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Os micro-organismos podem infectar seu hospedeiro por diferentes vias, sendo a principal o trato respiratório. O reconhecimento pela mucosa dessas vias pode desencadear inibição da proliferação e bloqueio da entrada microbiana, assim como estimular resposta direcionada a memória imunológica para prevenir posteriores infecções. Alguns micro-organismo, como as bactérias Neisseria meningitidis e Neisseria lactamica, são capazes de modular a resposta imune de mucosa diretamente, ou por meio das células epiteliais respiratórias. Este trabalho propôs, então, a avaliação das porinas B provenientes destas bactérias como moduladoras da produção de IL-8 nas linhagens BEAS-2B e Detroit 562. Também foi avaliada a dependência deste estímulo ao receptor TLR2. Ambas as porinas se ligaram a TLR2 e por este receptor estimularam a produção de IL-8. O perfil de produção foi dependente da expressão de TLR2 pelas células. A porina lactâmica induziu menos IL-8 por regular negativamente a expressão de TLR2, mas sua afinidade pelo receptor se mostrou maior que a da porina meningocócica. As porinas são então moduladoras das células de mucosa, fato que somado a atividade adjuvante destas proteínas por via parenteral estimulou a avaliação destas como adjuvantes de mucosa. O modelo escolhido para a avaliação foi o de inoculação intranasal de camundongos, utilizando como antígeno o lipopolissacarídio pouco imunogênico de Franciscella tularensis atenuada (Ft-LPS). A análise foi baseada no título de anticorpos IgG e IgM séricos. A porina meningocócica se mostrou a mais imunogênica, mas por ser originária de patógeno acarreta maior risco biológico em sua produção. Para viabilizar a porina meningocócica como adjuvante, a mesma foi substituída por porina homóloga produzida de modo recombinante em Escherichia coli não patogênica. A porina recombinante foi avaliada pelo mesmo sistema in vivo e comparada a adjuvantes experimentais de ação conhecida (rCTB, QS-21 e ODN 1826). A porina apresentou o melhor desempenho entre todos os adjuvantes, principalmente dois meses após o fim do esquema vacinal. O mesmo adjuvante foi adicionado ao vírus da raiva para caracterizar a amplitude de antígenos para sua aplicação e o efeito biológico dos anticorpos induzidos. Os resultados obtidos por via intranasal com antígeno da raiva confirmaram a propriedade de adjuvante de mucosa da porina recombinante, aumentando os títulos de IgG séricos. O ensaio biológico dos anticorpos por RFFIT comprovaram a funcionalidade dos anticorpos gerados, neutralizando a infectividade viral em células BHK-21. O uso da porina por via subcutânea não aumentou o nível de anticorpos neutralizantes, mas aumentou o de IgG. Não foi detectada imunidade celular específica de linfócitos do baço ao vírus da raiva nos parâmetros avaliados, independente da adição de adjuvantes. Em resumo, as porinas foram caracterizadas como relevantes na imunomodulação de células da mucosa respiratória por infecção meningocócica. A modulação também foi relevante para o aumento de resposta humoral frente a diferentes antígenos, por diferentes vias de administração, o que demonstra a eficiência e versatilidade da porina recombinante como adjuvante imunológico. / Microorganisms can invade the host through many routes, specially the respiratory tract. The respiratory mucosa is responsible for recognition, inhibition, proliferation and entry blockade of microorganisms, besides incitation of immunological memory to prevent further infections. Some microorganisms, such as Neisseria meningitidis and Neisseria lactamica, can modulate the mucosa immune response directly or through stimulation of respiratory epithelial cells. The present work proposed the evaluation of porin B proteins, derived from these microorganisms, as modulators of IL-8 production on respiratory epithelial cell strains BEAS-2B and Detroit 562. TLR2 receptor dependency for the modulation was also evaluated. Both porins bounded to TLR2 and through this receptor were able to stimulate IL-8 production, whereas this profile was correlated with TLR2 expression. Lactamica porin (Nlac PorB) induced less IL-8 and TLR2 expression, also for a shorter period of time. The effect caused by Nlac PorB was attributed to TLR2 down regulated expression, since its binding affinity to the receptor is greater than meningococcal porin (Nmen PorB). Porins were therefore able to immune modulate mucosal cells, fact that allied with their parenteral adjuvant activity incited evaluation of porins as potential mucosal adjuvants. The model chosen for the evaluation was intranasal immunization of mice, using as the antigen a low immunogenic lipopolysaccharide extracted from attenuated Franciscella tularensis (Ft-LPS). The evaluation was based on IgG and IgM serum titers. After the immunization scheme, Nmen PorB induced higher IgG and IgM titers than Nlac PorB. Although Nmen PorB was more efficient, it comes from a pathogen. To overcome the risk of its production, it was replaced by recombinant porin (rPorB) produced by Escherichia coli. rPorB was evaluated by the same model and compared with well known experimental adjuvants (rCTB, QS-21 e ODN 1826). rPoB had the highest IgM and IgG titers among all adjuvants tested, specially two months after vaccination. The same adjuvant was also combined with a viral antigen to characterize its application wideness and biological function of incited antibodies. Results obtained with rabies antigen by intranasal route confirmed the mucosal adjuvant properties of rPorB, increasing IgG titers induced by the antigen. These antibodies were also capable of virus neutralization, as demonstrated in RFFIT assays. rPoB didn´t raise neutralizing antibody titers by subcutaneous route, but increased IgG titers. Cellular immunity was undetectable in spleen lymphocytes with the screening method used, regardless of adjuvant addition. In conclusion, porins were characterized as revelant for immunomodulation of the respiratory mucosal cells, caused by infection with meningococcus. The immunomodulation was also revelant for increase of humoral reponse to different antigens and by different routes, pointing out recombinant porin B as an efficient and versatile immunological adjuvant.

Os micro-organismos podem infectar seu hospedeiro por diferentes vias, sendo a principal o trato respiratório. O reconhecimento pela mucosa dessas vias pode desencadear inibição da proliferação e bloqueio da entrada microbiana, assim como estimular resposta direcionada a memória imunológica para prevenir posteriores infecções. Alguns micro-organismo, como as bactérias Neisseria meningitidis e Neisseria lactamica, são capazes de modular a resposta imune de mucosa diretamente, ou por meio das células epiteliais respiratórias. Este trabalho propôs, então, a avaliação das porinas B provenientes destas bactérias como moduladoras da produção de IL-8 nas linhagens BEAS-2B e Detroit 562. Também foi avaliada a dependência deste estímulo ao receptor TLR2. Ambas as porinas se ligaram a TLR2 e por este receptor estimularam a produção de IL-8. O perfil de produção foi dependente da expressão de TLR2 pelas células. A porina lactâmica induziu menos IL-8 por regular negativamente a expressão de TLR2, mas sua afinidade pelo receptor se mostrou maior que a da porina meningocócica. As porinas são então moduladoras das células de mucosa, fato que somado a atividade adjuvante destas proteínas por via parenteral estimulou a avaliação destas como adjuvantes de mucosa. O modelo escolhido para a avaliação foi o de inoculação intranasal de camundongos, utilizando como antígeno o lipopolissacarídio pouco imunogênico de Franciscella tularensis atenuada (Ft-LPS). A análise foi baseada no título de anticorpos IgG e IgM séricos. A porina meningocócica se mostrou a mais imunogênica, mas por ser originária de patógeno acarreta maior risco biológico em sua produção. Para viabilizar a porina meningocócica como adjuvante, a mesma foi substituída por porina homóloga produzida de modo recombinante em Escherichia coli não patogênica. A porina recombinante foi avaliada pelo mesmo sistema in vivo e comparada a adjuvantes experimentais de ação conhecida (rCTB, QS-21 e ODN 1826). A porina apresentou o melhor desempenho entre todos os adjuvantes, principalmente dois meses após o fim do esquema vacinal. O mesmo adjuvante foi adicionado ao vírus da raiva para caracterizar a amplitude de antígenos para sua aplicação e o efeito biológico dos anticorpos induzidos. Os resultados obtidos por via intranasal com antígeno da raiva confirmaram a propriedade de adjuvante de mucosa da porina recombinante, aumentando os títulos de IgG séricos. O ensaio biológico dos anticorpos por RFFIT comprovaram a funcionalidade dos anticorpos gerados, neutralizando a infectividade viral em células BHK-21. O uso da porina por via subcutânea não aumentou o nível de anticorpos neutralizantes, mas aumentou o de IgG. Não foi detectada imunidade celular específica de linfócitos do baço ao vírus da raiva nos parâmetros avaliados, independente da adição de adjuvantes. Em resumo, as porinas foram caracterizadas como relevantes na imunomodulação de células da mucosa respiratória por infecção meningocócica. A modulação também foi relevante para o aumento de resposta humoral frente a diferentes antígenos, por diferentes vias de administração, o que demonstra a eficiência e versatilidade da porina recombinante como adjuvante imunológico. / Microorganisms can invade the host through many routes, specially the respiratory tract. The respiratory mucosa is responsible for recognition, inhibition, proliferation and entry blockade of microorganisms, besides incitation of immunological memory to prevent further infections. Some microorganisms, such as Neisseria meningitidis and Neisseria lactamica, can modulate the mucosa immune response directly or through stimulation of respiratory epithelial cells. The present work proposed the evaluation of porin B proteins, derived from these microorganisms, as modulators of IL-8 production on respiratory epithelial cell strains BEAS-2B and Detroit 562. TLR2 receptor dependency for the modulation was also evaluated. Both porins bounded to TLR2 and through this receptor were able to stimulate IL-8 production, whereas this profile was correlated with TLR2 expression. Lactamica porin (Nlac PorB) induced less IL-8 and TLR2 expression, also for a shorter period of time. The effect caused by Nlac PorB was attributed to TLR2 down regulated expression, since its binding affinity to the receptor is greater than meningococcal porin (Nmen PorB). Porins were therefore able to immune modulate mucosal cells, fact that allied with their parenteral adjuvant activity incited evaluation of porins as potential mucosal adjuvants. The model chosen for the evaluation was intranasal immunization of mice, using as the antigen a low immunogenic lipopolysaccharide extracted from attenuated Franciscella tularensis (Ft-LPS). The evaluation was based on IgG and IgM serum titers. After the immunization scheme, Nmen PorB induced higher IgG and IgM titers than Nlac PorB. Although Nmen PorB was more efficient, it comes from a pathogen. To overcome the risk of its production, it was replaced by recombinant porin (rPorB) produced by Escherichia coli. rPorB was evaluated by the same model and compared with well known experimental adjuvants (rCTB, QS-21 e ODN 1826). rPoB had the highest IgM and IgG titers among all adjuvants tested, specially two months after vaccination. The same adjuvant was also combined with a viral antigen to characterize its application wideness and biological function of incited antibodies. Results obtained with rabies antigen by intranasal route confirmed the mucosal adjuvant properties of rPorB, increasing IgG titers induced by the antigen. These antibodies were also capable of virus neutralization, as demonstrated in RFFIT assays. rPoB didn´t raise neutralizing antibody titers by subcutaneous route, but increased IgG titers. Cellular immunity was undetectable in spleen lymphocytes with the screening method used, regardless of adjuvant addition. In conclusion, porins were characterized as revelant for immunomodulation of the respiratory mucosal cells, caused by infection with meningococcus. The immunomodulation was also revelant for increase of humoral reponse to different antigens and by different routes, pointing out recombinant porin B as an efficient and versatile immunological adjuvant.

Les protéines lipidées purifiées à partir d’extraits de pathogènes ou produites de façon recombinante ont démontré leur pouvoir immunogène. Malheureusement, ce type de protéines est généralement difficile à purifier et/ou à produire. Afin de résoudre ces difficultés, l’objectif de cette thèse a été de mettre au point un procédé de lipidation chimique d’antigènes protéiques d’intérêt vaccinal, présents naturellement sous forme lipidée mais produits de façon recombinante sous forme non lipidée. La lipidation chimique a ainsi été réalisée sur 2 protéines, à savoir rTbpB-dl provenant de N. meningitidis et rNWMN_A issue de S. aureus, par des lipopeptides synthétiques de structure minimale, Pam2CAG et Pam3CAG, ciblant respectivement les récepteurs TLR2/6 et TLR2/1. Ces lipopeptides ont été préalablement fonctionnalisés par des groupes réactifs aux fonctions thiols (maléimide ou bromoacétyle) afin de réaliser le couplage chimique. Des analyses physico-chimiques et biochimiques ont démontré que la modification des protéines antigéniques a bien été réalisée. La lipidation de rTbpB-dl et rNWMN_A par les lipopeptides a induit in vitro l’activation des cellules immunitaires murines et humaines via TLR2 et a également augmenté in vivo l’immunogénicité de ces protéines recombinantes, en présence ou non d’un adjuvant. De plus, ces protéines lipidées ont joué in vivo le rôle d’adjuvant en augmentant l’immunogénicité d'une protéine antigénique co-administrée. Notre procédé de lipidation chimique a été simple, rapide, de faible coût et répétable. Au final, ce procédé pourrait s’appliquer sur des antigènes protéiques d’intérêt vaccinal et de faible immunogénicité provenant de différents pathogènes et/ou sur des antigènes lipoprotéiques rencontrant des problèmes de production/purification. Il pourraitreprésenter un choix pertinent dans la mise au point et dans le développement de candidatsvaccins, en présence ou non d’adjuvant(s) et/ou d’autre(s) antigène(s) d’intérêt. / Lipidated proteins, purified from pathogens extracts or produced recombinantly, have demonstrated their immunogenic power. Unfortunately, this type of proteins is generally difficult to purify and/or to produce. To solve these difficulties, the objective of this thesis was to develop a process of chemical lipidation of protein antigens of vaccine interest, which are present naturally inlipidated form but produced recombinantly in non-lipidated form. The chemical lipidation was carried out on two proteins, namely rTbpB-dl from N. meningitidis and rNWMN_A from S. aureus, by minimum structure synthetic lipopeptides, Pam2CAG and Pam3CAG targeting receptors TLR2/6 and TLR2/1 respectively. These lipopeptides have been functionalized beforehand with thiolreactive groups (maleimide or bromoacetyl) for performing chemical coupling. Physicochemical and biochemical analysis have shown that the modification of the antigenic proteins has been achieved. The lipidation of rTbpB-dl and rNWMN_A by these lipopeptides induced in vitro activation of murine and human immune cells through TLR2. This lipidation also increased in vivo immunogenicity (mainly humoral) of the recombinant protein, in the presence or absence of an adjuvant. Furthermore, these lipidated proteins acted in vivo as an adjuvant by increasing immunogenicity of a co-administered antigen protein. Our process of chemical lipidation was simple, rapid, low-cost and repeatable. Taken together, this process could apply to antigens of protein nature, of vaccine interest and poorly immunogenic from different pathogens and/or to lipoprotein antigens encountering production/purification problems. It could be a relevant choice for the development of candidate vaccines, in the presence or absence of adjuvant(s) and/or other antigen(s) of interest.

L’invasion de l’hôte par un pathogène induit l’activation séquentielle des réponses immunitaires innées et adaptatives. La reconnaissance du pathogène par des récepteurs tels que les récepteurs de type Toll (TLRs) initie la réponse innée qui repose sur l’activation des lignées myéloïdes, la production de cytokines, de chémokines et de médiateurs pro-inflammatoires qui contribuent à l’éradication du pathogène. L’amplification incontrôlée de la réaction inflammatoire est délétère pour l’organisme. Afin de mieux comprendre les mécanismes de régulation des réponses dépendant de TLR2, récepteur impliqué dans la reconnaissance de bactéries, parasites ou champignons, nous avons étudié la composition des complexes multimoléculaires d’activation au sein des radeaux lipidiques. En utilisant des approches protéomiques complémentaires, nous avons mis en évidence le rôle de la Src kinase Lyn et de la déshydrogénase IMPDHII après engagement des hétérodimères TLR2/TLR1 ou TLR2/TLR6. La tyrosine kinase Lyn est indispensable à l’activation de NF-kB après engagement de TLR2 et agit en phosphorylant la sous-unité p110 de la PI3-kinase (PI3-K). IMPDHII, cible de l’acide mycophénolique, est un régulateur négatif de la voie TLR2-NF-kB. IMPDHII interagit avec SHP1 pour inhiber la phosphorylation sur tyrosine de p85α, la sous-unité régulatrice de PI3-K, et prévenir la transactivation de NF-kB. Enfin, nous avons étudié le rôle d’un polymorphisme de IMPDHII dans la gravité du choc septique. Ces travaux affinent la compréhension de la régulation de la réponse dépendant de TLR2 et permettent d’identifier de nouvelles cibles thérapeutiques pour la prise en charge des infections graves. / The host invasion by pathogen results in an adaptive immune response subsequent to innate immunity. Pathogens recognition by receptors as Toll Like Receptors (TLRs) initiates activation of innate immune myeloid synchronized with cytokines, chemokines and pro-inflammatory mediators production that contribute to pathogen eradication. Exuberant inflammation should lead to acute organ dysfunction and death. To understand mechanisms of TLR2-dependent response regulation, we studied composition of multi-molecular complexes in lipid rafts. TLR2 recognizes bacterial, parasitic and fungal agents. Using complementary proteomic approaches, we show the roles of Lyn Src kinase and IMPDHII dehydrogenase following involvement of TLR2/TLR1 or TLR2/TLR6. Lyn tyrosine kinase is indispensable in TLR2-dependent activation of NF-kB and phosphorylates p110 subunit of PI3-Kinase (PI3-K). IMPDHII, the mycophenolic acid substrate, is a negative regulator of TLR2-NF-kB pathway. IMPDHII acts on SHP1 to inhibit tyrosine phosphorylation on p85α, regulatory subunit of PI3-K, and prevent NF-kB transactivation. Finally, by a translational approach, we investigate the role of a genetic polymorphism of IMPDHII in the septic shock outcome. These findings strengthen the knowledge of mechanisms in TLR2-dependent control and should predict immunomodulation strategies.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les nanoparticules à base d’acide hyaluronique (AH) sont utilisées pour de nombreuses applications pharmaceutiques. Elles peuvent cibler les tumeurs par interaction avec le CD44,qui est un récepteur biologique surexprimé à la surface de certaines cellules cancéreuses. Dans ce projet nous explorons l’application potentielle de ces nanoparticules dans les domaines cosmétiques, car l’AH est aussi un ingrédient important pour l’hydratation et le renouvellement de la peau. Les copolymères à bloc à base de polypeptides et de polysaccharides ont été synthétisés par une combinaison de polymérisation par ouverture de cycle et de couplage par chimie « click ». Les nanoparticules ont été obtenues par l’auto assemblage de ces copolymères en utilisant un procédé de nano precipitation, dont la taille et la morphologie sont contrôlées par les paramètres expérimentaux. L’interaction entre les nanoparticules d’AH et le CD44 a été quantifiée par la résonance de plasmon de surface(RPS). En comparant avec l’AH libre en solution, les nanoparticules d’AH ont montré une interaction plus efficace avec le CD44, mettant ainsi en évidence un effet de multivalence des nanoparticules. Finalement, la dégradation enzymatique de ces nanoparticules d’AH a été évaluée avec deux types de hyaluronidases, HYAL1 et SPAM-1. La digestion des nanoparticules de l’AH a été significativement ralentie par rapport à l’AH libre. De manière très surprenante, ces nanoparticules de AH ont pu inhiber l’activité de l’enzyme HYAL1 et protéger l’AH libre dans la solution. Enfin, des ligands du récepteur TLR2 de type lipopeptide ont été synthétisés et leurs performances via TLR2 ont été évaluées par RPS. / Nanoparticles based on hyaluronic acid (HA) are widely used in pharmaceutics. They can target the tumor by the interaction with CD44, a biological receptor overexpressed in some cancer cells. In this project, we investigate the potential applications of these nanoparticles in cosmetics, since HA is also an important ingredient for the skin hydration and renewing. Block copolymers based on polypeptides and polysaccharides were synthesized using a combination of ring opening polymerization and “click chemistry”. The nanoparticles were formed by the self-assembly of these block copolymers using a nanoprecipitation process, and their size and morphology were controlled by the experimental conditions. The interaction between nanoparticles and CD44 were measured by surface plasmon resonance(SPR). Compared to free hyaluronic acid chains in solution, the HA-based nanoparticles could interact more efficiently with CD44, thus demonstrating a multivalent effect. The enzymatic degradation of these HA nanoparticles was then evaluated with twohyaluronidases: HYAL1 and SPAM-1. The digestion of the HA nanoparticles was significantly slower than that of free hyaluronic acid. Surprisingly, these HA nanoparticles could even inhibit the activity of the enzyme HYAL1 and protect free HA chains in the solution. Finally, lipopeptide-based ligands of the biological receptor TLR2 were also synthesized and their performances were evaluated by SPR.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Made available in DSpace on 2014-12-17T14:16:26Z (GMT). No. of bitstreams: 1 MarcelaAGU_DISSERT.pdf: 6376152 bytes, checksum: 6d7d86fec335062b8c283cdea3878878 (MD5) Previous issue date: 2009-03-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Inflammation has been pointed out as an important factor in development of chronic diseases, as diabetes. Hyperglycemia condition would be responsible by toll-like receptors, TLR2 and TLR4, and, consequently by local and systemic inflammation induction. Thus, the objective of present study was to evaluate type 1 Diabetes mellitus (T1DM) pro-inflammatory state through mRNA expression of TLRs 2 and 4 and proinflammatory cytokines IL-1?, IL-6 and TNF-? correlating to diabetic nephropathy. In order to achieve this objective, 76 T1DM patients and 100 normoglycemic (NG) subjects aged between 6 and 20 years were evaluated. T1DM subjects were evaluated as a total group DM1, and considering glycemic control (good glycemic control DM1G, and poor glycemic control DM1P) and considering time of diagnosis (before achieving 5 years of diagnosis DM1< 5yrs, and after achieving 5 years of diagnosis DM1 <5yrs). Metabolic control was evaluated by glucose and glycated hemoglobin concentrations; to assess renal function serum urea, creatinine, albumin, total protein and urinary albumin-to-creatinine ratio were determined and to evaluate hepatic function, AST and ALT serum activities were measured. Pro-inflammatory status was assessed by mRNA expression of TLRs 2 and 4 and the inflammatory cytokines IL-1?, IL-6 and TNF-?. Except for DM1G group (18.4%), DM1NC patients (81.6%) showed a poor glycemic control, with glycated hemoglobin (11,2%) and serum glucose (225,5 md/dL) concentrations significantly increased in relation to NG group (glucose: 76,5mg/dL and glycated hemoglobin: 6,9%). Significantly enhanced values of urea (20%) and ACR (20,8%) and diminished concentrations of albumin (5,7%) and total protein (13,6%) were found in T1DM patients, mainly associated to a poor glycemic control (DM1P increased values of urea: 20% and ACR:49%, and diminished of albumin: 13,6% and total protein:13,6%) and longer disease duration (DM1 <5yrs - increased values of urea: 20% and ACR:20,8%, and diminished of albumin: 14,3% and total protein:13,6%). As regarding pro-inflammatory status evaluation, significantly increased mRNA expressions were presented for TLR2 (37,5%), IL-1? (43%), IL-6 (44,4%) and TNF-? (15,6%) in T1DM patients in comparison to NG, mainly associated to DM1P (poor glycemic control TLR2: 82%, IL-1?: 36,8% increase) and DM1 <5yrs (longer time of diagnosis TLR2: 85,4%, IL-1?: 46,5% increased) groups. Results support the existence of an inflammatory state mediated by an increased expression of TLR2 and pro-inflammatory cytokines IL-1?, IL-6 and TNF-? in T1DM / A inflama??o tem sido descrita como um fator importante no desenvolvimento de doen?as cr?nicas como o diabetes, e a condi??o da hiperglicemia seria a respons?vel pela ativa??o dos receptores toll-like (TLRs), TLR2 e TLR4, e, conseq?entemente, pela indu??o da inflama??o local e sist?mica. Nesse sentido, o presente estudo teve como objetivo de avaliar o estado pr?-inflamat?rio do Diabetes mellitus tipo 1 (DM1) atrav?s da express?o g?nica de TLRs 2 e 4 e das citocinas pr?-inflamat?rias IL-1?, IL-6 e TNF- ?, e correlacionar com o desenvolvimento da nefropatia diab?tica. Foram estudados 76 pacientes diab?ticos tipo 1 e 100 indiv?duos normoglic?micos NG, na faixa et?ria de 6 a 20 anos. Os indiv?duos diab?ticos foram avaliados como um grupo total DM1, e subdivididos em fun??o do controle glic?mico (diab?ticos compensados DM1C, e n?o-compensados DM1NC) e em fun??o do tempo de diagn?stico (diab?ticos com menos de 5 anos de diagn?stico DM1< 5anos, e a partir de 5 anos de diagn?stico DM1 <5 anos). Para a avalia??o do controle metab?lico foram determinadas as concentra??es de glicose e de hemoglobina glicada; para avaliar a fun??o renal as concentra??es s?ricas de ur?ia, creatinina, albumina, prote?na total e a rela??o albumina/creatinina (RAC) urin?ria; e para fun??o hep?tica a atividade s?rica de AST e ALT. O estado pr?-inflamat?rio foi avaliado a partir da express?o do mRNA dos TLRs 2 e 4, e das citocinas IL-1?, IL-6 e TNF-?. Com exce??o do grupo DM1C (18,4%), os pacientes DM1NC (81,6%) apresentaram um controle glic?mico insatisfat?rio, com valores de mediana para glicose (225,5mg/dL) e hemoglobina glicada (11,2%) significativamente superiores ao grupo NG (glicose: 76,5mg/dL e hemoglobina glicada: 6,9%). Foram obtidos valores aumentados para a ur?ia s?rica (20%) e RAC urin?ria (20,8%); e diminu?dos para albumina (5,7%) e prote?na total (13,6%) nos indiv?duos diab?ticos; e associados a um controle glic?mico insatisfat?rio (DM1NC aumento de 20% para ur?ia e 49% para RAC; e diminui??o de 8,6% para albumina e 12,1% para prote?na total) e a um maior tempo de diagn?stico (DM1 <5anos aumento de 20% para ur?ia e 20,8% para RAC; e diminui??o de 14,3% para albumina e 13,6% para prote?na total). No tocante ? avalia??o do estado pr?-inflamat?rio, as express?es de mRNA se apresentaram elevadas para TLR2 (37,5%), IL-1? (43%), IL-6 (44,4%) e TNF-? (15,6%) nos indiv?duos diab?ticos em rela??o aos NG, sendo principalmente associadas aos grupos DM1NC (controle glic?mico insatisfat?rio TLR2: 82%, IL-1?: 43% de aumento) e DM1 <5 anos (maior tempo de diagn?stico TLR2: 85,4%, IL-1?: 46,5% de aumento). O conjunto de resultados suporta a exist?ncia de um quadro inflamat?rio mediado pelo aumento da express?o do TLR2 e das citocinas pr?-inflamat?rias IL-1?, IL-6 e TNF-? no diabetes tipo 1

Orientador: Luciane Alarcão Dias-Melicio / Resumo: A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, prevalente na América Latina, que tem por agentes etiológicos diferentes espécies de Paracoccidioides. Entre eles encontra-se o Paracoccidioides brasiliensis (P. brasiliensis), espécie composta por um complexo de agrupamentos geneticamente isolados, classificados como espécies filogenéticas: S1, PS2, PS3 e PS4. O P. brasiliensis possui distribuído pela parede fúngica e dentro de vesículas citoplasmáticas uma glicoproteína de 43KDa, a gp43. Esta molécula é considerada como o principal componente antigênico produzido e secretado pelo fungo, a qual é encontrada no soro de pacientes acometidos pela PCM e, está associada aos fatores de virulência e escape do patógeno. Sabendo disso, tornou-se extremamente importante a avaliação da interação dessa proteína com as células do sistema imune inato e sua consequente atuação sobre os mecanismos moduladores celulares. Assim, nosso estudo avaliou os neutrófilos polimorfonucleares (PMNs) que estão presentes em abundância desde o início e durante a infecção pelo Paracoccidioides, e que apresentam além de suas ações efetoras diretas contra o fungo, uma importante ação moduladora da resposta imune. Estas ações envolvem o reconhecimento do fungo por Receptores de Reconhecimento de Padrão (PRRs) que culminam em ativação celular com consequente produção e liberação de diferentes mediadores inflamatórios. Dessa maneira, o presente trabalho avaliou o envolvimento dos receptores cel... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, prevalent in Latin America and has as etiological agents Paracoccidioides spp. One of them is Paracoccidioides brasiliensis (P. brasiliensis) composed by a complex of genetically isolated clusters, classified as phylogenetic species: S1, PS2, PS3 and PS4. P. brasiliensis present along the cell wall and inside cytoplasmic vesicles a 43 KDa glycoprotein, known as gp43. This molecule is recognized as the main antigenic component produced and secreted by the fungus into sera of PCM patients and is associated with the escape and virulence factors of the pathogen. Thereby, becomes extremely important to evaluate the interaction mechanisms of this protein with cells from the innate immune system and their consequent action on cellular functions. Polymorphonuclear neutrophils (PMNs) has prominent participation in PCM, once these cells are present in a great amount at the beginning and during Paracoccidioides infection. PMNs perform effector actions directly against the fungi as well as an important immune modulatory function. Such actions involve the recognition of fungal components by pattern recognition receptors (PRRs) that leads to cell activation and consequent production of inflammatory mediators. So, the present study evaluated the involvement of TLR2 and TLR4 on cytokines and eicosanoids release by PMNs from healthy human stimulated with gp43. Cells were initially incubated in the presence or absence of monoclo... (Complete abstract click electronic access below) / Doutor