In the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.

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No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_PapelOxidoNitrico.pdf: 1177312 bytes, checksum: 3e68e77c84db988c269f620ff0cec7eb (MD5) Previous issue date: 2013 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / Malária é uma das mais incidentes doenças infecciosas do mundo. Na Amazônia existem muitos casos de malária causados principalmente por duas espécies de protozoários, o Plasmodium vivax e o Plasmodium falciparum, sendo este último responsável pela maioria dos casos de malária grave, que geralmente levam a morte devido ao acometimento de múltiplos órgãos, como o cérebro. Um dos mediadores químicos amplamente estudados nessa patogênese é o Óxido Nítrico (NO), o qual apresenta papel controverso. Atualmente duas hipóteses principais são apontadas como potencializadoras na patogênese. Uma, que a MC é causa da superprodução de NO, produzido pela Óxido Nítrico Sintase Neuronal (nNOS), após um quadro de hipóxia. Outra, diz que a MC é a causa da resposta exacerbada do sistema imunológico com produção de NO pela Óxido Nítrico Sintase Induzida (iNOS), presente nos macrófagos quando ativados pro determinantes antigênicos. Devido grande relevância da doença e dificuldade em enteder a patologia, modelos experimentais têm sido estabelecidos com a finalidade de esclarecer vias potenciais da evolução para MC, dentre eles o modelo de malária aviária causada pelo Plasmodium gallinaceum. Pouco se sabe sobre o seu papel do NO em modelos de malária aviária, principalmente devido inexistência de marcadores específicos para avaliar expressão das enzimas de síntese. Diante disso é importante estabelecer protocolos de purificação da iNOS de galinhas para a produção de um possível marcador. Para tanto se faz necessário investigar o papel do NO durante a malária aviária, em modelo experimental in vivo e in vitro, com linhagens de macrófagos de galinha HD11. Animais infectados com P. gallinaceum tratados com aminoguanidina (AG), um inibidor da produção de NO, tiveram maior sobrevida, além de menores níveis de nitrito no plasma e em macrófagos derivados de monócitos do sangue periférico, sugerindo a inibição da iNOS. Nos experimentos in vitro, células HD11 tratadas com LPS mostraram produção aumentada de NO, inferindo aumento na expressão e atividade da iNOS. Na separação proteica, observamos padrões diferentes que podem ser associados a uma elevada expressão da iNOS nos macrófagos ativados com LPS. Esse estudo proporcionará o melhor entendimento do modelo de malária aviária em galinhas, incluindo a cerebral, e envolvimento do sistema nitrérgico em galinhas infectadas com P. gallinaceum. / Malaria is a severe infectious disease caused by parasites of the genus Plasmodium that infect different types of vertebrate’s hosts and is responsible for a huge number of deaths. The severe Malaria can lead to death and involves different pathophysiological signs as well as anemia and inflammation. Experimental models are necessary to improve the knowledge about mechanisms involved at the pathogenesis of the disease and the developing new protocols of treatment. Chickens infected with P. gallinaceum are a good model of malaria due to phylogenetic relatedness with human Plasmodium and because both species presents common clinical signs as cerebral malaria. The Nitric Oxide (NO) is an important effector molecule, but little is known about their role in malaria on chickens, meanly due to the lack of specific markers to evidence the NO production in this model. It is known that chickens infected with P. gallinaceum has a high mortality and causes an overproduction of nitrite by macrophages. The animals, when treated with aminoguanidine (AG), an inhibitor of inducible oxide nitric synthase (iNOS), showed a higher level of parasitemia. However, the rate of survivor was superior, beyond the clinical manifestations, as milder anemia. It is necessary to achieve a better comprehension about physiological aspects of avian malaria with the inhibition of NO’s production by the AG. In the context , the present study aims to investigate NOS activity and the role of NO during the avian malaria infection, with in vivo models, using P. gallinaceum as pathological agent , and in vitro with chicken’s macrophage of HD11 strain. This research will give a better understanding of avian malaria in chickens, including cerebral, and the involvement of nitrergic system in infected chickens.

Avian pathogenic Escherichia coli (APEC) is a bacterium that is responsible for colibacillosis in birds. However, information about broiler breeder APEC isolates is limited, but the data is critical due to the transfer of this bacteria down the production pyramid to progenies resulting in high mortality. Therefore, we evaluated the phenotypic virulence characteristics of 28 isolates using embryo lethality and day-old chick challenge assays. Also, the in vitro adhesion and invasion potential of selected nine isolates were identified. Results showed more than 1/3rd of the isolates were highly virulent and the virulence increased as the number of virulence-associated genes increased. High adhesion and invasion rates were observed among the isolates. Overall, the study helped us to evaluate the virulence characteristics of APEC from broiler breeders. However, future studies based on whole genome approach would help to identify the specific targets which can be used to develop effective interventions.