molecular networks of the hybrids compared to the homozygous parents. Partial correlations were used to assess this difference in the global interaction structure of regulatory networks between the hybrids and the homozygous genotypes. This network hypothesis for heterosis was tested on metabolite profiles as well as gene expression data of the two parental Arabidopsis thaliana accessions C24 and Col-0 and their reciprocal crosses. These plants are known to show a heterosis effect in their biomass phenotype. The hypothesis was confirmed for mid-parent and best-parent heterosis for either hybrid of our experimental metabolite as well as gene expression data. It was shown that this result is influenced by the used cutoffs during the analyses. Too strict filtering resulted in sets of metabolites and genes for which the network hypothesis for heterosis does not hold true for either hybrid regarding mid-parent as well as best-parent heterosis. In an over-representation analysis, the genes that show the largest heterosis effects according to our network hypothesis were compared to genes of heterotic quantitative trait loci (QTL) regions. Separately for either hybrid regarding mid-parent as well as best-parent heterosis, a significantly larger overlap between the resulting gene lists of the two different approaches towards biomass heterosis was detected than expected by chance. This suggests that each heterotic QTL region contains many genes influencing biomass heterosis in the early development of Arabidopsis thaliana. Furthermore, this integrative analysis led to a confinement and an increased confidence in the group of candidate genes for biomass heterosis in Arabidopsis thaliana identified by both approaches. / Als Heterosis-Effekt wird die Überlegenheit in einem oder mehreren Leistungsmerkmalen (z.B. Blattgröße von Pflanzen) von heterozygoten (mischerbigen) Nachkommen über deren unterschiedlich homozygoten (reinerbigen) Eltern bezeichnet. Dieses Phänomen ist schon seit Beginn des letzten Jahrhunderts bekannt und wird weit verbreitet in der Pflanzenzucht genutzt. Trotzdem sind die genetischen und molekularen Grundlagen von Heterosis noch weitestgehend unbekannt. Es wird angenommen, dass heterozygote Individuen mehr regulatorische Möglichkeiten aufweisen als ihre homozygoten Eltern und sie somit auf eine größere Anzahl an wechselnden Umweltbedingungen richtig reagieren können. Diese erhöhte Anpassungsfähigkeit führt zum Heterosis-Effekt. In dieser Arbeit wird ein systembiologischer Ansatz, basierend auf molekularen Netzwerkstrukturen verfolgt, um zu einem besseren Verständnis von Heterosis beizutragen. Dazu wird eine Netzwerkhypothese für Heterosis vorgestellt, die vorhersagt, dass die heterozygoten Individuen, die Heterosis zeigen, mehr regulatorische Interaktionen in ihren molekularen Netzwerken aufweisen als die homozygoten Eltern. Partielle Korrelationen wurden verwendet, um diesen Unterschied in den globalen Interaktionsstrukturen zwischen den Heterozygoten und ihren homozygoten Eltern zu untersuchen. Die Netzwerkhypothese wurde anhand von Metabolit- und Genexpressionsdaten der beiden homozygoten Arabidopsis thaliana Pflanzenlinien C24 und Col-0 und deren wechselseitigen Kreuzungen getestet. Arabidopsis thaliana Pflanzen sind bekannt dafür, dass sie einen Heterosis-Effekt im Bezug auf ihre Biomasse zeigen. Die heterozygoten Pflanzen weisen bei gleichem Alter eine höhere Biomasse auf als die homozygoten Pflanzen. Die Netzwerkhypothese für Heterosis konnte sowohl im Bezug auf mid-parent Heterosis (Unterschied in der Leistung des Heterozygoten im Vergleich zum Mittelwert der Eltern) als auch auf best-parent Heterosis (Unterschied in der Leistung des Heterozygoten im Vergleich zum Besseren der Eltern) für beide Kreuzungen für die Metabolit- und Genexpressionsdaten bestätigt werden. In einer Überrepräsentations-Analyse wurden die Gene, für die die größte Veränderung in der Anzahl der regulatorischen Interaktionen, an denen sie vermutlich beteiligt sind, festgestellt wurde, mit den Genen aus einer quantitativ genetischen (QTL) Analyse von Biomasse-Heterosis in Arabidopsis thaliana verglichen. Die ermittelten Gene aus beiden Studien zeigen eine größere Überschneidung als durch Zufall erwartet. Das deutet darauf hin, dass jede identifizierte QTL-Region viele Gene, die den Biomasse-Heterosis-Effekt in Arabidopsis thaliana beeinflussen, enthält. Die Gene, die in den Ergebnislisten beider Analyseverfahren überlappen, können mit größerer Zuversicht als Kandidatengene für Biomasse-Heterosis in Arabidopsis thaliana betrachtet werden als die Ergebnisse von nur einer Studie.

Os objetivos deste estudo foram: re-identificar 70 isolados de Candida em micoteca por método tradicional e API 20C; identificar a presença de C. dubliniensis; determinar e comparar as concentrações inibitórias mínimas (CIMs) das amostras, frente a sete antifúngicos (E-test); caracterizar molecularmente as amostras seqüenciais de Candida pela técnica de PFGE; estudar e comparar o perfil genotípico de isolados de C. albicans e C. parapsilosis, através da análise de marcadores microsatélites e cariotipagem. O nível de concordância entre o método tradicional e o API 20C foi de 93%. Nenhum isolado de C. dubliniensis foi identificado no estudo. Em relação à sensibilidade antifúngica, a maioria das amostras apresentou alta porcentagem de sensibilidade às sete drogas testadas. A análise molecular pela técnica de PFGE, mostrou seis perfis de cariótipos diferentes em 24 amostras seqüenciais. Em relação à comparação das técnicas de PFGE e microssatélites, observamos que a última mostrou maior poder discriminatório para as amostras de estudadas. / This study was aimed to: identify the 70 isolates of Candida in a culture collection by the traditional method and by API 20C; identify the presence of C. dubliniensis; determine and compare the minimum inhibitory concentrations (MICs) of these samples in regard to the seven drugs (E-test); molecularly characterize sequential samples from the same patient by the PFGE technique; study the genotypic profile of all the isolates of C. albicans and C. parapsilosis by means of the microsatellite technique and compare the results with those obtained by the PFGE technique. The level of agreement between the traditional method and the API 20C was 93%. No isolate of C. dubliniensis was identified in the study. In relation to antifungal susceptibility, most of the samples presented a high percentage of susceptibility to the seven drugs tested. The molecular analysis by the PFGE technique revealed six different karyotype profiles among 24 sequential samples. In the comparison between the PFGE and microsatellite, the latter showed a greater discriminatory power for samples.

Os objetivos deste estudo foram: re-identificar 70 isolados de Candida em micoteca por método tradicional e API 20C; identificar a presença de C. dubliniensis; determinar e comparar as concentrações inibitórias mínimas (CIMs) das amostras, frente a sete antifúngicos (E-test); caracterizar molecularmente as amostras seqüenciais de Candida pela técnica de PFGE; estudar e comparar o perfil genotípico de isolados de C. albicans e C. parapsilosis, através da análise de marcadores microsatélites e cariotipagem. O nível de concordância entre o método tradicional e o API 20C foi de 93%. Nenhum isolado de C. dubliniensis foi identificado no estudo. Em relação à sensibilidade antifúngica, a maioria das amostras apresentou alta porcentagem de sensibilidade às sete drogas testadas. A análise molecular pela técnica de PFGE, mostrou seis perfis de cariótipos diferentes em 24 amostras seqüenciais. Em relação à comparação das técnicas de PFGE e microssatélites, observamos que a última mostrou maior poder discriminatório para as amostras de estudadas. / This study was aimed to: identify the 70 isolates of Candida in a culture collection by the traditional method and by API 20C; identify the presence of C. dubliniensis; determine and compare the minimum inhibitory concentrations (MICs) of these samples in regard to the seven drugs (E-test); molecularly characterize sequential samples from the same patient by the PFGE technique; study the genotypic profile of all the isolates of C. albicans and C. parapsilosis by means of the microsatellite technique and compare the results with those obtained by the PFGE technique. The level of agreement between the traditional method and the API 20C was 93%. No isolate of C. dubliniensis was identified in the study. In relation to antifungal susceptibility, most of the samples presented a high percentage of susceptibility to the seven drugs tested. The molecular analysis by the PFGE technique revealed six different karyotype profiles among 24 sequential samples. In the comparison between the PFGE and microsatellite, the latter showed a greater discriminatory power for samples.

Made available in DSpace on 2014-08-20T14:37:53Z (GMT). No. of bitstreams: 1 dissertacao_talita_tejada.pdf: 1229434 bytes, checksum: 00aa0198737634a5ddf797dd569f628f (MD5) Previous issue date: 2013-02-04 / Salmonella is one of the main causative agents of foodborne diseases, and chicken-based products play a prominent role in this context, serving as vehicles to the microorganism. The present study aimed to provide data on the Salmonella spread in the poultry chain, check the occurrence of Salmonella and its different serotypes isolated from chicken stool, chicken products and human stool, as well as to verify the similarity between DNA profiles of Samonella isolated. Literature on the occurrence of Salmonella in the poultry chain was reviewed; parallel to it, a project in which 600 samples (200 chicken meat, 200 chicken stool and 200 human stool samples) were analyzed for Salmonella presence was developed. DNA profiles of isolated strains were obtained by PFGE and REP-PCR. The microorganism was isolated from 16 samples, 8 (8/200 4%) from chicken products, 4 of which (4/200 2%) from chicken stool and 4 (4/200 2%) from human stool. Salmonella serotype Schwarzengrund was found to prevail both in chicken meat and chicken stool, followed by serotype Mbandaka, whereas serotype Panama prevailed in humans. Strains with indistinguishable genotypes were found to be present both in chicken stool and chicken products, suggesting that the chicken contamination on the farm remained in the processed product. In humans, the isolated strains were indistinguishable between one another, suggesting an outbreak occurrence; however, the isolated serotypes in humans were not the same as those in chickens, which is probably related to different contamination sources. / Salmonella é um dos principais agentes causadores de doenças transmitidas por alimentos e os produtos a base de frango tem destaque importante neste contexto, podendo servir de veículo desse micro-organismo. O presente trabalho teve como objetivo apresentar dados quanto à propagação de Salmonella na cadeia avícola, verificar a ocorrência de Salmonella e de suas diferentes sorotipos isoladas de fezes de frango, produtos de frango e fezes humanas, bem como para verificar a similaridade entre os perfis de DNA de Salmonella isolados no extremo sul do Brasil. Foi realizada uma revisão bibliográfica discorrendo sobre Salmonella na cadeia aviária e paralelamente foi desenvolvido um projeto, no qual foram analisadas 600 amostras (200 de carne de frango, 200 de fezes de frango e 200 de fezes de humanos), quanto à presença de Salmonella. Os perfis de DNA das cepas isoladas foram obtidos em PFGE e REP-PCR. O micro-organismo foi isolado de 16 amostras, sendo 8 (8/200 4%) de produtos de frango, 4 (4/200 2%) de fezes de frango e 4 (4/200 2%) de fezes de humanos. Observou-se que, tanto em carne de frango como fezes de frango, o sorotipo predominante foi Schwazengrund, seguido de Mbandaka. Em humanos, predominou S. Panama. Foi constatado que de cepas com genótipos indistinguíveis estavam presentes tanto em fezes de frango como produtos de frango, sugerindo que a contaminação do frango no aviário permaneceu no produto processado. Em humanos, as cepas isoladas foram indistinguíveis entre si sugerindo que tenha ocorrido um surto, no entanto, os sorotipos isolados não foram os mesmos dos isolados de frango, o que sugere outra fonte de contaminação.

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