Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-05-21T13:12:10Z No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-05-28T17:29:36Z (GMT) No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) / Made available in DSpace on 2018-05-28T17:34:08Z (GMT). No. of bitstreams: 1 Vers?o completa da disserta??o-stefaniemuraro.pdf: 4426933 bytes, checksum: 5733e09060e6e08135de26c11374b171 (MD5) Previous issue date: 2018-03-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is a major cause of diseases of the respiratory tract in humans being mainly associated with bronchiolitis, chronic obstructive pulmonary disease (COPD) and asthma exacerbation. RSV infection occurs primarily in pulmonary epithelial cells and, once infection is established, an innate immune response is triggered and mainly neutrophil recruitment is induced. Neutrophils can extrude neutrophil extracellular traps (NETs) capable of entrapping and inactivate a multitude of microorganisms because of its composition and due to the stringy nature of DNA fibers. Recently, was demonstrated that RSV particles and its fusion (F) protein were able to induce the release NETs coated with neutrophil elastase and myeloperoxidase, both antimicrobial peptides. Also, was observed that the excessive formation of NETs can have negative consequences to the host, such as airway obstruction during RSV infection. Therefore, the aim was to evaluate the mechanisms involved in NET formation induced by RSV infection of neutrophils, alveolar epithelial cells (A549) or lung fibroblasts (MRC5). Human neutrophils were infected with RSV and were able to induce NETs release only after 3 hours of stimulation indicating classical NETosis. Next was characterized NETs formation during infection associating DNA extrusion with MPO, NE and F protein of RSV. Was also observed NADPH oxidase and PAD4 dependence and PI3K/AKT, ERK and p38 MAPK pathways during infection. The inhibition of these signaling pathways, PAD4 and ROS production abolished NET formation. Considering a possible involvement of necroptosis during NETs production, were tested MLKL and RIPK inhibitors and evaluated LDH release in the supernatant of infected neutrophils. Neutrophils released LDH and depend on necroptosis induction to produce NETs. Likewise, neutrophils were co-cultured with A549 or MRC5 cells infected with RSV. Both A549 and MRC5 cells triggered NET release by human neutrophils in a virus concentration-dependent manner, the opposite occurs when used UV-inactivated virus. Briefly, RSV induces the classical/ROS-dependent NETosis by human neutrophils, and this effect relies on specific kinases activity. Furthermore, neutrophils are able to recognize pulmonary cells infected by RSV, releasing NETs. Thus, NETs release control could be crucial for minimizing tissue inflammation caused by RSV infection. / O v?rus sincicial respirat?rio (VSR) ? uma das principais causas de doen?as do trato respirat?rio em humanos sendo associado principalmente com bronquiolite, doen?a pulmonar obstrutiva cr?nica (DPOC) e exacerba??o de asma. O VSR infecta principalmente c?lulas epiteliais pulmonares e, uma vez que a infec??o ? estabelecida, uma resposta imune inata ? desencadeada e ocorre o recrutamento de c?lulas do sistema imune, principalmente neutr?filos. Os neutr?filos podem liberar redes extracelulares de neutr?filos (NETs) capazes de capturar e inativar uma grande quantidade de microrganismos devido ? sua composi??o e natureza fibrosa das fibras de DNA. Recentemente, foi demonstrado que part?culas do VSR al?m da prote?na de fus?o (F) do v?rus foram capazes de induzir a libera??o de NETs revestidas com elastase neutrof?lica e mieloperoxidase, ambos pept?deos com atividade antimicrobiana. Al?m disso, observou-se que a forma??o excessiva de NETs pode ter consequ?ncias negativas para o hospedeiro, como a obstru??o das vias a?reas durante a infec??o por VSR. Portanto, o objetivo foi avaliar os mecanismos envolvidos na forma??o de NET induzida pela infec??o por RSV em neutr?filos humanos, c?lulas epiteliais alveolares (A549) ou fibroblastos pulmonares (MRC5). Neutr?filos humanos foram infectados com VSR e foram capazes de induzir a libera??o de NETs somente ap?s 3 horas de infec??o, indicando uma NETose cl?ssica. Em seguida, foi caracterizada a forma??o de NETs durante a infec??o associando a extrus?o de DNA com as prote?nas MPO, NE e com a prote?na F do VSR. Tamb?m se observou a depend?ncia de NADPH oxidase e PAD4 e das vias de sinaliza??o PI3K / AKT, ERK e p38 MAPK durante a infec??o. A inibi??o dessas vias de sinaliza??o, da produ??o de PAD4 e de EROs aboliu a forma??o de NET. Considerando um poss?vel envolvimento da necroptose na produ??o de NETs, foram utilizados inibidores de MLKL e RIPK1 e foi avaliada a libera??o de LDH no sobrenadante de neutr?filos infectados. Os neutr?filos liberaram LDH e dependeram da ativa??o da necroptose para produzir NETs. Do mesmo modo, os neutr?filos foram co-cultivados com c?lulas A549 ou MRC5 infectadas com VSR. Ambas as c?lulas A549 e MRC5 desencadearam a libera??o de NET por neutr?filos humanos de uma maneira dependente da concentra??o de v?rus, o oposto ocorreu quando usado um v?rus UV-inativado. Resumidamente, o VSR induz a NETose cl?ssica / dependente de EROs em neutr?filos humanos, e este efeito depende de atividade espec?fica de quinases. Al?m disso, os neutr?filos s?o capazes de reconhecer c?lulas pulmonares infectadas pelo VSR, induzindo a libera??o NETs. Assim, o controle de libera??o de NETs pode ser crucial para minimizar a inflama??o do tecido causada pela infec??o por VSR.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-04-27T15:00:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-05-16T19:24:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Made available in DSpace on 2017-05-16T19:24:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) Previous issue date: 2016 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O excesso de gordura corporal caracter?stico da obesidade est? relacionado a diversas altera??es metab?licas, que incluem a resist?ncia ? insulina. Dentre as medidas n?o farmacol?gicas empregadas para a melhora da sensibilidade ? insulina est? o treinamento f?sico aer?bio, como o treinamento intervalado de alta intensidade (HIIT, do ingl?s high intensity interval training). Sendo assim, esse estudo avaliou os efeitos do HIIT em componentes bioqu?micos, celulares e moleculares relacionados ? resist?ncia ? insulina em obesos. Indiv?duos obesos sens?veis (n=9) e resistentes ? insulina (n=8) foram submetidos a 8 semanas de HIIT, em cicloerg?metro, realizado 3 vezes por semana, com intensidade e volume progressivos (8 a 12 est?mulos; 80 a 110% da pot?ncia m?xima). Amostras de sangue venoso e do m?sculo vasto lateral foram obtidas antes e ap?s o programa de HIIT. Ap?s o programa de treinamento houve aumento da sensibilidade ? insulina nos obesos resistentes ? insulina, mas n?o houve redu??o da massa de gordura. A concentra??o de citocinas no soro, o estresse oxidativo sist?mico e frequ?ncia das c?lulas imunes n?o foram modificadas ap?s o treinamento. No m?sculo esquel?tico, o HIIT promoveu aumento da fosforila??o do substrato do receptor de insulina (IRS) (Tyr612), da Akt (Ser473) e da prote?na quinase dependente de c?lcio/calmodulina (CAMKII) (Thr286), e aumento do conte?do da ?-hidroxiacil-CoA desidrogenase (?-HAD) e citocromo C oxidase (COX-IV). Houve ainda, redu??o da fosforila??o da quinase regulada por sinal extracelular (ERK1/2) nos obesos resistentes ? insulina. Conclu?mos que 8 semanas de HIIT promoveram melhora da sensibilidade ? insulina, modificou componentes da via de sinaliza??o da insulina e do metabolismo oxidativo no m?sculo esquel?tico. Essas altera??es ocorreram independentes de mudan?as na gordura corporal total e de par?metros inflamat?rios sist?micos. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / Obesity is characterized by excess of body fat, and its development can lead to a variety of metabolic disorders, including insulin resistance. Exercise is recognized as a non-pharmacological approach to increasing skeletal muscle insulin sensitivity, although the mechanisms are not elucidated. Additionally, the understanding of high intensity interval training (HIIT, high intensity interval training) treat insulin resistance is less understood. Therefore, this study evaluated the effects of HIIT on biochemical, molecular, and cellular markers related to insulin resistance in sedentary obese individuals. Sensitive (n=9) and insulin resistant (n=8) obese individuals (body mass index ? 30 kg/m-2) were engaged in 8 weeks of HIIT using a cycle ergometer. The HIIT was performed 3 times a week, and its intensity and volume progressively increased throughout the training period (from 8 to 12 stimuli; from 80 to 110% of the maximum power). Venous blood and the vastus lateralis muscle samples were obtained before and after the HIIT. HIIT enhanced insulin sensitivity in insulin-resistant obese individuals without changing body fat mass. Cytokine concentration in serum, blood oxidative stress, and frequency of some immune cells were not altered by HIIT. In skeletal muscle, HIIT increased the phosphorylation of insulin receptor substrate (IRS) (Tyr612), Akt (Ser473), and protein kinase dependent calcium/calmodulin (CaMKII) (Thr286). HIIT also increased the expression of ?-hydroxyacyl-CoA dehydrogenase (?-HAD) and cytochrome C oxidase (COX-IV). A reduction of the kinase phosphorylation of extracellular signal-regulated (ERK1/2) was only seen in obese insulin resistant individuals. The results show that 8 weeks of HIIT enhanced insulin sensitivity, modified components of the insulin-signaling pathway, and improved skeletal muscle oxidative metabolism. These changes were independent of alterations in body fat and inflammatory parameters.