The gastrointestinal tract is colonized by an abundant and diverse community of microorganisms which has a profound impact on the health of the host. The profiling of these microbial communities with traditional culture-based methods identifies only a fraction of microbes present with limited specificity, high labour costs and limited sample throughput. To overcome these limitations, a molecular hybridization assay was developed and characterized using the target gene chaperonin 60 (cpn60). The interspecies discriminatory ability of the hybridization assay was determined by hybridizing cpn60 gene fragments from a known species to a series of cpn60 gene fragments derived from related species with distinct but similar cpn60 sequences. Species with less than 85% cpn60 sequence identity to the probe DNA were effectively distinguished using the hybridization approach. To characterize complex microbial communities, universal PCR primers were used to amplify a fragment of 549-567 nucleotides from cpn60 (the cpn60 universal target (UT)) using template DNA extracted from the ileal contents of pigs fed diets based on corn (C), barley (B), or wheat (W), or from plasmids containing the cpn60 UT selected from a clone library generated from these contents. The intensity of hybridization signals generated using labelled probes prepared from library clones designated B1 (Bacillales-related), S1 (Streptococcus-related), C1 (Clostridiales-related), and L10 (Lactobacillales-related) and targets prepared from ileal contents of C, W, or B-fed pigs correlated closely with the number of genomes of each bacterial group as determined by quantitative PCR. Universal PCR primers were also used to amplify genomic DNA extracted from jejeunal contents of pre- and post-weaning piglets. Labelled probe DNA was prepared from S1, L10, LV (Lactobacillus vaginalis-related) and EC (E.coli) library clones. The resulting signal intensities correlated with quantitative polymerase chain reaction (qPCR) data for L10 and LV, but minimal correlation was observed for the S1 and EC groups. A cpn60- based macroarray has potential as a tool for identification and semi-quantification of shifts in colonization abundance of bacteria in complex communities, providing a similar amount of data as techniques such as denaturation gradient gel electrophoresis or terminal restriction fragment length polymorphism analysis.

The gastrointestinal tract is colonized by an abundant and diverse community of microorganisms which has a profound impact on the health of the host. The profiling of these microbial communities with traditional culture-based methods identifies only a fraction of microbes present with limited specificity, high labour costs and limited sample throughput. To overcome these limitations, a molecular hybridization assay was developed and characterized using the target gene chaperonin 60 (cpn60). The interspecies discriminatory ability of the hybridization assay was determined by hybridizing cpn60 gene fragments from a known species to a series of cpn60 gene fragments derived from related species with distinct but similar cpn60 sequences. Species with less than 85% cpn60 sequence identity to the probe DNA were effectively distinguished using the hybridization approach. To characterize complex microbial communities, universal PCR primers were used to amplify a fragment of 549-567 nucleotides from cpn60 (the cpn60 universal target (UT)) using template DNA extracted from the ileal contents of pigs fed diets based on corn (C), barley (B), or wheat (W), or from plasmids containing the cpn60 UT selected from a clone library generated from these contents. The intensity of hybridization signals generated using labelled probes prepared from library clones designated B1 (Bacillales-related), S1 (Streptococcus-related), C1 (Clostridiales-related), and L10 (Lactobacillales-related) and targets prepared from ileal contents of C, W, or B-fed pigs correlated closely with the number of genomes of each bacterial group as determined by quantitative PCR. Universal PCR primers were also used to amplify genomic DNA extracted from jejeunal contents of pre- and post-weaning piglets. Labelled probe DNA was prepared from S1, L10, LV (Lactobacillus vaginalis-related) and EC (E.coli) library clones. The resulting signal intensities correlated with quantitative polymerase chain reaction (qPCR) data for L10 and LV, but minimal correlation was observed for the S1 and EC groups. A cpn60- based macroarray has potential as a tool for identification and semi-quantification of shifts in colonization abundance of bacteria in complex communities, providing a similar amount of data as techniques such as denaturation gradient gel electrophoresis or terminal restriction fragment length polymorphism analysis.

Paracoccidioides brasiliensis, um fungo termodimórfico, é o agente causador da paracoccidioidomicose (PCM), a micose sistêmica prevalente da América Latina. A patogenicidade aparenta estar intimamente relacionada com a transição dimórfica da forma de micélio para a de levedura, que é induzida pela mudança da temperatura do ambiente pela temperatura do hospedeiro mamífero. Há poucas informações disponíveis sobre genes de P. brasiliensis que são necessários durante a fase patogênica. Nós, então, realizamos as técnicas de SSH (“Suppression Subtraction Hybridization") e de “Macroarray" com o objetivo de identificar genes que sejam preferencialmente expressos na fase leveduriforme do isolado Pb18. Genes identificados em ambos os procedimentos estão mais expressos na fase leveduriforme e estão envolvidos em metabolismo básico, transdução de sinal, crescimento e morfogênese e metabolismo do enxofre. Para testar se as mudanças observadas na expressão gênica refletem as diferenças entre as condições de crescimento usadas para obter as duas formas morfológicas preferivelmente às diferenças intrínsecas dos tipos celulares, nós realizamos experimentos com RT-PCR em tempo real utilizando preparações de RNA derivadas de ambas as fases, micélio e levedura, crescidas a 26°C e 37°C nos meios de cultura completos (YPD e Sabouraud) e meio mínimo. Vinte genes, incluindo AGS1 ( -1,3-glucan synthase) e TSA1 (thiol-specific antioxidant), foram mostrados como mais expressos na levedura patogênica em relação ao micélio. Embora a expressão de RNA mensageiro foi bastante diferente em relação aos meios completos e meio mínimo, mostramos uma tendência geral para que esses genes serem mais expressos nas células leveduriformes patogênicas de P.x brasiliensis. Além disso, mostramos a complementação dos genes METR e SCONC de P. brasiliensis e uma cepa com estes genes deletados de Aspergillus nidulans, sugerindo uma possível homologia entre eles. Mostramos também a análise de genes da via do metabolismo do enxofre foram mais expressos na levedura patogênica de P. brasiliensis em relação ao micélio saprofítico. / Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNA derived from both yeast cells and mycelia that had been cultured at 37 and 26°C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 ( 1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells. Moreover, complementation of P. brasiliensis METR and SCONC genes in strains of Aspergillus nidulans with these genes deleted suggested a possible homology between them. We show the analyses of genes involved in the xii sulphur metabolism pathway and these genes were more expressed in the pathogenic yeast than saprophytic mycelia of P. brasiliensis.

Paracoccidioides brasiliensis, um fungo termodimórfico, é o agente causador da paracoccidioidomicose (PCM), a micose sistêmica prevalente da América Latina. A patogenicidade aparenta estar intimamente relacionada com a transição dimórfica da forma de micélio para a de levedura, que é induzida pela mudança da temperatura do ambiente pela temperatura do hospedeiro mamífero. Há poucas informações disponíveis sobre genes de P. brasiliensis que são necessários durante a fase patogênica. Nós, então, realizamos as técnicas de SSH (“Suppression Subtraction Hybridization”) e de “Macroarray” com o objetivo de identificar genes que sejam preferencialmente expressos na fase leveduriforme do isolado Pb18. Genes identificados em ambos os procedimentos estão mais expressos na fase leveduriforme e estão envolvidos em metabolismo básico, transdução de sinal, crescimento e morfogênese e metabolismo do enxofre. Para testar se as mudanças observadas na expressão gênica refletem as diferenças entre as condições de crescimento usadas para obter as duas formas morfológicas preferivelmente às diferenças intrínsecas dos tipos celulares, nós realizamos experimentos com RT-PCR em tempo real utilizando preparações de RNA derivadas de ambas as fases, micélio e levedura, crescidas a 26°C e 37°C nos meios de cultura completos (YPD e Sabouraud) e meio mínimo. Vinte genes, incluindo AGS1 ( -1,3-glucan synthase) e TSA1 (thiol-specific antioxidant), foram mostrados como mais expressos na levedura patogênica em relação ao micélio. Embora a expressão de RNA mensageiro foi bastante diferente em relação aos meios completos e meio mínimo, mostramos uma tendência geral para que esses genes serem mais expressos nas células leveduriformes patogênicas de P.x brasiliensis. Além disso, mostramos a complementação dos genes METR e SCONC de P. brasiliensis e uma cepa com estes genes deletados de Aspergillus nidulans, sugerindo uma possível homologia entre eles. Mostramos também a análise de genes da via do metabolismo do enxofre foram mais expressos na levedura patogênica de P. brasiliensis em relação ao micélio saprofítico. / Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNA derived from both yeast cells and mycelia that had been cultured at 37 and 26°C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 ( 1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells. Moreover, complementation of P. brasiliensis METR and SCONC genes in strains of Aspergillus nidulans with these genes deleted suggested a possible homology between them. We show the analyses of genes involved in the xii sulphur metabolism pathway and these genes were more expressed in the pathogenic yeast than saprophytic mycelia of P. brasiliensis.

La détection, voire l'identification, de bactéries dans l'air trouve une large application dans le domaine civil (diagnostic médical, surveillance de la qualité des aliments, de l'eau, de l'air ou du sol,...) et militaire (défense biologique). L'objectif principal de cette thèse a été, d'une part, d'étudier la diversité génétique de Bacillus anthracis et, d'autre part, d'analyser la composition bactérienne de prélèvements atmosphériques. L'identification précise de souches de B. anthracis (étude de la diversité de cette espèce bactérienne) repose, d'une part, sur une approche par typage de répétitions en tandem et, d'autre part, sur une approche par typage de mutations ponctuelles. Les résultats obtenus par ces deux méthodes indépendantes sont en accord. Cependant, les répétitions en tandem apportent une bien meilleure résolution. L'étude de la composition bactérienne de l'air participe à la validation d'un système de détection. Celui-ci doit être capable de caractériser en moins d'une heure, la présence de quelques bactéries par litre d'air de l'environnement. Le protocole développé ici permet de détecter 1 pg d'ADN pur en moins de deux heures, sans faire appel à une étape d'amplification génique. L'analyse du bruit de fond atmosphérique tire parti de ce protocole, à l'exception de quelques modifications qui permettent d'analyser un plus grand nombre d'échantillons. Les résultats de quatre collectes soulignent que le bruit de fond atmosphérique peut varier de demi-heure en demi-heure. Le seuil de sensibilité de la méthode est d'environ 100 bactéries par litre d'air.

Advances in DNA micro and macroarray technologies made these high-throughput systems good candidates for the development of cheaper, faster and easier qualitative and quantitative detection methods. In this study, a simple and cost effective sandwich hybridization-based method has been developed for the rapid and sensitive detection of various unmodified recombinant elements in transgenic plants. Attention was first focused on the optimization of conditions such as time, concentration and temperature using commercial ssDNA, which in turn could be used for real sample detection. In this sandwich-type DNA chip platform, capture probes complementary to the first half of recombinant element (target adapter) were immobilized onto poly-L-lysine covered conventional microscope slides. PCR-amplified un-purified target adapter and biotin labeled detection probe, which is complementary to the second half of target adapter, were hybridized in solution-phase to complementary capture probes to create a sandwiched tripartite complex. Later, hybridization signal was visualized by the attachment of streptavidin conjugated Quantum Dot to the sandwiched complex under UV illumination. Sandwich based array system that has been developed in this study allows multiplex screening of GMO events on a single DNA chip platform. 35S promoter, NOS terminator, CRY1Ab and BAR target sequences were successfully detected on the same DNA chip platform. The platform was able to detect unlabeled PCR amplified DNA fragments of CaMV 35S promoter sequence and NOS terminator and BAR transgene sequences from transgenic potato plants and NK603 Certified GMO Reference material, respectively. The DNA-chip platform developed in this study will allow multiple detection of label-free PCR-amplified transgenic elements from real GMO samples on a single slide via a cost effective, fast, reliable and sensitive sandwich hybridization assay.

Orientador: Sonia Marli Zingaretti / Banca: Julio Cezar Franco de Oliveira / Banca: Mozart de Azevedo Marins / Banca: Janete Apparecido Desiderio Sena / Banca: Eliana Gertrudes Macedo Lemos / Resumo: A seca é o principal estresse abiótico que afeta a produtividade das plantas. Para detectar os genes expressos sob condições de deficiência hídrica foi desenvolvido um estudo de expressão gênica usando plantas de cana-de-açúcar tolerantes (cv. SP83-5073) e sensíveis (cv. SP90-1638) à seca. A metodologia de macroarranjos de DNA foi empregada para monitorar a expressão gênica de 3.575 clones de folhas de cana-de-açúcar e os resultados foram confirmados por PCR quantitativo. Na cultivar tolerante 165 genes foram identificados em resposta ao déficit hídrico severo, em contraste à cultivar sensível, na qual um maior número de genes (n = 432) foi diferencialmente expresso ao longo do tratamento de deficiência hídrica. A cultivar tolerante ativou um menor número de genes em função do déficit hídrico aplicado. No entanto, 94% destes genes foram induzidos, enquanto na cultivar sensível 45% dos genes diferencialmente expressos foram reprimidos sob condições de deficiência hídrica. Comparando os perfis de expressão identificados em cada planta verifica-se que aproximadamente 55% dos genes expressos na cultivar tolerante também foram comuns à cultivar sensível, a maioria exibindo um perfil de expressão muito similar entre os genes induzidos. A classificação funcional dos genes foi realizada com base no papel exercido pela proteína no metabolismo celular, e mostrou que importantes vias metabólicas relacionadas ao déficit hídrico foram reprimidas na resposta das plantas sensíveis à seca. Além disso, 50% dos transcritos identificados em cada cultivar representam novos genes, os quais não estão descritos nos bancos de dados ou cuja função ainda não foi caracterizada / Abstract: Drought is a major abiotic stress that affects the plant productivity. To detect expressed genes under drought conditions, a gene expression study using tolerant (SP83-5073) and sensitive (SP90-1638) sugarcane plants was performed. Macroarray methodology was used to monitor the gene expression profile of the 3,575 cDNA clones from sugarcane leaves and the results were confirmed by real time PCR analysis. For the tolerant cultivar 165 genes were identified in response to severe water stress, contrasting with sensitive cultivar, in which a higher number of genes (n = 432) were responsive to the stress-treatment. Tolerant cultivar expressed a few genes in stress conditions. However 94% of them were induced, while for sensitive cultivar 45% of the differentially expressed genes were down-regulated under water stress conditions. Comparing the gene expression profiles identified in each cultivar, it is possible to verify that 55% of the expressed genes in tolerant cultivar were also observed in sensitive cultivar, the majority showing a very similar gene expression pattern. Functional gene classification was performed according to roles in cellular metabolism, and showed that important drought-pathways were repressed in sensitive plants response. Besides, 50% of the identified transcripts in each cultivar represent novel genes, which were not described in databases or whose functions were not characterized yet / Doutor

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-18Bitstream added on 2014-06-13T20:43:08Z : No. of bitstreams: 1 rodrigues_fa_dr_jabo.pdf: 1200881 bytes, checksum: de16e9283bd6c69ea47cb6957b6c81fb (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A seca é o principal estresse abiótico que afeta a produtividade das plantas. Para detectar os genes expressos sob condições de deficiência hídrica foi desenvolvido um estudo de expressão gênica usando plantas de cana-de-açúcar tolerantes (cv. SP83-5073) e sensíveis (cv. SP90-1638) à seca. A metodologia de macroarranjos de DNA foi empregada para monitorar a expressão gênica de 3.575 clones de folhas de cana-de-açúcar e os resultados foram confirmados por PCR quantitativo. Na cultivar tolerante 165 genes foram identificados em resposta ao déficit hídrico severo, em contraste à cultivar sensível, na qual um maior número de genes (n = 432) foi diferencialmente expresso ao longo do tratamento de deficiência hídrica. A cultivar tolerante ativou um menor número de genes em função do déficit hídrico aplicado. No entanto, 94% destes genes foram induzidos, enquanto na cultivar sensível 45% dos genes diferencialmente expressos foram reprimidos sob condições de deficiência hídrica. Comparando os perfis de expressão identificados em cada planta verifica-se que aproximadamente 55% dos genes expressos na cultivar tolerante também foram comuns à cultivar sensível, a maioria exibindo um perfil de expressão muito similar entre os genes induzidos. A classificação funcional dos genes foi realizada com base no papel exercido pela proteína no metabolismo celular, e mostrou que importantes vias metabólicas relacionadas ao déficit hídrico foram reprimidas na resposta das plantas sensíveis à seca. Além disso, 50% dos transcritos identificados em cada cultivar representam novos genes, os quais não estão descritos nos bancos de dados ou cuja função ainda não foi caracterizada / Drought is a major abiotic stress that affects the plant productivity. To detect expressed genes under drought conditions, a gene expression study using tolerant (SP83-5073) and sensitive (SP90-1638) sugarcane plants was performed. Macroarray methodology was used to monitor the gene expression profile of the 3,575 cDNA clones from sugarcane leaves and the results were confirmed by real time PCR analysis. For the tolerant cultivar 165 genes were identified in response to severe water stress, contrasting with sensitive cultivar, in which a higher number of genes (n = 432) were responsive to the stress-treatment. Tolerant cultivar expressed a few genes in stress conditions. However 94% of them were induced, while for sensitive cultivar 45% of the differentially expressed genes were down-regulated under water stress conditions. Comparing the gene expression profiles identified in each cultivar, it is possible to verify that 55% of the expressed genes in tolerant cultivar were also observed in sensitive cultivar, the majority showing a very similar gene expression pattern. Functional gene classification was performed according to roles in cellular metabolism, and showed that important drought-pathways were repressed in sensitive plants response. Besides, 50% of the identified transcripts in each cultivar represent novel genes, which were not described in databases or whose functions were not characterized yet

Neste trabalho apresenta-se uma revisão da metodologia de experimentos de microarray relativas a sua instalação e análise estatística dos dados obtidos. A seguir, aplica-se essa metodologia na análise de dados de expressão gênica em citros, gerados por um experimento de macroarray, utilizando modelos lineares de efeitos fixos considerando a inclusão ou não de diferentes efeitos e considerando ajustes de modelos para cada gene separadamente e para todos os genes simultaneamente. Os experimentos de macroarray são similares aos experimentos de microarray, porém utilizam um menor número de genes. Em geral, são utilizados devido a restrições econômicas. Devido ao fato de terem sido utilizados poucos arrays no experimento analisado neste trabalho foi utilizada uma abordagem bayesiana empírica que utiliza estimativas de variância mais estáveis e que leva em consideração a correlação entre as repetições do gene dentro do array. Também foi utilizado um método de análise não paramétrico para contornar o problema da falta de normalidade para alguns genes. Os resultados obtidos em cada um dos métodos de análise descritos foram então comparados. / This paper presents a review of the methodology of microarray experiments for its installation and statistical analysis of data obtained. Then this methodology is applied in data analysis of gene expression in citrus, generated by a macroarray experiment, using linear models with fixed effects considering the inclusion or exclusion of different effects and considering adjustments of models for each gene separately and for all genes simultaneously. The macroarray experiments are similar to the microarray experiments, but use a smaller number of genes. In general, are used due to economic restrictions. Because they have been used a few arrays in the experiment analyzed in this study it was used a empirical Bayes approach that uses estimates of variance more stable and that takes into account the correlation among replicates of the gene within array. A non parametric analysis method was also used to outline the problem of the non normality for some genes. The results obtained in each of the described methods of analysis were then compared.

Neste trabalho apresenta-se uma revisão da metodologia de experimentos de microarray relativas a sua instalação e análise estatística dos dados obtidos. A seguir, aplica-se essa metodologia na análise de dados de expressão gênica em citros, gerados por um experimento de macroarray, utilizando modelos lineares de efeitos fixos considerando a inclusão ou não de diferentes efeitos e considerando ajustes de modelos para cada gene separadamente e para todos os genes simultaneamente. Os experimentos de macroarray são similares aos experimentos de microarray, porém utilizam um menor número de genes. Em geral, são utilizados devido a restrições econômicas. Devido ao fato de terem sido utilizados poucos arrays no experimento analisado neste trabalho foi utilizada uma abordagem bayesiana empírica que utiliza estimativas de variância mais estáveis e que leva em consideração a correlação entre as repetições do gene dentro do array. Também foi utilizado um método de análise não paramétrico para contornar o problema da falta de normalidade para alguns genes. Os resultados obtidos em cada um dos métodos de análise descritos foram então comparados. / This paper presents a review of the methodology of microarray experiments for its installation and statistical analysis of data obtained. Then this methodology is applied in data analysis of gene expression in citrus, generated by a macroarray experiment, using linear models with fixed effects considering the inclusion or exclusion of different effects and considering adjustments of models for each gene separately and for all genes simultaneously. The macroarray experiments are similar to the microarray experiments, but use a smaller number of genes. In general, are used due to economic restrictions. Because they have been used a few arrays in the experiment analyzed in this study it was used a empirical Bayes approach that uses estimates of variance more stable and that takes into account the correlation among replicates of the gene within array. A non parametric analysis method was also used to outline the problem of the non normality for some genes. The results obtained in each of the described methods of analysis were then compared.