No description available.

Après exposition par inhalation, les nanoparticules (NPs) peuvent atteindre les alvéoles pulmonaires, se retrouver au niveau de la barrière alvéolo-capillaire (BAC), et induire une toxicité locale et / ou franchir cette barrière pour se retrouver dans la circulation sanguine. Dans ce contexte, l’objectif de ce travail a été de développer des modèles de co-cultures in vitro simples à mettre en œuvre (utilisation de lignées cellulaires humaines), pour étudier les effets des NPs au niveau de la BAC. Dans un premier temps, des co-cultures de cellules épithéliales alvéolaires ou de phénotype proche (lignées A549 ou NCI-H441), et de macrophages (lignée THP-1), ont permis l’étude des effets pro-inflammatoires des NPs de SiO2 et de TiO2. Avec ces modèles nous avons montré l’importance de la coopération cellulaire mise en jeu lors des processus inflammatoires liés aux NPs, mais aussi le rôle du ratio cellulaire employé dans ces réponses. Dans un second temps, des co-cultures tridimensionnelles en chambres bicamérales associant des macrophages (lignée THP-1), des cellules épithéliales bronchiques (lignée Calu-3), et des cellules endothéliales pulmonaires microvasculaires (lignée HPMEC-ST1.6R), ont permis l’étude de l’impact de NPs fluorescentes de polystyrène sur l’intégrité de la BAC, et leur passage à travers cette barrière. Les cellules épithéliales Calu-3 permettent d’établir une barrière de qualité mais la membrane microporeuse servant de support aux cellules doit être optimisée pour ne pas être un frein au passage des NPs. Ce travail montre qu’un seul modèle ne permet pas d’étudier de façon optimale à la fois la toxicité et la translocation des NPs, et qu’une approche adaptée doit être envisagée en fonction du paramètre que l’on souhaite étudier. / After inhalation, nanoparticles (NPs) can reach the alveoli and the alveolo-capillary barrier (ACB), and consequently induce local toxicity and / or cross this barrier to reach the bloodstream. In this context, the aim of this work was to develop co-culture in vitro models simple to implement (using human cell lines), to study effects of NPs on the ACB. In a first time, pro-inflammatory effects of SiO2 and TiO2 NPs were studied on co-cultures of alveolar epithelial cells (A549 and NCI-H441 cell lines), and macrophages (THP-1 cell line). We demonstrated the importance of cell cooperation during inflammatory processes caused by these NPs, and the role of the cellular ratio in these inflammatory responses. In a second time, effects of fluorescent polystyrene NPs on the ACB integrity, and their translocation were studied on three-dimensional co-cultures in bicameral chambers involving macrophages (THP-1 cell line), bronchial epithelial cells (Calu-3 cell line), and micro-vascular pulmonary endothelial cells (HPMEC ST1.6R cell line). The use of Calu-3 has provided a good barrier, but further investigations on microporous membranes are still needed to not interfere with NPs translocation. Altogether, these results show that a tailored approach should be considered in order to study toxicity or translocation of NPs.

Les cellules du système immunitaire sont programmées pour reconnaître et éliminer les cancéreuses, pourtant les cellules tumorales ont la capacité de mettre en œuvre les divers moyen pour échapper à la mort induite par les effecteurs du système immunitaire. Au cours de cette thèse, nous avons étudié plus particulièrement PD-L1, qui est impliqué dans la protection des cellules tumorales contre une attaque du système immunitaire et induit au cours de la TEM. / EMT foster cancer progression by acting on mechanisms allowing tumors to exide immune surveillance. Recent clinical advances immunotherapy demonstrated that some cancer with established lymphocyte infiltrates, express immune checkpoint inhibitory molecules, such as PD-L1, to allow their progression. During this thesis, another link between EMT and immune escape, through the regulation of PD-L1 in non-smal cell lung caricinoma was established. A new role of IKKƐ in the regulation of PD-L1 during EMT was also been shown.

Paclitaxel is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Paclitaxel, the development of drug resistance hampers its clinical applicability. Paclitaxel is used in malignant tumors in the present research, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), head-neck scale epitheliomatous, urinary bladder cancer, tumor of the reproduction organ and so on. Clinical treatment of paclitaxel be injected 250mg/m2 to previously non-treated patients of small lung cancer its effect about 34% and previously non-treated patients of non-small lung cancer its effect about 21% to 24% . On the other hand we had established a taxol-resistant human lung carcinoma subline A549R by paclitaxel to compare the different proteins with A549 wild type and treat the different concention of paclitaxel with MTT assay so as to observe the tolerance dosage of subline growth.We obtained the patient¡¦s specimens of lung cancer to treat with paclitaxel some of resistant and some of non-resistant to compare differentially expressed proteins between normal and tumor. When we cultured taxol-resistant human lung carcinoma subline in which paclitaxel and calcium regulate growth, owing to the proteins of changes result to resistance. The extraction cell subline and specimens were analyzed by 2-D electrophoresis patterns and we found that interact paclitaxel with calcium it is the important factor of drug resistant. Verified from clinical treatment might have hypercalcemia in malignant tumors and calcium ion may increase paclitaxel drug resistance and hypercalcemia patient will be more insensitive to paclitaxel treatment.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.

No description available.

No description available.

Cancer is a leading cause of mortality worldwide, responsible for nearly one in six deaths. Thus, there is a need for a greater understanding of cancer for the development of novel therapeutics. This master thesis project aims to compare the proteome signatures between dying and surviving cancer cells treated with diverse anticancer drugs. The first aim is to investigate if drug targets behave similarly and have the same sign (up- or down-regulation) in dying versus surviving cells. The second aim is to validate that combining the dying cancer cell’s proteome with the surviving cell’s can help improve drug target rankings for anticancer treatments. The third aim is to identify proteins and pathways involved in life and death decisions by comparing dying and surviving states in response to the anticancer drugs in different cell lines. First, we demonstrate that drug target behaviour in dying versus surviving cells is almost identical for nine diverse anticancer compounds with a correlation of 0.93. To identify drug targets, orthogonal partial least squares-discriminant analysis (OPLS-DA) modelling was performed to contrast the proteome signature of one anticancer drug against all other drugs and rank the proteins based on the magnitude of the model’s predictive component. There were occasions when the dying cells gave better rankings than the surviving ones. In some cases, the best target rankings were obtained when combining the data from both surviving and dying cells. To identify proteins and pathways involved in life and death decisions, OPLS-DA modelling contrasting the two states was performed, and heatmaps and scatterplots of dying and surviving log2 fold changes were made. As a result, several pathways involved in cell survival and cell death were identified. In addition, at least six proteins consistently differentially regulated between the surviving and dying cells were identified. Such proteins can be considered as putative survival (resistance) or sensitivity biomarkers and serve as potential drug targets for the development of novel anticancer agents.

No description available.