Made available in DSpace on 2015-04-14T14:51:32Z (GMT). No. of bitstreams: 1 458474.pdf: 1757493 bytes, checksum: c5379d3dddf546f5888748177b083560 (MD5) Previous issue date: 2014-03-07 / Acute viral bronchiolitis is the most common respiratory illness in children in the first years of life and at least half of patients are diagnosed with respiratory tract infection by viruses such as Respiratory Syncytial Virus (RSV). Because RSV is extremely common disease it generates large number of hospitalizations and large costs to health systems. The RSV Fusion (F) Protein is essential for the infective cycle of the virus. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease due to viral infection. Neutrophil Extracellular Traps (NETs) are formed by the release of the inner content of neutrophils (proteins from granules and DNA) in the extracellular space in response to different stimuli. They are essential to prevent the spread of microorganisms, which are killed by antimicrobial proteins that are anchored in networks of DNA, such as elastase and myeloperoxidase. The objective of this work is to characterize the effect of the RSV F protein on the generation of NETs. The F protein was capable of inducing the formation of NETs in vitro in a dose-dependent way with co-expression of neutrophil elastase and myeloperoxidase. The production of NETs in response to F protein was mediated by the TLR-4-receptor and was dependent on ROS production, p38 and ERK MAPK. Together, these data provide evidence that support the activation of specific signaling pathways by the F protein to induce the production of NETs. Excessive production of NETs can aggravate the inflammatory symptoms induced by infection with RSV. / A bronquiolite viral aguda ? a doen?a respirat?ria mais frequente em crian?as nos primeiros anos de vida, sendo que ao menos a metade dos pacientes ? diagnosticado com infec??o respirat?ria por v?rus como o V?rus Sincicial Respirat?rio (RSV). Por ser uma doen?a extremamente comum gera grande n?mero de interna??es e grandes custos aos sistemas de sa?de. A prote?na de fus?o (F) do RSV ? essencial para o ciclo infectivo do v?rus. Neutr?filos e seus produtos est?o presentes nas vias a?reas de pacientes infectados por RSV que desenvolveram doen?a pulmonar aumentada devido a infec??o viral. As Redes Extracelulares de Neutr?filos (NETs) s?o formadas pela libera??o do conte?do nuclear dos neutr?filos no espa?o extracelular em resposta a diferentes est?mulos, sejam patog?nicos ou n?o. Elas s?o fundamentais para impedir a dissemina??o de microrganismos, que s?o mortos pelas prote?nas antimicrobianas que ficam ancoradas nas redes de DNA, como elastase e mieloperoxidase. O objetivo desta disserta??o ? caracterizar o efeito da prote?na F do RSV sobre a gera??o de NETs. A prote?na F foi capaz de induzir a forma??o de NETs in vitro de forma dose-dependente e com a co-express?o de elastase neutrof?lica e mieloperoxidase. A produ??o de NETs pela prote?na F foi mediada pelo TLR-4, e dependente da produ??o de ROS e de ERK-p38 MAPK. Juntos, estes dados fornecem evid?ncias que suportam a ativa??o de vias de sinaliza??o espec?ficas pela prote?na F para induzir a produ??o de NETs. A produ??o excessiva de NETs pode agravar os sintomas inflamat?rios induzidos pela infec??o com RSV.

Made available in DSpace on 2015-04-14T14:51:32Z (GMT). No. of bitstreams: 1 459170.pdf: 2176701 bytes, checksum: a35f9eb08768c0d90b70f0dca32cbe46 (MD5) Previous issue date: 2014-03-31 / The mycobacterial DNA-binding protein 2 (MDP2) of Mycobacterium tuberculosis is a small, basic protein known to bind to DNA in a non-specific manner and to anchor the nucleoid to the plasma membrane, promoting its unpacking. Motivated by a prior intrinsic disorder pre-diction in silico, we have used a complementary set of techniques to characterize this pro-tein, such as heat and chemical stability, gel filtration, limited proteolysis and electrophoret-ic mobility. Our results suggest that the MDP2 is structurally disordered, since it (1) was pre-dicted to be an IDP, having 86 % of its structure disordered; (2) resisted to denaturation in-duced by boiling temperature and to the chemical thrichloroacetic acid (TCA); (3) migrated anomalously on SDS-PAGE, appearing to be 1.4-fold higher its calculated molecular weight; and (4) was highly sensitive to proteolysis performed by proteinase K in comparison to glob-ular proteins. We also accomplished two experiments to determine the quaternary structure of the MDP2, such as gel-filtration and glutaraldehyde crosslinking. These two experiments showed that the protein has a tendency of self-aggregation, forming large clusters of pro-tein. We also performed the site-directed mutagenesis by allelic exchange in the hns gene, in order to develop a M. tuberculosis strain lacking the protein MDP2. The result of this knock-out showed that the hns gene is not essential for the survival of the mycobacteria, confirm-ing previous results performed by transposon mutagenesis. Combined with previous results related to MDP2 from other works, we speculate that this protein uses its highly disorder N-terminal region to bind to DNA through its KAAK and PAKK sequences in a non-specific man-ner. Thereby the MDP2 may act as a promiscuous protein inside the cell, binding to different regions of nucleoid by forming large clusters of proteins, in order to perform the DNA un-packing and to regulate several genes of the mycobacteria. We hope this work may contrib-ute to a better understanding of the mycobacterial metabolism, since the M. tuberculosis still stands a major global threat. / A prote?na micobacteriana ligadora de DNA 2 (MDP2) de Mycobacterium tuberculosis ? uma prote?na pequena e de car?ter b?sico, conhecida por se ligar ao DNA de uma forma n?o es-pec?fica e de ancorar o nucle?ide ? membrana plasm?tica promovendo o seu desempacota-mento. Motivados por uma predi??o de desordem intr?nseca in silico pr?via, n?s usamos um conjunto de t?cnicas complementares para caracterizar esta prote?na, tais como determina-??o da estabilidade ao calor e a desnaturantes qu?micos, gel filtra??o, prote?lise limitada e mobilidade eletrofor?tica. Nossos resultados sugerem que a MDP2 ? estruturalmente de-sordenada, uma vez que ela (1) foi predita por ser uma prote?na intrinsecamente desorde-nada (IDP), possuindo 86 % da sua estrutura desordenada; resistiu ? desnatura??o induzida por temperatura de fervura e pela a??o qu?mica do ?cido tricloroac?tico (TCA); (3) migrou anomalamente em gel de poliacrilamida (SDS-PAGE), aparentando ser 1,4 vezes maior que o seu peso molecular calculado; e (4) foi altamente sens?vel ? prote?lise realizada pela protei-nase K, em compara??o ?s prote?nas globulares. N?s tamb?m realizamos dois experimentos para determinar a estrutura quatern?ria da prote?na, como a gel filtra??o e o crosslinking por glutaralde?do. Estes dois experimentos mostraram que a prote?na tem uma tend?ncia a auto agrega??o, formando grandes aglomerados em solu??o. Tamb?m foi realizada a muta-g?nese s?tio-dirigida por troca al?lica no gene hns, com o intuito de desenvolver uma cepa de M. tuberculosis sem a prote?na MDP2. O resultado deste nocaute mostrou que o gene hns n?o ? essencial para a sobreviv?ncia da micobact?ria. Combinado com resultados pr?vios de outros trabalhos relacionados ? MDP2, n?s especulamos que esta prote?na usa sua regi?o N-terminal altamente desordenada para se ligar ao DNA por meio das suas sequ?ncias repetiti-vas KAAK e PAKK de uma forma n?o espec?fica. Desta forma, a MDP2 deve atuar como uma prote?na prom?scua dentro da c?lula, ligando-se a diferentes regi?es do nucle?ide pela for-ma??o de grandes aglomerados de prote?na, com o objetivo de realizar o desempacotamen-to do DNA e de regular diversos genes micobacterianos. N?s esperamos que este trabalho possa contribuir para um melhor entendimento do metabolismo micobacteriano, uma vez que o M. tuberculosis ainda ? uma grande amea?a global.