The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed April 27, 2010). Thesis advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 116-150.

Thesis (M.A.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 78-87). Also available via the World Wide Web.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A infecção pulmonar de etiologia bacteriana é um dos principais problemas que levam a morbi-mortalidade na fibrose cística (FC). Staphylococcus aureus se destaca como um dos micro-organismos mais frequentes e com um agravante para a terapêutica quando se apresentam resistentes à oxacilina (MRSA). Amostras MRSA podem ser classificadas tanto genotipicamente quanto fenotipicamente em MRSA adquiridas na comunidade (CA-MRSA) ou adquiridas no hospital (HA-MRSA). Fenotipicamente, essa classificação é muito controversa, podendo se basear em critérios epidemiológicos ou ainda pelo perfil de susceptibilidade aos antimicrobianos. Por outro lado, a classificação genotípica consiste na determinação dos cassetes cromossômicos (SCCmec), local de inserção do gene mecA (que confere resistência a meticilina). Atualmente são reconhecidos 11 tipos de SCCmec, sendo os de tipo I ao III e VIII relacionados ao genótipo HA-MRSA e IV ao XI ao genótipo CA-MRSA. Classicamente CA-MRSA é capaz de produzir a toxina Panton-Valentine leukocidin (PVL), codificada pelos genes luk-S e luk-F que está associada à pneumonia necrotizante e infecções de tecidos moles em pacientes com FC com quadros de exacerbação pulmonar. No Brasil, raros são os trabalhos envolvendo caracterização de SCCmec em amostras de pacientes com FC. Diante disso, este estudo teve como objetivo principal a caracterização dos tipos de SCCmec e ainda a determinação do perfil de susceptibilidade a antimicrobianos em uma população de MRSA recuperada de pacientes com FC assistidos em dois centros de tratamento no Rio de Janeiro, Hospital Universitário Pedro Ernesto (HUPE) e Instituto Fernandes Figueira (IFF). Foram estudadas 108 amostras de MRSA isoladas do período de 2008 a 2010, sendo 94 oriundas de 28 pacientes adultos atendidos no IFF e 14 de 2 pacientes adultos atendidos no HUPE. Foram encontradas altas taxas de resistência para os antimicrobianos oxacilina, cefoxitina e eritromicina. Todas as amostras foram sensíveis à vancomicina e a linezolida quando determinada as Concentrações Inibitórias Mínimas (CIM). Através da técnica de PCR foi possível a tipificação dos SCCmec em 82,4% das amostras, sendo 64% destas compatíveis ao genótipo CA-MRSA. Não houve diferença estatística nas taxas de susceptibilidade aos antimicrobianos entre as amostras CA-MRSA e HA-MRSA. Foram encontrados os SCCmec dos tipos I, III, IV e V, sendo os tipos I e IV os mais frequentes. O gene que codifica a toxina PVL foi encontrado em 34,2% das amostras e foi observado em amostras CA-MRSA e HA-MRSA. Nosso estudo se destaca por apresentar um alto percentual de amostras CA-MRSA e ainda por ser o primeiro do país a detectar a presença do gene que codifica a toxina PVL em pacientes com FC. Além disso, de forma inédita na literatura, encontramos o gene luk-S, em amostras classificadas como HA-MRSA em pacientes com FC. Os poucos estudos nacionais, bem como as diferenças encontradas entre trabalhos, refletem a necessidade de conhecimento mais aprimorado do MRSA envolvido nas infecções pulmonares dos pacientes com FC. / Bacterial lung infections are the major cause of morbidity and mortality in CF patients. Staphylococcus aureus is one of the most common pathogens isolated from colonization/infection in pediatric patients particulary when it shows methicillin resistance (MRSA), causing problems to a choice of antimicrobial therapy. This samples MRSA are classified either genotypically or phenotypically in CA-MRSA (community acquired MRSA) or HA-MRSA (hospital acquired MRSA). However, this phenotypic classification is controverse and is based on criteria epidemiological features or antimicrobial susceptibility. In the other hand, genotypic classification based on the diversity of the cassette chromosomal, wich harbours gene mecA (responsible for methicillin resistant). Currently, there are 11 SCCmec. types Types I to III and VIII are related with HA-MRSA genotype and types IV to XI with CA-MRSA genotype. Classicaly, CA-MRSA is able to produce the Panton-Valentine Leukocidin toxin that is encoded by genes luk-S and luk-F. This toxin is associated to necrotizant pneumonia and soft tissue infections and in FC patients with pulmonary exacerbation. In Brazil, the are few studies envolving SCCmec characterization. Thus the main purpose of this study was to determine the presence of SCCmec types and the antimicrobial susceptibility profiles of MRSA strains recover from patients with CF attended at Hospital Universitário Pedro Ernesto (HUPE) - UERJ and Instituto Fernandes Figueiras (IFF) FIOCRUZ. One hundred and eight MRSA isolates from FC patients were studied in a period of two years (2008-2010). Ninety and four isolates belongs to pediatric patients treated in IFF and 14 from adults patients from HUPE. We found high rates of resistance to antimicrobials such as oxacillin, cefoxitin and erythromycin. All isolates were susceptible to vancomycin and linezolid by the microdilution testing. 82.4% of the isolates where typed by multiplex PCR and 64 % belonged to CA-MRSA. No statistical difference were detected between samples CA-MRSA and HA-MRSA. The SCCmec types I, IIII, IV and V were detected, and the types I and IV were the most frequent. 34,2% of the isolates harbored the PVL gene. This is the first in Brazil detecting the PVL toxin gene and high rates of CA-MRSA isolates among CF patients. Furthermore, we find luk-S gene HA-MRSA isolates from CF patients. The few national studies, as well as the differences between studies, reflecting the need for more improved knowledge of MRSA involved in lung infections of CF patients.

abstract: Cystic Fibrosis, one of the most severe childhood life-shortening illnesses, places demands on a child's life conceivably interfering with his or her academic success. It is possible that the medically related activities in which individuals with CF partake interfere with academic activities and the motivation, specifically beliefs, expectancies, and values held, toward those activities. These issues encouraged the investigation of academic achievement and achievement motivation in children with CF through exploration of three research questions. Question one concerns differences in academic achievement between children with CF and a healthy comparison group for 1) reading and 2) math. Question two explored differences in aspects of motivation including ability beliefs, outcome expectancies, and task values between the groups for the two academic subjects. Finally, question three examined the relationship between motivational components and academic achievement. Evidence is provided for differences in math achievement between the two groups. Differences in motivation between children with CF and healthy children remain unsubstantiated. / Dissertation/Thesis / M.A. Educational Psychology 2010

Introdução: A audiometria de altas frequências pode contribuir para a detecção precoce de alterações auditivas causadas por medicações ototóxicas. No tratamento dos pacientes com fibrose cística, existem muitos fármacos ototóxicos que são amplamente utilizados. A detecção precoce de alterações auditivas deve permitir que estas sejam identificadas antes que o dano atinja as frequências da fala. A lesão causada pela ototoxicidade é irreversível, trazendo importantes consequências sociais e psicológicas. Nas crianças, a perda auditiva, mesmo restrita às altas frequências, pode afetar o desenvolvimento da linguagem. Objetivo: Investigar a eficácia e a efetividade do monitoramento da audição por meio da audiometria de altas frequências em pacientes pediátricos com fibrose cística. Método: Foram consultadas as bases de dados eletrônicas PubMed, MEDLINE, Web of Science e LILACS, de janeiro a novembro de 2015. Foram selecionados os estudos em que foi realizada audiometria de altas frequências em pacientes com fibrose cística em tratamento com medicamentos ototóxicos, e publicados em português, inglês e espanhol. Para a avaliação da qualidade metodológica dos artigos optou-se pela utilização do Sistema GRADE. Resultados No processo de busca realizado de Janeiro de 2015 à Novembro de 2015 foram encontradas 512 publicações, sendo 250 na PubMed, 118 na MedLine, 142 na Web Of Science e dois na LILACS. Desses, foram selecionados nove artigos. Conclusões: Identificou-se a ocorrência de perda auditiva em altas frequências, em pacientes com fibrose cística sem queixas auditivas. Admite-se que audiometria em altas frequências possa ser um método de diagnóstico precoce a ser recomendado para investigação auditiva de pacientes em risco de ototoxicidade. / Introduction: High frequency audiometry may contribute to the early detection of hearing loss caused by ototoxic medications. In the treatment of patients with cystic fibrosis, there are many ototoxic drugs that are widely used. Early detection of hearing loss should allow them to be identified before the damage reaches frequencies of speech. The damage caused by ototoxicity is irreversible, bringing important social and psychological consequences. In children, hearing loss, even restricted to high frequencies, can affect language development. Objective: Investigate the efficacy and effectiveness of hearing monitoring by high frequency audiometry in pediatric patients with cystic fibrosis. Methods: Electronic databases were searched PubMed, MEDLINE, Web of Science and LILACS, from January to November 2015 were consulted. We selected only the studies that was carried out high-frequency audiometry in patients with cystic fibrosis and treatment with ototoxic drugs, published in Portuguese, English and Spanish. For the evaluation of the methodological quality of the items we chose to use the GRADE system. Results: In the search process carried out from January to November 2015 were found 512 publications, and 250 of PubMed, MedLine 118, 142 Web of Science and 2 from LILACS. Of these, nine articles were selected. Conclusion: It was identified the occurrence of hearing loss in high frequencies, in cystic fibrosis patients without hearing complaints. It is assumed that high frequency audiometry may be an early diagnostic method to be recommended for hearing investigation of patients at risk for ototoxicity.

Introdução: A fibrose cística também conhecida como mucoviscidose, é uma doença genética autossômica recessiva, crônica, com manifestações sistêmicas, sendo o transplante pulmonar uma das alternativas para o tratamento quando a doença se apresenta em fase terminal. Objetivos: Avaliar a função pulmonar, condicionamento físico e qualidade de vida em pacientes com fibrose cística após trinta meses do transplante pulmonar seguido de um programa de reabilitação do cardiopulmonar. Metodologia: Estudo de coorte ambispectivo, foram estudados 8 pacientes com fibrose cística (5 mulheres 3 homens com média de idade 27 ± 4,62 anos) no período de dezembro de 2006 a dezembro de 2010. Havido perda de 2 pacientes ao longo do estudo. Foram analisados o teste de caminhada de seis minutos(TC6M), testes de função pulmonar e o questionário SF-36 no pré-transplante, pós-transplante imediato, pósreabilitação cardiopulmonar e após trinta meses após o transplante. Resultados: Tempo de lista de espera de 883 ± 571 dias tempo de internação total 30,14 ± 12,6 dias. Os pacientes apresentaram em média no pré-transplante VEF1 25,1% e CVF 38,4%, no pós transplante imediato VEF1 52,6% e CVF 54,6%. após reabilitação VEF1 60,8% e CVF 65,2% e 30 meses pós transplante VEF1 66,6% e CVF 67,2% . No TC6M a média de distância percorrida antes do transplante foi de 488 metros, pós-transplante imediato 510 metros, pós-reabilitação 603 metros e 30 meses pós-transplante 462 metros. Quanto à qualidade de vida os pacientes apresentaram melhora nos momentos pós-alta hospitalar e após reabilitação e piora em alguns domínios 30 meses após o transplante. Conclusão: O transplante de pulmão permanece sendo um procedimento de alto risco, no entanto, é uma estratégia terapêutica viável para pacientes com fibrose cística em estágio avançado da doença. Os dados encontrados no estudo sugerem uma tendência positiva a curto prazo na capacidade funcional e qualidade de vida, contudo a médio prazo perecem diminuir, enquanto a função pulmonar apresenta crescente melhora a curto e médio prazo. São necessários mais estudos com um maior número de pacientes para se afirmar com propriedade os benefícios a longo prazo do transplante de pulmão para essa população. / Introduction: Cystic Fibrosis, also known as mucoviscidosis, is a chronic autosomal recessive genetic disorder with systemic manifestations, lung transplantation being one of the alternatives for treatment when the disease is in its terminal phase. Objective: Evaluate the lung function, physical conditions and the quality of life of the cystic fibrosis patient after 30 months from the lung transplantation following a rehabilitation program of cardiopulmonary therapy. Methodology: The study of the ambispective cohort, involved eight patients with cystic fibrosis (5 women and 3 men aged 27 ± 4.6 years) for the period from December 2006 to December 2010, two patients died during the study. Analysis was made of the Six Minutes Walk Test (6MWT), lung function and the SF-36 questionnaire for the pre-transplant, immediately post- transplant, post cardiopulmonary rehabilitation and 30 months after the transplant. Results: The waiting list was 883±571 days, hospitalization time totalled 30.14 ±12.6 days. The patients showed, on average, a pretransplant VEF1 25.1% and CVF 38.4%, immediately posttransplant VEF1 66.6% and CVF 54.6%, post cardiopulmonary rehabilitation VEF1 60.8% and CVF 65.2% end 30 months post-transplant VEF1 66.6% and CVF 67.2%. In the TC6M, the average distance done before the transplant was 488 meters, immediately post-transplant it was 510 meters, post cardiopulmonary rehabilitation it was 603 meters and 30 months after transplant it was 462 meters. The quality of life of the patients showed improvement from the moment of leaving the hospital and after rehabilitation but did deteriorate in some domains after 30 months from the transplant. Conclusion: The lung transplantation continues to be a high risk procedure, however it still is a therapeutic strategy available to patients with cystic fibrosis at the advance stage of the disease. The findings of the study suggest there is a positive trend in the short term functional capacity and quality of life in the medium term however perish decrease, while increasing lung function has improved in the short and medium term. Although it would require further study with a higher number of patients to affirm with confidence the benefit for the long term of lung transplants for this population.

La mucoviscidose est une maladie génétique dont l’atteinte respiratoire est responsable de 90 % de la morbidité et de la mortalité et est caractérisée par une infection chronique et une inflammation persistante. Cette inflammation non contrôlée participe de manière importante à la dégradation du tissu pulmonaire. Malgré les progrès récents, les thérapies actuelles ne permettent pas un traitement efficace de l’atteinte respiratoire. Il est donc indispensable d’identifier de nouveaux mécanismes moléculaires et cellulaires impliqués dans l’inflammation pulmonaire. Dans ce but, nous nous sommes intéressés au canal calcique "Transient Receptor Potential Vanilloid 4" (TRPV4) exprimé au niveau de l’épithélium respiratoire. A l’aide d’approches in vitro et in vivo, nous avons démontré que l’activation du TRPV4 déclenche la sécrétion de médiateurs inflammatoires cytokiniques et lipidiques et un recrutement leucocytaire dans les poumons. Nous avons également observé une altération de la signalisation dépendante du TRPV4 dans le contexte de la mucoviscidose, suggérant que le TRPV4 pourrait constituer une cible prometteuse pour le développement de nouvelles thérapies anti-inflammatoires applicables en santé respiratoire. / Cystic fibrosis (CF) is due to mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). The pulmonary consequence of the disease accunts for over 90 % of the morbidity and mortality and is characterized by chronic infection and persistent inflammation. This uncontrolled inflammation participates significantly to the degradation of the lung tissue. Despite recent progress, current therapies do not allow effecgive treatment of CF lung disease. It is therefore necessary to characterize nex cellular and molecular mechanisms that could contribute to lung inflammation. In that purpose, we focused on the calcium channel "Transient Receptor Potential Vanilloid 4" (TRPV4) expressed by respiratory epithelium. Using in vitro and in vivo approaches, we found that TRP4 activation triggers the secretion of inflammatory mediators (including cytokines and lipids) and leukocytes recruitment into the lungs. We also observed a significant alteration of TRPV4-dependent signalling in the CF context, suggesting that TRPV4 could constitue a promising target for the development of new anti-inflammatory therapies in lung diseases such as CF.

La mucoviscidose est une maladie génétique caractérisée par une obstruction des voies respiratoires, des infections et une inflammation pulmonaire résultant du recrutement massif de neutrophiles qui sécrètent des protéases : l’élastase, la protéase 3 et la cathepsine G. Ces protéases peuvent être sécrétées selon deux voies, la dégranulation ou la sécrétion de NETs (Neutrophil Extracellular Traps), qui sont des fibres de chromatine auxquelles elles sont associées et décrites comme des structures antimicrobiennes. Dans le milieu extracellulaire, la dérégulation du contrôle de l’activité des protéases par leurs inhibiteurs conduit à la dégradation progressive du tissu pulmonaire. Nous avons montré que cette dérégulation était modulée par l’interaction des protéases avec l’ADN présent dans les sécrétions bronchiques des patients et que le ciblage de ces protéases par des inhibiteurs exogènes pouvait être amélioré in vitro par de la DNase ou de la polylysine qui compacte l’ADN. Ce polypeptide est également bactéricide vis-à-vis des pathogènes majeurs de la mucoviscidose, S. aureus et P. aeruginosa. Nos travaux montrent également que les NETs sont sécrétés dans les poumons des patients où ils constituent un réservoir de protéases actives potentiellement délétère et n’ont pas d’effet bactéricide vis-à-vis de S. aureus et P. aeruginosa. Nos travaux montrent que les voies de signalisation conduisant à la sécrétion des NETs varient selon le stimulus, générant des structures aux propriétés différentes. / Cystic fibrosis (CF) is a hereditary disease characterized by the obstruction of the airways, infections and a chronic lung inflammation due to a massive recruitment of neutrophils that secrete proteases: the elastase, the proteinase 3 and the cathepsin G. These proteases can be secreted by two mechanisms, namely degranulation and the secretion of NETs (Neutrophil Extracellular Traps), which are chromatin fibers to which they are bound and that have been described as antimicrobial structures. In the extracellular environment, the dysregulation of these proteases control by their inhibitors leads to progressive lung tissue degradation. We have shown that this dysregulation was influenced by the interaction of the proteases with the DNA found in the lung secretions of CF patients and that targeting these proteases with exogenous inhibitors could be improved in vitro by DNase or polylysine, which compacts DNA. This polypeptide also presents a bactericidal effect towards the major CF-associated pathogens, S. aureus and P. aeruginosa. Our work also shows that NETs are secreted in the lungs of CF patients, where they are a potentially deleterious reservoir of active proteases, and that they do not display any bactericidal effect towards S. aureus and P. aeruginosa. Our work shows that the signalization pathways leading to NETs secretion vary depending on the stimulus, generating structures that present different properties.

The Multiple Breath Washout (MBW) test is increasingly being recognised as a sensitive method of detecting early small airways lung disease. Indices of MBW include lung clearance index (LCI), Scond and Sacin. Factors that affect MBW variability have not been fully established. This thesis presents five studies which examine MBW repeatability in children with and without cystic fibrosis (CF) or asthma. MBW was performed using 0.2% sulphur hexafluoride and the modified Innocor (Innovision). Testing was performed at the Clinical Research Facility of the Royal Hospital for Sick Children in Edinburgh. (1) MBW and spirometry were performed in children with and without CF (n=20 in each group), initially while sitting and then 30 minutes after assuming a supine posture. LCI was found to significantly rise on lying supine in healthy children (p < 0.01) and children with CF (p=0.03). (2) Thirty two children with CF performed MBW and spirometry on four study visits, results were correlated with findings from high resolution chest computed tomography scans taken on the first visit. LCI showed the strongest correlation with extent and severity of bronchiectasis (r=0.66, p < 0.01 and r=0.69, p < 0.01 respectively). Variability of LCI was similar to FEV1 over the 4 visits. (3) MBW and spirometry of 66 healthy children were compared to 63 children with stable asthma; lung function of asthmatic children was related to symptoms and medication use. LCI was higher in the asthmatic group (6.7 vs 6.3, p < 0.01); within the asthmatic group LCI was significantly higher if asthma was less well controlled (p=0.02). (4) Children with and without asthma (n=21 in each group) performed MBW and spirometry before and after exercise and again after salbutamol, symptom data was collected from asthmatic children. Baseline LCI was abnormal in the asthmatic group who had severe exercise induced bronchospasm during testing. (5) Asthmatic children admitted to hospital due to exacerbation performed MBW and spirometry. Mean (SD) LCI was abnormally high at 8.5 (1.7) in the nine patients recruited and returned to normal 6.7 (0.6) in three patients who attended follow up. I have presented evidence that LCI is repeatable and sensitive to early disease in CF and asthma. I have described for the first time the effects of exercise and exacerbation on MBW indices in asthmatic children. MBW is potentially a very useful tool in paediatrics; standardisation of testing and equipment may enable clinical use.