Inhaled drug delivery is currently the gold standard for treating many respiratory diseases. However, improved treatments are needed for lung diseases like Cystic Fibrosis (CF) and Acute Respiratory Distress Syndrome (ARDS), where mucus or fluid build-up in the lung limits ventilation and, thus, delivery of inhaled drugs. Delivery is most needed in the diseased or damaged regions of the lung, but if an area is not ventilated, inhaled drug will simply not reach it. To overcome this, this research proposes delivering drugs to the lungs within a perfluorocarbon (PFC) liquid. The lungs will be filled with a reverse emulsion containing a disperse phase of aqueous drugs within the bulk PFC and then ventilated. The PFC functions as both a respiratory medium, providing gas exchange, and as a delivery vehicle, providing a more uniform deposition of drugs. After treatment, the highly volatile PFCs are exhaled, returning the patient to normal respiration. This technique improves upon current therapies as follows. First, drugs are delivered directly to where they are needed, yielding higher concentrations in the lung and lower systemic concentrations. Second, PFCs are ideal for washing out lung exudate and mucus. The low surface tension and high density of PFC allows it to easily penetrate plugged or collapsed alveoli, detach infected mucus from the airway walls, and force these fluids to the top of the lungs where they can then be removed via suction. Mucus and exudate removal should allow drugs to penetrate previously plugged airways during emulsion delivery and subsequent treatment with inhaled therapies. Thus, drug delivery via emulsion would be used as a pre-treatment to enhance inhaled or systemic drug therapy. Third, PFC’s anti-inflammatory properties help return to normal lung function. This research examines two applications of this technology: delivery of antibiotics to combat respiratory infections (antibacterial perfluorocarbon ventilation, APV) or delivery of growth factors to enhance alveolar repair (perfluorocarbon emulsions for alveolar repair, PEAR). This work represents an in-depth analysis of the emulsions used during APV and PEAR. Initial efforts evaluated emulsion efficacy under in vitro setting that better simulated lung in vivo antibiotic delivery. The subsequent studies utilized an in vivo rat model of bacterial respiratory infection to validate the effects of emulsion on pharmacokinetics and to assess APVs potential treatment benefits. Lastly, in vitro methods of cellular response assessed the utility of delivering growth factors in PEAR. Significant advancements were made in optimizing the emulsion as a viable means of pulmonary drug delivery. Final efforts resulted in a promising emulsion formulation that overcame the quick transport of tobramycin away from the lung and successfully reduced pulmonary bacterial load in vivo. In vitro applications of PEAR showed the emulsions posed a significant barrier to the availability and, thus, the biological effect of lysophosphatidic acid growth factors. Further in vivo work is required to improve APV’s efficacy over conventional treatments and to determine PEAR’s feasibility and efficacy in promoting lung repair.

The purpose of this thesis was to extend our understanding of the assessment and interpretation of aerobic exercise function of paediatric patients with cystic fibrosis (CF). The first investigation sought to establish (1) the validity of traditional criteria to verify maximal oxygen (V ̇O2max) during a maximal cardiopulmonary exercise test (CPET); and (2) the utility of supramaximal verification (Smax) to confirm V ̇O2max. Traditional criteria significantly underreported V ̇O2max, whilst Smax was shown to provide a valid measurement in this patient group. The reproducibility of this CPET protocol, over the short- (48 h) and medium- (4-6 weeks) term, was then established in study two. V ̇O2max was repeatedly determined with no learning effect over 48 h (typical error (TE): ∆150 mL; ∆9.3%) and 4-6 weeks (TE: ∆160 mL; ∆13.3%). Supplementary maximal and submaximal CPET parameters should be incorporated for a comprehensive evaluation of a patient, however they are characterised by greater variability over time. The influence of mild-to-moderate CF on aerobic exercise function and the matching of muscle O2 delivery-to-O2 utilisation during ramp incremental exercise to exhaustion were then examined in study three. Aerobic function was impaired in CF, indicated by very likely reduced fat-free mass normalised V ̇O2max (mean difference, ±90% CI: -7.9 mL∙kg-1∙min-1, ±6.1), very likely lower V ̇O2 gain (-1.44 mL∙min-1∙W-1, ±1.12) and a likely slower V ̇O2 mean response time (MRT) (11 s, ±13). Arterial oxygen saturation was lower in CF, supporting the notion that centrally mediated O2 delivery may be impaired during ramp incremental exercise. Although a faster rate of fractional O2 extraction would be expected in the face of reduced O2 delivery, this was not observed, suggesting additional impairment in O2 extraction and utilisation at the periphery in CF. The fourth study then demonstrated the clinical utility of CPET to assess the response to 12 weeks treatment with Ivacaftor, using a case-based design. Whilst one patient with relatively mild disease demonstrated no meaningful change in V ̇O2max, the second demonstrated a 30% improvement in V ̇O2max, due to increased O2 delivery and extraction. Furthermore, changes in aerobic function were detected earlier than spirometric indices of pulmonary function. This study demonstrated that CPET represents an important and comprehensive clinical assessment tool and its use as an outcome measure in the functional assessment of patients is encouraged. Study five investigated the V ̇O2 kinetics in this patient group. During moderate intensity cycling, the phase II V ̇O2 time constant (τ) (p = 0.84, effect size (ES) = 0.11) and overall MRT (p = 0.52, ES=0.33) were not slower in CF. However, both were slowed during very heavy intensity cycling (p = 0.02, ES = 1.28 and p = 0.01, ES = 1.40, respectively) in CF. Cardiac output and muscle deoxygenation dynamics were unaltered in CF, however, the arterial-venous O2 content difference (C(a-v ̅)O2) was reduced (p=0.03) during VH and ∆C(a-v ̅)O2 correlated with the phase II τ (r= -0.85; p=0.02) and MRT (r = -0.79; p=0.03) in CF. This study showed that impaired oxidative muscle metabolism in this group is exercise intensity-dependent and mechanistically linked to an intrinsic intramuscular impairment, which limits O2 extraction and utilisation. In conclusion, this thesis has provided guidelines for a valid and reproducible CPET protocol for children and adolescents with mild-to-moderate CF, demonstrated the utility of CPET as clinical outcome measure and furthered our understanding of the factors responsible for impaired aerobic exercise function in this patient group.

La mucoviscidose (CF pour Cystic Fibrosis) est la conséquence de la mutation du gène codant pour le canal chlorure CFTR. Une des stratégies thérapeutiques proposées pour compenser la déficience de CFTR serait de stimuler une voie alternative à CFTR de sécrétion d'ions chlorures. En 2008, le canal ANO1 a été identifié comme CaCC (canal chlorure activé par le calcium) et alors proposé comme cible thérapeutique dans le contexte de la mucoviscidose. Des travaux précédents de notre laboratoire ont montré que l'activité et l'expression d'ANO1 étaient diminuées en contexte CF par rapport au contexte non-CF. Les mécanismes de régulation d'ANO1 n'étant pas connus, les objectifs principaux de ce travail étaient d'étudier la régulation d'ANO1 par les microARN. Nous avons donc, dans un premier temps, identifié un microARN, miR-9 qui est surexprimé dans les cellules CF et qui régule directement ANO1. Nous avons montré que la régulation d’ANO1 par miR-9 entraîne une diminution d’expression et d’activité d’ANO1 ainsi que de la vitesse de migration des cellules. Dans le contexte de la mucoviscidose, il nous a semblé plus intéressant de pouvoir augmenter l’expression d’ANO1 dans le but d’augmenter les efflux chlorures, nous avons donc fait synthétiser une molécule qui empêche la fixation de miR-9 à ANO1, un TSB (Target Site Blocker) ANO1. Nous avons alors démontré que l’utilisation de notre TSB ANO1 permettait d’augmenter l’expression d’ANO1, son activité chlorure et la migration cellulaire dans différents modèles in vitro et in vivo. L’ensemble de ses résultats suggère que notre TSB ANO1 pourrait être une cible thérapeutique intéressante chez les patients atteints de mucoviscidose. / Cystic Fibrosis (CF) is the consequence of the mutation of the chloride channel CFTR. One of the therapeutic strategy proposed in CF to compensate the CFTR deficiency is to stimulate others chlorides channels. In 2008, the channel ANO1 was identified as CaCC (calcium-activated chloride channel) and then proposed as a therapeutic target in CF. Previous works from our lab have shown that ANO1 activity and expression are reduced in the CF context compared to non CF. Mechanisms of ANO1 regulation being unknown, the objectives of this work were to study ANO1 regulation by microRNA. First, we identified a microRNA, miR-9, which is overexpressed in CF cells and directly regulates ANO1. We have then shown that ANO1 regulation by miR-9 induces decreases of ANO1 expression and activity, and migration rate of cells. In the context of CF, it seems more interesting to increase ANO1 expression in order to increase the chloride efflux, we thus designed a target site blocker (TSB ANO1) which prevents miR-9 fixation on ANO1. In different models in vitro and in vivo, we demonstrated that our TSB ANO1 increases ANO1 expression, ANO1 activity and migration rate of cells. These results suggest that ANO1 TSB could be considered as an interesting therapeutic target in CF.

La mucoviscidose est une maladie génétique due à des mutations du gène CFTR (Cystic Fibrosis Transmembrane conductance Regulator), conduisant à un défaut d'adressage de la protéine CFTR à la membrane apicale des cellules épithéliales, ou à un déficit de sa fonction de canal à ions chlorure. Ce travail a consisté à étudier les vésicules extracellulaires (EV), microvésicules (MV) et exosomes (Exo), comme vecteurs de la protéine CFTR et de son ARN messager. La preuve de concept du transfert de matériel biologique d'intérêt par l'intermédiaire d'EV, d'abord apportée sur un modèle de cellules animales (CHO), a été validée en cellules humaines. Les EV ont été isolées à partir de surnageant de Calu-3, cellules exprimant la protéine CFTR de manière endogène, et de A549 transduites par le vecteur adenoviral Ad5-GFP-CFTR, surexprimant la protéine de fusion GFP-CFTR. Les cellules cibles choisies, A549 et CF15, étaient déficientes en CFTR. Le transfert s'est révélé plus efficace en système homologue (A549/A549) qu'en système hétérologue (A549/CF15). Par ailleurs, l'utilisation d'inhibiteurs métaboliques suggère que les EV ne suivent pas une voie d'internalisation cellulaire unique, mais que plusieurs mécanismes sont mis en jeu, dont l'endocytose clathrine dépendante et la macropinocytose. Les deux types d'EV sont capables de rétablir la fonction canal associée au CFTR dans les cellules CF15 de façon dose-dépendante, mais avec un effet de seuil minimum. L'activité CFTR reste stable pendant 3 jours, et à un niveau encore détectable après 5 jours. Notre travail démontre l'intérêt potentiel des MV et Exo comme vecteurs de biothérapie de pathologies génétiques / Cystic fibrosis is a genetic disease in which its prognosis depends on the lung damage. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), resulting in a dysfunctional CFTR protein normally located at the plasma membrane of epithelial cells. This thesis is a study of a novel therapeutic approach to use extracellular vesicles (EVs), microvesicles and exosomes, as transfer vectors for CFTR mRNA and protein to target cells. The proof of concept for the transfer of CFTR mRNA and protein was first done in the CHO hamster model. To validate this concept on human cells, we used human bronchial Calu-3 cells, which express the endogenous CFTR protein, and A549 lung epithelial cells transduced by the adenoviral vector Ad5-GFP-CFTR to overexpress the fusion exogenous protein GFP-CFTR. We show that EVs produced by these cells could transfer a new functionality to CF15 target cells carrying the CFTRdeltaF508 mutation and the transfer seems to be more efficient in a homologous cell system versus a heterologous system. Interestingly, the exosomes seem to be more efficient in CFTR transfer than the microvesicles. A study of the mechanism of EVs cellular uptake show that it is temperature dependent and that endocytosis and macropinocytosis are implicated. Collectively, this study demonstrates the potential application of EVs for CFTR transfer and functional correction of the genetic defect in human CF cells

Four studies investigating the assessment of glycaemic control in cystic fibrosis are presented within this thesis. The first was a validation study of continual glucose monitoring (CGM) in cystic fibrosis (CF). 50 stable adults with CF underwent home CGM for 3 days, during which time they attended the CF centre for OGTT. Gold standard fasting (0 hour) plasma glucose and 2 hour plasma glucose values during OGTT were compared with concurrent CGM sensor glucose values using a 'limits of agreement' analysis. CGM was found to be valid in adults with CF, with its accuracy being consistent with that published in non-CF populations. The next investigation compared OGTT with CGM with several objectives: to determine whether OGTT is a relevant and adequate measure of glycaemia in CF, find out whether CGM could offer a superior alternative to OGTT and explore whether OGTT and CGM results are associated with prior change in lung function and weight in adults with CF. Data from the first study was used to show that the OGTT can only identify abnormal glycaemic control in CF at a late stage, and that CGM is a more relevant reflection of everyday glycaemia in CF. No correlation was found between prior change in lung function and nutritional status in CF and glycaemia measured by OGTT or CGM. The subsequent study investigated whether CGM could identify early abnormal glycaemic control in CF. This involved ten non-CF healthy controls undergoing the same study protocol as the 50 stable adults with CF, to determine 'normal' glycaemic control parameters. Of 25 CF patients with normal glucose tolerance by OGTT, 19 (76%) had significantly higher mean and/or variability of CGM levels than healthy controls. This lead to changes in their management, including 2 subjects being commenced on insulin therapy. The final investigation was a questionnaire study, asking the 50 CF patients to provide information on their experience of undergoing CGM. 58% of patients responded, with replies indicating that they found CGM broadly acceptable, interfering little in their lives and that their experiences were generally positive. This insight into patients' experiences of CGM can be used to guide future clinical and research roles for this tool. These studies have provided novel data regarding the assessment of glycaemic in CF. Information captured by CGM has greater relevance to CF patients' daily lives than OGTT. CGM can identify early problems with glycaemic control leading to changes in management that may not be detected by conventional measures. CGM offers potential in further clinical application and research to improve the lives and outcomes for adults with CF.

La phospholipase A2 sécrétée de type IIA (sPLA2-IIA) est une protéine possédant une activité antibactérienne très élevée envers les bactéries à Gram positif. La mucoviscidose (MV) est une maladie génétique résultant de la mutation du gène Cftr. L'altération de la fonction du CFTR dans les poumons favorise la colonisation par des pathogènes bactériens, dont les plus retrouvés, S. aureus et P. aeruginosa, colonisent les voies aériennes de manière séquentielle: S. aureus est principalement retrouvé chez les jeunes patients et P. aeruginosa chez les adultes. Les mécanismes impliqués dans ce changement de prévalence restent cependant mal connus. Dans ce travail, nous démontrons que l'expression de la sPLA2-IIA est plus importante dans les poumons de patients MV que dans les poumons de patients non MV et augmente avec l'âge des patients. La sPLA2-IIA présente dans les expectorations de patients MV est capable de tuer spécifiquement S. aureus. L'utilisation de modèles animaux nous a permis de démontrer sa spécificité d'action contre S. aureus et l'induction de son expression par P. aeruginosa contribuant à l'élimination de S. aureus des voies respiratoires. Enfin, nous avons identifié les cellules épithéliales comme une source majeure de sPLA2-IIA chez les patients MV. Dans ces cellules, P. aeruginosa induit l'expression de la sPLA2-IIA par un mécanisme dépendant de l'injection de la toxine ExoS et du facteur de transcription KLF2. L'ensemble de ces résultats indique i) que la sPLA2-IIA induite par P. aeruginosa, participe à l'élimination de S. aureus dans les voies aériennes des patients MV et ii) qu'une bactérie élimine une autre bactérie en utilisant la défense de l'hôte. / The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host defense protein endowed with antibacterial activity, especially against Gram positive bacteria. Cystic fibrosis (CF) is a genetic disease due to mutations of Cftr gene. In the lungs, CFTR mutation favored bacterial colonization by bacterial pathogens, of which S. aureus and P. aeruginosa are the most isolated. These two bacterial species sequentially colonized airways of CF patients: S. aureus is predominant in young patients and P. aeruginosa in adults. But the mechanisms involved in this switch of bacterial prevalence are still unknown. In this work we showed that sPLA2-IIA levels were increased in lungs of CF patients compared to lungs of non-CF patients and that sPLA2-IIA levels increased with age of patients. sPLA2-IIA recovered from CF patients expectorations was active and killed specifically S. aureus. Using animal models of lung infection, we demonstrated the selectivity of sPLA2-IIA against S. aureus and that P. aeruginosa induced sPLA2-IIA expression, the latter contributed to S . aureus elimination from the airways. Finally, we identified epithelial cells as a major source of sPLA2-IIA in CF airways. In these cells, P. aeruginosa induced sPLA2-IIA expression through injection of ExoS toxin and activation of KLF2 transcription factor. Taken together, these results indicate that i) P. aeruginosa-induced sPLA2-IIA expression in CF airways participated to S. aureus elimination and ii) a bacteria eliminate another bacteria by manipulating host innate immunity.

Four methods were investigated to determine their suitability for use in a CF screening programme for the province of British Columbia. A fecal trypsin method which measured trypsin activity by incubating dry stool samples on filter paper cards with the substrate p-tosyl-arginine methyl ester (TAME) and a pH sensitive dye was shown to be non-specific and therefore unsatisfactory. An attempt to combine a fecal albumin screen with a more specific quantitative immunodiffusion technique for albumin and alpha-1 antitrypsin was unsuccessful. A meconium albumin assay using the Boehringer-Mannheim Corporation (BMC) test-strip and a more specific fecal trypsin assay which uses the substrate benzoyl-arginine-p-nitroanilide (BAPNA) were incorporated into two pilot projects at Children's Hospital in Vancouver. The BMC test-strip was simple to use, reliable and inexpensive. Of 8,891 infants tested, 3 positives were diagnosed as suffering from cystic fibrosis and 1 CF patient tested negative. False positives were obtained on 1.3% of infants. The incidence of CF as determined by this screen was 1 in 2000. The meconium albumin screen was satisfactory as a local pilot project but the disadvantages of testing the unstable meconium specimens make the screen unsuitable for a province-wide application. The BAPNA fecal trypsin method devised by Crossley was used to test 4085 dry stool specimens collected in the hospital and at home. Out of a total number of 190 positive results, none was diagnosed as having CF, giving a false positive rate of 5.0% for the hospital collected specimens and 3.4% for the specimens collected at home. The false positive rate in the hospital collected specimens was due mostly to the large proportion of young infants (under 3 days). The false positive rate of the home collected specimens appeared to be due mostly to the thinner spread of stool sample on the card. Because the quantity of stool sample per test was significantly lower in the home than the hospital collected specimens a new cut-off point for the home collected specimens was considered. Its application/ however did not lower the false positive rate sufficiently. As a result, the high incidence of false positives and the difficulties encountered as a result of this incidence also makes the fecal trypsin screen unsuitable for the province of B.C. Difficulties encountered during the follow-up of positive results obtained in the two pilot projects are discussed and recommendations are made regarding the efficient and adequate implementation of a follow-up system. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) provoquent des infections sévères chez les personnes atteintes de mucoviscidose. L'infection peut varier d'une forme asymptomatique à une forme plus aiguë pouvant entraîner une pneumonie nécrosante et une septicémie, connue sous le nom de syndrome cepacia. Afin d'étudier les infections causées par le Bcc, nous avons développé un nouveau modèle in vivo, l'embryon de poisson zèbre. Nous avons montré que B. cenocepacia K56-2 pouvait se répliquer dans les macrophages et causer une infection aiguë mortelle pour les embryons. En revanche, B. stabilis LMG14294 induit une infection persistante chez les embryons. Dans cette étude, nous avons montré que les macrophages jouaient un rôle-clé dans la multiplication de K56-2 et dans l'induction d'une réponse inflammatoire MyD88-dépendante, caractérisée par la surexpression des gènes codant pour Cxcl8 (ou IL-8) et l'IL-1b. En l'absence de macrophages, les bactéries sont incapables de se multiplier durant les premières 24h de l'infection, ce qui donne un avantage pour la survie des embryons. L'absence de MyD88 induit aussi l'augmentation de la survie des embryons infectés par K56-2. Mais de manière paradoxale, les bactéries se multiplient mieux chez les embryons myd88-/- mutants que chez les embryons sauvages. Ceci suggère que ce n'est pas le nombre de bactéries qui est important pour l'infection, mais que c'est la réponse inflammatoire excessive causée par cette infection qui entraîne la mort des embryons. Afin d'avoir une vision globale des changements d'expression des gènes de l'hôte durant l'infection, nous avons effectué une expérience de RNAseq. Comme attendu, l'infection aiguë se caractérise par une importante modulation du transcriptome de l'hôte qui augmente avec le temps. A l'opposé, l'infection persistante n'induit que très peu de changements. La réponse immunitaire innée, et en particulier la voie des TLR, ainsi que l'apoptose sont très fortement activées durant une infection aiguë. Pour sa part, B. stabilis module essentiellement les gènes codant pour le système du complément.Le rôle critique des macrophages lors d'une infection par Bcc chez les poissons zèbre est en accord avec les récentes observations cliniques. Ceci suggère que le stade intracellulaire de B. cenocepacia et la réponse inflammatoire qui s'ensuit peuvent être des cibles pour le développement de nouvelles thérapies permettant de lutter contre cette infection. / Bacteria belonging to the Burkholderia cepacia complex (Bcc) can cause chronic infection with periods of acute exacerbation and sometimes fatal necrotizing pneumonia (“cepacia syndrome”) in individuals with cystic fibrosis (CF), and are associated with poor prognosis. Here, we exploited the exciting possibilities for in vivo non-invasive imaging of Bcc infection in transparent zebrafish embryos, with an innate immune system with remarkable similarity to that of humans, and numerous genetic and genomic tools to study the role of host phagocytes and the innate immune response in the pro-inflammatory character of the infection.We show that macrophages play a critical role in intracellular multiplication of B. cenocepacia K56-2 and induction of a MyD88-dependent fatal inflammatory response, characterised by high levels of cxcl8 and il1b expression. Surprisingly, in sharp contrast to the situation found for infections with other pathogens including Mycobacterium marinum and Staphylococcus aureus, in the absence of macrophages, K56-2 survived but was unable to replicate in the first 24 h, which resulted in a significant pro-survival advantage to the host compared to wild type embryos that died within 2 to 3 days. The Toll-like receptor (TLR) pathway is a major arm of the cell-mediated innate immune response with MyD88 as a key adaptor protein involved in the production of pro-inflammatory cytokines. We found that the absence of MyD88 also provided a pro-survival effect to the embryos after infection with K56-2. Paradoxically, the bacteria replicated better in myd88-/- mutant than wild type embryos, suggesting that it is not bacterial burden per se, but the inflammatory response that kills the embryos. Interestingly, cxcl8 and il1b expression were not significantly induced during the first 7 hours in the myd88-/- mutant while a strong induction was seen in control embryos, suggesting that a Myd88-dependent inflammatory response during early macrophage stages significantly contributes to fatal infection.Next, we performed RNAseq to analyse global changes in host gene expression during acute and persistent infection induced by K56-2 and B. stabilis LMG14294 respectively. Whereas acute infection was characterised by strong modulation of host gene expression increasing over time, persistent infection showed modulation of only a small set of genes. TLR and apoptosis signaling pathways were amongst the strongly activated groups during acute infection, in line with the strong inflammatory character of K56-2. During persistent infection, the major differentially expressed gene set concerned genes encoding complement proteins. The critical role for macrophages in Bcc infection in zebrafish is in agreement with recent clinical observations. We suggest that the intracellular stages of B. cenocepacia and the ensuing inflammatory response are essential targets to explore for the development of new therapies to combat this infection.

La mucoviscidose (Cystic Fibrosis, CF) est caractérisée par une symptomatologie variée, dominée par la gravité de l'atteinte pulmonaire et une réponse inflammatoire inadaptée. Elle est due à des mutations du gène CFTR codant un canal chlorure AMPc-dépendant à la surface des cellules épithéliales. L'inflammation des voies aériennes de type neutrophilique est caractérisée au niveau cellulaire par une surproduction de cytokines pro-inflammatoires telles que l'interleukine-8 (IL-8) et une activation anormale des facteurs de transcription impliqués dans les voies de signalisation de l'inflammation comme NF-κB, AP-1 et PPARγ.Notre projet est d'identifier des agents à effet anti-inflammatoire dans le contexte de la mucoviscidose et d'en décrypter les mécanismes d'action. Nous avons pour cela étudié l'effet de molécules anti-inflammatoires avec une autorisation de mise sur le marché mais jamais testées pour la mucoviscidose et l'effet de COMMD1 une protéine pléïotropique impliquée dans l'inhibition de l'inflammation et identifiée au laboratoire pour interagir avec CFTR.Dans un premier temps nous avons montré l'activité anti-inflammatoire de COMMD1 dans le contexte de la mucoviscidose via son action anti-NF-κB. Dans un second temps nous avons montré que le sulindac et l'amlexanox permettent de moduler des facteurs dérégulés dans des cellules CF. Ces molécules inhibent l'activité de NF-κB et activent PPARγ. Le sulindac permet de diminuer la sécrétion d'IL-8 in vitro sur des cellules épithéliales bronchiques et in vivo sur modèles d'inflammation pulmonaire murin. De plus nous avons montré que le sulindac augmente le transport des ions chlorures via CFTR sur des cellules épithéliales nasales humaines non CF suggérant que le sulindac a un double effet anti-inflammatoire et potentiateur.Ce projet de recherche nous a permis de proposer de nouveaux candidats pour les thérapies anti-inflammatoires dans la mucoviscidose et d'initier l'étude de leur mécanisme d'action. / Cystic fibrosis (CF) is characterized by a varied symptomatology dominated by the lung injury severity and inappropriate inflammatory response. It is caused by mutations in the CFTR gene that encodes a cAMP-dependent chloride channel on the surface of epithelial cells. Airway inflammation with neutrphilic profil is characterised by increased Interleukine-8 (IL-8) secretion with dysregulation of transcription factors implicated in the inflammatory pathway such as NF-κB, AP-1 and PPARγ.Our aim is to evaluate agents with anti-inflammatory effect in CF context and to decrypt their mechanisms of action. We have study the effect of anti-inflammatory FDA approved drugs having a yet undefined effect in CF airway epithelial cells and the effect of COMMD1 a pleiotropic protein involved in the inhibition of inflammation and identified in the laboratory to interact with CFTR.In a first time we have shown the anti-inflammatory activity of COMMD1 mediated by its anti-NF-kB activity in the CF context. In a second time we have shown that sulindac and amlexanox can modulate factors dysgulated in CF cells. Both inhibit NF-κB and activate PPARγ. Sulindac reduces IL-8 secretion in vitro on airway epithelial cells and in vivo in mouse model of lung inflammation. Additionally, sulindac increases CFTR-dependent chloride currents in non-CF primary human nasal epithelial cells suggesting that sulindac has a dual anti-inflammatory and potentiator effect.This research has allowed us to propose good anti-inflammatory agents for CF context and to initiate the study of their molecular targets.

This diploma thesis describes the issue of rare diseases in terms of their essence and the situation in the Czech Republic nowadays. As a disease model was chosen cystic fibrosis. The main goal of this work is to quantify the cost of treatment of cystic fibrosis for three consecutive years and to analyse their structure in the cohorts of patients.