Le syndrome de détresse respiratoire aiguë (SDRA) se développe suite à une atteinte pulmonaire lésionnelle, induisant un œdème et une inflammation excessive, généralement suivis d’une réparation atypique menant à la fibrose. Malgré de signifiants progrès dans les traitements, la mortalité reste élevée : ~ 40 %. Mon hypothèse de travail est que l’atténuation de l’œdème ou de la réponse inflammatoire pourrait freiner le développement ou la sévérité de la phase exsudative. Nous avons évalué cette hypothèse à l’aide d’un modèle de phase exsudative du SDRA, i.e. instillation intra-trachéale de bléomycine, chez les souris.  La modulation des fluides alvéolaires est étudiée avec des souris transgénique (Tg) pour le canal ENaC, qui sont sensibles à la formation d’un œdème. Cependant, ces souris Tg ne sont pas plus sensibles au développement de la phase exsudative en condition lésionnelle (bléomycine). Nous avons déterminé par une étude électrophysiologique des cellules épithéliales alvéolaires de type II (AT II) que ce n’est pas lié à une inhibition par la bléomycine de la fonction du canal ENaC.  Le traitement de la réponse inflammatoire associée au SDRA par des glucocorticoïdes est une thérapie potentielle mais controversée. Les glucocorticoïdes dans notre modèle murin ne réduisent pas la sévérité des lésions. Nous avons pu déterminé lors d’expériences in vitro que ce serait dû à une réduction de la capacité de réparation des AT II. En résumé :  La modulation du canal ENaC ne modifie pas le développement de la phase exsudative, suggérant que la régulation de l’œdème n’est pas suffisante pour modifier l’évolution du SDRA.  La modulation de l’inflammation par les glucocorticoïdes est ineffective, possiblement à cause d’une altération de la réparation. Mon étude suggère que le traitement de la phase exsudative du SDRA est complexe. En effet, la régulation de l’œdème ou de l’inflammation de façon isolée ne peut pas modifier l’évolution du SDRA. L'hétérogénéité des sources du SDRA et la redondance des mécanismes cellulaires impliqués dans l’évolution des lésions pulmonaires suggèrent que le traitement nécessitera une approche visant plusieurs cibles mécanistiques afin d’en accélérer la résolution. / Although much has been learned about the mechanisms leading to acute respiratory distress syndrome (ARDS), mortality remains high: ~ 40%. This syndrome is associated with lung injury where alveolar edema and excessive inflammatory response can progress to abnormal epithelial repair and fibrosis. The hypothesis of the work presented in this thesis is that attenuation of edema or of the inflammatory response in the initial stage of the acute lung injury would decrease the severity of injury. I evaluated this hypothesis in an ARDS acute phase, modeled by an intratracheal instillation of bleomycin in mice, using two distinct experimental strategies.  The importance of edema clearance was studied in a transgenic (Tg) ENaC mouse, a mouse known to be sensitive to the formation of edema. However, our results show that these Tg mice were not more susceptible to the development of the ARDS acute phase induced by bleomycin. Furthermore, we have been able to show that bleomycin itself did not interfere with the ENaC channel function of alveolar epithelial cells type II (AT II).  The treatment of the inflammatory response associated with ARDS by glucocorticoid therapy is subject to controversy. In our mouse model, glucocorticoids decrease the level of cytokine in the alveolar milieu but did not decrease the severity of lung injury. Using in vitro experiments, we show that this lack of response could be secondary to the impact of the treatment on the epithelial repair capacity of AT II. In summary:  The ENaC channel expression did not have an impact on the development of the exudative phase, suggesting that the regulation of edema is not sufficient to alter the course of ARDS.  The modulation of inflammation by glucocorticoids was ineffective, possibly because of impaired repair of the epithelium. These results suggest that the control of edema or inflammation separately does not modify the evolution of lung injury. The heterogeneity of the ARDS origins and the redundancy of cellular mechanisms involved in lung injury will require therapy aimed at multiple pathophysiological targets to permit the resolution of lung injury.

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

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-07-07T15:09:57Z No. of bitstreams: 1 arquivototal.pdf: 1854233 bytes, checksum: 535d273c615cebce265419e27f74439a (MD5) / Made available in DSpace on 2017-07-07T15:09:57Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1854233 bytes, checksum: 535d273c615cebce265419e27f74439a (MD5) Previous issue date: 2016-10-17 / Ouabain is a cardiotonic steroid initially described as a substance of plant origin. In 1991, the endogenous production of higher mammals was identified and since then their physiological actions have been studied. Work of our group have demonstrated that ouabain modulates the acute inflammatory response induced by different phlogistic agents, also being able to interfere negatively in inflammatory profile triggered by Leishmania (L.) amazonensis. Acute lung injury (ALI) is a serious inflammatory disease characterized by acute inflammation, extensive accumulation of polymorphonuclear leukocytes and accumulation of proinflammatory mediators, which culminates with diffuse alveolar damage and which may cause the patient died due to severe hypoxemia. There is no data in the literature on the effects of ouabain in ALI. Objectives: Evaluate the immunomodulatory effect of ouabain in a murine model of ALI induced by LPS. Methods: BALB / c mice were treated intraperitoneally with ouabain at a dose of 0.56 mg / kg for a period of three consecutive days, 1 hour after the last treatment the animals were challenged intranasally with 40μl of an LPS solution (2 5 mg / kg); 24 hours after challenge, the animals were euthanized, the collected biological sample and inflammatory parameters, including cell migration, protein exudates, cytokine production, TLR4 expression and histopathological changes were then evaluated. Data were analyzed by PRISMA software. Results: The treatment with ouabain decreased total leukocytes migration to the inflamed site (48,84%), this event associated with decreased neutrophil migration (70,71%) and independent of macrophage migration. The ouabain also decreased the exudate protein in the broncho-alveolar region (26,32%) and production of the cytokines TNF-α (14,80%), IL-6 (47,07%) and IL1-β (33,59%), however this reduction in the production of these mediators was not related to the expression of TLR4. Additionally, the ALI histopathology changes were also reduced by treatment with ouabain. Conclusions: The results show that ouabain has anti-inflammatory action in ALI induced by LPS. / A Ouabaína é um esteroide cardiotônico inicialmente descrito como uma substância de origem vegetal. Em 1991, a sua produção endógena por mamíferos superiores foi identificada e desde então suas ações fisiológicas vêm sendo estudadas. Trabalhos do nosso grupo demonstraram que a ouabaína modula a resposta inflamatória aguda induzida por diferentes agentes flogísticos, sendo também capaz de interferir negativamente no perfil inflamatório desencadeado pela Leishmania (L.) amazonensis. A lesão pulmonar aguda (LPA) é uma doença inflamatória caracterizada por inflamação aguda e extenso acúmulo de polimorfonucleares e de mediadores pró-inflamatórios, que culmina com dano alveolar difuso podendo levar o paciente a óbito por hipoxemia severa. Não há dados na literatura sobre os efeitos da ouabaína na LPA. Objetivos: Avaliar o efeito imunomodulador de ouabaína em modelo murino de LPA induzida por LPS. Métodos: Camundongos BALB/c machos foram tratados via intraperitoneal com ouabaína na dose de 0,56 mg/Kg por um período de três dias consecutivos, 1h após o último tratamento os animais foram desafiados via intranasal com LPS (2,5 mg/Kg); 24h após o desafio, os animais foram eutanásiados, as amostras biológicas coletadas e os parâmetros inflamatórios, incluindo migração celular, exsudato proteico, produção de citocinas, expressão de TLR4 e alterações histopatológicas foram então avaliados. Os dados foram analisados pelo software PRISMA. Resultados: O tratamento com a ouabaína diminuiu a migração de leucócitos totais para o sítio inflamado (48,84%), evento este, associado a diminuição da migração de neutrófilos (70,7%) e independente da migração de macrófagos. Ouabaína também diminuiu o exsudato proteico na região bronco-alveolar (26,32%) e a produção das citocinas TNF-α (14,80%), IL-6 (47,07%) e IL1-β (33,59%), entretanto essa redução na produção desses mediadores não mostrou relação com a expressão do TLR4. Adicionalmente, as alterações histopatológicas características da LPA também foram reduzidas pelo tratamento com ouabaína. Conclusões: Os resultados obtidos demonstram que ouabaína possui ação anti-inflamatória na LPA induzida por LPS.

Submitted by Rafaelle Batistella Pires (rafaelle.pires@gmail.com) on 2018-03-07T15:41:30Z No. of bitstreams: 1 Tese Rafaelle Batistella Pires.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) / Approved for entry into archive by Luciana Pizzani null (luciana@btu.unesp.br) on 2018-03-08T20:07:06Z (GMT) No. of bitstreams: 1 pires_rb_dr_bot.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) / Made available in DSpace on 2018-03-08T20:07:06Z (GMT). No. of bitstreams: 1 pires_rb_dr_bot.pdf: 2978722 bytes, checksum: f79b8076fd69934911d1a338cd131aa3 (MD5) Previous issue date: 2018-02-20 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Síndrome do Desconforto Respiratório Agudo (SDRA) cursa com alta morbi-mortalidade apesar dos avanços no entendimento de sua fisiopatologia e tratamento. A terapia ventilatória baseia-se na proteção pulmonar, sendo a ventilação oscilatória de alta frequência (VOAF) uma opção de método protetor. A posição prona (PP) é terapia adjuvante que possibilita homogeneização da distribuição do volume corrente (VC) e promove recrutamento alveolar. O objetivo do estudo foi investigar o efeito da posição prona associada à VOAF e ventilação mecânica convencional (VMC) protetora sobre a oxigenação, inflamação, dano oxidativo e histologia pulmonares, comparando-a à posição supina em ambos os modos ventilatórios. Foram instrumentados 75 coelhos com traqueostomia e acessos vasculares. A lesão pulmonar aguda (LPA) foi induzida por lavagem traqueal de salina aquecida (30mL/Kg, 38°C). Os animais foram então aleatorizados em cinco grupos (n=15): 1) GC (Controle): animais sadios em VMC protetora basal; 2) GVMS: animais com LPA em VMC protetora e posição supina; 3) GVMP: animais com LPA em VMC protetora e posição prona; 4) GVAFS: animais com LPA em VOAF e posição supina; 5) GVAFP: animais com LPA em VOAF e posição prona. Após, foram submetidos a quatro horas de VMC protetora (modo pressão regulada-volume controlado, PEEP 10 cmH2O, VC 6mL/kg, Ti 0,5s, FR 40 rpm e FiO2 1) ou VOAF (MAP 15 mmHg, FR 10Hz, amplitude 22 e FiO2 1). O nível de significância foi de 5%. Após a indução, os grupos apresentaram comportamentos semelhantes, com diminuição da relação PaO2/FiO2 e da complacência pulmonar, e aumento do índice de oxigenação (IO) e da pressão média de via aérea (p > 0,05). Ao final do experimento, houve aumento da PaO2/FiO2 nos grupos VOAF comparado aos grupos em VMC (p < 0,05). Houve queda do IO para os grupos em VOAF comparados ao GVMS (p < 0,05), porém o GVMP não diferiu deles (p > 0,05). Não houve diferença estatística quanto à contagem de células polimorfonucleares no lavado broncoalveolar (BAL) nos grupos com LPA. Não houve diferença estatística entre os grupos com lesão para a medida de TNF-alfa no plasma e para sua expressão gênica em tecido pulmonar. Entretanto, a medida de TNF-alfa no lavado broncoalveolar (BAL) e no tecido pulmonar no grupo GVMP foi menor, assemelhando-se ao controle (p > 0,05). Não houve diferença no dano oxidativo avaliado no tecido pulmonar entre os grupos (p > 0,05) e, também, na comparação entre regiões ventral e dorsal dos pulmões. O escore de lesão histológica foi menor nos grupos em VOAF, efeito potencializado no grupo em prona quando comparado aos grupos em VMC (GC = GVAFP < GVMS = GVMP), sem diferença na regionalização pulmonar. Concluimos que, em modelo de LPA por lavagem alveolar com salina aquecida em coelhos: a VOAF melhora a oxigenação quando comparados à VMC; na VMC, a PP atenua a lesão inflamatória avaliada pela medida de TNF-alfa no BAL e tecido pulmonar; os modos ventilatórios e as posições não modificam o grau de estresse oxidativo quando avaliados pelo método de malondialdeído; a VOAF melhora o escore histopatológico de lesão pulmonar, independemente da posição, mas a associação de VOAF e PP atenua a lesão histopatológica quando comparada com a VMC protetora, seja em posição prona ou supina. / Acute Respiratory Distress Syndrome (ARDS) presents with high morbidity and mortality despite advances in the understanding of its pathophysiology and treatment. Ventilatory therapy is based on the intention of injuring less, with high frequency oscillatory ventilation (HFOV) being a protective method option. Prone position (PP) is an adjuvant therapy that enables homogenization of volume tidal (VT) distribution and promotes alveolar recruitment. The aim of this study was to investigate the effects of prone position associated with HFOV and protective conventional mechanical ventilation (CMV) on oxygenation and lung inflammation, oxidative damage and histology, comparing it with the supine position in both ventilatory modes. Seventy five rabbits were submitted to tracheostomy and vascular accesses. ALI was induced by tracheal infusion of heated saline (30mL/kg, 38° C). The subjects were then ramdomized in five groups (n=15): 1) CG (Control): healthy animals in basal protective CMV; 2) MVSG: animals with ALI in protective CMV and supine position; 3) MVPG animals with ALI in protective CMV and prone position; 4) HFSG: animals with ALI in HFOV and supine position; 5) HFPG: animals with ALI in HFOV and prone position. After that, they were submitted to four hours of protective VMC (PRV mode, PEEP 10 cmH2O, VC 6ml/kg, Ti 0,5s, FR=40 rpm and FiO2 1) or HFOV (MAP 15 mmHg, FR 10 Hz, amplitude 22 and FiO2 1). The level of significance was 5%. After induction, the groups presented similar behaviors, with a decrease in the PaO2/FiO2 ratio and lung compliance, and an increase in oxygenation index (OI) and mean airway pressure (p > 0.05). At the end of experimental time, PaO2/FiO2 increased in the HFOV groups compared to the CMV groups (p < 0.05). There was a decrease in OI for HFOV groups compared to MVSG (p < 0.05), but MVPG did not differ from them (p > 0.05). There was no statistically significant difference in polymorphonuclear cell counts in bronchoalveolar lavage (BAL) in the groups with ALI. There was no difference between ALI groups regarding the TNF-alfa dosage in plasma and its gene expression in lung tissue. However, TNF-alpha measurement in BAL and in lung tissue was smaller, resembling control (p > 0.05). There was no difference in the oxidative damage assessed in the lung tissue between the groups (p > 0.05), nor between the lung regions. The histological damage score was lower in the HFOV groups, potentiated effect in the prone group when compared to the CMV groups (CG = HFPG < MVSG = MVPG), no difference in pulmonary regionalization. We conclude that, in the model of ALI induced by alveolar lavage with heated saline in rabbits: HFOV improves oxygenation if compared to CMV; PP in CMV attenuates lung inflammation, evaluated by TNF-alfa dosage in BAL and in lung tissue; ventilatory modes and positions don’t modify the oxidative stress whan evaluated by malondialdehyde method; HFOV improves histopathological lung lesion score, regardless of position, but HFOV and prone position association attenuates histopathological injury compared to protective CMV, either in the prone or supine positions. / FAPESP: 2010/06242-8

A isquemia/reperfusão intestinal (I/R-i) se associa ao desenvolvimento de inflamação pulmonar aguda, que pode ser modulada por mediadores inflamatórios presentes na linfa. Avaliamos os efeitos da I/R-i sob a inflamação pulmonar e a participação do sistema linfático. Wistar machos foram submetidos a 45 min de isquemia intestinal e 24, 72 ou 120 h de reperfusão. Outro grupo teve o ducto linfático bloqueado antes da isquemia. Os resultados revelaram maior inflamação pulmonar nos animais reperfundidos por 120 h em relação aos outros períodos de reperfusão estudados. Os animais apresentaram aumento de MPO e permeabilidade. Aumento de VEGF e de IL-1<font face=\"Symbol\">&#946 e diminuição de IL-10 no explante pulmonar. Diminuição de vWf e aumento de integrina <font face=\"Symbol\">&#9461, PECAM-1 e colágeno I e IV no endotélio pulmonar. Os dados indicam que mecanismos temporais modulam a resposta inflamatória decorrente da I/R-i. Mediadores na linfa e na circulação participam do desencadeamento/manutenção da inflamação pulmonar alterando a integridade do endotélio e ativando o pulmão que libera mediadores adicionais. / Intestinal ischemia/reperfusion (i-I/R) is associated with the development of acute lung inflammation, which can be modulated by inflammatory mediators present in the lymph. We evaluated the effects of i-I/R in lung inflammation and the involvement of the lymphatic system. Wistar rats were subjected to 45 min of intestinal ischemia and 24, 72 or 120 h of reperfusion. Another group had the lymphatic duct blocked before ischemia. The results revealed greater lung inflammation in animals reperfused for 120 h in comparison to other periods studied. These animals showed increased MPO and permeability. Increased VEGF and IL-1<font face=\"Symbol\">&#946 and decreased IL-10 in lung explants. Decreased vWf and increased <font face=\"Symbol\">&#9461 integrin, PECAM-1 and collagen I and IV in the pulmonary endothelium. These data indicate that temporal mechanisms modulate the inflammatory response due to i-I/R. Mediators in the lymph and circulation participate in the initiation / maintenance of lung inflammation by altering the integrity of the endothelium and activating the lung which release additional mediators.

Le syndrome de détresse respiratoire aiguë (SDRA) est une forme sévère de défaillance respiratoire qui se caractérise par la présence de dommages alvéolaires, d’un oedème pulmonaire et d’une réponse inflammatoire exacerbée. C’est une condition pour laquelle il n’existe à ce jour aucun traitement pharmacologique efficace. Lors des dernières années, des antagonistes des récepteurs de l’IL-1 (Kineret) et de l’IL-6 (tocilizumab) ont fait preuve d’une efficacité modérée pour le traitement du SDRA causé par la COVID-19. Cependant, leur potentiel thérapeutique en SDRA clinique non causé par la COVID reste à démontrer et les résultats obtenus dans les modèles animaux sont mitigés. Nous avons émis l’hypothèse que le tocilizumab et le Kineret pourraient améliorer la résolution des différents paramètres du SDRA non causé par la COVID-19. Nous avons aussi posé l’hypothèse que des peptides, antagonistes des récepteurs de l’IL-1 (rytvela) ou de l’IL- 6 (HSJ633) et permettant de préserver certaines voies aux propriétés cytoprotectrices en aval de ces récepteurs, pourraient potentiellement être plus efficaces que le Kineret et le tocilizumab pour le traitement des paramètres du SDRA. L’objectif de ma maîtrise était donc de tester ces deux hypothèses dans un modèle murin d’atteinte pulmonaire aiguë (ALI) induite par la bléomycine, qui mime pendant sa phase aiguë les principaux paramètres du SDRA. Mes travaux montrent qu’aucun des quatre antagonistes n’a permis d’améliorer significativement les paramètres observés à jour 7 post-bléomycine (état général, dommages alvéolaires, oedème et inflammation pulmonaire). Ainsi, mes données suggèrent que dans notre modèle d’ALI induit par la bléomycine, la réponse inflammatoire induite via le IL-1R ou le IL-6R ne semble pas constituer un des mécanismes principaux engendrant les différentes atteintes, puisqu’elles ne sont pas prévenues par les antagonistes de ces récepteurs. En plus de contribuer à mieux comprendre ce modèle animal, mes résultats permettent de mettre en lumière que la réparation des dommages ainsi que la résorption secondaire de l’oedème sont cruciales pour la résolution du SDRA et que de viser seulement la voie inflammatoire est insuffisant. / Acute respiratory distress syndrome (ARDS) is a form of severe lung failure characterized by the presence of a pulmonary edema, an inflammatory response, and alveolar damage. There is currently no effective pharmacological treatment for ARDS. In recent years, IL-1 and IL-6 receptor antagonists Kinerert and tocilizumab, respectively, have shown some efficacy as a treatment of ARDS caused by COVID-19. However, their therapeutic potential in non-COVID ARDS remains to be proven and the results obtained in animal models are conflicting. We thus tested the hypothesis that tocilizumab and Kineret could improve the resolution of key parameters of non-COVID ARDS. We also hypothesized that two peptides, rytvela and HSJ633, IL-1 and IL-6 receptor antagonists, respectively, which preserve some of the cytoprotective downstream pathways, could potentially be more effective than Kineret and tocilizumab in treating the various parameters of ARDS. The goal of my master thesis was therefore to test these two hypotheses in a mouse model of acute lung injury (ALI) induced by bleomycin instillation, which, during its acute phase, mimics the main parameters of ARDS. My work has shown that none of the antagonists were able to significantly improve the parameters observed on day 7 post-bleomycin (general condition of the mice, alveolar damages, pulmonary edema and inflammation). Thus, my data suggest that in our bleomycin-induced ALI model, the inflammatory response triggered via IL-1R or IL-6R does not appear to be the principal mechanism generating the main damaging outcome, since they are not prevented by the antagonists of these receptors. In addition to contributing to a better understanding of this animal model of ALI, my research has highlighted the fact that targeting inflammation alone is insufficient and that repairing alveolar damages, and secondary resorbing lung edema, are cornerstones for the resolution of ARDS.

Avant propos L'objectif de cette thèse est d'explorer l'inflammation et l'infection au niveau du transcriptome, à l'aide de la technologie des puces à ADN. Pour cela, nous avons dans un premier temps travaillé sur des données publiques. Nous avons construit une base de données de signatures transcriptionnelles annotées, et développé un logiciel modulaire d'analyse. Ce logiciel permet d'explorer aisément les données publiques en effectuant des recherches par nom de gène ou par mots-clés. Nous avons ensuite exploré la modulation temporelle de l'expression des gènes du parenchyme pulmonaire dans un modèle murin d'inflammation aiguë par injection d'acide oléique. Dans un second modèle murin d'infection par Coxiella burnetii, nous avons analysé le rôle du sexe dans la modulation de la réponse transcriptionnelle hépatique, et identifié des voies métaboliques impliquées dans le contrôle de l'infection. Dans un troisième modèle in-vitro d'infection par différentes souches du virus de la grippe, nous avons identifié une signature transcriptionnelle commune de réponse à l'infection. Par une approche bio-informatique originale, cette signature a conduit à l'identification de nouveaux anti-viraux à large spectre, dont l'efficacité a été démontrée in-vitro sur les souches utilisées pour l'analyse, et sur la souche H1N1, responsable de la dernière pandémie grippale. Enfin, nous avons analysé les modulations du transcriptome lors de pneumonies associées à la ventilation mécanique compliquant l'évolution de sujets traumatisés graves admis en réanimation.

Les complications respiratoires restent la première cause des complications postopératoiresen chirurgie thoracique oncologique. Le développement des ces complications sont le plussouvent de nature infectieuse. Leur fréquence reste élevée (30 %) et représente la premièrecause de mortalité hospitalière. Des données récentes suggèrent que ces complicationsrespiratoires soient liées à une colonisation périopératoire des voies aériennes. Plusieurstravaux fondés sur l’analyse phénotypique de mise en culture traditionnelle démontrentl’existence d’une colonisation bronchique proximale chez près de 40 % des malades.Néanmoins, les liens entre colonisation et complications respiratoires restent controversés.Une des principales limites demeure les méthodes de cultures employées qui ne permettentl’identification que d’une faible partie (< 1%) des espèces microbiologiques potentiellementexistantes dans la biosphère. Nous avons formulé l’hypothèse que des techniques debiologie moléculaire d’amplification universelle des ADN présents dans les échantillonssuivies du clonage des produits de PCR et du séquençage de ces clones, appliquées à deséchantillons obtenus des bronches distales et de biopsies pulmonaires, permettraientl’identification de pathogènes bactériens, viraux ou émergents. Nos résultats suggèrent quel’identification précise et exhaustive de ces colonisations ne peut être réalisée que par uneapproche moléculaire moderne, innovante et systématique. Cette approche permetd’envisager, d’une part, un lien plus précis entre colonisation et complications respiratoires etd’autre part, l’identification de pathogènes difficilement cultivables ou émergents. / Postoperative respiratory complications remain the most frequent and seriouscomplications, as well as being the primary cause of hospital death after thoracic oncologicsurgery. Their incidence is relatively high and concern near 30 % of patients submitted forsurgery. These complications are notoriously infectious and airways colonizations (AWC)have been suggested to be an essential first step in the pathogenesis of this respiratorymorbidity. Previous studies have documented that AWC are presents in near 40 % of cases.However, correlation between AWC and respiratory complications remains controversial.One of the limits is the traditional phenotypic methods of cultures that precludes for definitiveconclusions when considering that majority of microbiological species required modern andinnovating techniques of culture to be identified. Recent data have demonstrated that 99% oforganisms seen microscopically are not cultivated by routine techniques and requiredmolecular techniques to be identified. We have postulated that instead of culture test,molecular detection (DNA genes amplification and sequencing of the bacterial 16S ribosomalRNA) applied to distal bronchial samples or to lung biopsies, should allow identifyingbacteria, virus or emerging pathogens. Our results suggest that molecular cultureindependenttechniques applied in the context of AWC will provide in the future a greatopportunity to precise correlation between colonization and respiratory complications and tothe other hand, to discover new and/or emerging pathogens that are currently unknown.

Dans mon projet de doctorat, j’ai étudié des fonctions primordiales de l’épithélium respiratoire telles que la régulation du transport ionique, la clairance liquidienne et la réparation épithéliale. J’ai particulièrement mis l’emphase sur le rôle des canaux potassiques qui interviennent dans ces trois fonctions de l’épithélium respiratoire. J’ai tout d’abord prouvé que la modulation des canaux potassiques régulait l’activité du promoteur de αENaC, en partie via la voie de signalisation ERK1/2, dans des cellules alvéolaires. Cette régulation entraîne une variation de l’expression génique et protéique du canal ENaC. Physiologiquement, il en résulte une augmentation du phénomène de clairance liquidienne suite à l’activation des canaux K+, tandis que l’inhibition de ces canaux la diminue sévèrement. J’ai aussi pu démontrer que l’absence de canal KvLQT1 entraînait une diminution du courant (ENaC) sensible à l’amiloride, dans les cellules de trachée en culture primaire, isolées de souris KO pour kcnq1. Dans la seconde partie de mon étude, j’ai évalué l’impact de l’hyperglycémie sur la capacité de transport ionique et de réparation de cellules épithéliales bronchiques saines ou Fibrose Kystique. Mes résultats montrent que l’hyperglycémie diminue le transport transépithélial de chlore et le transport basolatéral de potassium. Des études préalables du laboratoire ayant montré que les canaux K+ et Cl- contrôlent les processus de réparation, j’ai donc évalué si ceux-ci étaient modifiés par l’hyperglycémie. Et en effet, l’hyperglycémie ralentit la vitesse de réparation des cellules issues des voies aériennes (CFBE-wt et CFBE-ΔF508). J’ai donc démontré que le transport de potassium intervenait dans des fonctions clés de l’épithélium respiratoire, comme dans la régulation génique de canaux ioniques, le contrôle de la clairance liquidienne alvéolaire, et que l’hyperglycémie diminuait le transport ionique (K+ et Cl-) et la réparation épithéliale. / During my Ph.D. training, I studied 3 important functions of respiratory epithelium : regulation of ion transport, liquid clearance and epithelial repair. I focused on potassium channels, because they control these three respiratory epithelial functions. First, I proved that αENaC promoter activity was regulated following K+ channel modulation, in alveolar cells. This regulation of αENaC promoter which might be through a modification of ERK1/2 phosphorylation, was followed by ENaC mRNA and protein expression regulation. I then showed that activation of KvLQT1 and KATP channels increased alveolar liquid clearance, whereas inhibition of these K+ channels decreased the alveolar clearance. I showed that the absence of KvLQT1 channel inhibited the amiloride-sensitive current (ENaC), in tracheal epithelial cells isolated from KvLQT1-KO mice. In the second part of my Ph.D. project, I studied the impact of hyperglycemia on Cystic Fibrosis (CF) and non-CF epithelial cells. I first observed that K+ and Cl- currents were reduced by hyperglycemia. Because we have previously shown that wound-healing process was dependant on K+ and Cl- channels, I then evaluated the impact of hyperglycemia on wound-healing. As expected, hyperglycemia slowed the repair rate of non-CF (CFBE-wt) and CF (CFBE-ΔF508) cell monolayers.

Le syndrome de détresse respiratoire aiguë (SDRA) est caractérisé par des lésions alvéolairesdiffuses qui résultent d’une lésion de la membrane alvéolo-capillaire entrainant entre autresune réaction inflammatoire intense et une perte massive et hétérogène du volume pulmonaireaéré. La tomographie par émission de positons (TEP) et la tomographie par impédanceélectrique (TIE) sont deux techniques d’imagerie fonctionnelle permettant l’étude noninvasive, quantitative et régionale du poumon.Ce travail présente le résultat d’études expérimentales conduites dans le SDRA. Tout d’abord,nous avons comparé positivement la TIE à la TEP pour la mesure de la ventilation pulmonaireet du volume aéré. Nous avons ensuite décrit et validé une technique robuste d’obtention duvolume aéré et de la ventilation spécifique en TEP sans prélèvement invasif. Enfin, nousavons étudié en TEP l’influence de la pression expiratoire positive (PEP) et du décubitusventral (DV) sur la répartition de la ventilation, de la perfusion et de l’inflammationpulmonaires. Les poumons agressés par l’acide chlorhydrique inhalé ont une inflammationsignificativement plus élevée que le groupe contrôle. Aucune différence significatived’inflammation n’a été trouvée entre les groupes expérimentaux malgré des modificationsimportantes de la répartition de la ventilation et de la perfusion régionales lors de la mise enDV. Ces études donc ont permis le développement d’un modèle porcin stable d’agressionpulmonaire aiguë et la validation de techniques d’imagerie permettant l’étude non invasive deparamètres physiologiques importants pouvant aider au réglage de la ventilation mécanique aucours du SDRA. / Acute respiratory distress syndrome (ARDS) is characterized by diffuse alveolar damage andresulting from an increased permeability of the alveolar-capillary membrane. Of notice, thereis an intense lung inflammation. Positron emission tomography (PET) and electricalimpedance tomography (EIT) allow noninvasive assessment of pulmonary ventilation,perfusion and inflammation. We use these techniques to decipher the impairments ofventilation and inflammation throughout the lungs in an experimental model of acute lunginjury by hydrochloric acid inhalation in pigs.In a first study, we compared EIT to PET in quantifying pulmonary aerated volume andventilation, using PET as a gold standard. We found that lung ventilation and volume wereaccurately measured with EIT over a wide range of lung volume and minute ventilation. Wehave then described and validated a new model to obtain lung aerated volume and ventilationwith PET, without the requirement of gas sampling in the respiratory circuit. Finally, weconducted a controlled study with PET to evaluate the effects of positive end-expiratorypressure and body position on regional lung inflammation, ventilation and perfusion.Inflammation was significantly higher in injured groups than in control. However, there wasno significant change in inflammation across ALI groups despite significant differencebetween ventilation and perfusion repartition.We have developed in this work a stable experimental model of acute lung injury andvalidated noninvasive imaging tools allowing studying of important physiologic parametersthat could help setting up mechanical ventilation.