Orientador: Maria Marluce dos Santos Vilela / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-07-19T08:26:02Z (GMT). No. of bitstreams: 1 Silva_MarcosTadeuNolascoda_M.pdf: 3136388 bytes, checksum: 55e005bb4732584ad379e5d556b31897 (MD5) Previous issue date: 1994 / Resumo: Foi avaliada a imunidade humoral para o Haemophilus influenzae do tipo b (Hib) em 68 crianças brasileiras saudáveis com idades entre 15 e 19 meses. Foram analisados os títulos naturais de anticorpos contra o polissacáride capsular do Hib (PRP) bem como os títulos estimulados por uma dose de 0,5 ml por via intramuscular da vacina conjugada do PRP com o toxóide diftérico (PRP-D) contendo 25ug de PRP e 18 ug de toxicóide diftérico por dose).Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Humoral immunity to Haemophilus influenzae type b HIB) was studied in 68 healthy Brazilian children between 15 and 19 month of age. Ehe evaluation was done by the measure, by radioiminoassay, of the levels of antibodies against the capsular polysaccharide of Hib (PRP). The children enrolled in the trial received one intramuscular dose of the conjugate vaccine of the capsular polysaccharide of HIB and diphtheria toxoid. Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Mestre em Pediatria

Orientador: Marcelo Lancellotti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:32:22Z (GMT). No. of bitstreams: 1 Cury_GiseleCristianeGentile_D.pdf: 5956781 bytes, checksum: b28217e4d95beb4734f0357ed4181696 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital quando for liberada / Abstract: The abstract is available with the full electronic document when available / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T07:21:21Z (GMT). No. of bitstreams: 1 Varela_JuliaNogueira_M.pdf: 1464845 bytes, checksum: 930cccae996588333b99f1aaa50988c0 (MD5) Previous issue date: 2013 / Resumo: Haemophilus influenzae é uma bactéria causadora de doenças tipicamente associadas ao trato respiratório superior e inferior. Tal bactéria é classificada em linhagens capsuladas e não capsuladas - as não tipadas. As grandes responsáveis por patogenias mais severas são as capsuladas, especialmente as do sorotipo b, a existência de uma vacina para somente esse sorotipo, faz com que ocorra uma emergência de casos com H. influenzae não tipado - NTHi. A crescente resistência a antibióticos dessa bactéria está associada à plasmídios de resistência, bem como sua competência natural. A presença desses patógeno é maior em países nos quais não existe acesso a vacina, devido ao alto custo da mesma, que acabam utilizando antibióticos mais acessíveis como o cloranfenicol no tratamento. Esse trabalho estudou a transferência horizontal do gene ompP2 em diversas cepas de H. influenzae com a ajuda de nanopartículas de óxido de grafeno. Essas nanopartículas mimetizam uma atmosfera rica em partículas suspensas como as grandes cidades e zonas de agricultura precoce, já que, nesses locais ocorrem com maior frequência mutações e adaptações desse patógeno. Quando as nanopartículas encontravam-se no meio de cultura, verificou-se um aumento da taxa de transformação dessas bactérias. Assim como uma modificação no padrão de adesão celular das bactérias mutadas quando comparadas com as selvagens em linhagens celulares distintas e expostas ao antibiótico de resistência, levando a um aumento da taxa de adesão das cepas mutadas com relação às cepas selvagens. Como esse gene é e de possível aquisição entre cepas de H. influenzae em seu ambiente natural seria possível utilizá-lo para obtenção de uma proteína recombinante, com possível antigenicidade. Uma vez que a taxa de adesão aumenta com a presença do mesmo, levando a uma possível nova vacina que também protegeria contra cepas não tipadas e não somente capsuladas / Abstract: Haemophilus influenzae is a bacteria that causes diseases typically associated with the upper and lower respiratory tract. Their strains are divided in capsulated and non-capsulated - the non typable. The major responsible for more severe cases are the capsulated types, specially the b type. The existence of a vaccine for the serotype b, allows the emergence of cases of non typable H. influenzae - NTHi. The growing resistance is associated with resistance plasmids, and with its natural competence, that enables the bacteria to acquire DNA fragments between it's' species. Since this pathogen is common in countries that there is no access to this vaccine, therefore the use of accessible and cheaper antibiotics, such as chloramphenicol for treatment is. This work studied the horizontal transference of the ompP2 gene from multiresistant strains of H. influenzae, with the aid of grafen oxide nanoparticles, that mimesis an atmosphere rich in suspended particles, such as great urban areas and ancient agricultural zones. In these environments a great frequency in mutation and adaptations of these bacteria is verified. When we look at the adhesion patterns of these bacteria we can see that it is modified when they are mutated and exposed to the resistance antibiotic. Leading to an augmentation of the adhesion patterns when we compare to the wild strains. Since this gene was present in all strains and it was of easy acquisition between strains, it would be possible to use it to obtain a recombinant protein with likely antigen properties. Because the adhesion tax enhances with the presence of this gene. Leading to a possible new vaccine target, for NTHi and capsulated strains also / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestra em Biociências e Tecnologia de Produtos Bioativos

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T23:59:18Z (GMT). No. of bitstreams: 1 Andrade_AliceTavaresReis_M.pdf: 3292132 bytes, checksum: ee5318f5b87992964c9d97bedc343f00 (MD5) Previous issue date: 2013 / Resumo: Em 1998, foi relatada a transferência lateral do gene sodC do gênero Haemophilus para a espécie Neisseria meningitidis. Sabe-se que, nestes dois grupos a dinâmica deste gene é bastante distinta. Este trabalho tem por objetivo estimar árvores filogenéticas que possam apontar qual a espécie do gênero Haemophilus compartilhou o gene sodC com a espécie N. meningitidis. Testes de seleção positiva foram empregados no intuito de avaliar quais forças evolutivas estão subjacentes ao processo de diversificação molecular do gene nestas espécies ao longo do tempo. Além disso, foi realizada uma modelagem protéica computacional por homogia para avaliar quais substituições de aminoácidos tinham impacto no processo adaptativo da enzima nas espécies consideradas. Ao se reconstruir uma filogenia para o gene sodC, foi constatado que a origem deste gene na espécie H. influenzae é distinta. Um grupo de linhagens recebeu o gene, provavelmente por transferência lateral, da espécie H. haemolyticus, enquanto o outro grupo recebeu o gene da espécie H. parainfluenzae. Neste grupo, o gene sofreu pseudogeneização. Foi observado também que as sequências de N. meningitidis agrupam com as sequências que compartilham um ancestral comum com a espécie H. haemolyticus, porém as sequências do meningococo formam um ramo distinto dentro deste clado. Dada à alta clonalidade das sequências de N. meningitidis, foi constatado que o evento de transferência lateral de genes foi muito recente na escala do tempo. O teste de seleção positiva demonstrou que seleção positiva está atuando especificamente no ramo da árvore que compartilha um ancestral comum com a espécie H. haemolyticus, através da modificação de uma alanina por uma serina na posição 72, embora a nota geral da árvore tenha sido menor que 1. Sabe-se que pseudogenes, por não codificarem uma proteína ativa e, portanto, por não estarem sob nenhum tipo de restrição funcional, estão sob uma ação maior da deriva genética. Portanto, diferentes forças evolutivas estão governando a evolução deste gene nas espécies consideradas. A modelagem protéica concluiu que tal modificação contribuiu para o aumento do potencial redox do sítio ativo. Desta forma, a ação da seleção positiva sob um único resíduo de aminoácido foi benéfica para a função da enzima como um todo / Abstract: In 1998, it was reported the lateral transfer of the sodC gene from the genus Haemophilus to Neisseria meningitidis. It is known that this two groups show a quite distinct dynamics of this gene. This study aims to estimate phylogenetic trees that might point to which species of the genus Haemophilus shared the sodC gene with N. meningitidis. In addition, tests of positive selection were employed in order to assess which evolutionary forces are governing the process of molecular diversification of the gene in these species through time. Moreover, we performed a computational protein modeling by homology to asses which amino acids substitutions had an impact on the adaptative process of the enzyme in the species considered. A phylogeny of the sodC gene was reconstructed and it was found that this gene in H. influenzae has two different origins. A group of lineages has received the gene, probably by lateral transfer, from H. haemolyticus, whereas the other group has received the gene from H. parainfluenzae. In the latter, the gene has become a pseudogene. It was also observed that the sequences from N. meningitides group together with those sequences that share a common ancestor with H. haemolyticus, but they form a distinct branch within this clade. Given the high clonality of the sequences from N. meningitidis, it was found that the lateral gene transfer event is very recent in the time scale. A test of positive selection showed that positive selection is acting specifically in the branch that shares a common ancestor with H. haemolyticus through the substitution of an alanine to a serine at position 72, though the overall score of the tree is less than one. It is known that pseudogenes do not encode active proteins and therefore they are not under any kind of functional constraints, so they are under greater influence of genetic drift. Thus, it was concluded that different forces are driving the evolution of this gene in the species considered here. Protein modeling concluded that this modification contributed to the increase in the redox potencial of the active site. Thus the action of positive selection under a single amino acid residue was beneficial to the function of the enzyme as whole / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

La BPCO se traduit rapidement par l'apparition d'une susceptibilité aux infections liées aux atteintes des mécanismes de défense du poumon. Les travaux antérieurs de l’équipe montrent qu'une altération de la réponse IL-17/IL-22 et de la fonction des cellules dendritiques (DC) participe au développement de l’exacerbation de la BPCO par les bactéries. Les mécanismes responsables de ce défaut de réponse ne sont pas élucidés. Au cours de cette thèse, nous nous sommes intéressés aux points suivants :1-Mécanismes cellulaires responsables du défaut de production d'IL-17 et d'IL-22 au cours de l'infection.Les cellules présentatrices d'antigène (APC) et en particulier, les DC jouent un rôle essentiel dans la réponse antimicrobienne, par leur fonction de phagocytes et par l’activation et la polarisation de cellules immunitaires innées et adaptatives. Sur des modèles murins d’exacerbation de la BPCO par Streptococcus pneumoniae ou Haemophilus influenzae non typable (NTHi), nous avons réalisé des tris de macrophages, DC et monocytes inflammatoires du poumon par cytométrie en flux. Ces analyses montrent que les cellules APC pulmonaires présentent des altérations fonctionnelles aboutissant à une limitation de leur capacité à polariser la réponse Th17 de Lymphocytes T CD4+. Une analyse transcriptomique est également effectuée sur les ARN des APC triées afin de préciser les altérations fonctionnelles de ces cellules par rapport aux souris contrôles.2-Rôle des cytokines IL-20 dans la susceptibilité à l'infection et l'exacerbation de la BPCO Myles et al ont montré en 2013 que les cytokines IL-20 (IL-19, IL-20 et IL-24) jouent un rôle délétère dans la réponse immunitaire cutanée contre Staphylococcus aureus par un mécanisme impliquant une inhibition indirecte d’IL-17 produite par les cellules T. La fonction des DC peut être affecté par l'environnement cytokinique. Comme les cytokines IL-20 sont surexprimées chez les souris BPCO, notre objectif a été de définir leur rôle au cours de l'exacerbation de la BPCO et l'impact de ces cytokines sur les DC dans ce contexte.Dans notre modèle d’exacerbation de la BPCO, nous avons bloqué cette voie en neutralisant l'IL-20RB qui est commune aux 2 récepteurs de ces cytokines afin d’étudier leur impact sur l'exacerbation et sur la réponse immune associée, notamment la réponse IL-17/IL-22. En parallèle, nous avons analysé la modulation de la fonction des DC humaines par ces cytokines dans un contexte d'infection bactérienne. Nos résultats montrent que le traitement avec l'anticorps bloquant anti-IL-20RB permet de bloquer le développement de l'exacerbation de la BPCO en diminuant la charge bactérienne et l'inflammation associée. Cet effet est associé à une diminution importante de la mobilisation des DC dans le poumon mais sans affecter sur la réponse IL-17/IL-22. In vitro, les cytokines IL-20 sont produites par les DC dans un contexte infectieux. De plus, ces cytokines sont capables de diminuer l'activation des ces cellules par les bactéries et de réduire leur capacité à activer les Lymphocytes T dans ce contexte.3-Capacité d'un immunostimulant à restaurer la réponse IL-17/IL-22, et à limiter le développement de l'exacerbation.L’utilisation d’immunostimulant dont la flagelline (agoniste du TLR-5, principale composante du flagelle bactérien) est souvent proposé comment pouvant promouvoir la réponse immunitaire des muqueuses et en particulier la réponse IL-17/IL-22. Nous avons analysé la capacité de cet agoniste du TLR-5 à améliorer la réponse à S. pneumoniae et NTHi dans notre modèle d’exacerbation de la BPCO.Le traitement par la flagelline permet de limiter les conséquences de l'infection bactérienne chez les souris BPCO en diminuant l'inflammation et les lésions pulmonaires associées. L'effet de ce ligand de TLR est au moins en partie dépendant de la production d'IL-22._A terme, ces données permettent d'envisager de nouvelles options thérapeutiques pour le traitement des exacerbations de la BPCO. / Patients with COPD often presented a susceptibility to respiratory infections. Previous works in our lab have showed that a defect of IL-17/IL-22 response and an altered dendritic cell (DC) function is involved in COPD exacerbation with bacteria. However, the mechanism responsible for this defect is not elucidated yet. In order to better define these mechanisms and to develop new therapeutic approaches against COPD exacerbation, we focused on the following points during this PhD project:1-Cellular mechanisms responsible of the defect of IL-17 and IL-22 production during infection in COPD.Antigen presenting cells (APC) particularly, DC are essential in antimicrobial immune response since they are professional phagocytes able to engulf and kill bacteria, but also by their essential role in the polarization of innate and adaptive immune responses. We worked on COPD exacerbation mice model infected with Streptococcus pneumoniae or Nontypeable Haemophilus influenzae (NTHi). APC including macrophages, DC and inflammatory monocytes were sorted by flow cytometry for phenotype and functional analysis. We found an altered function of these lung APC showing limited capacity of Th17 polarization. Transcriptional analysis on total RNA from sorted APC is performed to decipher the mechanisms involved in this functional alteration regarding to control mice.2-The role of IL-20 cytokines in the susceptibility to infection during COPD exacerbation.Myles et al showed in 2013 that IL-20 cytokines (IL-19, IL-20 and IL-24) are deleterious in skin immune response against Staphylococcus aureus. Indeed, these IL-20 cytokines indirectly inhibited IL-17 produced by T cells. The function of DC is also controlled by the presence of cytokines in the microenvironment. Because IL-20 cytokines are overexpressed in COPD, we aimed to determine their role during COPD exacerbation and the impact on DC.We used IL-20RB (common subunits of the 2 receptors) neutralizing antibodies to blocked IL-20 cytokines in COPD exacerbation mice model. We analyzed the impact of this treatment on the immune response, more particularly IL-17/IL-22 response. In addition, we analyzed the modulation of human monocyte derived DC (MDDC) function by IL-20 cytokines in the context of bacterial infection.Our results shows that treatment with IL-20 RB neutralizing antibodies limited COPD exacerbation by reducing the bacterial burden and the associated inflammatory response. This process was associated to reduced number of DC in the lung without impacting IL-17 and IL-22 production. In vitro, MDDC produced IL-20 cytokines upon bacterial infection. Additionally, these cytokines impaired MDDC activation following bacterial infection, which was associated to a reduced capacity of MDDC to activate T lymphocytes.3-The possibility to restore IL-17 and IL-22 response with immuno-stimulants in order to limit the development of COPD exacerbation.Flagellin (a TLR-5 agonist, the main component of bacterial flagellum) is an immuno-stimulant often used to promote mucosal immune response. This activity is related to its ability to promote IL-17 and IL-22 production. In this PhD work, we analyzed the capacity of this TLR-5 agonist to improve the immune response during S. pneumoniae and NTHi infection in COPD exacerbation mice model.Flagellin treatment reduced the bacterial burden and limited the consequences of bacterial infection in COPD mice, by lowering the inflammation and the associated lung remodeling. We also found that the immunomodulatory effect of flagellin was at least partially IL-22 dependent.Finally, these data allow to identify new therapeutic tools potentially useful for the treatment of COPD exacerbations.