La mucoviscidose est une maladie génétique autosomique causée par une mutation dans le gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Mon travail s’est décomposé en deux parties principales : d’une part j’ai réalisé une revue de la littérature sur l’analyse des génomes bactériens isolés de patients mucoviscidosiques comparativement aux génomes des mêmes espèces isolées dans d’autrescontextes et d’autre part j’ai analysé les génomes de trois espèces bactériennes (Microbacterium yannicii, Chryseobacterium oranimense et Haemophilus parahaemolyticus). L’analyse exhaustive des génomes bactériens issus de patients atteints de mucoviscidose a révélé une extraordinaire évolution de ces génomes en fonction du temps et des traitements reçus par ces patients qui témoigne de la capacité qu’ont ces bactéries à s’adapter à leur écosystème notamment par l’acquisition de nouveaux gènes par transfert latéral de gènes. Ce travail montre l’extraordinaire plasticité des génomes bactériens dans un milieu donné et à ce titre le poumon de patients atteints de mucoviscidose représente un modèle unique pour comprendre l’évolution des génomes bactériens. De plus, notre travail a permis d’identifier leurs mécanismes moléculaires de résistance aux antibiotiques. Les travaux à venir sur l’étude des métagénomes de prélèvements chez ces patients pourrait permettre de répondre à ces questions dans le futur. La découverte de nouvelles espèces et / ou émergentes va nous permettre d’avoir une image plus complète de la mucoviscidose qui pourrait conduire à une meilleure connaissance de la maladie et donc à une meilleure prise en charge thérapeutique. / Cystic fibrosis is an autosomal genetic disorder caused by a mutation in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. Pulmonary infection is the major problem faced by patients with cystic fibrosis. My work is divided into two main parts: first I made a review of the literature on the analysis of bacterial genomes isolated from CF patients compared to the genomes of the same species isolated in autrescontextes and other part I analyzed the genomes of three species of bacteria (Microbacterium yannicii, Chryseobacterium oranimense and Haemophilus parahaemolyticus). The comprehensive analysis of bacterial genomes from cystic fibrosis patients revealed an extraordinary evolution of these genomes with time and treatment received by these patients reflects the ability of these bacteria to adapt to their particular ecosystem the acquisition of new genes by lateral gene transfer. This work shows the extraordinary plasticity of bacterial genomes in a given environment and as the lungs of patients with cystic fibrosis represents a unique model for understanding the evolution of bacterial genomes. In addition, our work has identified their molecular mechanisms of resistance to antibiotics. Future work on the study of metagenomes sampling in these patients could help to answer these questions in the future. The discovery of new species and / or emerging will allow us to have a more complete picture of cystic fibrosis which could lead to a better understanding of the disease and thus a better therapeutic management.

Objetivo: Diversas estratégias de erradicação da Pseudomonas aeruginosa (Pa) em pacientes com fibrose cística (FC) têm sido propostas, mas poucas realizaram o tratamento em fases e incluíram crianças na primeira colonização por Pa na vida. O objetivo desse estudo é descrever a efetividade do protocolo de erradicação em fases, em crianças brasileiras com FC no primeiro aparecimento de Pa. Métodos: Estudo de vida real, retrospectivo, que avaliou os prontuários dos pacientes pediátricos submetidos ao protocolo de erradicação em um período de 8,5 anos. O protocolo de 3 fases era guiado pela cultura de via aérea, e utilizou colistimetato nebulizado e ciprofloxacino oral. Foram avaliadas as taxas de sucesso após cada fase e a acumulada. Resultados: 47 episódios de colonização por Pa foram elegíveis à erradicação. A fase 1 do protocolo foi aplicada em 29 pacientes (mediana 2,7 anos, 59% masculino, 65% com no mínimo um alelo F508del), a fase 2 em 12 e a fase 3 em 6 pacientes. A taxa de sucesso foi de 58,6% (IC 95%: 40,7 a 74,5), 50,0% (IC 95%: 25,4 a 74,6) e 66,7% (IC 95%:30,0 a 90,3), após as fases 1, 2 e 3 respectivamente. A taxa de sucesso acumulado foi de 93,1% (IC: 78,0 a 98,1). Apenas 2 pacientes tiveram falha do tratamento de erradicação. Conclusão: O primeiro aparecimento da Pa ocorreu em crianças de baixa idade. O protocolo de erradicação em fases foi efetivo com alta taxa de sucesso / Background: Although several Pseudomonas aeruginosa (Pa) eradication strategies have been proposed for Cystic Fibrosis (CF) patients, only a few have used a stepwise treatment and enrolled children in their first-onset Pa colonization. The purpose of this study is to describe the effectiveness of a multistep eradication protocol in CF Brazilian children in their first-onset Pa. Methods: This was a real life retrospective study that evaluated medical records of pediatric patients who underwent the eradication protocol in a timeframe of 8.5 years. The 3-step protocol was guided by airway culture, using inhaled colistimethate and oral ciprofloxacin. The success rate after each step, and also the cumulative success rate were evaluated. Results: During the study period, 47 Pa colonization episodes were eligible to eradication. All the 29 patients underwent protocol\'s step 1 (median 2.7 years old, 59% male, 65% with at least one F508del allele), twelve patients step 2, and six patients step 3. Success rate was 58.6% (CI 95%: 40.7 to 74.5), 50.0% (CI 95%: 25.4 to 74.6) and 66.7% (CI 95%:30.0 to 90.3), after step 1, 2 and 3 respectively. Cumulative success rate was 93.1% (CI: 78.0 to 98.1). Failure of the eradication protocol occurred in only 2 patients. Conclusion: First-onset Pa colonization occurred in very young children. The stepwise eradication protocol was effective with a high success rate

Contexte : Les maladies pulmonaires comme l'asthme ou la mucoviscidose représentent des problèmes majeurs de santé publique. Elles se manifestent par une inflammation chronique avec une production accrue de cytokines pro-inflammatoires et à terme une dégradation de la fonction respiratoire. Les efforts thérapeutiques tentent, d'un côté, de contrôler la réaction inflammatoire et aussi d'améliorer la biodisponibilité médicamenteuse. Objectif : Notre objectif est d'explorer l'implication des phospholipases dans l'inflammation et le rôle des transporteurs peptidiques dans le transport des antibiotiques dans la mucoviscidose. Nous avons aussi cherché à comprendre l'effet d'une supplémentation en cobalamine sur l'efficacité de la dexaméthasone dans un contexte inflammatoire. Méthodes : Des techniques immunologiques, électrophorétiques, de culture cellulaire, d'immunoprécipitation et d'expression génique sont utilisées sur des lignées bronchiques humaines normales ou mucoviscidosiques. Résultats : 1) La PC-PLC est constitutivement suractivée dans les cellules mucoviscidosiques et conduit à une surproduction d'arachidonate, à une surexpression de Cox-2, une surproduction de PGE2, une surexpression d'interleukine-8, et au défaut de régulation beta-adrénergique de la sécrétion. L'inhibition de cette enzyme par le D609 permet de corriger tous ces défauts. 2) L'activité du transporteur peptidique, impliquée dans le transport d'antibiotiques, PEPT2, a été caractérisée dans les cellules bronchiques normales (Vm = 115 pmol/106 cellules/min ; Km = 15µM). Ce transporteur n'est pas influencé par un contexte inflammatoire. Ce transporteur est inactif dans les cellules CF. 3) La cobalamine potentialise l'effet de la déxaméthasone sur la sécrétion et l'expression des cytokines pro-inflammatoires induite par le TNFa et l'histamine. Conclusions/perspectives : Cette étude devrait permettre 1) de mettre en lumière l'importance de la PC-PLC comme cible pharmacologique potentielle dans la mucoviscidose. 2) de comprendre la relative faible efficacité de l'antibiothérapie dans cette maladie et 3) de mettre en évidence une possible participation du cycle de la méthionine dans le processus inflammatoire / Background: Lung diseases such as asthma or cystic fibrosis are major public health problems. They are manifested by chronic inflammation with increased production of proinflammatory cytokines leading to respiratory failure. Current therapeutic is aimed at controling the inflammatory response and also at improving drug bioavailability. Objective: The objective is to explore the involvement of phospholipases in inflammation and the role of peptide transporters in the transport of antibiotics in cystic fibrosis. We also sought to understand the effect of cobalamin supplementation on the effectiveness of dexamethasone in an inflammatory context. Methods: immunological techniques, electrophoresis, cell culture, immunoprecipitation and gene expression are used on normal or cystic fibrosis human bronchial cell lines. Results : 1) PC-PLC is constitutively overactivated in cystic fibrosis cells and leads to overproduction of arachidonate, to overexpression of Cox-2 , an overproduction of PGE2 , an interleukin -8 overexpression , and to alteration of beta-adrenergic secretion. Inhibition of this enzyme by D609 corrects these defects. 2) The activity of the dipeptide carrier involved in the transport of antibiotics, PEPT2, was characterized in normal bronchial cells (Vm = 115 pmol/106 cells / min, Km = 15µM). This transporter is not affected by an inflammatory context. However, it was shown to be inactive in CF cells. 3) Cobalamin potentiates the effect of dexamethasone on the expression and secretion of pro-inflammatory cytokines induced by TNFa and histamine. Conclusions : This study should help 1) to highlight the importance of PC-PLC as a potential pharmacological target in cystic fibrosis. 2) to understand the relative ineffectiveness of antibiotics in this disease , and 3) to highlight a possible involvement of methionine cycle in the inflammatory process

Mycobacterium abscessus est une mycobactérie responsable principalement d’infections pulmonaires, en particulier chez les patients atteints de mucoviscidose ou de dilatation des bronches. M. abscessus est naturellement résistante aux antituberculeux, laissant peu d’options thérapeutiques. Le traitement de référence associait classiquement un aminoside, un macrolide (clarithromycine) et une β-lactamine (céfoxitine ou imipénème), avec un taux de succès d’environ 50 %. Cependant, des souches résistantes à la clarithromycine sont fréquemment isolées, remettant en cause l’utilisation de cet antibiotique. M. abscessus produit naturellement une β-lactamase à large spectre (BlaMab) mais les mécanismes d’action des β-lactamines n’ont pas été étudiés chez cette espèce, ce qui constitue une entrave à l’optimisation des traitements par cette classe d’antibiotiques. Le premier objectif était d’identifier et de caractériser les cibles des β-lactamines chez cette espèce. Inhibant la dernière étape de polymérisation du peptidoglycane, les cibles potentielles des β-lactamines sont trois familles d’enzymes : les D,D-transpeptidases et les D,D­carboxypeptidases appartenant à la famille des protéines de liaison à la pénicilline (PLP), ainsi que les L,D-transpeptidases qui sont majoritairement responsables de cette dernière étape chez cette espèce. Pour identifier les cibles, des mutants résistants aux β-lactamines ont été sélectionnés à partir de la souche de référence M. abscessus CIP104536 et d’un dérivé portant une délétion du gène blaMab (∆blaMab). Pour les deux souches, l’émergence de la résistance aux β-lactamines a requis de multiples étapes, ce qui constitue un atout pour leur utilisation thérapeutique. Pour les mutants obtenus à partir de la souche CIP104536, les analyses phénotypiques ont montré que la résistance aux β-lactamines n'est pas due à une augmentation de l’efficacité catalytique de BlaMab, à une surproduction de cette enzyme, ou à une diminution de la perméabilité. Le séquençage des génomes de mutants résistants n’a pas révélé de mutations dans les gènes codant pour les L,D-transpeptidases, mais des mutations ont été trouvées dans des gènes codant pour deux PLP. D’autres mutations se situent dans des gènes codant en particulier pour des protéines non caractérisées. L’acquisition de la résistance pourrait donc dépendre de mutations affectant des facteurs essentiels à l’activité des cibles des β­lactamines. Le deuxième objectif était d’étudier et de comparer l’activité in vitro des β-lactamines sur M. abscessus. Des expériences de bactéricidie et d’activité intracellulaire chez le macrophage infecté ont été effectuées pour les souches CIP104536 et ∆blaMab. Parmi les antibiotiques étudiés (amikacine, céfoxitine, imipénème, ceftaroline, et amoxicilline), l’imipénème est le plus efficace sur les deux souches. Sur la souche ∆blaMab, l’association d’imipénème et d’amikacine est bactéricide. En l’absence de BlaMab, l’amoxicilline est aussi efficace que l’imipénème. L’avibactam augmente l’activité de la ceftaroline mais l’inhibition de BlaMab est seulement partielle en intracellulaire. Les résultats obtenus in vitro montrent que l’imipénème est supérieur à la céfoxitine pour des concentrations atteignables dans le sérum. L’inhibition de BlaMab pourrait augmenter l’efficacité de l’imipénème et d’autres composés utilisés pour traiter les infections pulmonaires à M. abscessus. / Mycobacterium abscessus is an important pathogen responsible for pulmonary infections in cystic fibrosis patients or in patients suffering from bronchiectasis. The treatment of infections due to M. abscessus is complicated since this bacterium is naturally resistant to the anti­tuberculous agents. The recommended treatment includes an aminoglycoside, a macrolide (clarithromycin) and a β-lactam (cefoxitin or imipenem), with a success rate of about 50 %. However, strains resistant to clarithromycin are frequently isolated, questioning the use of this antibiotic. M. abscessus naturally produces a broad spectrum β-lactamase (BlaMab) but the mechanisms of action of the β-lactams have not been studied in this species, impairing the optimization of the treatment by these antibiotics. The first objective was to identify and characterize the targets of β-lactams antibiotics in this species. Inhibiting the final stage of the peptidoglycan polymerization, the potential targets of β-lactams are three families of enzymes: the D,D-transpeptidases and D,D­carboxypeptidases belonging to the family of penicillin-binding proteins (PBP), and the L,D-transpeptidases which are mainly responsible for this final stage in this species. To identify the targets, mutants resistant to β-lactams have been selected from the reference strain M. abscessus CIP104536 and from its β-lactamase-deficient derivative ΔblaMab. For both strains, the emergence of resistance to β­lactams has required multiple steps, which is an advantage for the therapeutic use of these antibiotics. For the mutants derived from the strain CIP104536, phenotypic analyzes showed that the resistance to β-lactams is not due to an increase in the catalytic efficiency of BlaMab, to an overproduction of this enzyme, or to a decrease in permeability. Genomes sequencing of the resistant mutants did not reveal mutations in the genes encoding the L,D-transpeptidases, but mutations have been found in genes encoding two PBPs. Other mutations have been detected in genes encoding uncharacterized proteins. Acquisition of resistance could therefore depend on mutations affecting key factors essential for the activity of β-lactams targets. The second objective was to study and compare the in vitro activities of β-lactams against M. abscessus. Bactericidal experiments and intracellular activity in the infected macrophage were performed for the strains CIP104536 and ΔblaMab. Among the antibiotics tested (amikacin, cefoxitin, imipenem, ceftaroline, and amoxicillin), imipenem is the most effective agent against the two strains. Combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. In the absence of BlaMab, amoxicillin was as active as imipenem. Avibactam increased the intracellular activity of ceftaroline but inhibition of BlaMab was only partial intracellularly. Evaluation of the killing and intracellular activities of β-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drug potentially useful to treat pulmonary infections.

INTRODUÇÃO: Em contraste aos bons resultados do transplante pulmonar no tratamento de pacientes com doença supurativa pulmonar avançada, a colonização por Burkholderia cepacia complex (BCC), sobretudo o genomovar III, vem sendo relacionada a pior prognóstico e, por conseguinte, uma contraindicação ao procedimento em alguns centros transplantadores. O objetivo deste estudo foi avaliar o impacto em sobrevida após o transplante pulmonar de pacientes com doença pulmonar supurativa colonizados por BCC, além de determinar a incidência da colonização e suas variantes genômicas no Instituto do Coração/HC-FMUSP. MÉTODOS: Foram analisados prospectivamente dados clínicos e amostras de culturas do trato respiratório dos pacientes que realizaram transplante pulmonar por doença supurativa entre janeiro de 2008 e dezembro de 2013. A tipagem molecular para estudar os diferentes genótipos da BCC foi realizada a partir de janeiro de 2012 por método de sequenciamento genético e análise do gene RecA. RESULTADOS: Foram realizados 132 transplantes pulmonares, 62 pacientes com doença pulmonar supurativa, sendo 28 em pacientes com Bronquiectasias e 34 com Fibrose Cística. Observou-se a colonização por BCC em 16 pacientes; em 7 amostras identificados os seguintes subtipos: três cepas B. metallica e quatro cepas B. cenocepacia. A incidência de BCC nos pacientes com Fibrose Cística foi de 38,2%, enquanto nos pacientes com Bronquiectasias foi 10,7%. Dentre os 16 pacientes colonizados por BCC, ocorreram 2 óbitos, nenhum deles relacionados à infecção pelo agente. Um óbito foi atribuído a sepse por Acinetobacter baumannii resistente a múltiplas drogas e o outro, a disfunção orgânica múltipla. O estudo desenvolvido demostrou que a colonização por BCC não gerou impacto em mortalidade nos pacientes após o transplante pulmonar, mesmo quando colonizados pelo subtipo B. cenocepacia / INTRODUCTION: Notwithstanding the good results of lung transplantation for treatment of patients with advanced lung suppurative disease, colonization by Burkholderia cepacia complex (BCC), especially genomovar III has been related to a worse prognosis in these patients and therefore contraindication to the procedure certain centers. The aim of this study was to evaluate the impact on survival after lung transplantation in patients with suppurative lung disease colonized with BCC to determine the incidence of colonization and its genomic variants at the Heart Institute / HC -FMUSP. METHODS: We prospectively analyzed clinical data and respiratory tract samples of suppurative lung disease patients that performed lung transplantation from January-2008 through November-2013. From January-2012 through December-2013, we also subtyed the different B. cepacia genotypes by DNA sequencing primers of the gene RecA. RESULTS: 132 lung transplantation were performed, 62 patients with suppurative lung disease, 28 patients with Bronchiectasis and 34 with Cystic Fibrosis. BCC was observed in 16 patients; in 7 samples we identified the following subtypes: three strains B. metallica and four strains B. cenocepacia. The incidence of BCC in patients with Cystic Fibrosis was 38.2% while in patients with Bronchiectasis was only 10.7%. Among the 16 patients colonized with BCC, there were two deaths, none of them related to infection by the agent. One death due to sepsis Acinetobacter baumannii resistant to multiple drugs and the other, multiple organ dysfunction. The study demonstrated that colonization by BCC developed no impact on the mortality rate of patients after lung transplantation, even when colonized by the subtype B. cenocepacia

Introdução: A tolerância ao exercício e os níveis de atividade física tendem a ser reduzidos em pacientes com fibrose cística (FC). Estudos trazem que o nível de atividade física pode estar associado com melhor estado nutricional e com um maiordeclínio da função pulmonar. Com a evolução da doença,o declínio da função pulmonar associado a distúrbios metabólicos e desnutrição, provocam alterações na mecânica respiratória, distúrbios musculoesqueléticos e deformidades torácicas, trazendo prejuízos na qualidade de vida destes indivíduos. Objetivo: Verificar a associação entre gravidade funcional pulmonar, nível de atividade física e variáveis de avaliação postural em adultos com FC. Secundariamente, verificar a correlação entre os parâmetros de atividade física observados pelo acelerômetro e pelo questionário internacional de atividade física (IPAQ) e, verificar a correlação entre os parâmetros de atividade física observados pelos dois instrumentos com a distância percorrida no teste de caminhada de seis minutos (DTC6M) e os parâmetros de avaliação postural.Métodos: Estudo de caráter transversal, aprovado pelo Comitê de Ética em Pesquisa do Hospital de Clínicas de Porto Alegre. Para a verificação de parâmetros de função pulmonar foi utilizado o exame de espirometria, para a verificação do nível de atividade física foi utilizado o questionário IPAQ e um acelerômetro, para avaliação postural foi utilizada a fotogrametria com auxílio do Software de avaliação postural (SAPO) e, para comparações complementares foi utilizado o teste de caminhada de seis minutos (TC6M). Resultados: Participaram do estudo 28 indivíduos adultos com FC, idade média de 25,1 anos e VEF1 (%) com média de 47,1. O VEF1 se associou com os parâmetros obtidos pelo acelerômetro, avaliação postural e TC6M, porém, não houve associação com os dados obtidos pelo IPAQ. Conclusão: Este estudo demonstrou que o declínio da função pulmonar dos pacientes adultos com FC está associado com maior cifose torácica, menor tempo em atividade física moderada e vigorosa e menor distância percorrida no TC6M. O acelerômetro demonstrou-se ser o melhor instrumento para avaliação da atividade física neste público. / Introduction: Exercise tolerance and levels of physical activity tend to be reduced in patients with cystic fibrosis (CF). Studies suggest that the level of physical activity may be associated with better nutritional status and with larger decline in lung function. With the evolution of the disease, the decline of lung function associated with metabolic disorders and malnutrition, causes alterations in respiratory mechanics, musculoskeletal disorders and thoracic deformities, bringing injury to the individual’s quality of life.Objective: To verify the association between pulmonary functional severity, physical activity level and postural evaluation variables in adults with CF. Secondly, to verify the correlation between the physical activity parameters observed by the accelerometer and the international physical activity questionnaire (IPAQ) and to verify the correlation between the parameters of physical activity observed by the two instruments with the six-minute walking distance (6MWD) and the parameters of postural evaluation. Methods:Cross-sectional study, approved by the Research Ethics Committee of the Hospital de Clínicas, Porto Alegre.The spirometry test was used to verify pulmonary function parameters. The IPAQ questionnaire and an accelerometer were used to assess the level of physical activity, for postural evaluation the photogrammetry was used with the aid of the Postural Evaluation Software (SAPO) and, for further comparisons, the six-minute walk test (6MWT) was used. Results: 28 adult subjects with CF, mean age of 25.1 years and FEV1 (%) with a mean of 47.1 participated in the study. FEV1 was associated with the parameters obtained by the accelerometer, postural evaluation and 6MWT, but there was no association with the data obtained by IPAQ. Conclusion: This study demonstrated that the decline in lung function in adult patients with CF is associated with higher thoracic kyphosis, shorter time in moderate and vigorous physical activity, and shorter distance walked on 6MWT. The accelerometer has been shown to be the best instrument for assessing physical activity in this public.

Estudos para a avaliação da resistência das vias aéreas de pré-escolares com fibrose cística através da técnica do interruptor (Rint) são escassos. Foi realizado um estudo tranversal, com 38 pacientes acompanhados no ambulatório de Fibrose Cística do Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul. Foram realizadas medida da Rint, seguida da avaliação espirométrica em todos os pacientes, repetindo os testes após o uso de salbutamol para a aferição da resposta ao broncodilatador. Nos resultados, foram encontradas forte correlação entre o inverso da Rint e o volume expiratório forçado no 1º segundo (VEF1), (r=0,8 p<0,001) e moderadas correlações entre o inverso da Rint e o fluxo expiratório forçado entre 25% e 75% da capacidade vital (FEF 25-75), (r=0,74 p<0,001) e entre o inverso da Rint e o índice de massa corporal (IMC), (r=0,62 p<0,001). A acurácia da avaliação da resposta ao broncodilatador pela Rint, foi testada através da curva ROC (Receiver Operator Characteristic Curve),comparando-se com resposta ao broncodilatador na espirometria. Foi obtida uma área de 0,75 para o ponto de corte de -28%, correspondendo a uma sensibilidade de 66% e uma especificidade de 82%. Os achados indicam que a Rint apresenta boa correlação com a espirometria, no entanto a técnica não possui uma acurácia suficiente para substituir a Espirometria na avaliação da resposta ao broncodilatador. / Few studies have been published on airway resistance measurements using the interrupter technique (IT). We performed a cross-sectional study, evaluating 38 children and adolescents with Cystic Fibrosis (CF), followed at the outpatient CF clinic of Hospital São Lucas from Pontifícia Universidade Católica do Rio Grande do Sul. Airway resistance (Rint) was measured by the IT, followed by spirometry in all patients. Measurements were repeated after inhalation of salbutamol in order to evaluate bronchodilator response. There was a strong corelation between inverse Rint and forced expiratory volume in one second (FEV1) (r=0.8, p<0.001) and fair correlations between the inverse Rint and mid expiratory flow (MEF) (r=0.74 p<0.001) and between inverse Rint and body mass index (BMI) (r=0.62 p<0.001). The accuracy of bronchodilator response by the IT was tested through the ROC (reciever operating curve), comparing results with spirometry bronchodilator response. An area of 0.75 under the curve was obtained, for the cutoff point of -28% of Rint, achieving a sensitivity of 66% and a specificity of 82%. The findings suggest that Rint shows good correlation with spirometry parameters, although the IT is not sufficiently acurate to replace spirometry in the evaluation of bronchodilator response.

La mucoviscidose est une maladie monogénique, caractérisée par des mutations survenant au niveau du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Le clonage en 1989 du gène CFTR a permis d’envisager de traiter cette maladie par thérapie génique. Cela consiste à transférer à l’aide d’un vecteur, une version normale du gène CFTR dans les cellules atteintes des patients. En raison de la gravité des complications pulmonaires, c’est l’épithélium respiratoire qui constitue aujourd’hui le tissu cible pour le transfert de gènes. Le principe de la thérapie génique est évidemment très séduisant et un certain nombre d’essais cliniques ont d’ores et déjà été réalisés. La thérapie génique nécessite des outils de vectorisation efficaces et compatibles avec une utilisation répétée en clinique.Mon sujet de thèse a porté donc sur le développement, la biodistribution et l’optimisation de vecteurs synthétiques (lipides cationiques) pour le transfert de gènes dans l’épithélium respiratoire. Au cours de mes travaux, nous avons donc pu mettre au point des lipophosphoramidates KLN47 fluorescents utiles pour les études de biodistribution in vivo. Comparés au KLN47 non fluorescent, ces nouveaux composés présentent les mêmes propriétés physicochimiques, à savoir une taille relativement petite et un potentiel zêta positif. Sur lignées cellulaires, nous avons montré que les nouvelles formulations étaient aussi efficaces que le KLN47, et pas ou peu toxiques. Ensuite, sur modèle animal, les profils de biodistribution de lipoplexes pégylés et non-pégylés ont été comparés après injection systémique. Les profils de biodistribution des lipoplexes pégylés et non-pégylés étaient similaires, cependant, la pégylation des complexes a conduit à une circulation prolongée dans la circulation sanguine, alors que l’expression du transgène (luciférase) était équivalente dans les deux cas. De plus, l’activité luciférase était similaire à celle obtenue avec le KLN47 non fluorescent. Nous avons ainsi démontré que l’ajout des sondes lipidiques fluorescentes dans la solution liposomale du KLN47, ne modifie pas ses propriétés physicochimiques et transfectantes. L’ensemble des résultats montre que nous disposons d’outils prometteurs pour les études de biodistribution in vivo. D’autres molécules ont également été testées avec succès. / Cystic fibrosis is a monogenic disease characterized by mutations occurring at the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The clonining in 1989 of the CFTR gene has enabled to consider treating this disease by gene therapy. This consists of transferring a normal version of the CFTR gene in the affected patients’ cells, using a vector. Due to the severity of pulmonary complications, it is obvious that the respiratory epithelium constitutes the target tissue for the gene transfer. The principle of gene therapy is indeed very attractive and a number of clinical trials have already been made. Gene therapy requires vectorization tools that are efficient and compatible with repeated clinical use.My thesis has focused on the development, biodistribution and optimization of synthetic vectors (cationic lipids) for gene transfer in the respiratory epithelium. During my work, we were able to develop useful fluorescent KLN47 lipophosphoramidates for in vivo biodistribution studies. Compared to non fluorescent KLN47, these new compounds exhibit the same physicochemical properties: a relatively small size and a positive zeta potential. On cell lines, we found that the new formulations were as effective as the KLN47, with little or no toxicity. Then, in animal models, the biodistribution profiles of pegylated and non-pegylated lipoplexes were compared after systemic injection. The biodistribution profiles of pegylated and non-pegylated lipoplexes were similar. However, the pegylation of the complex resulted in prolonged circulation in the bloodstream, whereas transgene expression (luciferase) was equivalent in both cases. In addition, luciferase activity was similar to that obtained with the non-fluorescent KLN47. We have demonstrated that the addition of fluorescent lipid probes in the liposomal solution KLN47, does not change its physicochemical and transfectant properties. The overall results show that we have promising tools for in vivo biodistribution studies. Other molecules have also been tested successfully.

Introdução: Patógenos emergentes são isolados nas vias respiratórias de pacientes com fibrose cística (FC), entre eles a Burkholderia cepacia. Atualmente, é conhecida como um conjunto de nove espécies relacionadas (\"genomovares\"), referidas coletivamente como complexo B. cepacia. A identificação fenotípica do complexo B. cepacia é difícil, e métodos de análise do genoma bacteriano, como a reação em cadeia da polimerase, que exploram diferenças no gene recA, têm mostrado grande eficácia na caracterização dos genomovares. Alguns Centros de tratamento de FC demonstraram infecções cruzadas entre os pacientes e marcadores de virulência foram identificados com freqüência em alguns deles. Métodos baseados em biologia molecular são capazes de realizar a genotipagem das cepas e têm sido utilizados na avaliação epidemiológica. Objetivos: Identificar o genomovar e a presença de marcadores de virulência entre as cepas do complexo Burkholderia cepacia isoladas de pacientes com fibrose cística atendidos no ICr e analisar as cepas do complexo Burkholderia cepacia através de genotipagem pela técnica de RAPD. Métodos: Foram coletadas 672 amostras de escarro e esfregaço de orofaringe de 140 pacientes com fibrose cística (6 meses a 19 anos) atendidos na nossa Unidade nos períodos de set/2000 a abr/2001 e jun/2003 a jun/2004. As amostras foram cultivadas em meios seletivos, incluindo meio para B. cepacia, e a identificação realizada por sistema automatizado (1º período) e por testes fenotípicos clássicos (2º período). Após a extração do DNA, as cepas foram submetidas a uma série de reações de PCR para a determinação dos genomovares (I a VII), utilizando primers direcionados à amplificação de diferentes trechos da seqüência do gene recA, sendo, em seguida, submetidas ao seqüenciamento do DNA deste gene. Os genes de virulência pesquisados foram o cblA (que codifica o pili) e o esmR (marcador de uma cepa epidêmica). A genotipagem foi realizada pela técnica do RAPD, que analisa todo o genoma bacteriano. Resultados: Foram isoladas 41 cepas do complexo B. cepacia, obtidas de 21 pacientes com fibrose cística. O método de PCR identificou o genomovar de 32/41 (78%) das cepas e todos os resultados foram confirmados através do seqüenciamento do DNA. B. cenocepacia foi o genomovar mais prevalente (n = 17), seguido da B. multivorans (n = 12), B. vietnamiensis (n = 2) e B. cepacia (n = 1). As nove cepas não caracterizadas foram submetidas ao seqüenciamento, tendo sido encontradas 5 cepas de B. gladioli, 2 cepas de X. campestris e 2 cepas permaneceram sem identificação. O gene cblA não foi identificado em nenhuma cepa, mas o gene esmR foi encontrado em 2 amostras (pacientes não relacionados). A genotipagem detectou 23 padrões distintos, sem identificar padrões idênticos entre pacientes diferentes. Conclusões: O método de PCR baseado na amplificação do gene recA mostrou ser eficaz para a determinação do genomovar. B. cenocepacia e B. multivorans foram as espécies mais prevalentes entre nossos pacientes. A prevalência de marcadores de virulência foi baixa entre as cepas isoladas. Infecção cruzada pelo complexo B. cepacia não parece ter ocorrido na nossa Unidade durante os períodos estudados. / Introduction: Emerging pathogens are found in the respiratory tract of the cystic fibrosis (CF) patients, including the bacterium Burkholderia cepacia. At the present moment, it is recognized as a group of nine related species (\"genomovars\"), collectively referred as B. cepacia complex. Phenotypical identification of B. cepacia complex is difficult, and molecular based methods such as PCR, exploring differences in recA gene sequence, showed high efficacy for genomovar determination. B. cepacia complex cross infections among CF patients were previously described in some CF treatment Centers, and virulence markers were identified in a high frequency in some of them. Molecular based methods are suitable for strain genotyping and have been used for epidemiological evaluation. Aims: To identify genomovar status and virulence markers among Burkholderia cepacia complex isolates obtained from cystic fibrosis patients attending in the ICr, and to analyze the isolates through genotyping by RAPD. Methods: 672 sputum or oropharyngeal samples were obtained from 140 cystic fibrosis patients (6 months to 19 years) attending our Unit from sep/2000 to apr/2001 and jun/2003 to jun/2004. The samples were cultivated in selective media, including B. cepacia medium, and bacterial identification obtained by automated system (first period) and by classical phenotypic tests (second period). After DNA extraction, B. cepacia complex strains were submitted to sequential PCR reactions targeting recA gene in order to determine genomovar status (I to VII), and after that, submitted to automated DNA sequencing of this gene. Virulence genes screened were cblA (cable pilus) and esmR (epidemic strain marker). Genotyping was performed by whole bacterial genome fingerprinting using RAPD. Results: 41 isolates of B. cepacia complex were obtained from 21 cystic fibrosis patients. The PCR method identified genomovar status of 32/41 (78%) isolates and all results were confirmed by DNA sequencing. B. cenocepacia was the main genomovar (n=17), followed by B. multivorans (n = 12), B. vietnamiensis (n = 2) and B. cepacia (n = 1). The nine isolates uncharacterized were submitted to sequencing and we found 5 isolates as B. gladioli, 2 isolates as X. campestris and 2 isolates remained unidentified. The cblA gene was not identified in all isolates, but esmR gene was found on 2 strains (unrelated patients). Genotyping depicted 23 patterns, without identical patterns among different patients. Conclusions: The PCR method targeting recA gene showed to be a valuable tool for determination of genomovar status. B. cenocepacia and B. multivorans were the most prevalent specie among our patients. The prevalence of virulence markers was low among the isolates. Cross infection by B. cepacia complex does not seem to have occurred in our Unit during the two studied periods.

Introdução: Patógenos emergentes são isolados nas vias respiratórias de pacientes com fibrose cística (FC), entre eles a Burkholderia cepacia. Atualmente, é conhecida como um conjunto de nove espécies relacionadas (\"genomovares\"), referidas coletivamente como complexo B. cepacia. A identificação fenotípica do complexo B. cepacia é difícil, e métodos de análise do genoma bacteriano, como a reação em cadeia da polimerase, que exploram diferenças no gene recA, têm mostrado grande eficácia na caracterização dos genomovares. Alguns Centros de tratamento de FC demonstraram infecções cruzadas entre os pacientes e marcadores de virulência foram identificados com freqüência em alguns deles. Métodos baseados em biologia molecular são capazes de realizar a genotipagem das cepas e têm sido utilizados na avaliação epidemiológica. Objetivos: Identificar o genomovar e a presença de marcadores de virulência entre as cepas do complexo Burkholderia cepacia isoladas de pacientes com fibrose cística atendidos no ICr e analisar as cepas do complexo Burkholderia cepacia através de genotipagem pela técnica de RAPD. Métodos: Foram coletadas 672 amostras de escarro e esfregaço de orofaringe de 140 pacientes com fibrose cística (6 meses a 19 anos) atendidos na nossa Unidade nos períodos de set/2000 a abr/2001 e jun/2003 a jun/2004. As amostras foram cultivadas em meios seletivos, incluindo meio para B. cepacia, e a identificação realizada por sistema automatizado (1º período) e por testes fenotípicos clássicos (2º período). Após a extração do DNA, as cepas foram submetidas a uma série de reações de PCR para a determinação dos genomovares (I a VII), utilizando primers direcionados à amplificação de diferentes trechos da seqüência do gene recA, sendo, em seguida, submetidas ao seqüenciamento do DNA deste gene. Os genes de virulência pesquisados foram o cblA (que codifica o pili) e o esmR (marcador de uma cepa epidêmica). A genotipagem foi realizada pela técnica do RAPD, que analisa todo o genoma bacteriano. Resultados: Foram isoladas 41 cepas do complexo B. cepacia, obtidas de 21 pacientes com fibrose cística. O método de PCR identificou o genomovar de 32/41 (78%) das cepas e todos os resultados foram confirmados através do seqüenciamento do DNA. B. cenocepacia foi o genomovar mais prevalente (n = 17), seguido da B. multivorans (n = 12), B. vietnamiensis (n = 2) e B. cepacia (n = 1). As nove cepas não caracterizadas foram submetidas ao seqüenciamento, tendo sido encontradas 5 cepas de B. gladioli, 2 cepas de X. campestris e 2 cepas permaneceram sem identificação. O gene cblA não foi identificado em nenhuma cepa, mas o gene esmR foi encontrado em 2 amostras (pacientes não relacionados). A genotipagem detectou 23 padrões distintos, sem identificar padrões idênticos entre pacientes diferentes. Conclusões: O método de PCR baseado na amplificação do gene recA mostrou ser eficaz para a determinação do genomovar. B. cenocepacia e B. multivorans foram as espécies mais prevalentes entre nossos pacientes. A prevalência de marcadores de virulência foi baixa entre as cepas isoladas. Infecção cruzada pelo complexo B. cepacia não parece ter ocorrido na nossa Unidade durante os períodos estudados. / Introduction: Emerging pathogens are found in the respiratory tract of the cystic fibrosis (CF) patients, including the bacterium Burkholderia cepacia. At the present moment, it is recognized as a group of nine related species (\"genomovars\"), collectively referred as B. cepacia complex. Phenotypical identification of B. cepacia complex is difficult, and molecular based methods such as PCR, exploring differences in recA gene sequence, showed high efficacy for genomovar determination. B. cepacia complex cross infections among CF patients were previously described in some CF treatment Centers, and virulence markers were identified in a high frequency in some of them. Molecular based methods are suitable for strain genotyping and have been used for epidemiological evaluation. Aims: To identify genomovar status and virulence markers among Burkholderia cepacia complex isolates obtained from cystic fibrosis patients attending in the ICr, and to analyze the isolates through genotyping by RAPD. Methods: 672 sputum or oropharyngeal samples were obtained from 140 cystic fibrosis patients (6 months to 19 years) attending our Unit from sep/2000 to apr/2001 and jun/2003 to jun/2004. The samples were cultivated in selective media, including B. cepacia medium, and bacterial identification obtained by automated system (first period) and by classical phenotypic tests (second period). After DNA extraction, B. cepacia complex strains were submitted to sequential PCR reactions targeting recA gene in order to determine genomovar status (I to VII), and after that, submitted to automated DNA sequencing of this gene. Virulence genes screened were cblA (cable pilus) and esmR (epidemic strain marker). Genotyping was performed by whole bacterial genome fingerprinting using RAPD. Results: 41 isolates of B. cepacia complex were obtained from 21 cystic fibrosis patients. The PCR method identified genomovar status of 32/41 (78%) isolates and all results were confirmed by DNA sequencing. B. cenocepacia was the main genomovar (n=17), followed by B. multivorans (n = 12), B. vietnamiensis (n = 2) and B. cepacia (n = 1). The nine isolates uncharacterized were submitted to sequencing and we found 5 isolates as B. gladioli, 2 isolates as X. campestris and 2 isolates remained unidentified. The cblA gene was not identified in all isolates, but esmR gene was found on 2 strains (unrelated patients). Genotyping depicted 23 patterns, without identical patterns among different patients. Conclusions: The PCR method targeting recA gene showed to be a valuable tool for determination of genomovar status. B. cenocepacia and B. multivorans were the most prevalent specie among our patients. The prevalence of virulence markers was low among the isolates. Cross infection by B. cepacia complex does not seem to have occurred in our Unit during the two studied periods.