A floresta plantada de eucalipto representa a maior porcentagem entre as florestas plantadas no Brasil, principalmente para a producao de papel e celulose. Porem, durante todo o seu desenvolvimento, o eucalipto sofre o ataque de diferentes patogenos, e entre eles esta o fungo Puccinia psidii causador da ferrugem, principal doenca do eucalipto em regioes tropicais. Assim, com o intuito de estudar alguns mecanismos de defesa da planta contra a infeccao do fungo, este trabalho visou analisar a expressao genica de enzimas relacionadas ao estresse oxidativo e a atividade das mesmas; e o proteoma de clones de eucalipto resistentes e suscetiveis, ao longo do processo de infeccao, colonizacao e multiplicacao do fungo nas plantas. Foi possivel observar diferencas na expressao dos genes em todos os horarios estudados, principalmente as 24 horas apos inoculacao com o fungo. Alem disso, com a analise do proteoma pode-se observar algumas proteinas de grande relevancia para este trabalho, como algumas relacionadas a estresse oxidativo e a resposta de defesa da planta. / The eucalyptus planted forest represents the major percentage among all planted forests in Brazil, mainly for the production of paper and cellulose. However, during its development, the eucalyptus can be attacked by a large number of different pathogens, like the fungus Puccinia psidii, the causer of the eucalyptus rust, main disease that affects the eucalyptus in tropical regions. Thus, in order to study some plant defense mechanisms against the infection of the fungus, this study aimed to analyse the gene expression of some enzymes related to oxidative stress and their activities, and the proteome of resistant and susceptible eucalyptus clones, during the process of infection, colonization and multiplication of the fungus in the plant. We could observe differences in the gene expression at all times studied, mostly at 24 hours after inoculation with the fungus. Besides, with the proteome analysis we could find the very relevant proteins for this study, for instance some proteins related to the oxidative stress and to the plant defense response.

A significativa prevalência da infecção pelo HTLV-1/2 no Brasil tornou compulsória a triagem sorológica em bancos de sangue desde 1993. A realização de um diagnóstico eficaz e seguro desta infecção tem importância no correto aconselhamento dos pacientes, bem como na adoção de medidas preventivas em relação à transmissão do HTLV-1/2. Entretanto, a ineficiência dos testes confirmatórios disponíveis somado ao alto custo, são fatores limitantes para a conclusão segura do status clínico do paciente. O objetivo deste trabalho foi desenvolver, padronizar e validar uma plataforma molecular (NAT) multiplex utilizando a metodologia de PCR em tempo real para o diagnóstico confirmatório e discriminatório da infecção pelo HTLV-1/2. Para tanto, foi padronizada a PCR em tempo real utilizando o sistema de sondas de hidrólise (TaqMan®) e a região gênica viral pol foi utilizada como alvo dessas reações. As reações foram otimizadas e validadas em amostras de DNA de controles positivos e negativos, amostras de DNA obtidas a partir do sangue total de indivíduos infectados pelo HTLV-1/2, candidatos à doação de sangue e de pacientes infectados por outras viroses (HIV, HBV e HCV). Após a padronização, a plataforma molecular foi validada em relação aos seguintes parâmetros: sensibilidade analítica, sensibilidade diagnóstica, especificidade analítica, especificidade diagnóstica, precisão e robustez. O limite de detecção do teste (LoD) foi de 3,85 cópias/reação para HTLV-1 e 10,88 cópias/reação para HTLV-2. Para a especificidade, não foi observada reação cruzada com os vírus HAV, HBV, HCV, HIV-1 e HIV-2 e B19V obtidos a partir de um painel de referência viral e nem com amostras de indivíduos portadores de HIV, HBV ou HCV. A sensibilidade diagnóstica foi de 94,6% para HTLV-1 e 78,6% para HTLV-2. O coeficiente de variação encontrado no parâmetro precisão foi de até 0,475%. A metodologia desenvolvida é uma ferramenta simples, de baixo custo, sensível e altamente específica, portanto, adequada para detecção e discriminação da infecção por este retrovírus. A padronização desta plataforma tem um importante impacto no fortalecimento da capacitação tecnológica nacional. Além disso, propõe seu uso no algoritmo diagnóstico, o qual permitirá a conclusão segura do diagnóstico do HTLV-1/2. / The significant prevalence of HTLV-1/2 infection in Brazil has turned the serological testing for this virus mandatory in national blood banks since 1993. The performance of efficient and safe diagnosis of this infection has importance on the correct counseling of the patients and the prevention of the transfusion/hemoderivatives transmitted HTLV-1/2 infection. However, the inefficiency of the available confirmatory tests combined with their high cost present limiting factors for adequate conclusions over the clinical status of the patient. A safe diagnosis of the HTLV-1/2 infection is of crucial importance for the correct counseling of the patients and prevention of the transmission of the infection by blood transfusions, breastfeeding and solid organ transplantations. Therefore, the objective of this study was to develop, optimize and validate a molecular multiplex platform (NAT) utilizing the real-time PCR methodology for confirmatory and discriminatory diagnosis of the HTLV-1/2 infection. DNA samples obtained from HTLV-1/2 positive patients, blood donor candidates, and HIV, HBV and HCV infected patients were used in this study. The real-time PCR was optimized using the TaqMan® system (hydrolysis probes) and the pol gene was a target for real-time amplification. After optimization, the molecular platform was validated by analysis of the analytic and diagnostic sensitivity, analytic and diagnostic specificity, precision, and robustness. The detection limit (LoD) of the test was 3.85 copies/reaction for HTLV-1 and 10.88 copies/reaction for HTLV-2. Evaluation of the specificity did not demonstrate cross reaction with human viruses like HAV, HBV, HCV, HIV-1, HIV-2 and B19V obtained from a diagnostic panel and also with samples obtained from HIV, HBV or HCV positive individuals. The diagnostic sensitivity was 94.6% for HTLV-1 and 78.6% for HTLV-2. The estimated variation coefficient of the precision analysis was not more than 0.475%. Therefore, this methodology presents simple, inexpensive, sensitive and highly specific test, and can be used adequately for the detection and discrimination of this retroviral infection. The optimization of this molecular platform have important impact on the improvement of the technologic capability and it can be used for the definition of a national diagnostic algorithm which will permit a safe conclusion of the HTLV-1/2 diagnosis.

O cultivo de células humanas para fins experimentais e terapêuticos se firmou como um dos pilares da biologia celular e tem se tornado uma prática laboratorial cada vez mais disseminada. Associada a esse processo está a contaminação por microrganismos, dentre os quais se destacam as bactérias do gênero Mycoplasma spp., os quais são os contaminantes detectados com maior frequência in vitro. Um dos pontos críticos no processo de garantia da qualidade, pureza e segurança para uso destas células é a adoção de uma metodologia analítica, para detecção de Mycoplasma spp., que seja simples e de rápida execução, com sensibilidade e especificidade adequada e economicamente viável. O objetivo desse trabalho foi desenvolver uma plataforma molecular (PCR em tempo real) para detecção de Mycoplasma spp. em culturas celulares. Para tanto, foram testados diversos conjuntos de primers (gene 16S rDNA) dentre os quais um foi selecionado para a plataforma molecular. Foram utilizadas amostras de DNA obtidas a partir do sobrenadante de cultura. A PCR em tempo real in house foi padronizada utilizando o intercalante fluorescente de DNA dupla fita BRYT Green® e produziu um fragmento de 270 pares de bases. A temperatura de melting para as amostras positivas foi definida no intervalo de 81 a 84°C. A plataforma molecular foi validada por meio dos parâmetros sensibilidade analítica e diagnóstica, especificidade analítica, potencial de arraste, precisão e robustez. O limite de detecção do teste (LOD) foi de 5 cópias/5 ?L. Para a especificidade, foi observada reação cruzada com bactérias de relação filogenética próxima e com DNA de célula humana. A sensibilidade diagnóstica foi de 100%. Não foi houve contaminação por arraste. O coeficiente de variação encontrado no parâmetro precisão foi de 0,64% e 1,80% para a avaliação intra-ensaio e inter-ensaio, respectivamente. O coeficiente de variação da análise robustez foi de 5,42%. Ao comparar a plataforma molecular com o teste comercial, observou-se que 29,85% (n=40) das amostras apresentaram resultados falso-negativos pelo teste comercial. Determinou-se ainda que a PCR em tempo real in house foi mais sensível do que o teste comercial. A análise por microscopia eletrônica de varredura demonstrou a presença de estruturas sugestivas de espécies de Mycoplasma spp. na superfície das células. A análise filogenética permitiu a classificação das espécies encontradas. Os resultados obtidos neste trabalho permitiram propor o algoritmo para aplicação de um método de detecção simples e rápido para Mycoplasma spp.; e fazer deste uma ferramenta para o controle de qualidade das células cultivadas, garantindo a eficiência destas células para uso em pesquisa e maior segurança para uso em terapia celular. / The culture of human cells for experimental and therapeutic purposes has been consolidated as one of the foundations of cell biology and has increasingly become a widely used laboratory practice. The contamination by microorganisms is associated with this process, among them we can highlight bacteria of the genus Mycoplasma spp., which are contaminants more frequently detected in vitro. One of the critical points in the process of assuring quality, purity, and safety for the use of these cells is the adoption of an analytical methodology for the detection of mycoplasma that is simple and fast to conduct, with proper sensitivity and specificity and that is also economically viable. The objective of this work was to develop a molecular platform (real time PCR) for the detection of mycoplasmas in cell cultures. Therefore, we tested several sets of primers (16S rDNA gene), among them one was selected for the molecular platform. DNA samples used were obtained from the culture supernatant. In house real time PCR was standardized using BRYT Green® double-stranded DNA intercalating fluorescent and produced a fragment of 270 pair bases. The melting temperature for the positive samples was set in the range 81 to 84°C. The molecular platform was validated through the parameters: analytical and diagnostic sensitivity, analytical specificity, carry-over contamination, precision and robustness. The limit of detection of the test (LOD) was 5 copies/5 ?L. For specificity, it was observed a cross reaction with bacteria of close phylogenetic relationship and with human cell DNA. Diagnostic sensitivity was 100%. There was no carry-over contamination. The coefficient of variation was found in the precision values of 0.64% and 1.80% for intra-assay and inter-assay assessment, respectively. The variance coefficient of the robustness analysis was 5.42%. By comparing the molecular platform with the commercial test, we observed that 29.85% (n=40) of the samples showed false negative results by the commercial test. It has been determined further that the real-time PCR in house was more sensitive than the commercial test. The analysis by scanning electron microscopy demonstrated the presence of suggestive structures of Mycoplasma spp. species in the surface of cells. Phylogenetic analysis allowed the classification of the species found. The results obtained in this work allowed us to propose the algorithm for application of a simple and fast method for mycoplasma detection and making from this tool for quality control for cultured cells, assuring the efficiency of these cells for use in research and greater safety for use in cell therapy more safety to patients subjected to cell therapy.

Respiratory diseases in cattle can cause economic losses due to the decreased dairy and meat production. Virus is the main reason for these diseases. Symptoms can be fever, cough and nasal discharge.     Influenza are a group of viruses belonged in the Ortomyxoviridae family. The big influenza groups are influenza A, B and C. The viruses can cause respiratory signs, and mammals can be affected. Recently a new influenza virus was found in the United States. The influenza virus was found in swine, but the natural host was later considered to be cattle. The virus was named influenza D. Different studies worldwide have confirmed the virus in a variety of regions. Antibodies have also been reported.     In this study, virologic and serologic methods were used to detect if influenza D circulates among cattle in Sweden. The serologic method performed was indirect ELISA. Serum and milk samples were investigated in the ELISA method. For the virologic detection a real-time RT-PCR was made, with a variety of study material.     Antibodies against influenza D were found in both serum and milk samples. No virus was found in the real-time RT-PCR. In Sweden the animal keeping is different compared to several other nations. For instance, the conditions of health and hygiene are better in Sweden, this may be an important cause of a system more resistant against spreading of infections. Influenza D could be more common in Sweden, but in that case further researches are needed to determine the prevalence.

O Brasil é o maior produtor mundial de feijão com produção média anual de 3,5 milhões de toneladas, entretanto, um dos maiores problemas enfrentados por essa cultura é o déficit hídrico, que leva a uma redução considerável em seu rendimento. Dessa forma, a identificação de genes que controlam os mecanismos de defesa e adaptação do feijoeiro à falta de água durante o seu desenvolvimento é de grande utilidade. E, como a tolerância à seca é um caráter multigênico, genótipos com diferentes graus de tolerância apresentam expressão gênica diferencial, com ativação e/ou repressão de determinados genes. Diante disso, esse trabalho teve como objetivos: (i) identificar genes diferencialmente expressos em dois genótipos de feijoeiro comum, sendo um tolerante (BAT 477) e o outro suscetível (IACCarioca 80SH) à seca; (ii) verificar a expressão gênica espacial (em raízes, caules e folhas) e temporal (cinco níveis crescentes de déficit hídrico - 24 h, 48 h, 72 h, 96 h e 120 h de exposição ao estresse) por meio de RT-qPCR; (iii) predizer a função dos genes identificados, com base nos genes ortólogos de Arabidopsis thaliana, usando dados públicos de microarranjo disponíveis na plataforma Genevestigator. Para atingir esses objetivos, foi conduzido um experimento em casa-de-vegetação, sendo induzido o déficit hídrico, por meio da suspensão da irrigação, quando as plantas atingiram o estádio fenológico R5, mantendo sob suprimento hídrico adequado as plantas-controle. Foi então utilizada a técnica de cDNA-AFLP para isolar os transcritos diferencialmente expressos entre os dois genótipos, sob seca, a qual aliada ao sequenciamento possibilitou a identificação e anotação de 45 transcritos, sendo 21 exclusivamente expressos no genótipo tolerante e 24 no genótipo suscetível. Dentre os transcritos identificados no genótipo tolerante, podem ser listados pelo menos 11, com potencial de serem usados para transformação genética (chlorophyll A-B binding protein, HSP40, HSP70, glycosyl hydrolase, serine/threonine protein kinase, trehalose-6-phosphate synthase, E3 ubiquitin ligase, fructose biphosphate aldolase, mediator complex subunit 13, aquaporin nodulin MTN-3-related e TCP transcription factor), e no genótipo suscetível, podem ser listados nove (coatomer protein complex, monoamine-oxidase A repressor R1, synaptobrevin, haloacid dehalogenase-like hydrolase, ADP-ribosylation factor, mTERF, serine protease S1C HtrA-related, legume lectin e SWI/SNF-related chromatin binding). Na análise de expressão gênica espacial e temporal, os transcritos mais promissores para uso em trabalhos futuros de transformação genética foram: aquaporin nodulin MTN3, E3 ubiquitin ligase, serine/threonine protein kinase, glycosyl hydrolase e HSP 70 protein, uma vez que tiveram uma expressão bastante pronunciada no genótipo tolerante. Através das análises in silico, baseadas nos genes ortólogos de A. thaliana, foram descobertos processos e vias metabólicas que podem estar envolvidos na resposta do feijoeiro comum ao déficit hídrico. Além disso, foram identificados genes associados com a tolerância à seca, corroborando os dados experimentais. Sendo assim, os resultados obtidos nesse trabalho fornecem o entendimento necessário ao desenvolvimento de ferramentas moleculares (marcadores para genes diferencialmente regulados) para serem utilizados em programas de melhoramento, bem como para informação genética básica na anotação funcional do genoma do feijoeiro e também para utilização desses genes candidatos em trabalhos de transformação genética para obtenção de plantas mais tolerantes à seca. / Brazil is the largest producer of common beans, with average annual production of 3.5 million tonnes; however, one of the biggest problems faced by this crop is drought, which leads to a considerable reduction in their yield. Thus, the identification of genes that control the defense mechanisms and adaptation of common bean to drought during its development is very useful. Drought tolerance is a multigenic character, so genotypes with different degrees of water deficit tolerance exhibit differential gene expression, with activation and/or repression of certain genes. Therefore, this study aimed to: (i) identify differentially expressed genes in two common bean genotypes, one tolerant (BAT 477) and other susceptible (IAC-Carioca 80SH) to drought, (ii) verify the spatial (root, stem and leaves) and temporal (five increasing levels of water deficit - 24 h, 48 h, 72 h, 96 h and 120 h of stress exposition) gene expression by RTqPCR (iii) predict the genes function, based on Arabidopsis thaliana orthologous genes, using available data in the public microarray platform Genevestigator. To achieve these objectives, an experiment was conducted in a green house, being induced water deficit, by withholding water when the plants reached growth stage R5, maintaining adequate water supply under the control plants. To isolate transcripts differentially expressed between the two genotypes under drought was used the cDNA-AFLP technique, which coupled with sequencing enabled the identification and annotation of 45 transcripts, 21 exclusively expressed in the tolerant genotype and 24 in the susceptible one. Among the transcripts identified in the tolerant genotype, may be listed at least 11, with potential to be used in genetic transformation (chlorophyll A-B binding protein, HSP40, HSP70, glycosyl hydrolase, serine/threonine protein kinase, trehalose-6-phosphate synthase, E3 ubiquitin ligase, fructose biphosphate aldolase, mediator complex subunit 13, aquaporin nodulin MTN-3-related and TCP transcription factor), and in the susceptible genotype, can be listed nine (coatomer protein complex, monoamine-oxidase A repressor R1, synaptobrevin, haloacid dehalogenase-like hydrolase, ADP-ribosylation factor, mTERF, serine protease S1C HtrA-related, legume lectin and SWI/SNF-related chromatin binding). In the spatial and temporal gene expression analysis, the transcripts that stood out for use in genetic transformation future studies were: aquaporin nodulin MTN3, E3 ubiquitin ligase, serine/threonine protein kinase, glycosyl hydrolase and HSP 70 protein, since it had an expression quite pronounced in the tolerant genotype. Through the in silico analysis, based on orthologous genes of A. thaliana, was discovered processes and metabolic pathways that may be involved in the common bean response to drought. In addition, we identified genes associated with drought tolerance, corroborating the experimental data. Thus, the present results provide the necessary understanding to develop molecular tools (markers for differentially regulated genes) to be used in breeding programs, as well as basic genetic information in the common bean functional genome annotation and also to use these candidate genes for genetic transformation to obtain drought-tolerant plants.

O ciclo estral das cadelas difere das demais espécies domésticas. Estudos demonstraram que o aumento da concentração plasmática de P4 durante a fase luteínica das cadelas pode levar a alterações metabólicas como a resistência insulínica, acarretando complicações como Diabetes mellitus. A glicose é uma molécula transportada, na maioria das células, por proteínas transportadoras. O processo de instalação da resistência insulínica é caracterizado por alterações teciduais da expressão de algumas proteínas transportadoras de glicose, como o GLUT4. Atualmente 13 isoformas de proteínas transportadoras já foram seqüenciadas (GLUT1 ao GLUT13). O GLUT4 está presente nos músculos e no tecido adiposo, principalmente. Para avaliar se a expressão do GLUT 4 está presente nas células luteínicas e se esta expressão relaciona-se à produção de P4 e E2, 28 cadelas foram divididas em 7 grupos de acordo com os dias após a ovulação -p.o. (de 10 à 70 dias, n = 4 por grupo). Os ovários foram dissecados e congelados em nitrogênio líquido, o RNA extraído e o cDNA confeccionado e submetido ao PCR em tempo real. O gene GAPDH foi utilizado como controle endógeno para padronização da expressão do gene alvo. Foi coletado sangue para dosagem da glicemia, insulinemia, progesterona e estradiol. Para avaliar a regulação positiva do GLUT4 avaliamos também a expressão do mRNA do HIF-1α, destas mesmas cadelas. A expressão do GLUT4 apresentou tendência a aumento de expressão aos 20 dias (p. o.), quando comparado aos 10, 30 e 40 dias, pico de expressão aos 50 dias (p.o.), e então apresentou tendência a queda aos 60 e 70 dias p.o. Já a expressão do HIF-1α manteve-se muito semelhante através dos dias, tendendo a queda aos 10 e aos 40 dias pós ovulação, quando comparado com os demais grupos. Os resultados de dosagem de P4 e E2 variaram dentro do esperado para o diestro e não apresentaram correlação com a expressão de GLUT 4; a glicemia e insulina, aqui expressas através do índice HOMA (insulina x glicose % 22,5), apresentou pico aos 40 dias. Sabe-se que quanto mais alto o índice HOMA, menos este animal é sensível à insulina, ou seja, mais resistente à ela. Observou-se que o índice HOMA apresentou-se mais alto aos 40 dias, associado aos menores valores de expressão do GLUT4. Por outro lado, obtivemos o pico de expressão de GLUT4 aos 50 dias, quando o índice HOMA apresentou valores baixos. Sugere-se que a queda da P4 associada à elevação do estradiol plasmático possa influenciar o índice HOMA. Pode-se concluir que a expressão do GLUT4 no corpo lúteo de cadelas segue o padrão observado para tecidos sensíveis à insulina, nos quais existe uma maior expressão durante a fase de maior sensibilidade à insulina e diminuição drástica em fase de pré ou já instalada resistência insulínica. / The canine estral cycle differs from other domestic species. Some studies demonstrated that the increase of the plasmatic concentration of progesterone during canine luteinic phase can lead to metabolic alterations, such as insulinic resistance and may cause complications such as Diabetes mellitus. Glucose is a molecule that is transported in most cells by transporting proteins. The process of installation of the insulinic resistance is characterized by tissue alterations of the expression of some glucose transporting proteins, as GLUT4. Currenly, 13 isoforms of transporting proteins were sequenced (GLUT1 to GLUT13). GLUT4 is present mainly in muscle and fat tissue. In order to assess if GLUT4 expression is present in luteal cells, and if this expression is related to P4 and E2 production, 28 bitches were divided into 7 groups, in accordance with the days after the ovulation -p.o. (from 10 to 70 days, n=4 for group). The ovaries were dissected and frozen in liquid nitrogen. The RNA was extracted and the cDNA was made and submitted to real time PCR. GAPDH gene was used as endogenous conntrol to standardization of target gene expression. Blood was collected to glycemia, insulinemia, P4, and E2β dosage. To assess the positive regulation of GLUT4, we also assessed HIF-1α mRNA expression of the same bitches. GLUT4 expression showed a tendency to increase the expression on the twentieth day (p.o.), when compared to the 10th, 30th, and 40th days, expression top on the 50 th day (p.o.) , and then, it showed a tendency to foll on the 60th and 70th days p.o. HIF-1α expression was very similar over the days, tending to fall on the 10th and 40th days post ovulation, when compared to other groups. P4 and E2β dosage results varied according to thr expectations in diestrus and have not shown correlation with GLUT4 expression; glycemia and insulin, here expressed by HOMA index (insulin x glucose % 22,5) showed crest (highest point) on the 40 th day. It is knows that the higher the HOMA index, the less sensitive this animal is to insulin, it is, more resistant to it. It was observed that HOMA index was higher on the 40th day, associated to small values of the GLUT4 expression. Otherwise, the got the top of GLUT4 expression on the 50th day, when HOMA index showed low values. It has been suggested that P4 fall associated to the plasmatic E2 increase may influence HOMA index. We may conclude that GLUT4 expression into the corpus luteum of bitches follows the standard observed in insulin-sensitive tissues, in which there is a higher expression over the phase of higher sensitiveness to insulin and remarkable decrease in pre or even installed insulinic resistance.

A diarréia neonatal é o principal problema sanitário que acomete os bezerros nas primeiras semanas de vida, causando grandes prejuízos devido à morbidade, mortalidade, custos com tratamento e atraso no desenvolvimento. Os rotavírus são os mais importantes agentes virais causadores de gastroenterites em crianças e diarreia em diferentes espécies animais. Além do seu impacto econômico na produção animal, os bovinos podem atuar como reservatórios da diversidade genética e antigênica para humanos. Assim, o diagnóstico deste agente é fundamental para o desenvolvimento de medidas profiláticas mais especificas visando o seu controle. O objetivo deste trabalho foi desenvolver um método de PCR em Tempo Real para a detecção de rotavírus bovinos em sistema Taqman® tendo como alvo, a proteína não-estrutural 5 (NSP5). Para isso, 113 amostras de fezes bovinas foram coletadas de reabanhos do estado de São Paulo, e previamente testadas por PCR convencional. Para a padronização da PCR em Tempo Real, a estirpe padrão foi clonada, gerando um plasmídeo com 3x1010 cópias/reação. Como controle exógeno foi utilizado β-actina. O limite de detecção foi determinado por diluições seriadas na base 10, detectando até 6x101 cópias/µl. A curva padrão da PCR em Temo Real para a detecção do segmento codificador da proteína NSP5 teve como resultados, uma eficiência de 100,47%; com slope igual a -3,18 e R2 de 1,0. De um total de 113 amostras testadas pela PCR convencional 63 delas (55,7%) foram consideradas positivas para rotavírus. Dessas mesmas amostras testadas, 5 não amplificaram para β-actina e não foram incluídas nas análises posteriores. Para a PCR em Tempo Real o limite de detecção foi considerado o valor de 6x101 cópias/reação, sendo definido o ponto de corte o ciclo (Ct) de número 36 para o teste com a amostra viral a partir do DNA ligado em vetor plasmidial. Considerando-se o ponto de corte de 60 (6x101) cópias/reação, das 108 amostras testadas, 63 (58,3%) foram consideradas positivas ao teste. O valor de concordância obtido através do teste Kappa, a um intervalo de confiança de 95%, a partir dos resultados gerados entre os testes de PCR convencional e PCR em foi de 0.279 (baixa concordância). Os resultados obtidos nesse estudo demonstrou que o teste foi eficiente podendo ser utilizado para um diagnóstico rápido e eficiente do rotavirus do grupo A, aumentando assim o repertório dos testes já estabelecidos / Neonatal diarrhea is the main health problem affecting the calves in the first weeks of life, causing major losses due to morbidity, mortality, treatment costs and delayed development. Rotaviruses are the most important causative agents of viral gastroenteritis in children and in different animal species. In addition to its economic impact on livestock, cattle can act as reservoirs for genetic and antigenic diversity for human samples. Therefore, the diagnosis of this agent is critical to the development of more specific preventive measures for their control. The objective of this work is to develop a method of PCR for rotavirus detection in cattle using TaqMan® system targeting the 5 nonstructural protein (NSP5). For this, 113 samples of cattle feces were collected from farms of São Paulo, and previously tested by convencional PCR. To PCR standardization, the standard sample was cloned, generating plasmid 3x1010 copies/reaction. The β-actin was usede as exogenous control. The limit of detection was determined by serial dilutions, to detect 6 x101 copies/µl. The standard curve of PCR to encoding segment detection NSP5 protein had as a result, an efficiency of 108.5%; with slope equal to -3.18 and R2 equal 1.. From a total of 113 samples tested by conventional PCR 63 (55.7%) were positive for rotavirus. From these samples 5 not amplifyed for β-actin gene and were not included in subsequent analyzes. For detection limit of Real-Time PCR was considered the amount of 6x101 copies / reaction, the cut-off being defined cycle (Ct) number 36 to the test with a viral sample from the DNA ligated into plasmid vector. Considering the cut-off 60 (6x101) copies/reaction of the 108 samples tested, 63 (58.3%) were positive to the test. The correlation value obtained by the Kappa test, at a 95% confidence interval, based on results generated between the conventional and PCR testing PCR was 0.279 (low agreement). The results of this study demonstrated that the probe can be efficiently used for a fast and efficient diagnosis of rotavirus of group A, thereby enhancing the repertoire of the established tests

Seafood substitution is a worldwide problem due to factors such as limited monitoring coupled with complex supply chains. Red snapper (Lutjanus campechanus) is a highly valued and overfished species that is commonly substituted with other fish, such as tilapia, rockfish, and other snapper species. DNA barcoding is typically used by regulatory agencies to detect seafood substitution; however, it is expensive and time-consuming. A rapid, real-time PCR assay targeting red snapper was developed previously for use in fisheries management; however, it has not been tested for its ability to detect red snapper species substitution. The objective of this study was to assess the ability of the real-time PCR assay to identify red snapper fillets and differentiate red snapper from common substitute fish species in combination with DNA barcoding. A total of 21 fresh/frozen fillets labeled as “red snapper” were tested with real-time PCR, along with 57 fresh/frozen fillets representing 15 of the most common categories of fish mislabeled as red snapper. All samples were tested with DNA barcoding to confirm the identity of fish species. Real-time PCR parameters were optimized to reduce background signals associated with cross-reactivity. Overall, real-time PCR identified 4 samples as red snapper: 3 were authenticated as red snapper with DNA barcoding and 1 was identified as mahi-mahi. Overall, 40% of all samples and 91% of “red snapper” samples were considered mislabeled according to DNA barcoding. Red snapper was substituted with other snapper species (e.g., Lutjanus malabaricus, Lutjanus peru, Ocyurus chrysurus, and Rhomboplites aurorubens), rockfish (Sebastes flavidus and Sebastes brevispinis), sea bream (Pagrus major/Pagrus auratus), and mahi-mahi (Coryphaena hippurus). The real-time PCR assay tested in this study can serve as a rapid screening test for the detection of mislabeled species, which can then be confirmed with sequencing techniques. This species identification technique has the potential to be used by regulatory agencies to rapidly determine the authenticity of red snapper on-site.

The emergence of MNAzymes (Multi-component nucleic acid enzymes) provides a new approach for detection of target analytes in various applications. In this thesis, three novel MNAzyme-based methodologies were developed to expand the range of the applications of MNAzymes. MNAzymes can be coupled with DNA or RNA ligands called aptamers to generate an apta-MNAzyme system, which can be used for the detection of non-nucleic target analytes such as small molecules and proteins. Direct detection using apta-MNAzyme system is performed in a format, which was isothermal, fluorescent, rapid, and requires no protein enzymes. Apta-MNAzymes can be coupled with a signal amplification cascade to increase the sensitivity of the reaction. Another MNAzyme-based methodology termed truncated MNAzyme arm system was developed to discriminate the presence of a single base mismatch of two closely related sequences. The system employs a partzyme with a truncated sensor arm and a stabiliser oligonucleotide that binds adjacently to the truncated sensor arm to stabilise the active MNAzyme structure. Truncated MNAzyme real-time PCR system is capable of discriminating the presence of a single base mismatch in a target DNA with high specificity and sensitivity (down to approximately 10 gene copies). The generic nature of the system enables simultaneous detection of three SNP targets in a multiplex format. MNAzymes was also investigated with various strategies to discriminate DNA sequences that are either methylated or unmethylated. In this thesis, bisulphite-treated DNA samples present in as low as 0.032 % of methylated DNA in a background of unmethylated DNA were discriminated using MNAzyme real-time methylation specific PCR (MSP) system. Furthermore, the presence of 5-methylcytosines in a target sequence increases the melting temperature of the duplex DNA. This was exploited further to directly discriminate DNA methylation status of target sequences using the truncated MNAzyme arm system without the need for bisulphite modification. Findings in this thesis have broadened the scope of MNAzymes as versatile tools for many possible applications and flexible alternative to the current technologies.

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.