O biglicam é um proteoglicano é altamente expresso em células trofoblásticas de patologias placentárias com invasividade exacerbada. No entanto, as funções do biglicam no trofoblasto ainda não foram elucidadas. Sendo assim, verificamos a expressão e as funções de biglicam e seus receptores Toll-like (TLR)-2 e TLR-4 nas células trofoblásticas durante a gestação. As células do citotrofoblasto extraviloso (CTEV) foram positivas para todas as moléculas, menos para o biglicam em placentas a termo. Adição exógena de biglicam promoveu migração e invasão das células trofoblásticas. O biglicam estimulou a fosforilação de AKT nos sítios Thr308 e Ser473 nas células trofoblásticas. A migração e a invasão biglicam-dependentes e as fosforilações de AKT foram inibidas após a adição de anticorpos bloqueadores anti-TLR-2 e anti-TLR-4. O silenciamento gênico de AKT1 em células SGHPL-5 aboliu os efeitos do biglicam na motilidade. Em conclusão, o biglicam aumenta a motilidade de células trofoblásticas após sinalização por AKT através da ativação de TLR-2 e TLR-4. / Biglycan is a highly expressed proteoglycan in trophoblast cells from invasiveness-changed placental pathologies. However, biglycan functions in the trophoblast were not yet identified. Therefore, it was verified the expression and functions of biglycan and its receptors Toll-like (TLR)-2 and TLR-4 in trophoblast cells throughout pregnancy. The extravillous cytotrophoblast cells (EVT) were positive to all the molecules, although biglycan was negative in term placentas. Exogenous biglycan promoted migration and invasion of trophoblast cells. Biglycan stimulated AKT phosphorilation at Thr308 and Ser473 sites in trophoblast cells. The biglycan-dependent migration, invasion and AKT phosphorilation were inhibited upon addiction of anti-TLR-2 and anti-TLR-4 blocking antibodies. AKT1 genic silencing in SGHPL-5 cells abolished the motility effects. In conclusion, biglycan increases the motility of trophoblast cells after AKT signaliing throughout TLR-2 and TLR-4 activation.

Nanoparticles are promising platforms for biomedical applications ranging from diagnostic tools to therapeutic delivery agents. During the course of these applications, nanoparticles are exposed to a complex mixture of extracellular serum proteins that nonspecifically adsorb onto the surface. The resulting protein layer, or protein "corona," creates an interface between nanoparticles and the biological environment. Protecting the nanoparticle surface can reduce protein adsorption, but complete inhibition remains a challenge. As a result, the corona, rather than the nanoparticle itself, mediates the cellular response to the nanoparticle. The following dissertation describes the fundamental characterization of the cellular binding of charged nanoparticles, interactions of protein-nanoparticle complexes with cellular receptors, and the structural and thermodynamic properties of adsorbed corona proteins.

O biglicam é um proteoglicano é altamente expresso em células trofoblásticas de patologias placentárias com invasividade exacerbada. No entanto, as funções do biglicam no trofoblasto ainda não foram elucidadas. Sendo assim, verificamos a expressão e as funções de biglicam e seus receptores Toll-like (TLR)-2 e TLR-4 nas células trofoblásticas durante a gestação. As células do citotrofoblasto extraviloso (CTEV) foram positivas para todas as moléculas, menos para o biglicam em placentas a termo. Adição exógena de biglicam promoveu migração e invasão das células trofoblásticas. O biglicam estimulou a fosforilação de AKT nos sítios Thr308 e Ser473 nas células trofoblásticas. A migração e a invasão biglicam-dependentes e as fosforilações de AKT foram inibidas após a adição de anticorpos bloqueadores anti-TLR-2 e anti-TLR-4. O silenciamento gênico de AKT1 em células SGHPL-5 aboliu os efeitos do biglicam na motilidade. Em conclusão, o biglicam aumenta a motilidade de células trofoblásticas após sinalização por AKT através da ativação de TLR-2 e TLR-4. / Biglycan is a highly expressed proteoglycan in trophoblast cells from invasiveness-changed placental pathologies. However, biglycan functions in the trophoblast were not yet identified. Therefore, it was verified the expression and functions of biglycan and its receptors Toll-like (TLR)-2 and TLR-4 in trophoblast cells throughout pregnancy. The extravillous cytotrophoblast cells (EVT) were positive to all the molecules, although biglycan was negative in term placentas. Exogenous biglycan promoted migration and invasion of trophoblast cells. Biglycan stimulated AKT phosphorilation at Thr308 and Ser473 sites in trophoblast cells. The biglycan-dependent migration, invasion and AKT phosphorilation were inhibited upon addiction of anti-TLR-2 and anti-TLR-4 blocking antibodies. AKT1 genic silencing in SGHPL-5 cells abolished the motility effects. In conclusion, biglycan increases the motility of trophoblast cells after AKT signaliing throughout TLR-2 and TLR-4 activation.

Asbestos é um termo geral usado comercialmente para descrever minerais fibrosos de silicato. A fibra mais utilizada até hoje é denominada crisotila, com uso considerado seguro. Embora, fragmentos menores podem permanecer por longo tempo em tecidos pulmonares. As fibras de crisotila, assim como as demais fibras de asbestos, possuem sílica na sua composição e podem ser fagocitadas com a participação de receptores scavenger. Este trabalho teve como objetivo o estudo de mecanismos de interação e de internalização das pequenas fibras de crisotila em duas linhagens celulares. Uma análise por microscopia confocal de varredura a laser e microscopia eletrônica de transmissão foi realizada e observou-se que ambas as linhagens são capazes de internalizar fibras, que apresentavam livres ou envoltas pela membrana plasmática. A presença de elementos do citoesqueleto próximos às fibras de crisotila foi verificada, assim como alterações no nível de expressão de alguns desses elementos. Dentro deste contexto, a participação de receptores no processo de internalização de fibras de crisotila também foi estudada e verificamos que esses receptores podem estar envolvidos de alguma forma com o processo de internalização de fibras de crisotila. / Asbestos is a term used commercially to describe silicate minerals. Chrysotile is the most used fiber until today and it is considered safe. However, smaller fragments can be found in lung tissues for a long period of time. Chrysotile fibers, as other asbestos fibers, are composed by silica and may be phagocyted with the participation of scavenger receptors. This studys goal was to study the interaction an internalization mechanisms of small chrysotile fibers in two cell lineages. Confocal laser scan microscopy analysis and electronic microscopy transmission analysis were conducted in both lineages, that showed capable of internalize fibers, that were involved or not by cell membrane. Cytoeskeleton elements presence near the fibers was verified and there were also changes in expression levels these elements. In this context, the participation of scavenger receptors in the internalization process of chrysotile fibers was also studied, and we verified that these receptors may be associated to the internalization process of chrysotile fibers.

Receptores ativados por protease (PARs) pertencem à família de GPCRs. Desses, PAR-1, PAR-3 e PAR-4 são ativados por trombina, e PAR-2 por tripsina. Como as proteases, peptídeos sintéticos (PARs-AP) também ativam esses receptores. Estudamos o papel dos PARs na reatividade vascular de Wistar e SHR. In vitro, PAR-1 AP, promoveu maior vasoconstrição em aorta com endotélio (E+) de SHR vs Wistar. PAR-2 AP promoveu vasodilatação similar em aorta E+ de SHR e Wistar, enquanto PAR-4 AP e peptídeos reversos não causaram efeito. In vivo/in situ PAR-1 e PAR-2 AP mostraram intensa vasomotilidade em arteríolas mesentéricas. A expressão gênica de PAR-1 está aumentada em aorta e arteríolas de SHR, mas a expressão protéica está aumentada apenas em arteríolas. Demonstramos ainda que a vasoconstrição induzida por PAR-1 AP, é dependente de Ca++ e da liberação de Ang II, ET-1 e O2- pelo endotélio. Assim, sugerimos que PAR-1 pode ser um alvo terapêutico para novos antihipertensivos com efeito antitrombótico, já que este receptor também tem sido envolvido em eventos romboembólicos. / Protease activated receptors are a new GPCRs family. The PAR-1, PAR-3 and PAR-4 are activated by thrombin and PAR-2 by tripsin. Like proteases, synthetic peptides (PARs-AP) can also activate those receptors. We studied the role of PARs in vascular reactivity of Wistar and SHR. In vitro, PAR-1 promoted higher vasoconstriction to PAR-1 AP in SHR aorta with endothelium (E+) than the Wistar ones. PAR-2 AP produced similar vasodilation in Wistar and SHR aorta E+, while neither PAR-4 nor reverse peptides presented any effect. In vivo/in situ PAR-1 and PAR-2 showed an intensive vasomotion in mesenteric vessels. PAR-1 gene expression was increased in SHR aorta and arterioles, while the protein expression was increased only in the arterioles. We have also shown that the vasoconstriction induced by PAR-1 AP, is Ca++-dependent and Ang II, ET-1 and O2- release from endothelium. Thus, we suggest that PAR-1 might represent a therapeutic target to new antihypertensive drugs with antithrombotic effect, since this receptor has been involved in thromboembolics events.

Receptores ativados por protease (PARs) pertencem à família de GPCRs. Desses, PAR-1, PAR-3 e PAR-4 são ativados por trombina, e PAR-2 por tripsina. Como as proteases, peptídeos sintéticos (PARs-AP) também ativam esses receptores. Estudamos o papel dos PARs na reatividade vascular de Wistar e SHR. In vitro, PAR-1 AP, promoveu maior vasoconstrição em aorta com endotélio (E+) de SHR vs Wistar. PAR-2 AP promoveu vasodilatação similar em aorta E+ de SHR e Wistar, enquanto PAR-4 AP e peptídeos reversos não causaram efeito. In vivo/in situ PAR-1 e PAR-2 AP mostraram intensa vasomotilidade em arteríolas mesentéricas. A expressão gênica de PAR-1 está aumentada em aorta e arteríolas de SHR, mas a expressão protéica está aumentada apenas em arteríolas. Demonstramos ainda que a vasoconstrição induzida por PAR-1 AP, é dependente de Ca++ e da liberação de Ang II, ET-1 e O2- pelo endotélio. Assim, sugerimos que PAR-1 pode ser um alvo terapêutico para novos antihipertensivos com efeito antitrombótico, já que este receptor também tem sido envolvido em eventos romboembólicos. / Protease activated receptors are a new GPCRs family. The PAR-1, PAR-3 and PAR-4 are activated by thrombin and PAR-2 by tripsin. Like proteases, synthetic peptides (PARs-AP) can also activate those receptors. We studied the role of PARs in vascular reactivity of Wistar and SHR. In vitro, PAR-1 promoted higher vasoconstriction to PAR-1 AP in SHR aorta with endothelium (E+) than the Wistar ones. PAR-2 AP produced similar vasodilation in Wistar and SHR aorta E+, while neither PAR-4 nor reverse peptides presented any effect. In vivo/in situ PAR-1 and PAR-2 showed an intensive vasomotion in mesenteric vessels. PAR-1 gene expression was increased in SHR aorta and arterioles, while the protein expression was increased only in the arterioles. We have also shown that the vasoconstriction induced by PAR-1 AP, is Ca++-dependent and Ang II, ET-1 and O2- release from endothelium. Thus, we suggest that PAR-1 might represent a therapeutic target to new antihypertensive drugs with antithrombotic effect, since this receptor has been involved in thromboembolics events.

Asbestos é um termo geral usado comercialmente para descrever minerais fibrosos de silicato. A fibra mais utilizada até hoje é denominada crisotila, com uso considerado seguro. Embora, fragmentos menores podem permanecer por longo tempo em tecidos pulmonares. As fibras de crisotila, assim como as demais fibras de asbestos, possuem sílica na sua composição e podem ser fagocitadas com a participação de receptores scavenger. Este trabalho teve como objetivo o estudo de mecanismos de interação e de internalização das pequenas fibras de crisotila em duas linhagens celulares. Uma análise por microscopia confocal de varredura a laser e microscopia eletrônica de transmissão foi realizada e observou-se que ambas as linhagens são capazes de internalizar fibras, que apresentavam livres ou envoltas pela membrana plasmática. A presença de elementos do citoesqueleto próximos às fibras de crisotila foi verificada, assim como alterações no nível de expressão de alguns desses elementos. Dentro deste contexto, a participação de receptores no processo de internalização de fibras de crisotila também foi estudada e verificamos que esses receptores podem estar envolvidos de alguma forma com o processo de internalização de fibras de crisotila. / Asbestos is a term used commercially to describe silicate minerals. Chrysotile is the most used fiber until today and it is considered safe. However, smaller fragments can be found in lung tissues for a long period of time. Chrysotile fibers, as other asbestos fibers, are composed by silica and may be phagocyted with the participation of scavenger receptors. This studys goal was to study the interaction an internalization mechanisms of small chrysotile fibers in two cell lineages. Confocal laser scan microscopy analysis and electronic microscopy transmission analysis were conducted in both lineages, that showed capable of internalize fibers, that were involved or not by cell membrane. Cytoeskeleton elements presence near the fibers was verified and there were also changes in expression levels these elements. In this context, the participation of scavenger receptors in the internalization process of chrysotile fibers was also studied, and we verified that these receptors may be associated to the internalization process of chrysotile fibers.

Streptococcus suis est un important pathogène porcin et agent zoonotique responsable de méningites et de septicémies. À ce jour, les mécanismes impliqués dans la réponse immunitaire de l’hôte lors de l’infection par S. suis sont peu connus; et il en est de même pour les stratégies utilisées par S. suis afin de déjouer cette réponse. L’augmentation de l’incidence et de la sévérité des cas humains souligne le besoin d’une meilleure compréhension des interactions entre S. suis et le système immunitaire afin de générer une réponse immunitaire efficace contre ce pathogène. Les cellules dendritiques (DCs) sont de puissantes cellules présentatrices d’antigènes qui stimulent les lymphocytes T et B, assurant la liaison entre l’immunité innée et l’immunité adaptative. L’objectif principal de ce projet était d’évaluer le rôle joué par différents facteurs de virulence de S. suis sur la modulation de la fonction des DCs et de la réponse T-dépendante. Nous avons examiné l’effet des facteurs clés pour la virulence de S. suis, dont la capsule polysaccharidique (CPS), les modifications de la paroi cellulaire (D-alanylation de l’acide lipotéichoïque et N-déacétylation du peptidoglycane) et la toxine suilysine, sur l’activation et la maturation de DCs murines dérivées de la moelle osseuse (bmDCs). Suite à l’infection par S. suis, les bmDCs sont activées et subissent un processus de maturation caractérisé par l’augmentation de l’expression de molécules de co-stimulation et la production de cytokines pro-inflammatoires. La CPS est le principal facteur interférant avec la production de cytokines, même si les modifications de la paroi cellulaire et la suilysine peuvent également moduler la production de certaines cytokines. Enfin, la CPS, les modifications de la paroi cellulaire et la suilysine interfèrent avec la déposition du complément à la surface des bactéries et, en conséquence, avec le « killing » dépendant du complément. Les résultats ont été confirmés à l’aide de bmDCs porcines. Nous avons aussi voulu identifier les récepteurs cellulaires impliqués dans la reconnaissance de S. suis par les DCs. Nous avons démontré que la production de cytokines et l’expression des molécules de co-stimulation par les DCs sont fortement dépendantes de la signalisation par MyD88, suggérant que les DCs reconnaissent S. suis et deviennent activées majoritairement via la signalisation par les récepteurs de type Toll (TLRs). En effet, on remarque une diminution de la production de plusieurs cytokines ainsi que de l’expression de certaines molécules de co-stimulation chez les DCs TLR2-/- ou TLR2-/- et TLR9-/- double négatives. Finalement, le récepteur NOD2 semblait jouer un rôle partiel dans l’activation des DCs suite à une infection par S. suis.Enfin, nous avons évalué les conséquences de la modulation des fonctions des DCs sur le développement de la réponse T-dépendante. Les splénocytes totaux produisent plusieurs cytokines en réponse à S. suis. Des analyses in vivo et ex vivo ont permis d’observer l’implication des cellules T CD4+ et le développement d’une réponse de type « T helper » 1 (TH1) bien que la quantité de cytokines TH1 produites lors de l’infection in vivo par S. suis demeure assez basse. La CPS de S. suis interfère avec la production de plusieurs cytokines par les cellules T in vitro. Expérimentalement, l’infection induite par S. suis résulte en de faibles niveaux de production d’anticorps anti-S. suis, mais aussi d’anticorps dirigés contre l’ovalbumine utilisée comme antigène rapporteur. Cette interférence est corrélée avec la sévérité des signes cliniques, suggérant que S. suis interfère avec le développement d’une réponse immunitaire adaptative appropriée qui serait requise pour contrôler la progression de l’infection. Les résultats de cette étude mèneront à une meilleure compréhension de la réponse immunitaire de l’hôte lors de l’infection par S. suis. / Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis. Knowledge of host immune responses towards S. suis, and strategies used by this pathogen for subversion of these responses is scarce. Increased severity of S. suis infections in humans underscores the critical need to better understand the interactions between S. suis and the immune system to generate an effective immune response against this pathogen. Dendritic cells (DCs) are powerful antigen-presenting cells. Once activated, they stimulate T cells and B cells, linking innate and adaptive immunity. Thus, the main objective of this project was to evaluate the role of different S. suis virulence factors on the modulation of DC functions and the T cell-dependent response. Initially, we investigated the effect of S. suis key virulence factors, including the capsular polysaccharide (CPS), the cell wall modifications (D-alanylation of the lipoteichoic acid and N-deacetylation of the peptidoglycan) and the toxin suilysin, on the activation and maturation of mouse bone-marrow derived DCs (bmDCs). We observed that following S. suis infection, bmDCs are activated and go through a complex maturation process characterized by the up-regulation of the surface expression of costimulatory molecules and the production of pro-inflammatory cytokines. The CPS is the main virulence factor interfering with cytokine production, even if cell wall modifications and suilysin can also modulate the production of cytokines. Finally, CPS, cell wall modifications and suilysin were shown to interfere with complement deposition on S. suis, and consequently with complement-dependent killing. Results were confirmed using porcine bmDCs. We also aimed to identify the cellular receptors involved in S. suis recognition by DCs. Production of cytokines and expression of co-stimulatory molecules by DCs were shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize S. suis and become activated mostly through Toll-like receptor (TLR) signaling. Supporting this fact, TLR2-/- or double negative TLR2-/- and TLR9-/- DCs were severely impaired in the release of several cytokines and the surface expression of certain costimulatory molecules. In addition, NOD2 receptor also seems to play a partial role in DC activation by S. suis. Finally, we evaluated the consequences of the modulation of DC functions on T cell activation. In response to S. suis infection, total splenocytes readily produced several cytokines ex vivo. Ex vivo and in vivo analysis revealed the involvement of CD4+ T cells and development of a T helper 1 (TH1) response. Nevertheless, levels of TH1-derived cytokines during S. suis infection were very low. The bacterial CPS was shown to interfere with the release of several T cell-derived cytokines in vitro. As a consequence, a clinical infection resulted in low levels of not only anti-S. suis antibodies but also of those directed against ovalbumin, used as reported antigen. This interference was correlated with the presence of severe clinical signs of S. suis disease. These data suggest that S. suis impairs the development of an efficient adaptive immune response, which is required to control the infection progress. Overall, these results will permit a better comprehension of the host immune response during S. suis infection.