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1
Demolombe, Sophie. "Cftr : ou cystic fibrosis transmembrane conductance regulator." Paris 11, 1996. http://www.theses.fr/1996PA112463.
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La mucoviscidose, maladie genetique grave la plus frequente dans les populations europeenne et nord-americaine, est caracterisee par un defaut de transport d'ions chlore par les cellules epitheliales. Cette affection, transmise selon le mode autosomique recessif, est liee a la mutation du gene cf codant pour la proteine cftr (cystic fibrosis transmembrane conductance regulator) qui exerce un role de canal chlore regule par l'atp intracellulaire et l'ampc. La mutation la plus frequente (70% des alleles mutes) est une deletion de la phenylalanine en position 508 qui conduit a un defaut d'adressage de la proteine neoformee. Par des techniques d'immunomarquages faisant appel a differents anticorps monoclonaux, nous avons montre que l'anomalie moleculaire caracteristique de la mutation f508 est retrouvee dans la lignee cfpac-1 issue d'un carcinome pancreatique preleve chez un patient homozygote pour cette mutation. Cfpac-1 est une lignee cellulaire couramment utilisee pour les investigations biochimiques, physiologiques et pharmacologiques de l'insuffisance cellulaire responsable de la mucoviscidose. Nous avons developpe une nouvelle methode permettant d'evaluer l'efficacite de la complementation par transfert de gene de l'epithelium respiratoire mucoviscidosique. Le principe de cette methode consiste a detecter les proteines cftr recombinantes correctement adressees dans la membrane apicale a l'aide d'un anticorps dirige contre la premiere boucle extracellulaire et utilise sur des cellules vivantes. Cette methode est suffisamment sensible pour etre utilisee sur un petit nombre de cellules prelevees par simple curetage de la surface epitheliale. La fonction de la proteine cftr ne se limite pas a celle de canal chlore. Par un mecanisme inconnu, cftr exerce un role regulateur d'autres canaux ioniques, chlorures et sodiques epitheliaux. Nous avons mis en evidence la regulation par cftr de canaux potassiques epitheliaux rectifiant dans le sens entrant et dont le controle par la voie de l'ampc depend de la presence d'une proteine cftr fonctionnelle. Ces canaux k+ interviendraient dans le controle du potentiel membranaire et participeraient au maintien d'un gradient electro-chimique indispensable a la vectorisation transepitheliale du chlore
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Tucker, Stephen John. "Studies on the cystic fibrosis transmembrane conductance regulator." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357427.
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Marrs, Kevin L. "The cystic fibrosis transmembrane conductance regulator regulation by HSP-90 /." View the abstract Download the full-text PDF version, 2007. http://etd.utmem.edu/ABSTRACTS/2007-031-Marrs-index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.Title from title page screen (viewed on July, 18, 2008). Research advisor: Anjaparavanda Naren, Ph.D. Document formatted into pages (xv, 72 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 66-72).
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Glanville, Michael. "The molecular basis of renal tubular anion secretion." Thesis, University of Newcastle Upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273477.
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Bhura-Bandali, Farah. "The cystic fibrosis transmembrane conductance regulator in essential fatty acid metabolism." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq22572.pdf.
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GARCIA-FONKNECHTEN, NORIA. "Etude des transcrits du gene cftr. (cystic fibrosis transmembrane conductance regulator)." Paris 7, 1993. http://www.theses.fr/1993PA077156.
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Le gene cftr (cystic fibrosis transmembrane conductance regulator), constitue de 27 exons repartis sur 250 kb et dont l'alteration est responsable de la mucoviscidose, est essentiellement exprime dans les epitheliums respiratoires et digestifs. Cette expression est indetectable par northern-blot dans des cellules facilement accessibles comme les lymphocytes. Cependant, par la technique d'amplification de l'adnc par nested-pcr, nous avons mis en evidence et quantifie les transcrits cftr dans les lymphoblastes (transcription illegitime). Bien qu'ils soient en tres faible quantite, par cette methodologie nous obtenons du materiel en quantite suffisante pour l'analyse moleculaire de ces transcrits. C'est par cette analyse chez un patient homozygote pour la mutation majoritairement retrouvee dans cette maladie (f508), que nous avons prouve que ces transcrits etaient une source de materiel pathologique. Tres encourages par nos premiers resultats et afin d'etudier la totalite du transcrit cftr, nous avons developpe un protocole permettant d'amplifier l'adnc codant (4,2 kb) en six fragments chevauchants. Utilisant cette strategie nous exposons dans ce travail trois applications de cette methode sur l'arn obtenu a partir des lymphoblastes des patients atteints de mucoviscidose: (1) detection de mutations ponctuelles sur de grands fragments d'adnc, ce qui permet d'explorer plusieurs exons de facon simultanee; (2) mise en evidence de transcrits anormaux resultant d'une mutation d'epissage; (3) detection de transcrits anormaux, s'agissant le plus souvent de la perte d'exons, chez des malades ne presentant aucune mutation d'epissage. Ces transcrits seraient issus d'un epissage aberrant ou alternatif. Des transcrits depourvus des exons 9 ou 12 ont ete egalement detectes chez les sujets normaux. Enfin, pour mieux comprendre ce phenomene d'epissage alternatif ou aberrant nous avons etudie les transcrits depourvus de l'exon 9 dans le pancreas humain normal, l'un des sites d'expression du gene cftr. Cette etude nous a permis la mise en evidence de tels transcrits dans les tissus pancreatiques a differents ages du developpement embryonnaire, ainsi que chez l'adulte
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Qureshi, Emili Alia. "Expression and purification of transmembrane segments 3 and 4 of the cystic fibrosis transmembrane conductance regulator." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0007/MQ29199.pdf.
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Hull, Jeremy. "Mutation analysis and screening in the cystic fibrosis transmembrane conductance regulator gene." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260734.
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Holleran, John. "Fluorescence Platform Development for Detection of Cystic Fibrosis Transmembrane Conductance Regulator Trafficking." Research Showcase @ CMU, 2011. http://repository.cmu.edu/dissertations/97.
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Cystic fibrosis is caused by mutations in the membrane chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, disrupts protein folding resulting in premature degradation which precludes expression at the cell surface. Therapeutic strategies have been developed to rescue ΔF508 by using small molecules, called correctors, which promote folding and trafficking to the surface. Currently, the discovery and evaluation of these correctors requires indirect functional measurements or time intensive biochemical methods. In order to facilitate faster analysis of corrector compounds and provide a screening assay that directly monitors rescue of ΔF508 trafficking, we developed a rapid fluorescence detection platform using fluorogen activating proteins (FAPs) capable of labeling of CFTR at the cell surface. We created two chimeric reporter constructs by fusing the FAP to the N-terminus, or by insertion of the FAP into the fourth extracellular loop. We expressed these constructs in HEK293 cells and verified that the ion transport function, biochemical properties and cellular localization reproduced the native behavior of CFTR. Under normal conditions, CFTR ΔF508 is absent from the surface, however incubation in the presence of correctors restored trafficking to the plasma membrane that was robustly detected by FAP fluorescence. Using this approach we have characterized the efficacy of two new corrector compounds C548, C951 and the well-studied reference corrector, C4. The most potent corrector identified was C951 which performed 2 fold better than the previously described, C4 corrector. Other studies have shown that combinations of correctors exhibit an additive or synergistic effect, therefore, we tested combinations of correctors using FAP-CFTR constructs and found they had a synergistic effect on the rescue of ΔF508, improving the density of protein at the cell surface 4 fold greater than C4 alone. These results correlated closely with functional data obtained from polarized human bronchial epithelia that endogenously express ΔF508, suggesting that the FAP tagged CFTR reporters represent a physiologically faithful model of corrector rescue.
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Al, Salmani Majid Khamis Ali. "Functional studies of rare mutations in the cystic fibrosis transmembrane conductance regulator." Thesis, University of Bristol, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738550.
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Gokce, Isa. "Expression of CFTR and its transmembrane domains in E.coli and yeast." Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299633.
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Rowntree, Rebecca Kate. "Regulation of expression of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393265.
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Zeltwanger, Shawn. "Gating of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels by nucleoside triphosphates." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9924950.
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Meng, Xin. "Thermal stability and structural studies of the human Cystic Fibrosis Transmembrane Conductance Regulator." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/thermal-stability-and-structural-studies-of-the-human-cystic-fibrosis-transmembrane-conductance-regulator(67bcfde0-5a49-4fcf-bfd2-2a888e951bf8).html.
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The cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) is a member of the ATP-binding cassette (ABC) membrane transporter family that has evolved as an ion channel which is directly linked to the disease cystic fibrosis (CF). Cystic fibrosis, the most common inherited disease in populations of European descent, is due to mutations causing loss of CFTR function. Drugs targeting some channel-disrupting mutations such as G551D have recently been approved, but a structure for CFTR that could aid drug development is needed. In this thesis, G551D, the second most frequent CF missense mutation was chosen for study because of its affect on gating. The main aims were to compare the mutated protein with wild-type CFTR and other mutations; to use the protein as a negative control in assays of CFTR function and lastly to lock the CFTR protein in a closed state. The latter aim was pursued in order to test the hypothesis that a locked channel could be more stable and hence better suited for biophysical and structural studies including the crystallization of the protein. This thesis presents the generation, expression and purification of milligram quantities of the human G551D CFTR protein in a Saccharomyces cerevisiae (yeast) expression system. Two detergents, dodecylmaltoside (DDM) and lyso-phosphatidyl glycerol (LPG), were used to solubilize and extract CFTR and subsequent purification was by nickel affinity, FLAG affinity and size exclusion chromatography (SEC). In LPG, approximately 20 mg CFTR was recovered from an 18 L fermenter. Thermal stability of wild-type CFTR and the effects of different human CFTR mutations were studied using two different measurements, thermal gel analysis and coumarin maleimide binding (CPM binding). In the thermal stability studies, the additional effects of the two approved CFTR drugs, VX-770 and VX-809, were studied. G551D CFTR was shown to be more stable than WT and F508del CFTR. CFTR purified in the two detergents was also compared using the thermal stability assay and the relationship between stability and structure/function will be discussed. The CPM assay presented here was developed into a medium-throughput assay of potential use for CFTR modulator drug screening. The LPG-purified CFTR was a homogenous population of monomeric particles as judged by electron microscopy (EM) studies and could be concentrated to up to 30 mg/ml. A low resolution structure of G551D CFTR was initially generated by single-particle cryo-EM at about 13 A and a recently-obtained higher-resolution structure (at 6.5 A) will also be presented and discussed. The structures show that G551D CFTR displays an outward-facing conformation even in the absence of nucleotide (ATP), which is a novel finding for ABC transporters. The two nucleotide-binding domains interact closely over a wide interface. LPG-purified G551D CFTR was also used in the 3D crystallization trials and the prospects for using an X-ray crystallographic approach for structure determination are discussed.
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Zhang, Zhihui. "Assembly and Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator and Associated Proteins." UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/101.
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Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which is believed to affect the folding and stability of CFTR. The misfolded ΔF508-CFTR undergoes ER associated degradation (ERAD), causing the failure of ΔF508-CFTR trafficking to the cell surface. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. In this dissertation, real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however the amount of channel in each vesicle remained the same. Therefore, a 2 step therapeutic approach might be an ideal treatment for CF. The first step would be composed of a compound that mimics the mechanism of stabilization provided by low temperature or the I539T mutation, while the second step would be VX-809 or a similar corrector compound. These studies suggest that understanding how low temperature and second site suppressors alter ΔF508-CFTR could be key to the development of future therapeutics for the effective treatment of CF.
The precise pathophysiology of cystic fibrosis is not well studied. The involvement of another transport protein, epithelial sodium channel (ENaC), makes the situation more complicated. ENaC and CFTR are colocalized on the apical surface of epithelia cells. With our fluorescence microscopy techniques, we explored the effects of CFTR on the residence time of ENaC on the cell membrane. A reliable approach measuring the half-life of protein on the cell membrane is required for this study. We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Photoconversion of Dendra2 works as a pulse chase experiment by monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. The half-life of the protein on the plasma membrane was calculated upon imaging over time and quantifying the change in red fluorescence. Our method provides a unique opportunity to observe real-time protein turnover at the single cell level without addition of protein synthesis inhibitors. This technique will be valuable for the future protein half-life study.
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England, Alice. "The regulation of the cystic fibrosis transmembrane conductance regulator in human respiratory epithelia." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/3788/.
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Cystic fibrosis transmembrane conductance regulator (CFTR) is activated by cAMP-dependent phosphorylation, and functions as an ATP-dependent chloride channel involved predominantly in the movement of chloride ions to maintain cellular ionic homeostasis. Mutations in this channel cause cystic fibrosis, the most common lethal autosomal recessive disease in caucasians. The regulation of CFTR forms a large body of research; this thesis investigated the role of three potential components of the regulatory machinery – nucleoside diphosphate kinase B (NDPK-B), tyrosine phosphorylation and G proteins. This thesis aimed to: 1. Describe the functional relationship between NDPK-B and CFTR. 2. Describe the effect of altered tyrosine phosphorylation in 16HBE14o- cells on CFTR function. 3. Identify the tyrosine phosphatases involved in CFTR regulation. 4. Explain how alterations in tyrosine phosphorylation change CFTR function. 5. Describe the effect of G protein stimulation on CFTR channels which have already been activated by an increase in cellular cAMP. The whole cell patch clamp technique was used to examine ion channel function in two cell types - human bronchial epithelial cells (16HBE14o-) and baby hamster kidney cells (BHK-21). Due to alterations in the function of cultured cells, it was not possible to describe the functional relationship between the histidine kinase NDPK-B and CFTR. Further work is required in this area to elucidate the role of this protein in CFTR regulation. The use of general tyrosine phosphatase inhibitors resulted in a significant decrease in the CFTRinh-172-sensitive conductance in 16HBE14o- cells, and specific inhibitors ruled out the involvement of PTP1B and Shp1/2 phosphatases. Further work is required to explain how tyrosine phosphatase inhibition alters CFTR function. Finally, G protein stimulation in 16HBE14o- after increasing intracellular cAMP had no significant effect on channel function. This suggested that the cAMP-dependent activation of the channel is the predominant mechanism for stimulating channel function.
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Dal'Maso, Vinícius Buaes. "Contribuição da análise molecular do gene CFTR na investigação diagnóstica de pacientes com suspeita de fibrose cística leve ou doença atípica." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2012. http://hdl.handle.net/10183/79587.
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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.
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Davies, Lee. "The electrical manipulation of bio-formulations for delivery to the lung." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365799.
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Witt, William T. "The Expression and Characterization of Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Tobacco." Thesis, Virginia Tech, 2003. http://hdl.handle.net/10919/33504.
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The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by â leakyâ expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR.Master of Science
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Hinkson, Deborah Anne Rochelle. "The mechanism of protein kinase C regulation of the cystic fibrosis transmembrane conductance regulator /." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33407.
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane chloride channel expressed in epithelial tissues. While protein kinase A (PKA) is the main activator of CFTR, patch clamp data have suggested a permissive role for PKC in modulating PKA responses. To clarify the role of PKC in CFTR regulation we used PCR mutagenesis to construct a CFTR mutant (9CA) in which the serines or threonines in nine consensus sequences for PKC phosphorylation were replaced by alanines. Its expression in stably transfected baby hamster kidney (BHK) cells was consistently lower than that of wild-type (WT) CFTR. Nevertheless, 9CA cells displayed a robust cAMP-stimulated iodide efflux which was comparable in magnitude to that of WT CFTR cells when normalised for protein expression level. The cpt-cAMP concentration-response relationship in intact cells was similar for WT and 9CA CFTR (EC50 ≈ 100muM), however the onset of iodide efflux from cells expressing the mutant was markedly delayed at all cpt-cAMP concentrations tested. This delay was mimicked by pre-treating WT cells with the PKC inhibitor chelerythrine (10muM), and chelerythrine did not cause further slowing of iodide efflux from 9CA cells. In vitro phosphorylation by PKA and PKC was similar for WT and 9CA CFTR and phosphorylation in the presence of both kinases was additive.A major impediment to understanding CFTR structure/function has been the difficulty of obtaining sufficient purified CFTR protein for biochemical studies. In an attempt to overcome this problem we expressed a His-tagged CFTR construct in the methylotrophic yeast Pichia pastoris. The final yield of CFTRHis10 following solubilization of Pichia membranes in 0.5% lysophosphatidylglycerol (LPG) and nickel chelate chromatography was ∼20mug/litre. CFTRHis10 was not glycosylated in this yeast system. Both PKA and PKC phosphorylated semi-purified CFTRHis10 in vitro and phosphorylation in the presence of both kinases was additive.
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Seibert, Fabian S. "Structure-function relationships of the cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0005/NQ27718.pdf.
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Simpson, Janet Elizabeth. "The cystic fibrosis transmembrane conductance regulator and acid-base transporters of the murine duodenum." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4391.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2006" Includes bibliographical references.
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Le, Drévo Marie-Anne. "L'Annexine A5 : une nouvelle protéine régulatrice du canal CFTR (cystic fibrosis transmembrane conductance regulator)." Brest, 2007. http://www.theses.fr/2007BRES3201.
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Le cystic fibrosis transmembrane conductance regulator (CFTR) est un canal chlorure cAMPdependant dont la fonction peut-être régulée par des interactions protéine-protéines. Cependant, on connaît encore mal l’étendue et l’importance de ces intéractions. Sachant que I’ annexine A5 (anxA5) est surexprimée dans la mucoviscidose et que l’anxA5 et le CFTR partagent certaines propriétés, nous avons émis l’hypothèse selon laquelle ces deux protéines interagissent physiquement et/ou fonctionnellement. Dans la première partie de cette thèse, nous montrons pour la première fois que l’anxA5 se lie au domaine NBD1 (nucleotide-binding domain 1) du CFTR et que cette interaction est renforcée en présence de Ca2+ et d’ATP. Nous montrons également que la diminution de l’expression de l’anxA5 est corrélée avec une diminution de la fonction du canal CFTR. Nous concluons donc que l’anxA5 est nécessaire au fonctionnement normal du CFTR. Dans une seconde partie, nos résultats indiquent que la surexpression de l’anxA5 permet de corriger partiellement le défaut d’expression du canal CFTR dans la membrane plasmique induit par la mutation ΔF508, la mutation la plus fréquente. De plus, nous montrons que l’augmentation de la concentration intracytosolique en Ca2+, induite par la thapsigargine, permet de potentialiser l’effet de I ‘anxA5. L’ensemble de ces résultats suggère que l’anxA5 peut être envisagée comme une cible thérapeutique potentielleThe cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel which is regulated by protein-protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin A5 (anxA5) is overexpressed in cystic fibrosis (CF) and given the functional properties of anxA5 and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. In the first part of this thesis, we show for the first time that anxA5 is associated with nucleotide-binding domain 1 (NBD1) of CFTR and that this interaction is Ca2+ and ATP-dependent. The decreased anxA5 expression was correlated with a decreased CFTR chloride channel function. We concluded that anxA5 is necessary for normal CFTR chloride channel activity. In the second part of this thesis, our results indicated that the overexpression of anxA5 permitted to partially correct the ceIl surface expression defect of the misfolded ΔF508-CFTR, the most common mutation. Moreover, we show that the increase of intracytosolic Ca2+ concentration, induced by a thapsigargin treatment, allowed to increase the anxA5 effect. All these results suggest that anxA5 may be seen as a potential therapeutic target
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Jurkuvenaite, Asta. "Biogenesis, trafficking, and function of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR)." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2009r/jurkuvenaite.pdf.
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Guimbellot, Jennifer S. "Role of hypoxia in epithelial gene regulation." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/guimbellot.pdf.
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Singer, Thomas David. "The cloning and characterization of killifish Fundulus heteroclitus cystic fibrosis transmembrane conductance regulator (CFTR) homolog." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362034.
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Davies, W. L. "Molecular and functional investigation of the cystic fibrosis transmembrane conductance regulator (CFTR) in rabbit heart." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598353.
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The heterogeneous distribution of ion channels in the heart is critical for the co-ordinated conductance of electrical activity in the myocardium. Disturbances in the normal patterns of ion channel expression, in response to hypertrophic stimuli, are thought to underlie the markedly increased risk of arrhythmias in hypertrophied and failing hearts. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is expressed in the heart with an epicardial (higher) to endocardial (lower) gradient and is thought to contribute to the maintenance of the normal epicardial to endocardial gradient of repolarisation in the heart during adrenergic stress. CFTR can also modulate the activity of other ion channels. However, whether CFTR functions in this way in the heart remains unclear. CFTR exhibits many interesting features at a molecular level. In native tissue and cell lines, CFTR sustains diverse transcription start site utilisation, accompanied by differential 5' UTR usage. In adult rabbit left ventricle, the predominantly expressed CFTR isoform is the exon 5 minus alliteratively spliced variant. The objective of this study was to analysis the regional distribution of CFTR alternatively spliced variants in normal and hypertrophied hearts and to investigate its developmental regulation. Our results ascertain, for the first time, the CFTR transcripts in the heart possess multiple transcription start sites and display alternative splicing of the 5' UTR as well as exon 5. Furthermore, our results indicate that the resulting patterns of gene expression are tissue-specific, developmentally regulated and influenced by pathological stimuli. Additionally, the alternatively spliced variants display different functional characteristics that may have important implications for our understanding of CFTR function in both cardiac and epithelial tissues.
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Cai, Weisong, and 蔡蔚松. "Cystic fibrosis transmembrane conductance regulator is involved in therelease of ATP from contracting skeletal muscle." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49618088.
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Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions.
Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions.
Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction.
These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction.published_or_final_version
Physiology
Master
Master of Philosophy
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Rimington, Tracy L. "Expression, purification and characterisation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in Saccharomyces cerevisiae." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/expression-purification-and-characterisation-of-the-cystic-fibrosis-transmembrane-conductance-regulator-cftr-in-saccharomyces-cerevisiae(5c8c606b-8925-4627-91dc-67a896b9f286).html.
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Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.
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Huguet, Florentin. "Impact de la modulation de TRPM7 et ATF6 sur le cystic fibrosis transmembrane conductance regulator." Thesis, Brest, 2017. http://www.theses.fr/2017BRES0058/document.
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La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551DCystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D
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Woods, Parker. "THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) CHANNEL AS A HOST DETERMINANT OF INFLUENZA SEVERITY." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1459850990.
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Arruda, Leonardo Vicentini. "Incidenicia da fibrose cistica calculada atraves de portadores do alelo ?F508 no Nordeste e Sudeste do Brasil." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/308589.
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Orientador: Carmen Silvia BertuzzoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A incidência da fibrose cística no Brasil é significativamente variável, com diferenças de até 20 vezes de acordo com o grupo étnico e região geográfica estudada. A população brasileira é composta da mistura de muitos grupos étnicos. Os portugueses começaram a colonização no século XVI. Os holandeses invadiram o nordeste em 1630. Os africanos foram trazidos ao Brasil, numa contínua migração forçada, que perdurou do século XVI ao século XIX. No final do século XIX, tiveram início novos movimentos migratórios, principalmente da Alemanha, Itália, Arábia e Espanha. Durante as três primeiras décadas do século XX, nova corrente migratória ocorreu, principalmente da Itália, Espanha e Portugal Após a segunda guerra mundial, o Brasil recebeu novos imigrantes (japoneses, judeus) compondo esta população. Este estudo gerou os primeiros dados sobre a incidência da FC no nordeste e também foram obtidos novos dados para a região sudeste. Na época do estudo, na cidade de Campinas estão sob atendimento no ambulatório 70 pacientes não aparentados com dois testes de suor alterados. Nestes pacientes, foram triadas as seguintes mutações. ?F508 (50%), G542X (4,29%), R1162X (2,14%), N1303K (1,43%) e R553X (0,71%). A mutação G551D não foi encontrada. A mutação ?F508 também foi analisada em 1.138 mulheres saudáveis, sendo 694 da cidade de Campinas - SP e 444 de João Pessoa ¿ PB com idade média de 26,3 anos (15-39, ±6,8), que participaram voluntariamente de projeto de pesquisa anterior. Nas amostras coletadas em Campinas n=694 não foi encontrado nenhum alelo mutante 0/1.388, o que nos impediu de calcular a incidência nesta cidade através deste método. Dos 888 alelos analisados de João Pessoa, foram encontrados quatro alelos mutantes (p=0,0045). Sabendo que a mutação ?F508 corresponde a aproximadamente 50% dos alelos de indivíduos com FC no Brasil, a freqüência dos alelos causadores da FC foi estimada utilizando a proporção: (0,0045/0,5)=0,0090. Com isso, para a cidade de João Pessoa a incidência estimada desta doença autossômica recessiva é de 1:12.321 indivíduos. Esta incidência é similar à encontrada por afro-brasileiros, entretanto difere por exemplo, da encontrada na população do RS. Quando utilizamos o método de cruzamento de dados étnicos das duas regiões estudadas com dados literários da doença nos diferentes grupos étnicos, na cidade de Campinas a incidência da FC ficaria em 1/4.434 e na cidade de João Pessoa ficaria 1/6.087
Abstract: The incidence of the Cystic Fibrosis (CF) is significantly variable in Brazil, with differences larger than 20 fold, according with the ethnic group and geographic studied region. Brazilian population is composed by ethnic admixture. Portuguese started colonization in the 16th century. The Netherlander invaded the northeast in 1630. The Africans were brought to Brazil, in a continuous forced migration, which lasted from 16th to 19th centuries. In the 19th century, new migratory movements have begun from Germany, Italy, Arab and Spain. In the first three decades of the 20th century, started a new migratory flow, mainly from Italy, Spain and Portugal. After the World War II, Brazil received additional immigrants (Japanese, Jewish) compounding its population. These studies generated the first data about the CF incidence on the Brazilian northeast and also were obtained new data about the southeast region. At the time of this study, 70 non related patients were attended at the local CF center in Campinas, with two positive sweat tests in the city of Campinas-SP. On theses patients were screened the following mutations: ?F508 (50%), G542X (4.29%), R1162X (2.14%), N1303K (1.43%) and R553X (0.71%). The mutation G551D wasn¿t found. The ?F508 mutation was also analyzed in 1,138 healthy voluntary women, 694 from Campinas ¿ SP and 444 from João Pessoa ¿ PB, with average age of 26.3 years (15-39, ±6.8), who previously participated from another research. In the samples collected in Campinas ¿ SP n=694 wasn¿t found any mutated allele 0/1,388 and so, we wasn¿t able to make any incidence calculation through this method. In the 888 alleles from João Pessoa, four carry the ?F508 mutation (p=0.0045). Knowing that this mutation accounts for approximately 50% of the FC patients alleles in Brazil, the incidence of the CF in this region was estimated using the proportion: (0.0045/0.5)=0.009. Thus, the estimated incidence of this recessive disease in João Pessoa was 1:12,321. This incidence is similar to the found in African-Brazilians, although differs for example, to the found on the RS population. When we use the method of crossing ethnic data of both studied regions with literary data of the disease in the different ethnic groups, in the city of Campinas, the incidence of the CF would be in 1/4,434 and in the city of João Pessoa would be 1/6,087
Mestrado
Mestre em Farmacologia
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Tryansky, Jonathan N. "Characterization of the interaction between the cystic fibrosis transmembrane conductance regulator and protein phosphatase type-2C." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29482.
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Cystic Fibrosis transmembrane conductance regulator (CFTR) Cl - channels are rapidly and efficiently deactivated by a type 2C serine/threonine phosphatase (PP2C). The efficiency of this regulation suggests that PP2C and CFTR are associated within a regulatory complex. Previous work has shown that PP2C and CFTR can be co-immunoprecipitated and reversibly coupled by exposing cell lysates to the hydrophilic cross-linker dithiobis [sulfosuccinimidyl proprionate]. While it is clear that PP2C can associate with CFTR, the nature of the interaction between them has not been defined. The goal of this study was to identify specific region(s) within CFTR that mediate this interaction. The interaction between CFTR and PP2C was strong enough to be reproduced in the absence of cross-linking agents, supporting its physiological relevance. We then translated CFTR in vitro and incubated it with cell lysates containing endogenous PP2C, however interaction between the proteins was not observed under these conditions. Since the association seemed to require some factor present in cells, a distal fragment of CFTR was expressed in vivo and used for pull down assays. PP2C was co-purified under these conditions, suggesting an interaction between PP2C and the C-Tail of CFTR. To further narrow down the region of interaction, three residues within the C-Tail (Y1424, LL1430/1431) that had been shown previously to interact with the AMP-activated protein kinase (AMPK, a known PP2C substrate and possible targeting protein) were mutated to alanines. When this mutant was used in a pull-down assay, no association with PP2C was observed, consistent with a role for AMPK-binding residues and AMPK in the association of PP2C with CFTR.
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Alzahrani, Ateeq Ahmed Hassan. "Structural biology of Cystic Fibrosis Transmembrane Conductance Regulator, an ATP-binding cassette protein of medical importance." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/structural-biology-of-cystic-fibrosis-transmembrane-conductance-regulator-an-atpbinding-cassette-protein-of-medical-importance(b8d020d3-24d7-474a-afb5-a112e38ac027).html.
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that functions as an ion channel. Mutations in this protein cause Cystic Fibrosis. For this reason, it is important to study the structure and function of CFTR. In this study, constructs of CFTR (C-terminii), a CFTR-interacting protein and full-length CFTR were cloned, expressed and purified for structural and functional studies. The purified C-terminal polypeptides of CFTR were soluble and shown to interact with NHERF1 PDZ 1 (a CFTR-interacting protein). The CFTR C-terminus and NHERF1 PDZ 1 domain were co-expressed and co-purified. The purified complex showed a strong interaction that might induces a conformational change. Site-directed mutation of the C-terminus of CFTR was performed in order to examine the effect of removing a potentially flexible amino acid (Arginine) on protein crystallization. Pull-down assay experiments with full-length CFTR demonstrated an interaction between CFTR (in DDM detergent) and NHERF1 PDZ 1(+). No interaction was observed for CFTR in LPG (a relatively denaturing detergent) and NHERF1, implying that the interaction between the PDZ motive of CFTR and NHERF1 requires a stable folded structure for both proteins. In addition, full-length CFTR in DDM has been studied by electron microscopy and Single Particle Analysis in the presence of NHERF1 PDZ 1(+). A 3D structure was generated for the CFTR-NHERF1 PDZ 1(+) complex at a resolution of ~ 18 A. This 3D structure showed a new open conformation of CFTR (V shape). In comparable studies with CFTR alone, a 3D structure was generated at a resolution of 27 A and this structure showed a closed state as previously reported. This new data suggest a possible role for NHERF1 in terms of CFTR channel gating or activation.
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Wrennall, Joe Alexander. "An investigation of the role of the cystic fibrosis transmembrane conductance regulator in epithelial wound healing." Thesis, University of Bristol, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738548.
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Missaglia, Mariangela Tuzzolo. "Pesquisa de mutações no gene CFTR (Cystic Fribrosis Transmembrane Conductance Regulator) em homens brasileiros inférteis portadores de ausência congênita dos ductos deferentes (CAVD)." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-03062009-092152/.
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A Fibrose Cística (FC) é a doença autossômica recessiva mais freqüente em caucasianos e está associada, em seu amplo espectro de apresentação clínica, a mais de 1500 mutações no gene CFTR (Cystic Fibrosis Transmembrane conductance regulator). O papel de CFTR é especialmente relevante no desenvolvimento da porção reprodutiva dos ductos mesonéfricos. Em 98% dos pacientes masculinos portadores da FC, mutações em CFTR são responsáveis pela ausência bilateral congênita dos ductos deferentes (CBAVD), associada à anomalias variáveis das vesículas seminais, ductos ejaculatórios e da porção distal dos epidídimos. A ausência uni ou bilateral congênita dos ductos deferentes (CAVD), na ausência de outros sinais clínicos de FC, é conhecida causa de infertilidade masculina, presente em 1%-2% de todos os homens inférteis, e em cerca de 10% dos azoospérmicos. A reprodução assistida utilizando a injeção intracitoplasmática de espermatozóides (ICSI) obtidos preferencialmente por aspiração microcirurgica de espermatozóides do epidídimo (MESA) permite a paternidade biológica a esses pacientes. Em função da alta morbi-mortalidade da FC e da alta freqüência de portadores assintomáticos, estimada em 1:25, é recomendável que seja realizado teste para identificação de mutações em CFTR em todos os pacientes com CAVD antes de serem submetidos à ICSI. Em populações de etnia homogênea, a mutação F508 é identificada em 90% dos pacientes com FC e em 70% a 85% dos pacientes com CAVD. No Brasil, onde diferenças étnicas refletem a heterogeneidade genética, a freqüência da mutação F508 varia entre 23% e 50% em paciente com FC indicando que outras mutações devam estar envolvidas. Este dado levou ao estudo completo do gene CFTR de 20 homens inférteis com CAVD visando a identificação das mutações mais prevalentes em nossa população. Foram identificadas mutações em 17 pacientes (85%): três DF508 representando 15% (3/20), uma G542X, uma 875+1G>A e 4 mutações ainda não descritas na literatura, a S753R, G149W identificada em dois irmãos, V580F e a 712-1G>T. A variação no trato polipirimidínico em IVS8 (alelo 5T), seja como segunda mutação ou presente em homozigose, está diretamente relacionada com a CAVD, com freqüências em população caucasiana masculina infértil variando entre 21% e 30%. No presente estudo, 15 (15/20=75%) pacientes apresentaram o alelo a variante alélica 5T sendo que em 8 pacientes essa variante alélica foi identificada em heterozigose composta com outra mutação. Anomalias renais foram identificadas em 6 pacientes, todos com CBAVD. O presente estudo pode correlacionar o fenótipo da CAVD a alterações no genótipo de CFTR em 100% dos pacientes investigadosCystic Fibrosis (CF) is the most common autosomal recessive disorder in caucasians and is associated, in an wide variety of different clinical manifestatons. More than 1500 mutations in the CFTR gene (Cystic Fibrosis regulator Transmembrane conductance) have been described and an even growing number of mutations are being currently studied worldwide. The role of CFTR gene is especially important in reproductive tissues of the mesonephric tract sensitive to the expression of the CFTR gene. The great majority of infertile males with CF (98%) have clinical manifestations and mutations in CFTR are responsible for the congenital bilateral absence of the vas deferens (CBAVD), associated to the abnormalities of the seminal vesicles, ejaculatory ducts and/or the distal portion of the epididymis. The congenital absence, uni or bilateral, of the vas deferens (CAVD), in the absence of other clinical signals of CF is a known cause of male infertility present in 1%-2% of all men investigated and in about 10% of men with obstructive azoospermia. Serious considerations should be drawn about the lack of proper diagnosis of infetile males with CFTR that seek reproductive clinics for assisted reproductive techniques (ARTs), as well as the lack of proper consideratins of the existance of this disease as a potential cause of male infetility among male are takers, like urologistas, andrologistas and gynecologists that rush for the misuse of ARTs. The introduction of Intracytoplasmic Sperm Injection (ICSI), has given new reproductive potetntial for these couples, but again as in the majority of cases it is obstructive azoospermia, couples should be advised about proper microsurgical sperm retrieval, preferentialy microsurgical epydidymal sperm aspiration (MESA). As a consequence of the potential high mortality rate of the CF descendents and the high frequency of carriers, estimated in 1:25,it is highly recommended that tests for correct identification of mutations in CFTR gene are carried out for all patients with CAVD before considered being submitted to ICSI. In populations of homogeneous ethnic origin, the mutation F508 is identified in 90% of the patients with CF and between 70% and 85% of the patients with CAVD. In Brazil, where ethnic differences reflect the genetic heterogeneity, the frequency of the mutation in F508 varies between 23% and 50%, indicating that other mutations must have a role. Our data looked carefully in the CFTR gene of 20 infertile men with CAVD aiming at the identification of the most prevalent mutations in our population. Mutations had been identified in 17 patients (85%): three DF508 representing 15% (3/20), one G542X, one 875+1G>A and 4 mutations not yet described in literature, S753R, G149W identified in two brothers, V580F and 712-1G>T. In the literature the allelic variant in IVS8 (allele 5T), either as a second mutation or in homozygosis, is directly related with the CAVD, with reported frequencies in the infertile caucasian male population varying between 21% and 30%. In the present study, 15 (15/20=75%) patients presented the CFTR mutation in the IVS8/5T: eight of them in heterozygosis composed with another mutation. Regarding genitourinary tract malformations, kidney anomalies were identified in 6 patients, all with CBAVD. In the present study we could correlationate the phenotype of the CAVD with the genotype alterations of CFTR gene in 100% of the investigated patients
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Servidoni, Maria de Fátima Corrêa Pimenta 1961. "Diagnóstico clínico e laboratorial da fibrose cística = métodos clássicos e novas perspectivas = Clinical and laboratorial diagnosis of cystic fibrosis: classical methods and new perspectives." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/308375.
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Orientadores: Antônio Fernando Ribeiro, Jose Dirceu Ribeiro, Francisco Ubaldo Vieira JúniorTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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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
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Smith, Emily M. "The Three-Dimensional Structure of the Cystic Fibrosis Locus: A Dissertation." eScholarship@UMMS, 2011. http://escholarship.umassmed.edu/gsbs_diss/744.
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The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression.
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Smith, Emily M. "The Three-Dimensional Structure of the Cystic Fibrosis Locus: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/744.
Full textAbstract:
The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression.
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Ng, Ronny Tah Yen 1979. "Fibrose cística = avaliação diagnóstica através da diferença de potencial nasal e sua correlação com duas mutações genéticas." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309204.
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Orientador: Eulalia SakanoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A fibrose cística (FC) é uma doença genética autossômica recessiva, resultante da ausência total na proteína CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ou de alterações qualitativas ou quantitativas do gene que transcreve esta proteína, em células de diversos órgãos do corpo humano, resultando em inúmeros genótipos e fenótipos desta doença. Em muitos pacientes, o diagnóstico é difícil de ser definido, pelo método clássico de dosagem de sódio e cloro no suor, ou pelo sequenciamento genético, justificando a utilização de novas técnicas de auxílio diagnóstico, como a Diferença de Potencial Nasal (DPN). Este teste proporciona uma forma de avaliação direta e sensível, através do epitélio nasal, do transporte de sódio e cloro das membranas celulares, baseado nas propriedades bioelétricas transepiteliais. O objetivo deste trabalho foi verificar se existe diferença dos valores obtidos no exame de DPN em pacientes com FC em comparação com indivíduos controles saudáveis; e verificar se este teste permite diferenciar pacientes com FC das subclasses funcionais mais graves (I, II, III) das subclasses menos graves (IV, V, VI). Foram incluídos no estudo 15 pacientes FC, 10 com mutações mais graves (grupo A) e 5 com mutações menos graves (grupo B), e 21 controles saudáveis (grupo C). Foram considerados os seguintes parâmetros do teste da DPN: "Finger", PDMax, ?Amilorideo, ?Amilorídeo+livrecloreto e index de Wilchanski. Para a variável "Finger", foi encontrada diferença entre pacientes com FC grupo B - mutações menos graves (classe IV, V ou VI) e indivíduos saudáveis - grupo C. O valor do index de Wilchanski mostrou diferença entre pacientes com FC grupo A - mutações mais graves (classes I, II ou III) e indivíduos saudáveis - grupo C. No nosso estudo, a DPN mostrou valores estatisticamente diferentes entre FC com 2 mutações conhecidas e sujeitos saudáveis. Porém, não conseguiu diferenciar fibrocísticos com mutações mais graves (classes I, II e III) daqueles com mutações consideradas menos graves (classes IV, V e VI)
Abstract: Cystic fibrosis (CF) is an autosomal recessive genetic disease, due to the total absence of protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), or due to qualitative or quantitative changes in the gene that transcript this protein in cells of various organs of the human body, resulting in numerous genotypes and phenotypes of the disease. In several patients, the diagnosis is difficult to be defined by the classical method of sodium and chloride dosage in sweat, or by genetic sequencing, justifying the use of new techniques for diagnosis, as the Nasal Potential Difference (NPD). This test provides a way of direct and sensitive assessment of the transport of sodium and chloride ions in cell membranes, via the nasal epithelium, based on transepithelial bioelectric properties. The objective of this work was to verify the difference of the values obtained in the examination of NPD in patients with CF compared with healthy control subjects, and, to verify if this test allows differentiating patients with more severe CF functional subclasses (I, II , III) from patients with less severe CF subclasses (IV, V, VI). This study included 15 CF patients, 10 with more severe mutations (group A) and 5 with less severe mutations (group B), and 21 healthy controls (group C). We considered the following test parameters of NPD: "Finger", PDMax, ?Amiloride, ?Amiloríde+Chloridefree and Wilchanski index. For "Finger" values, it was found difference between patients with CF Group B - less severe mutations (class IV , V or VI) and healthy individuals - group C. The value of Wilchanski index showed difference between group A CF patients, with more severe mutations (class I, II or III) and healthy individuals - group C. In our study, NPD showed statistically different values between CF patients with two known mutations and healthy subjects. However, it was not able to distinguish between CF patients with more severe mutations (class I, II and III) of the CF patientswith less severe mutations (Class IV, V and VI)
Mestrado
Otorrinolaringologia
Mestre em Ciências Médicas
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Wheatley, Courtney M. "Endogenous and Exogenous Regulation of Exhaled Ions in Patients with Cystic Fibrosis." Diss., The University of Arizona, 2013. http://hdl.handle.net/10150/293489.
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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.
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Miranda, Margarida Sofia Quintanilha. "Transmembrane transport of anions by synthetic transporters." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22273.
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Mestrado em QuímicaThe chloride transport across the phospholipid bilayer assisted by small synthetic molecules were studied by computational methods including quantum calculations followed by molecular dynamics simulations and free energy calculations. A series of twelve tris-thiourea aryl substituted putative transporters, with three different six-membered rings as scaffolds (desmethyl cyclohexane, hexamethyl cyclohexane and benzene) and each of them with four possible aryl substituents (3,5-trifluoromethyl, p-trifluoromethyl, p-nitro and phenyl), were studied in silico in order to understand the experimental transport data previously reported for these molecules. The computational study started with the DFT optimization of twelve chloride complexes in gas phase and in DMSO at the M062X/6-31+G** theory level. The strength of the N-H···Cl- interactions were ascertained through the E2 values obtained from the second order perturbation theory, the Wiberg bond Indexes and the most positive value of the electrostatic potential (VS,max). Overall, these descriptors increases with the binding affinity constants reflecting the electron withdrawing character of the aryl substituents. Thus, the 3,5-trifluoromethyl substituted transporters presented the highest values for the quantum descriptors as well as the highest binding affinity for chloride anion, indicating that these molecules are able to successfully uptake the anion from the water phase and further proceed to its transport across the phospholipid bilayer. Subsequently, the passive diffusion of these chloride complexes were then investigated by MD simulations positioning them either in the bilayer core and water phase. Overall, the MD simulation runs reveal that the transporters were able to promote chloride uptake and release events, consistent with anion carrier transport mechanism. Furthermore, free energy profile for 3,5-trifluoromethyl hexamethyl cyclohexane complex and free chloride were constructed from the potential mean force calculations. The chloride complex has to surpass an energy barrier to cross the middle of the phospholipid bilayer of 2 kcal mol-1 while for the free chloride this energy increases to 19.5 kcal mol-1 indicating that the chloride transport assisted by this receptor is energetically favoured. Another remarkable feature from the MD simulations is that the chloride complexes fit comfortably well below the membrane’s interface which seems to indicate that the receptors are able to operate as a chloride shuttle without leaving the phospholipid bilayer.
O transporte de cloreto pela bicamada fosfolipídica, assistido por pequenas moléculas sintéticas, foi estudado através de métodos computacionais, incluindo cálculos quânticos, simulações de dinâmica molecular e cálculos de energia livre. As propriedades de transporte de um conjunto de doze tris-tioureias contendo um anel central de seis membros (cicloexano, cicloexano hexametilado e benzeno) derivatizado com grupos arilo (3,5-trifluorometilo, p-trifluorometilo, pnitro e fenilo), foram estudados in silico tendo como objetivo a compreensão de dados experimentais de transporte reportados. O estudo computacional iniciou-se com a otimização dos doze complexos em fase gasosa e em dimetilsulfóxido (DMSO), ao nível de teoria de M06-2X/6- 31+G**. A força das interações NH···Cl- foi determinada através dos valores de E2 obtidos da teoria de perturbação de segunda ordem, dos índices de ligação de Wiberg e do valor mais positivo de potencial electroestático (VS,max). Estes descritores aumentam com as constantes de afinidade, refletindo o carácter electroatractor dos substituintes arilo. Assim, os três transportadores com grupos 3,5-trifluorometilo apresentaram os valores mais altos para estes descritores e para afinidade ao cloreto, demonstrando que estas moléculas conseguem de facto capturar um anião da fase aquosa e proceder ao seu transporte através da bicamada fosfolipídica. Seguiu-se o estudo do processo de difusão passiva para estes três complexos por dinâmica molecular, tendo estes sido colocados tanto na fase aquosa como no centro da bicamada fosfolipídica. Estas simulações revelaram que os transportadores eram capazes de promover os processos de captura e libertação de cloreto. Além disso, os perfis de energia livre para o complexo do ligando cicloexano hexametilado derivatizado com 3,5-trifluorometilo e para o cloreto livre foram obtidos através de cálculos de potencial de força média. O complexo tem que superar uma barreira energética de 2 kcal mol-1 para atravessar o meio da bicamada enquanto o cloreto livre tem que ultrapassar uma barreira de energia livre de 19.5 kcal mol-1, i.e. o transporte assistido pelo recetor é energeticamente mais favorável. Outra caraterística importante das simulações está relacionada com o facto dos complexos de cloreto de inserirem confortavelmente abaixo da interface da membrana, indicando que os recetores conseguem operar como transportadores de cloreto sem sair da bicamada fosfolipídica.
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Gruis, Darren Ben. "The cystic fibrosis transmembrane conductance regulator : advancement of the structural model of the protein and development of a novel approach to understand defective protein processing related to cystic fibrosis /." free to MU campus, to others for purchase, 1999. http://wwwlib.umi.com/cr/mo/fullcit?p9946257.
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Colaço, Ana Rita Freitas. "Transmembrane transport of chloride by Squaramides : in silico study." Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12505.
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Mestrado em Biomedicina MolecularThe anion transport across cellular membranes is essential to the cell functioning and its regulation depends on transmembrane channels. The malfunction of ion channels leads to channelopathies. In particularly, the impairment of chloride ion channels is associated with cystic fibrosis. These diseases have motivated the supramolecular chemists for the development of new chloride synthetic transporters with potential use in channel replacement therapies. In this context, this thesis reports an in silico study performed to evaluate the ability of five squaramides to assist the chloride transport across a POPC bilayer. Indeed, earlier experimental studies have shown that these small molecules were able to mediate the chloride efflux across POPC vesicles more efficiently than their analogous thioureas and ureas, as mobile-carriers using an anion-exchange mechanism. This theoretical investigation was carried out by a combination of quantum calculations and Molecular Dynamics simulations in a POPC membrane model. The MD simulations were preceded by the development of specific bond term parameters for the squaramide moiety using the crystal data from an extensive series of squaramides. The remaining parts of these molecules were described with GAFF default parameters. The phospholipids were described with parameters taken from LIPID11. The passive diffusion of chloride complexes was investigated by placing each receptor in two different starting positions: in the water slab and in the bilayer core of the POPC membrane model. In both cases the receptor moved towards the water/lipid interface and accommodated themselves below the lipid head groups. In the first case, the chloride release occurred in the water slab before the receptor reaches the water/lipid interface. By contrast, in the second case the chloride is released concomitantly with the receptor approach to the interface. The squaramides interact with phospholipid head groups mainly via N-H···O hydrogen bonds as analyzed along the thesis.
O transporte de aniões através de membranas celulares é essencial para o funcionamento da célula e a sua regulação depende de canais transmembranares. O mau funcionamento destes canais leva a canalopatias, designadamente o dano dos canais de cloreto associado à fibrose quística. Estas doenças têm motivado os químicos supramoleculares para o desenvolvimento de novos transportadores sintéticos de cloreto visando uma potencial aplicação em terapias de substituição de canais. Neste contexto, esta dissertação reporta um estudo in silico em que se avalia a capacidade de cinco squaramides (amidas quadrangular planas) assistirem o transporte de cloreto através de uma bicamada de POPC. De facto, estudos experimentais anteriores demonstraram que estas pequenas moléculas são capazes de mediar o efluxo de cloreto de vesículas de POPC com maior eficiência do que os seus análogos tioureias e ureias, actuando como transportadores móveis através de um mecanismo de permuta de aniões. Esta investigação teórica foi realizada com base em cálculos quânticos e simulações de dinâmica molecular num modelo de membrana POPC. As simulações foram precedidas pelo desenvolvimento de parâmetros específicos para as ligações e ângulos da unidade central da squaramide, sendo que o resto das moléculas descritas com parâmetros de defeito do GAFF. Os fosfolipídos foram descritos com parâmetros do campo de forças LIPID11. A difusão passiva dos complexos de cloreto foi investigada colocando cada um dos receptores em diferentes posições de partida: na fase aquosa e no meio da bicamada de POPC. Em ambos os casos, os receptores moveram-se em direcção à interface da membrana tendo-se posicionado abaixo das cabeças dos lípidos. No primeiro caso, o cloreto foi libertado ainda na fase aquosa antes do receptor chegar à interface. Enquanto que no segundo caso a libertação do cloreto ocorreu concomitantemente com a aproximação do receptor à interface. Durante o tempo de simulação os receptores interactuaram principalmente com as cabeças dos lípidos via ligações de hidrogénio N-H···O.
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Wong, Melanie Hoi-Lee. "The role of the C-terminus in the cellular physiology of cystic fibrosis transmembrane-conductance regulator (CTFR)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0024/MQ50414.pdf.
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Walker, Jennifer Harriet. "The production, and characterisation of monoclonal, polyclonal and phage display antibodies to the cystic fibrosis transmembrane regulator." Thesis, University of Bristol, 1994. http://hdl.handle.net/1983/e64704e1-ced9-4cd9-a358-d6cab313ed52.
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Banasavadi-Siddegowda, Yeshavanth Kumar. "Functional Dissection of FKBP38 in the Biogenesis of Cystic Fibrosis Transmembrane Conductance Regulator in the Endoplasmic Reticulum." University of Toledo Health Science Campus / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=mco1321308181.
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Chen, Kathy. "Epitope-tagging of Cystic Fibrosis Transmembrane Conductance Regulator, CFTR, for the detection of interacting proteins during its biosynthesis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0015/MQ53137.pdf.
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Goldstein, Rebecca F. "Protein interaction and cell surface trafficking differences between wild-type and [Delta]F508 cystic fibrosis transmembrane conductance regulator." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007p/goldstein.pdf.
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Hegde, Ramanath Narayana. "Modulation of proteostasis for the efficient intracellular transport of the AF508 mutant of cystic fibrosis transmembrane conductance regulator." Thesis, Open University, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607442.
Full textAbstract:
The cystic fibrosis transmembrane conductance regulator (CFTR) L\F508 mutant (L\F508CFTR) contributes to 70% cystic fibrosis cases, and it undergoes aberrant proteostasis, misfolding, intracellular retention and degradation. Targeting L\F508CFTR proteostasis components has been successful in the partial rescue of L\F508CFTR at the plasma membrane. Drug screening has identified correctors which rescue a fraction of the L\F508CFTR at the plasma membrane. However, all these correctors are marginally effective and are not therapeutically viable. This thesis project used a systems-biology-based meta-analysis approach of gene expression induced by corrector drugs to infer their mechanisms of action, and this led to the identification of a group of genes that are commonly regulated by many of these drugs. These groups of genes were used to determine the networks/ pathways/ molecules that might be correlated with their correction. These include components of RNA processing, the cell cycle, ubiquitin ligases, and kinases, many of which have led to patiial rescue of L\F508CFTR when they have been depleted by RNA interference. FUlihermore, two of these pathways are characterised here: the MAP3K II-JNK cascade and the CaM kinase (CAMKK2) cascade. Several members of these cascades rescued L\F508CFTR. The MAP3Kll -initiated pathway has a role in ER-associated degradation and plasma-membrane stability of L\F508CFTR. MAP3K 11 also appears to link oxidative stress and inflammation to intracellular proteostasis of L\F508CFTR. Some upstream activators and downstream targets of MAP3K 11 can also rescue L\F508CFTR. Drugs that inhibit the MAP3K II-JNK cascade rescued functional L\F508CFTR at the plasma membrane. Combinations of these drugs with the previously clinically studied corrector pharmacochaperone YX-809 led to a high level of correction of L\F508CFTR, which was much greater than for YX-809 alone. Many candidate genes and drugs that are identified in the current study open the way to the development of the new efficient therapeutic agents for cystic fibrosis caused by the L\F508 mutation. .