Exercise has become a vital component of the therapy regimen prescribed to cystic fibrosis (CF) patients due to its systemic benefits, such as increased sputum expectoration, attenuation of the expected 2-3% annual decline in pulmonary function, and extended life expectancy. However, exercise still is not viewed as being as beneficial as pharmacological treatments by many CF patients and can be intimidating. My aims in this study were two-fold; first, to determine the ideal exercise intensity for individuals with CF; and second, to determine if exercise at this ideal intensity could provide improvements in ion regulation in the lungs, which was measured using exhaled breath condensate (EBC) collection and nasal potential difference (NPD), that were comparable to one of their standard pharmacological therapies, albuterol. I hypothesized that with moderate intensity exercise, Na⁺ absorption would decrease from baseline due to Na⁺ channel inhibition, rather than increase or remain unchanged, as was expected with albuterol, and cause an even greater increase Cl- secretion compared to albuterol due to activation of both CF-dependent and independent Cl- efflux with exercise. CF (n=14) and healthy (n=16) subjects completed three visits, a baseline screening and two treatment visits. I collected EBC at baseline, 30- and 60-minutes post-albuterol administration on one visit, and at baseline and during three separate 15 min exercise bouts at low, moderate and high intensity on the other visit. Following the EBC collection, NPD was performed at 30- and 80-minutes post albuterol or following moderate and high intensity exercise. We also measured spirometry and diffusing capacity of the lungs for nitric oxide (DLNO) during each visit at the various time points. In CF subjects, moderate intensity exercise resulted in greater improvements in DLNO (39 ± 29vs.15 ± 22% change from baseline, exercise vs. albuterol respectively), similar levels of bronchodilation compared to 60-minutes post-albuterol administration, no change in Na⁺ absorption, and a four-fold increase in Cl- secretion. Our results suggest that moderate intensity exercise is the best dose for CF patients, and can provide comparable changes as its pharmacological counterpart albuterol, when compared over a short term duration.

No description available.

Una de las principales cuestiones en farmacología molecular de los GPCR es entender los mecanismos estructurales de las siete hélices transmembrana (TM) que se producen para estabilizar ya sea Rg o los diferentes estados R*. Para entender el mecanismo que cambia el equilibrio del conjunto a un estado activo R* se construyeron tres de los receptores de la serotonina (5-HT4, 5-HT6, y 5 HT7) sobre la base de su información más reciente de cristalografía de rayos X. Dando lugar a dos modelos de cada receptor: una inactiva y otra activa. Los modelos, mejorados y evaluados con la ayuda de datos farmacológicos y químicos se utilizaron principalmente para comprender la interacción entre un ligando y su receptor y su mecanismo de acción. Estos hallazgos estructurales pueden a su vez resultar útiles para el diseño de nuevos fármacos más eficaces y selectivos. / One of the main questions in G protein coupled receptors (GPCRs) molecular pharmacology is to understand the structural arrangements of the seven transmembrane (TM) helices that occur to stabilize either the ground state (Rg) or different active states (R*) of the receptors. In order to understand the mechanism that shift the equilibrium of the ensemble to an active R* state models of the inactive and the active state of three serotonin receptors (5-HT4, 5-HT6, and 5-HT7) were built based on the latest information from X-ray crystallography. The resulting models were mainly used to understand the interaction between a ligand and its receptor and the mechanism of action. With the help of pharmacological and chemical data these models and complexes were improved and evaluated. These findings may prove valuable for structural based drug discovery efforts and facilitate the design of more effective and selective pharmaceuticals.

This thesis describes the molecular characterisation of Broome virus (BroV), a new fusogenic orthoreovirus species that was isolated from a little red flying-fox (Pteropus scapulatus) in Broome, Western Australia in 2002. The BroV genome consists of ten segments of dsRNA, each containing a plus-strand with a 3’ terminal pentanucleotide sequence that is conserved amongst all viruses in the genus Orthoreovirus, family Reoviridae, and a 5’ terminal pentanucleotide sequence that is unique to BroV. With the exception of S4, all genome segments are predicted to encode a single translation product producing a total of seven structural and four nonstructural proteins. All BroV proteins were identified as homologues of known orthoreovirus proteins and shown to have similar secondary structure and possess key conserved amino acid sequence motifs and structural features implicated in biological function. Notably, no cell-attachment protein gene homologue was identified in the BroV genome suggesting the use of an alternate cell entry mechanism to that employed by most orthoreoviruses. The amino acid sequence identity between cognate BroV proteins and those of other orthoreoviruses ranges from 13-50%, which is too low for BroV to be considered a new isolate of any established orthoreovirus species group. Phylogenetic analyses based on both structural and nonstructural proteins provide additional evidence to support this claim. It is proposed that BroV is the prototype member of a new sixth species group Broome virus, in the genus Orthoreovirus. The complete genome characterisation of BroV provided an opportunity to produce recombinant proteins in Escherichia coli and to generate polyclonal antibodies in rabbits for use in research and surveillance. Such reagents proved valuable in the experimental identification of the fusion-associated small transmembrane (FAST) protein p13 that is responsible for the syncytia observed in BroV-infected cells. Despite the low amino acid sequence identity between the FAST proteins of different orthoreovirus species they possess conserved structural features that have been implicated in biological function. Of these conserved features, the BroV p13 protein is predicted to possess one transmembrane domain, a C-terminal polybasic region, a C-terminal hydrophobic patch and an N-terminal myristoylation consensus sequence. The unique repertoire and arrangement of sequence-predicted structural features identified in p13 indicate that it is a novel fifth member of the FAST protein family. The BroV-specific immunological reagents were also used to develop an enzyme-linked immunosorbent assay (ELISA) suitable for serological screening. A survey of flying-foxes from Papua New Guinea (PNG) revealed that BroV or BroV-like viruses are currently circulating in these animals. This demonstrates that BroV is not limited to the Australian continent.

A fibrose cística (FC) é diagnosticada na presença de achados fenotípicos, história familiar ou triagem neonatal positiva acompanhada de evidência laboratorial de disfunção da CFTR, seja pelo teste do suor, diferença de potencial nasal ou pela identificação de duas mutações conhecidas como causa de FC nos genes da CFTR. Objetivos: Avaliar a contribuição da análise molecular do gene CFTR na investigação diagnóstica da fibrose cística em pacientes com suspeita de FC leve ou doença atípica. Secundariamente, comparar as características dos pacientes em 3 grupos: grupo com identificação de duas mutações conhecidas como causadoras da FC, grupo com identificação de apenas uma mutação e grupo sem mutação identificada. Métodos: Estudo transversal em adolescentes e adultos (≥14 anos). Os pacientes foram submetidos à avaliação clínica, laboratorial e radiológica; espirometria, microbiologia do escarro, ecografia hepática, teste do suor e análise molecular do gene CFTR. Resultados: Foram avaliados 37 pacientes com achados fenotípicos de FC, com ou sem confirmação pelo teste do suor. Houve predomínio do sexo feminino (75,7%) com média de idade de 32,5 ± 13,6 anos. A análise molecular contribuiu para o diagnóstico definitivo de FC em 3 casos (8,1%) dentre 37 pacientes em avaliação. Em 7 pacientes (18,9%) foram identificadas apenas uma mutação causadora de FC e em 26 pacientes (70,3%) não foram identificadas mutações. Nenhuma característica clínica estudada se associou com o diagnóstico genético. A mutação p.F508del foi a mais comum, encontrada em 5 pacientes. A associação de p.V232D e p.F508del foi encontrada em 2 pacientes. Outras mutações encontradas foram: p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P e p.R1066H e p.T351S. Conclusão: A análise molecular da região codificante do gene CFTR apresentou contribuição limitada para investigação diagnóstica de pacientes com suspeita de fibrose cística leve ou doença atípica. Além disso, não houve associação entre as características clínicas e o diagnóstico genético. / Cystic fibrosis (CF) is diagnosed in the presence of phenotypic findings, family history or positive neonatal screening accompanied by laboratory evidence of CFTR dysfunction, either by sweat test, nasal potential difference or the identification of two mutations known to cause CF in the CFTR gene. Objectives: To evaluate the contribution of molecular analysis of CFTR gene in cystic fibrosis diagnostic investigation in patients with suspected mild FC or atypical disease. Secondarily, to compare the characteristics of patients into 3 groups: group with identification of two mutations known to cause CF, group with identification of just one mutation and group without mutations. Methods: Cross-sectional study in adolescent and adult (≥ 14 years). The patient underwent clinical, laboratory and radiological spirometry, sputum microbiology, liver ultrasound, sweat test and molecular analysis of the CFTR gene. Results: We evaluated 37 patients with phenotypic findings of FC, with or without confirmation by the sweat test. There was a predominance of females (75.7%) with a mean age of 32.5 ± 13.6 years. Molecular analysis contributed to the definitive diagnosis of CF in 3 cases (8.1%) among 37 patients under evaluation. In 7 patients (18.9%) were identified only one mutation that causes CF and in 26 patients (70.3%) were not identified mutations. No clinical feature studied was associated with genetic diagnosis. The P.F508del mutation was the most common, found in 5 patients. The association p.V232D and p.F508del was found in 2 patients. Other mutations found were: p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, and p.P1290P p.R1066H and p.T351S. Conclusion: Molecular analysis of the CFTR gene coding region showed limited contribution to the diagnostic investigation of patients with suspected cystic fibrosis mild or atypical disease. Moreover, there was no association between clinical features and genetic diagnosis.

Chez Cupriavidus metallidurans CH34, le complexe CnrYXH contribue à réguler l'expression des gènes de régulation et de résistance au cobalt et au nickel en fonction de la concentration environnementale de ces cations. La fixation de cobalt ou de nickel sur le domaine périplasmique senseur de CnrX induit des modifications conformationnelles à l'origine de la transduction du signal transmembranaire. Cela conduit à rendre le facteur sigma CnrH disponible pour sa liaison à l'ARN polymérase (ARNP) dans le cytoplasme. Le complexe CnrH:ARNP se fixe alors spécifiquement sur les promoteurs des gènes cnrY et cnrC pour initier la transcription des gènes de régulation (cnrYXH) et de résistance (cnrCBAT). Dans le but de déterminer la nature de ce signal, mon projet de thèse visait à cartographier les interactions protéine:protéine au sein du complexe CnrYXH dans les trois compartiments concernés : le périplasme, la membrane plasmique et le cytoplasme. La documentation des déterminants d'interaction entre CnrX, CnrY et CnrH a permis d'élaborer un modèle structural et fonctionnel pour le complexe CnrYXH et ses homologues soulevant des hypothèses nouvelles sur le fonctionnement du système Cnr. / The CnrYXH complex contributes to regulate the expression of the regulatory genes and resistance genes involved in cobalt and nickel resistance in Cupriavidus metallidurans CH34. The binding of nickel or cobalt to CnrX in the periplasm induces conformational modifications that could trigger transmembrane signal transduction. As a result, CnrH is made available in cytoplasm for binding to RNA polymerase. The CnrH:RNA polymerase complex binds specifically the cnr promoters and initiates transcription of both the regulatory genes (cnrYXH) and resistance genes (cnrCBAT). In order to delineate the mechanism of signal transduction through the membrane, I studied the interactions between the three partners in the different cellular compartments: periplasm, plasmic membrane and cytoplasm. The identification of the interaction determinants between CnrX, CnrY and CnrH allowed us to propose a structural and functional model for the CnrYXH complex and its homologues. This model brings up new hypothesis on the function of Cnr system.

A fibrose cística (FC) é diagnosticada na presença de achados fenotípicos, história familiar ou triagem neonatal positiva acompanhada de evidência laboratorial de disfunção da CFTR, seja pelo teste do suor, diferença de potencial nasal ou pela identificação de duas mutações conhecidas como causa de FC nos genes da CFTR. Objetivos: Avaliar a contribuição da análise molecular do gene CFTR na investigação diagnóstica da fibrose cística em pacientes com suspeita de FC leve ou doença atípica. Secundariamente, comparar as características dos pacientes em 3 grupos: grupo com identificação de duas mutações conhecidas como causadoras da FC, grupo com identificação de apenas uma mutação e grupo sem mutação identificada. Métodos: Estudo transversal em adolescentes e adultos (≥14 anos). Os pacientes foram submetidos à avaliação clínica, laboratorial e radiológica; espirometria, microbiologia do escarro, ecografia hepática, teste do suor e análise molecular do gene CFTR. Resultados: Foram avaliados 37 pacientes com achados fenotípicos de FC, com ou sem confirmação pelo teste do suor. Houve predomínio do sexo feminino (75,7%) com média de idade de 32,5 ± 13,6 anos. A análise molecular contribuiu para o diagnóstico definitivo de FC em 3 casos (8,1%) dentre 37 pacientes em avaliação. Em 7 pacientes (18,9%) foram identificadas apenas uma mutação causadora de FC e em 26 pacientes (70,3%) não foram identificadas mutações. Nenhuma característica clínica estudada se associou com o diagnóstico genético. A mutação p.F508del foi a mais comum, encontrada em 5 pacientes. A associação de p.V232D e p.F508del foi encontrada em 2 pacientes. Outras mutações encontradas foram: p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P e p.R1066H e p.T351S. Conclusão: A análise molecular da região codificante do gene CFTR apresentou contribuição limitada para investigação diagnóstica de pacientes com suspeita de fibrose cística leve ou doença atípica. Além disso, não houve associação entre as características clínicas e o diagnóstico genético. / Cystic fibrosis (CF) is diagnosed in the presence of phenotypic findings, family history or positive neonatal screening accompanied by laboratory evidence of CFTR dysfunction, either by sweat test, nasal potential difference or the identification of two mutations known to cause CF in the CFTR gene. Objectives: To evaluate the contribution of molecular analysis of CFTR gene in cystic fibrosis diagnostic investigation in patients with suspected mild FC or atypical disease. Secondarily, to compare the characteristics of patients into 3 groups: group with identification of two mutations known to cause CF, group with identification of just one mutation and group without mutations. Methods: Cross-sectional study in adolescent and adult (≥ 14 years). The patient underwent clinical, laboratory and radiological spirometry, sputum microbiology, liver ultrasound, sweat test and molecular analysis of the CFTR gene. Results: We evaluated 37 patients with phenotypic findings of FC, with or without confirmation by the sweat test. There was a predominance of females (75.7%) with a mean age of 32.5 ± 13.6 years. Molecular analysis contributed to the definitive diagnosis of CF in 3 cases (8.1%) among 37 patients under evaluation. In 7 patients (18.9%) were identified only one mutation that causes CF and in 26 patients (70.3%) were not identified mutations. No clinical feature studied was associated with genetic diagnosis. The P.F508del mutation was the most common, found in 5 patients. The association p.V232D and p.F508del was found in 2 patients. Other mutations found were: p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, and p.P1290P p.R1066H and p.T351S. Conclusion: Molecular analysis of the CFTR gene coding region showed limited contribution to the diagnostic investigation of patients with suspected cystic fibrosis mild or atypical disease. Moreover, there was no association between clinical features and genetic diagnosis.

A fibrose cística (FC) é diagnosticada na presença de achados fenotípicos, história familiar ou triagem neonatal positiva acompanhada de evidência laboratorial de disfunção da CFTR, seja pelo teste do suor, diferença de potencial nasal ou pela identificação de duas mutações conhecidas como causa de FC nos genes da CFTR. Objetivos: Avaliar a contribuição da análise molecular do gene CFTR na investigação diagnóstica da fibrose cística em pacientes com suspeita de FC leve ou doença atípica. Secundariamente, comparar as características dos pacientes em 3 grupos: grupo com identificação de duas mutações conhecidas como causadoras da FC, grupo com identificação de apenas uma mutação e grupo sem mutação identificada. Métodos: Estudo transversal em adolescentes e adultos (≥14 anos). Os pacientes foram submetidos à avaliação clínica, laboratorial e radiológica; espirometria, microbiologia do escarro, ecografia hepática, teste do suor e análise molecular do gene CFTR. Resultados: Foram avaliados 37 pacientes com achados fenotípicos de FC, com ou sem confirmação pelo teste do suor. Houve predomínio do sexo feminino (75,7%) com média de idade de 32,5 ± 13,6 anos. A análise molecular contribuiu para o diagnóstico definitivo de FC em 3 casos (8,1%) dentre 37 pacientes em avaliação. Em 7 pacientes (18,9%) foram identificadas apenas uma mutação causadora de FC e em 26 pacientes (70,3%) não foram identificadas mutações. Nenhuma característica clínica estudada se associou com o diagnóstico genético. A mutação p.F508del foi a mais comum, encontrada em 5 pacientes. A associação de p.V232D e p.F508del foi encontrada em 2 pacientes. Outras mutações encontradas foram: p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P e p.R1066H e p.T351S. Conclusão: A análise molecular da região codificante do gene CFTR apresentou contribuição limitada para investigação diagnóstica de pacientes com suspeita de fibrose cística leve ou doença atípica. Além disso, não houve associação entre as características clínicas e o diagnóstico genético. / Cystic fibrosis (CF) is diagnosed in the presence of phenotypic findings, family history or positive neonatal screening accompanied by laboratory evidence of CFTR dysfunction, either by sweat test, nasal potential difference or the identification of two mutations known to cause CF in the CFTR gene. Objectives: To evaluate the contribution of molecular analysis of CFTR gene in cystic fibrosis diagnostic investigation in patients with suspected mild FC or atypical disease. Secondarily, to compare the characteristics of patients into 3 groups: group with identification of two mutations known to cause CF, group with identification of just one mutation and group without mutations. Methods: Cross-sectional study in adolescent and adult (≥ 14 years). The patient underwent clinical, laboratory and radiological spirometry, sputum microbiology, liver ultrasound, sweat test and molecular analysis of the CFTR gene. Results: We evaluated 37 patients with phenotypic findings of FC, with or without confirmation by the sweat test. There was a predominance of females (75.7%) with a mean age of 32.5 ± 13.6 years. Molecular analysis contributed to the definitive diagnosis of CF in 3 cases (8.1%) among 37 patients under evaluation. In 7 patients (18.9%) were identified only one mutation that causes CF and in 26 patients (70.3%) were not identified mutations. No clinical feature studied was associated with genetic diagnosis. The P.F508del mutation was the most common, found in 5 patients. The association p.V232D and p.F508del was found in 2 patients. Other mutations found were: p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, and p.P1290P p.R1066H and p.T351S. Conclusion: Molecular analysis of the CFTR gene coding region showed limited contribution to the diagnostic investigation of patients with suspected cystic fibrosis mild or atypical disease. Moreover, there was no association between clinical features and genetic diagnosis.

O fluido multifásico complexo (suco de açaí) tem uma forte interação com membranas poliméricas ou cerâmicas de microfiltração e a formação de incrustação depende da composição e das condições de dinâmica de fluidos. Neste estudo experimental foi investigada a influência dos dois principais ácidos graxos presentes no açaí, ácido oleico e ácido palmítico, em misturas com água e no processo de microfiltração tangencial com membranas cerâmicas de alumina com um tamanho de poro nominal de 0,2 \'mü\'m. Mediu-se o fluxo de permeado em função do tempo, nas pressões transmembranas de 300 kPa, 400 kPa e 500 kPa. Para o fluxo da corrente de alimentação foram encontrados valores de Reynolds numa faixa de 9500 a 31000. Cada amostra de misturas de água/ácido oleico, água/ácido palmítico e água/ácidos oleico e palmítico, foi estudada em três séries de ensaios realizados durante 180 minutos e 72 minutos para a mistura água/ácido palmítico, a temperatura em 25 ºC. Analisou-se as incrustações resultantes e as fortes interações fluido/membrana utilizando o modelo de resistência em série e imagens tomadas por microscopia eletrônica de varredura (MEV). Os melhores resultados de permeado encontrados para a mistura de água/ácido oleico foram para Re = 33000, no entanto, resultados satisfatórios foram encontrados para Re = 20000. Agora para a água/ácido palmítico foram encontrados para Re = 20000. Os melhores resultados de permeado para a mistura água/ácidos oleico e palmítico foram para Re = 31000. De acordo com os resultados das resistências, a mistura água/ácido causa um bloqueio dos poros da membrana, resultando em uma maior diminuição do fluxo transmembrana. A limpeza foi eficiente para reduzir a resistência associada com a polarização. / The complex fluid multiphase (açaí juice) has a strong interaction with polymeric or ceramic membranes for microfiltration fouling and its formation depends on the fluid composition and fluid dynamics conditions. In this experimental study was investigated the influence of two major fatty acids present in açaí, oleic acid and palmitic acid in mixtures with water and in the process of crossflow microfiltration with ceramic membranes. In the separation process is used alumina ceramic membrane with a nominal pore size of 0.2 micrometers. The permeate flux was measured in function of time using the 300 kPa, 400 kPa and 500 kPa for the transmembrane pressure. The flow of feed stream and its respective value of Reynolds were in range of: 8900-3300. For each sample of mixtures oleic acid/water and palmitic acid/water and palmitic acid, oleic acid/ water, three series of experiments were conducted for 180 minutes and 72 minutes for mixture palmitic acid/water at temperature in 25 Celcius. For analyze of fouling resulting from strong interactions fluid/membrane was used the model of resistance in series and images taken via scanning electron microscopy (SEM). The best results for mixing oleic acid/water were to Re = 33000, however, satisfactory results were found for Re = 20000. Now for the palmitic acid/water were found to Re = 20000. For mixture palmitic acid, oleic acid/ water were found to Re = 31000. According to the results of the resistances, the mixture oleic acid/water cause a blockage of the pores of the membrane resulting in a greater decrease of the transmembrane flow. The cleaning is efficient for reducing the resistance associated with the polarization.

Orientadores: Antônio Fernando Ribeiro, Jose Dirceu Ribeiro, Francisco Ubaldo Vieira Júnior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T21:55:40Z (GMT). No. of bitstreams: 1 Servidoni_MariadeFatimaCorreaPimenta_D.pdf: 12508528 bytes, checksum: b3b02333b5c69daaf09a90ff457cc849 (MD5) Previous issue date: 2014 / Resumo: A Fibrose Cística (FC) é uma doença genética autossômica recessiva, comum em caucasianos. Tem incidência de 1: 2.500 a 1: 6.000 nascidos vivos e 1: 25 em portadores sãos na Europa e EUA e no Brasil a incidência estimada é de 1:10.000 nascidos vivos. É causada pela presença de dois genes CFTR (do inglês Cystic Fibrosis Transmembrane Conductance Regulator) mutados, que codificam uma proteína também denominada CFTR. A CFTR é o principal canal de Cloro (Cl-), é expressa na membrana apical das células epiteliais dos tratos respiratório e digestório (pâncreas, fígado e intestino), nas glândulas sudoríparas e salivares, e no aparelho reprodutor masculino. Regula o transporte de iôns e de água. O comprometimento ou a ausência da função da CFTR promove a desidratação das mucosas com produção de um muco viscoso com consequente obstrução das vias respiratórias e ductos das glândulas exócrinas determinando o fenótipo da FC. O grau de função da CFTR será determinante da gravidade da doença. Até à data, já foram descritas cerca de 2000 mutações no gene CFTR. A F508del é a mutação mais prevalente, está presente em 85% dos pacientes a nível mundial e em 65% no Brasil. As mutações podem ser classificadas em 6 grupos de acordo com o defeito molecular e celular e determina o fenótipo da FC. Pode ser classificado em: clássico e não-clássico. O clássico é o mais conhecido e frequente e apresenta sintomas graves. O não-clássico ocorre em cerca de 15% dos doentes e apresenta sintomas mais brandos, com diagnóstico em geral complexo e tardio. A FC é assim um "espectro de doenças" e o seu rastreio precoce na triagem neonatal (TNN), antes mesmo dos primeiros sintomas, abre novas perspectivas de prognóstico por isso é emergente a necessidade de métodos acurados que determinem a função da CFTR, direcionando uma terapia individualizada, em busca da cura. A primeira parte deste trabalho procurou consolidar a medição da função do canal CFTR em biopsias retais como um marcador biológico para diagnóstico e prognóstico da FC; a segunda descreveu a realização da biópsia retal e suas particularidades sob a ótica dos pacientes e da técnica. A terceira abordou a realização do teste do suor (TS) no estado de São Paulo (SP) expressando o panorama brasileiro do TS. Desta forma, entre 2007 e 2010 foi realizado estudo prospectivo de pacientes atendidos no ambulatório de FC do Hospital das Clínicas (HC) da Universidade Estadual de Campinas (Unicamp) com e sem FC submetidos à biópsia retal. Em 2013 foi aplicado em 14 serviços (9 públicos, 5 privados) que realizam o TS, um questionário qualitativo através de visita às sete cidades que contam com Centros de Referência para atendimento de pacientes com FC em SP. Nossos resultados demonstraram que a determinação de Cl- em biópsias retais mediadas pela CFTR é um biomarcador robusto, sensível, preditivo e reprodutível para o diagnóstico e prognóstico da FC e com potencial uso para ensaios pré-clínicos de terapias moduladoras da CFTR. A pinça jumbo e a solução salina fisiológica determinaram as melhores amostras para os estudos bioquímicos e de eletrofisiologia, a grande maioria dos indivíduos entrevistados não relataram maiores desconforto (76%), sendo a técnica utilizada segura e reprodutível. O estudo do TS em SP demonstrou a necessidade urgente de equipamentos adequados de estimulação e dosagem do Cl- no suor, associado à normatização da técnica e treinamento de pessoal capacitado para a sua realização. Dando seguimento a este trabalho, estamos implementando novas ferramentas diagnósticas para a FC: a avaliação eletrofisiológica da CFTR em câmara de Ussing através da cultura de células nasais e/ou organoides e da unção da CFTR na glândula sudorípara pelo evaporímetro. Por fim, todos os métodos de avaliação diagnóstica devem respeitar procedimentos operacionais padrão (POP), sendo que alguns nomeadamente os de eletrofisiologia, ainda dispõem de aplicação limitada a poucos centros no mundo / Abstract: Cystic Fibrosis (CF) is an autosomal recessive genetic disease, common among Caucasians. In Europe and USA, it has an incidence of 1:2,500-1:6,000 in newborns and 1: 25 for healthy carriers. In Brazil, the estimated incidence is 1:10,000 in newborns. It is caused by the presence of two mutated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) genes encoding for CFTR protein, a Chloride (Cl-) channel expressed at the apical membrane of epithelial cells. CFTR is the main regulator of ion transport and water. Its defect leads to dehydrated epithelia and to the production of viscous mucus secretions that clogs the airways and ducts of exocrine glands, leading to the clinical manifestations of CF disease, mostly affecting the respiratory and digestive tracts (pancreas, liver and intestine). CFTR is also expressed in the sweat and salivary glands, and in the male reproductive system. The degree of CFTR function will determine the severity of the disease. About 2000 mutations have been already described in the CFTR gene. The F508del is the most prevalent, present in 85% of patients worldwide and 65% in Brazil. Mutations can be classified into six groups, depending on the molecular and cellular defect, and also determining the severity of the CF phenotype: Classical and Non-Classical. The Classical phenotype is best-known and frequent, presenting severe symptoms; but the Non-Classical phenotype, representing ~15 % of all CF patients, shows atypical symptoms, with variable organ involvement, which make the diagnosis difficult and often late. CF thus includes a "spectrum of diseases" and its early detection in newborn screening, even before the first symptoms, opens up new perspectives for prognosis. Since CF diagnosis requires proof of CFTR dysfunction, there is an emerging need for accurate methods capable of detecting CFTR function with high sensitivity and of directing CF therapy, in the quest for the most appropriate treatment. The first part of this study sought to consolidate the measurements of CFTR channel function in rectal biopsies as a biomarker for CF diagnosis and prognosis. The second part focused on the rectal biopsies procedure and its technical aspects and also on how it is perceived in the patients' perspective. The third part, approached how the sweat test (ST) procedure is carried out in CF centers in the state of São Paulo (SP), so as to assess the Brazilian scenario for the ST. To this end, between 2007 and 2010, we conducted a prospective study of patients seen at CF outpatient clinic, of the Clinical Hospital (HC) ¿ State University of Campimas (Unicamp) who underwent rectal biopsy and we also included non-CF subjects as controls. In 2013, a qualitative questionnaire was applied to 14 services (9 public, 5 private) which perform the ST by visiting the 7 cities of SP which have reference CF care centers. Data shown that determination of CFTR-mediated Cl- secretion in rectal biopsies proved to be a robust, sensitive, and reproducible predictive biomarker for CF diagnosis and prognosis, besides being a safe technique with the potential for use in preclinical trials of CFTR modulating therapies. The jumbo forceps and saline solution determined the best samples for electrophysiology and biochemical studies. Moreover, the great majority of the individuals tested by this procedure did not report major discomfort (76%). The work assessing the achievement of ST in SP, demonstrated an urgent need for adequate equipment for the stimulation of sweat and also for the measurement of Cl- in sweat, associated with standardization and training of specialized personnel for its implementation. As a follow up of this work, we are already implementing new diagnostic tools for CF, namely: the study of CFTR function in the sweat gland by the evaporimeter and in cultured nasal cells by Ussing chamber. Finally, all diagnostic methods must comply with strict standardized operation procedures (SOP) and some, including electrophysiology, still have limited use in few centers worldwide / Doutorado / Saude da Criança e do Adolescente / Doutora em Ciências