Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-02-15T11:56:11Z No. of bitstreams: 1 AnaBeatrizMedeirosLinsDeAlbuquerqueTavares_DISSERT.pdf: 11754321 bytes, checksum: 9e9a6f52cc32b8d2dcb05449e7c48e9b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-02-16T15:39:16Z (GMT) No. of bitstreams: 1 AnaBeatrizMedeirosLinsDeAlbuquerqueTavares_DISSERT.pdf: 11754321 bytes, checksum: 9e9a6f52cc32b8d2dcb05449e7c48e9b (MD5) / Made available in DSpace on 2018-02-16T15:39:16Z (GMT). No. of bitstreams: 1 AnaBeatrizMedeirosLinsDeAlbuquerqueTavares_DISSERT.pdf: 11754321 bytes, checksum: 9e9a6f52cc32b8d2dcb05449e7c48e9b (MD5) Previous issue date: 2017-12-08 / Quando ativado, o nosso sistema imunol?gico ? capaz de reconhecer e destruir c?lulas tumorais pela ativa??o dos chamados pontos de verifica??o imunol?gicos, essenciais para evitar eventos autoimunes, criando barreiras para a ativa??o de c?lulas T e rejei??o de tumores. Uma das principais vias de inibi??o ? feita pela prote?na PD-1, cuja ativa??o ? explorada por v?rios tipos de tumores cancer?genos, sendo considerado hoje em dia uma abordagem terap?utica nova e importante para o tratamento do c?ncer em geral incluindo o c?ncer de mama. V?rios anticorpos inibidores dos pontos de verifica??o imunol?gicos j? foram aprovados pela US-FDA (United States - Food and Drug Administration) para interromper a intera??o do receptor PD-1 com os seus ligantes PD-L1 e PDL2, atenuando os sinais inibit?rios e aumentando a resposta antitumoral.Entre eles, a droga pembrolizumab, um anticorpo inibidor do ponto de controle imune, est? sendo amplamente utilizada para bloquear eficientemente um mecanismo protetor do tumor, estimulando as c?lulas T para atacar e destruir as c?lulas cancer?genas. Dentro deste contexto, o objetivo deste trabalho ? descrever as energias de intera??o entre a prote?na PD-1, o receptor de morte celular programado, e seu inibidor, o f?rmaco pembrolizumab, usando m?todos de bioqu?mica qu?ntica, considerando-se a estrutura cristalina da prote?na PD-1 emcomplexo com seu inibidor. A energia de intera??o entre cada mol?cula de PD-1 e o f?rmaco foi calculada in silico atrav?s de abordagens qu?nticas, levando-se em conta res?duos de amino?cidos atrativos e repulsivos significativos. Foi observado poucas intera??es repulsivas, com forte predomin?ncia da contribui??o atrativa, apontando para uma forte inibi??o do receptor PD-1. Al?m disso, foram analisados v?rios aspectos bioqu?micos, especialmente ?queles relacionados aos pontos de verifica??o imune. Nossos resultados mostram que o m?todo computacional usado neste trabalho ? um primeiro passo, de baixo custo, para desvendar os res?duos de amino?cidos do f?rmaco que desempenham o papel mais importante na afinidade de liga??o do complexo pembrolizumab / PD-1. Al?m disso, pode ser considerado uma etapa qualitativa para que a imunoterapia se torne uma das ferramentas padr?o, levando ao desenvolvimento de novas e eficientes drogas farmac?uticas contra o c?ncer. / During immunosurveillance, the immune system is capable to recognize and destroy tumor cells by the activation of the so-called immunological checking points, essential to avoid autoimmune events, creating barriers to the T-cell activation and tumor rejection. One of the foremost inhibitory pathways is done by the PD-1 protein (Programmed cell death protein-1), which is activated by several types of cancerous tumors, and is now considered a new and important therapeutic approach for the treatment of cancer in general, including breast cancer. Several immune checkpoint inhibitor antibodies are already approved by the US-FDA (United States - Food and Drug Administration) to bind the protein PD-1, disrupting its interaction with the PD-L1 and PD-L2 ligands, thereby attenuating inhibitory signals and augmenting the host anti-tumor response. Among them, the drug pembrolizumab, an immune checkpoint inhibitor antibody, is being widely used to efficiently blocks a protective mechanism on cancer cells, triggering the T-cells to destroy them. The aim of this work is to describe the interaction energies between the protein PD-1 and its drug inhibitor pembrolizumab by using a quantum biochemistry calculation, taking advantage of the X-ray crystal structure of the protein PD-1 in complex with its inhibitor. The interaction energy between each PD-1 molecule and the drug was calculated in silico through quantum chemistry approaches considering any significant attractive and repulsive drug?s amino acid residues. Although it was observed few repulsive interactions, the attractive ones were predominant, pointing out to a strong inhibition of the programmed cell death receptor. Besides, several biochemical aspects were analyzed, especially those related to the immune checking points. Our results show that the computational method used in this work is a low cost efficient first step to unveil the drug?s amino-acids residues that play the most important role on the binding affinity of the pembrolizumab/PD-1 complex. Besides, it can be considered a great qualitative step for immunotherapy to become one of the standard tools leading to the development of new and effective cancer drugs.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-02-16T19:08:17Z No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-02-22T17:20:04Z (GMT) No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) / Made available in DSpace on 2018-02-22T17:35:15Z (GMT). No. of bitstreams: 1 Tese Vers?o Final Tiago Fazolo 18_01_2018.pdf: 3827531 bytes, checksum: b081e8333d16cb49ba00d0df25dff485 (MD5) Previous issue date: 2017-03-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Respiratory syncytial virus (RSV) is the most common etiologic agent in severe lower respiratory tract infections (LRTI) in children. RSV-associated LRTI is the main cause of bronchiolitis, pneumonia and exacerbation of asthma. This infection is responsible for the high rates of hospitalizations related to respiratory diseases worldwide, especially in children younger than 2 years. Currently, annual mortality rate due to RSV infections is worrying worldwide and is estimated at approximately two hundred thousand cases. The treatment strategies to RSV infections are limited. Ribavirin is an approved drug for use in RSV infections, but its use is limited due to adverse side-effects and risks posed to health professionals who handle it. Palivizumab is a monoclonal antibody which targets RSV F glycoprotein and its use is only indicated as a prophylactic measure. This treatment is already accepted in several countries for groups of high risk children (premature children, with chronic lung disease and with congenital heart disease). However, palivizumab has a high cost for public health and is not available in all countries. The development of an effective RSV vaccine to generate a long-lasting immunological memory response that prevents infection may be the best alternative because it will reduce high public health expenditures with antiviral drugs and monoclonal antibodies. The first attempt in the search for a vaccine against RSV was in the 1960s. This vaccine produced high levels of serum antibodies but could not protect against infection. Children who were vaccinated developed a more serious disease when later infected with the same virus. To date, there is no licensed vaccine for RSV, so the search for effective vaccines is an important focus of research. Natural RSV infections do not induce lasting protective memory, and multiple reinfections can occur lifetime. Nasal secretions from infected infants presented a small number of regulatory CD4 T cells (Treg) in peripheral blood, an increase in interleukin 4 (IL-4) production and T helper type 2 (Th2) response. Treg cells are important for controlling an exacerbated increase in immune responses. A reduction of the Tregs caused by the RSV infection generates an exacerbation of the pulmonary disease due to a Th2 response. The M209-223 RSV peptide was identified to increase IFN-? production by peptide-specific CD4 T cells after challenge with the virus. The treatment with this peptide also induced an increase in pulmonary Treg frequency in infected mice. Recently, it has also been shown that Tregs aid in the development of a T CD8+ effector response, which is crucial for the control of RSV viral load. Our hypothesis is that the RSV M209-223 peptide impacts in the differentiation of CD4 T cells, increasing the population of specific Treg, reducing lung inflammation and modulating the anti-RSV immune response. This peptide in animal model induces the differentiation of specific Treg. Our findings suggest that vaccination with M209-223 peptide results in the differentiation of specific CD4 T cells into conventional effectors and Treg cells. Vaccination with this peptide decreased the expansion of a Th2 response in animals infected with RSV, protecting both the infection site and systemically. We believe that this approach could be an important component in vaccination strategies against this virus. / O v?rus sincicial respirat?rio (VSR) ? o agente etiol?gico mais comum nas infec??es graves do trato respirat?rio inferior (TRI) em crian?as. As infec??es do TRI associada com o VSR s?o a principal causa de bronquiolite, pneumonia e exacerba??o da asma. As TRI causadas pelo VSR s?o respons?veis pelas altas taxas das hospitaliza??es relacionadas ?s doen?as respirat?rias em todo o mundo, principalmente em crian?as menores de dois anos. Atualmente a taxa de mortalidade anual mundial devido ?s infec??es pelo VSR ? preocupante e ? estimada em aproximadamente duzentas mil crian?as. As estrat?gias de tratamento contra o VSR utilizadas s?o limitadas. A ribavirina ? um f?rmaco aprovado no uso para infec??es pelo VSR, por?m sua utiliza??o ? limitada devido aos efeitos secund?rios adversos e aos riscos que representam para os profissionais da sa?de que o manipulam. O palivizumabe ? um anticorpo monoclonal dirigido contra a glicoprote?na F do v?rus e sua utiliza??o ? apenas como medida profil?tica. Este tratamento j? ? aceito em v?rios pa?ses nos grupos de crian?as de alto risco (crian?as prematuras, com doen?a pulmonar cr?nica e com cardiopatia cong?nita). Entretanto o palivizumabe tem um alto custo para sa?de p?blica, n?o sendo disponibilizado em todos os pa?ses. O desenvolvimento de uma vacina eficaz contra o VSR pode ser a melhor alternativa, pois ao gerar resposta de mem?ria duradoura que previne a infec??o e reduz, desta forma, os altos gastos com a sa?de p?blica, com os f?rmacos antivirais e com os anticorpos monoclonais. A primeira tentativa na busca de uma vacina contra o VSR foi na d?cada de 60. A vacina produzida estimulou n?veis moderadamente elevados de anticorpos no soro, mas n?o conseguiu proteger contra ? infec??o. As crian?as que foram vacinadas desenvolveram uma doen?a mais grave quando mais tarde infectados com o v?rus. At? o presente momento n?o existe nenhuma vacina licenciada para o VSR. Desta forma, a busca de vacinas eficazes constitui um importante foco de pesquisa em todo mundo. As infec??es naturais pelo VSR n?o induzem mem?ria protetora duradoura, ocorrendo m?ltiplas reinfec??es ao longo da vida. Em crian?as infectadas, observou-se um n?mero reduzido de c?lulas T CD4+ regulat?rias (Treg) no sangue perif?rico, um aumento na produ??o de interleucina 4 (IL-4) e uma resposta T helper do tipo 2 (Th2) nas secre??es nasais. As c?lulas Treg s?o importantes para controlar um aumento exagerado da resposta imunol?gica. Por este fato acredita-se que quando h? uma redu??o das Tregs causada pela infec??o do VSR ocorre uma exacerba??o da doen?a pulmonar devido uma resposta Th2. Foi identificado que o pept?deo M209-223 do VSR aumenta a produ??o de IFN-? nas c?lulas T CD4+ ap?s o desafio com VSR. O tratamento com este mesmo pept?deo tamb?m apresentou um aumento na frequencia de c?lulas Treg ap?s infec??o prim?ria pelo VSR. Recentemente tamb?m foi demonstrado que as Tregs auxiliam no desenvolvimento de uma resposta efetora T CD8+, que ? crucial para o controle da carga viral do VSR. Nossa hip?tese ? que o pept?deo M209-223 do VSR influencia na diferencia??o das c?lulas T CD4+, aumentando a popula??o de c?lulas T efetoras e regulat?rias espec?ficas, reduzindo a inflama??o pulmonar e modulando a resposta imune. Os nossos resultados sugerem que a vacina??o com pept?deo M209-223 resulta na diferencia??o de c?lulas T CD4+ espec?ficas em efetoras convencionais, que produzem mais IFN-? e em c?lulas Treg. A vacina??o com este pept?deo diminuiu a expans?o de uma resposta Th2 nos animais infectados com o VSR, protegendo da inflama??o exacerbada tanto no local da infec??o como sistemicamente. Acreditamos que esta abordagem pode constituir um componente importante nas estrat?gias de vacina??o contra este v?rus.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-02-29T22:55:50Z No. of bitstreams: 1 JoseNazarenoMoreiraDeAguiarJunior_TESE.pdf: 2810253 bytes, checksum: d577f52edf024bf1bd64c021a5bed745 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-03-02T19:49:24Z (GMT) No. of bitstreams: 1 JoseNazarenoMoreiraDeAguiarJunior_TESE.pdf: 2810253 bytes, checksum: d577f52edf024bf1bd64c021a5bed745 (MD5) / Made available in DSpace on 2016-03-02T19:49:24Z (GMT). No. of bitstreams: 1 JoseNazarenoMoreiraDeAguiarJunior_TESE.pdf: 2810253 bytes, checksum: d577f52edf024bf1bd64c021a5bed745 (MD5) Previous issue date: 2015-02-24 / A doen?a periodontal ? uma condi??o inflamat?ria cr?nica de car?ter infeccioso causada primariamente por bact?rias presentes em um biofilme dent?rio que interagem com o hospedeiro, determinando, assim, a natureza da doen?a resultante. Apesar de j? se conhecer muito sobre a patog?nese destas patologias, ainda n?o se sabe a composi??o exata do perfil de c?lulas T durante a fase ativa da doen?a (Th1, Th2 ou Th17). Este trabalho visou avaliar, atrav?s da express?o imuno-histoqu?mica, a presen?a dos marcadores (IL-17, IL-23 e ROR?t), envolvidos na resposta Th 17 em casos de gengiva clinicamente saud?veis (n=32), gengivite induzida pelo biofilme dental (n=30), periodontite cr?nica (n=32) e periodontite agressiva (n=25), objetivando analisar se a express?o e/ou distribui??o destas mol?culas em linf?citos e macr?fagos, presentes no infiltrado inflamat?rio dos tecidos periodontais, influencia na destrui??o tecidual observada nestas doen?as. Foi realizada a an?lise morfol?gica dos casos, onde avaliou-se a intensidade do infiltrado inflamat?rio em leve, moderado e intenso. Para cada caso, nas ?reas mais imunomarcadas, 5 campos foram escolhidos e analisados, tanto em rela??o a intensidade do infiltrado inflamat?rio quanto a quantidade de c?lulas imunomarcadas, baseando-se em escores predeterminados: escore 0 (aus?ncia de infiltrado inflamat?rio/imunomarca??o), escore 1 (o infiltrado/imunomarca??o abrangia menos de 25% da ?rea do campo), escore 2 (o infiltrado/imunomarca??o ocupava entre 25 e 50%) e escore 3 (infiltrado/imunomarca??o presente em mais de 50% da ?rea do campo). A partir disto, gerouse uma mediana que representava cada caso. A intensidade do infiltrado inflamat?rio foi correlacionada com a progress?o da doen?a, se mostrando crescente da gengiva clinicamente saud?vel at? a periodontite agressiva (p<0,001). Detectou-se a presen?a da IL-17, IL-23 e do ROR?t na maioria dos casos avaliados e a quantidade de c?lulas imunomarcadas foi correlacionada tanto com a intensidade do infiltrado inflamat?rio (P<0,001) quanto com os par?metros cl?nicos analisados (P<0,001), apresentando uma correla??o positiva, predominantemente moderada. A periodontite agressiva apresentou um maior percentual de imunomarca??o em rela??o ?s outras condi??es cl?nicas avaliadas, para todos os marcadores, sugerindo uma poss?vel associa??o destes marcadores com a progress?o desta doen?a, onde quanto maior a perda de suporte periodontal, maior a quantidade do infiltrado inflamat?rio e maior n?mero de c?lulas imunomarcadas. / Periodontal disease is a chronic inflammatory condition primarily caused by bacteria in dental biofilm, which interact with the host, thus determining the nature of the resulting disease. Despite the wide knowledge about the pathogenesis of these diseases, the exact composition of the T cell profile during the active phase of the disease (Th1, Th2 or Th17) remains unknown. This study aimed to evaluate by immunohistochemical expression, the presence of the markers (IL-17, IL-23 and ROR?t), involved in Th17 response in clinically healthy gingiva cases (n = 32), biofilm-induced gingivitis (n = 30), chronic periodontitis (n = 32) and aggressive periodontitis (n = 25), in order to analyze if the expression and/or distribution of these molecules in lymphocytes and macrophages, present in the inflammatory infiltrate of periodontal tissue, influences the tissue destruction observed in these diseases. The morphological analysis of cases was performed which assessed the intensity of the inflammatory infiltrate in mild, moderate and intense. For each case, in the area with the most representative immunostaining, 5 fields were chosen and analyzed, both for the intensity of the inflammatory infiltrate as for the quantity of immunostained cells, based on predetermined scores: score 0 (absence of inflammatory infiltrate/immunostaining), score 1 (the infiltrate/immunostaining covered less than 25% of the field area), score 2 (the infiltrate/immunostaining occupied between 25 and 50%) and score 3 (infiltrate/immunostaining present in over 50% of the field area). From this, a median was generated representing each case. The intensity of the inflammatory infiltrate correlated with the disease progression, in other words, it was crescent from clinically healthy gingiva to aggressive periodontitis (P <0.001). It was detected the presence of IL-17, IL-23 and ROR?t in most of the evaluated cases and the number of immunostained cells correlated with the intensity of the inflammatory infiltrate (P <0.001) and with the clinical parameters analyzed (P <0.001), showing a positive correlation, mainly moderate. Aggressive periodontitis showed a higher percentage of immunostaining for all markers in relation to other clinical conditions assessed, suggesting a possible association of these markers with the progression of this disease, in which the higher the loss of periodontal support, the greater the amount of inflammatory infiltrate and larger the number of immunostained cells.