Tesis sobre el tema "CFTR function"

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is δF508 and this causes cystic fibrosis (CF). Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiologic mechanisms in CF and developing therapies. New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse δF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human δF508-CFTR. The processing behavior of ferret wild-type (WT) and δF508-CFTR proteins remain unknown and such information is important to predicting the utility of a δF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and δF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells, as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret δF508-CFTR processing was negligible, while low levels of processing of δF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not δF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTRδF508/δF508 animal model may be useful. Furthermore, in the CFTR and CFTR+/+ ferret model we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine and gallbladder of newborn CF ferrets. Short circuit current (ISC) analysis of CF and WT tracheas revealed the following similarities and differences: 1) amiloride sensitive sodium currents were similar between genotypes, 2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) were ~4-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels, and 3) as expected, there was a lack of IBMX/forskolin-stimulated and GlyH-101-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA and protein was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. Interestingly, IBMX/forskolin-inducible intestinal ISC in WT animals was not inhibited by the CFTR inhibitor GlyH-101 or bumetanide. The luminal pH of the CF ferret stomach was significantly decreased relative to the controls, while both genotypes maintained near neutral pH along the length of the intestine. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

The genetic disease cystic fibrosis is the most common lethal genetic disease in Western countries. People born with cystic fibrosis suffer from many health issues including severe respiratory problems, inflammation and recurrent lung infections that can become fatal. The disease is caused by the loss of function of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an chloride ion channel and, in healthy people, its activity assures correct water and salt transport across the cell membrane. Most cases of cystic fibrosis are caused by a genetic defect that leads to the deletion of phenylalanine 508 (F508del) in the amino acid sequence of the protein. The molecular mechanism by which F508del leads to loss of function of the CFTR channel is still poorly understood. The mutation is found in the first nucleotide binding domain (NBD1) and studies have shown that it causes misfolding of CFTR and subsequent degradation of the protein by the cellular quality control system. It is established that the mutation affects stability and dynamics of NBD1 but does not alter its structure significantly. This destabilizing effect of F508del can be compensated by specific mutations distributed over different regions of NBD1, leading to recovery of membrane expression of a functional channel. A surprising example involves the regulatory insertion (RI), a 32-residue long segment found in all CFTR orthologs but not in related channels or transporters. The RI is not resolved in crystal structures of NBD1 nor cryo-EM structures of CFTR and has been described as intrinsically disordered. Its functional role in CFTR is unknown. Removal of the RI increases the stability of the NBD1 domain and, in the context of F508del-CFTR, this deletion restores maturation, cell surface expression and activity of the mutant channel. We probed the effect of the RI on NBD1 structure, dynamics and allostery using X-ray crystallography, single molecule FRET and hydrogen-deuterium exchange. We discovered that the RI enables an alternative NBD1 fold which departs markedly from the canonical fold previously observed for this domain and the NBDs of other ABC transporters. The conformational equilibrium between these states is regulated by ATP binding and affected by disease-associated conditions. Aside from clear alterations to structure and dynamics of NBD1, the RI also affects allostery, i.e. how NBD1 structure and dynamics respond to perturbations such as ligand binding. Finally, we show that the RI-enabled conformation is adopted in full-length CFTR and associated with increased channel activity in electrophysiological assays. We then identify an allosteric network that links the structural hotspots of the conformational changes to F508 and its surroundings. Lastly, we argue that these conformational changes lead to unfolding of NBD1 in the context of F508del, providing a new model for the molecular mechanism leading to pathogenesis.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Cystic fibrosis (CF) is the most common, fatal genetic disease in the Caucasian population caused by loss of function mutations in gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed at the apical surface of epithelial cells of different organs, such as: lungs, pancreas, gut, and testes. For this reason even if in CF the pulmonary manifestations are the most severe, CF is considered a multi-system disease, which affects several bodily districts. The new challenge for the CF therapy is based on the development of small molecules able to rescue the function of the mutated CFTR. Many pharmacological agents have been designed to increase the surface level of mutated CFTR (correctors), as well as its plasma membrane (PM) activity (potentiators). Recently, combined therapy that includes a corrector of the CFTR folding (lumacaftor or VX-809) and a potentiator of the channel activity (ivacaftor or VX-770) called Orkambi®, was approved for CF patients homozygous for the deletion of phenylalanine at position 508 (F508del), the most common CF-causing mutation. Unfortunately, clinical studies revealed that the effects of Orkambi® on lung function were modest, due to low stability of rescued F508del-CFTR at the PM level. Indeed, many factors contribute to PM CFTR stability, including its compartmentalization in PM macromolecular complexes composed of phospholipids, sphingolipids, with particular regards for monosialoganglioside 1 (GM1), and scaffolding proteins such as ezrin and NHERF-1. Interestingly, it has been proved that in bronchial epithelial cells the lack of CFTR in the cell PM, such as in the case of the patients carrying the mutation F508del, is associated with a decreased content of GM1. By performing photolabelling experiments, I demonstrated for the first time that GM1 and CFTR at PM level reside in the same microdomain, suggesting a direct interaction between them. Then I investigated on the potential effect of the exogenous administration of ganglioside GM1 on the PM stabilization and function of F508del-CFTR rescued by Orkambi® treatment. In particular, I proved that in CF bronchial epithelial cells GM1 antagonizes the negative effect of VX-770, increasing F508del-CFTR maturation and its channel activity by the recruitment of the scaffolding proteins NHERF-1 and ezrin. Taken together the results obtained during my PhD project pointed out the role of GM1 as possible adjuvant to Orkambi® therapy to restore the function of F508del-CFTR.

Cystic fibrosis (CF) and Primary Ciliary Dyskinesia (PCD) are chronic suppurative lung diseases (CSLD). CF is characterised by inherited mutations affecting the cystic fibrosis transmembrane regulator (CFTR) protein, which is thought to be normal in PCD, however the role of CFTR in disease is incompletely understood. This thesis investigates the relationship between CFTR, inflammation and airway health, firstly in the context of the CF gene therapy Multidose trial followed by contrasting CF, PCD and control patients. The first study explored the relationship between lower airway potential difference (LAPD) measurements performed in the Multidose trial as a measure of CFTR function, and physiological, radiographic and inflammatory markers of disease severity. At baseline, FEV1 correlated with basal LAPD measurements, however not between restored chloride secretion and change in airway disease following treatment; implicating the role of sodium transport, not chloride in disease pathogenesis. As no direct correlation was seen, I went on to explore an alternative theory that a bi-directional relationship exists between CFTR and inflammation; CFTR dysfunction triggers a hyper-inflammatory state and inflammation causes secondary CFTR dysfunction. Cell cultures were cultivated from the nasal epithelium of patients with CF, PCD and controls. Both at baseline and following stimulation with common respiratory pathogens, the levels of inflammatory mediators in the supernatant from each group of cells were comparable. The numbers involved with this study were small, however did not indicate that CF cells cultured in these conditions (in vitro) were hyper-inflammatory. The final study explored in vivo whether inflammation causes secondary CFTR dysfunction. Nasal potential difference (NPD) measurements were compared with localised levels of inflammation in subjects with CF, PCD and controls. PCD traces showed reduced chloride secretion, however it was not possible to differentiate secondary CFTR dysfunction from damage to epithelial cell integrity. Elevated levels of inflammatory mediators were detected in PCD nasal fluid, however the results were variable and these levels did not correlate with NPD measurements of ion channel function. These studies did not support the hypothesis that there is a direct relationship between CFTR function and airway disease, that in vitro CF cells are hyperinflammatory, or in vivo that inflammation leads to secondary CFTR dysfunction. The experiments performed in this thesis provide a basis for future work exploring this relationship, and may help guide future trials for novel therapies in CF.

Abdominal surgery inhibits gastrointestinal motility, a phenomenon referred to as postoperative ileus. Since the postoperative ileus disturbs duodenal physiology it is important to minimize the side effects of this condition. Recent experiments in our laboratory show that treatment of anaesthetised rats with parecoxib, a selective cyclooxygenase-2 inhibitor, prevents duodenal postoperative ileus, increases duodenal mucosal bicarbonate secretion and improves other functions as well. One aim of the thesis was to investigate whether removal of luminal chloride affect the parecoxib- and the vasoactive intestinal peptide (VIP)-induced stimulation of duodenal mucosal bicarbonate secretion. The proximal duodenum of anaesthetised Dark Agouti rats was perfused with isotonic solutions containing zero or low Cl- and the effect on luminal alkalinisation determined. The basal as well as the parecoxib-induced increase in alkalinisation, but not that stimulated by VIP, were markedly reduced in the absence of luminal Cl-. One important function of the duodenum is to adjust luminal osmolality towards that in the blood. It is believed that the adjustment of osmolality in the duodenum is achieved by osmosis and diffusion of electrolytes along their concentration gradients and that these processes occur predominately paracellularly. Another aim of the thesis was to examine whether prevention of postoperative ileus affects the duodenal response to luminal hypertonicity. The proximal duodenum of anaesthetised Dark Agouti and Sprague-Dawley rats were perfused with hypertonic solutions of different composition and osmolality and the effects on duodenal motility, alkaline secretion, transepithelial fluid flux, mucosal permeability and the adjustment of luminal osmolality were determined in absence and presence of parecoxib. It is concluded that COX-2 inhibition increases duodenal mucosal bicarbonate secretion by stimulating apical Cl-/HCO3- exchange in duodenocytes. Furthermore, pretreatment of anaesthetised rats with parecoxib improves a number of duodenal functions in both rat strains that contribute to improve the ability to adjust luminal osmolality. The choice of rat strain is another important feature to consider when interpreting the results because the DA strain was more responsive to luminal hypertonicity than the SD strain. Finally, several evidences are provided to suggest that the adjustment of luminal osmolality in the rat duodenum is a regulated process.

Class of 2012 Abstract
Specific Aims: The epithelial sodium channels (ENaC) found on the apical membranes of epithelial cells including those lining the respiratory tract are the rate limiting step of the absorption of excess fluid from the airspace of the alveoli. ENaC function is modulated by the effects of various physiologic signals such as the adrenergic and purinergic pathways, in addition to other local channels which control the flow of negatively charged ions such as the cystic fibrosis transmembrane conductance regulator (CFTR). We sought to determine the influence of genetic variation on the alpha subunit of ENaC at amino acid position 663 on baseline exhaled ions and pulmonary function in patients with CF. Methods: We assessed pulmonary function ( forced vital capacity[FVC], forced expiratory volume in one second [FEV1], forced expiratory flow maximum[FEFmax]) using a Medical Graphics cardiopulmonary testing device (Minneapolis, MN). Measures of exhaled sodium (Na+) and chloride (Cl-) were obtained using exhaled breathe condensate collected on a Jaeger Ecoscreen condenser unit (Cardinal Health, Yorba Linda, CA) with Na+ quantification using an atomic absorption spectrophotometer (Analyst 100; Perkin Elmer, Norwalk, CT) and Cl- anion quantification using a Dionex AS11 HC column. Healthy n=31 (n=18[58%], 9[29%], and 4[13%] subjects; Body mass index (BMI)=23±1, 25±2, and 25±2kg/ m2 for AA, AT and TT groups respectively). CF n= 42 (n=33[79%], 7[16%], and 2[5%] subjects; BMI equals 23±7, 19±0.4, and 20±2.2kg/m2 for AA, AT and TT groups respectively). Main Results: We found that the distribution of genotypes in CF differed from healthy subjects, with the AA genotype in 80% of CF and 59% in healthy. No significant difference were demonstrated in healthy subjects between genotype groups for pulmonary function and exhaled chloride while the genotypes did differ in exhaled Na (Na=2.9±0.4, 1.7±0.3, and 3.7±1.1mmol/L for AA, AT, and TT respectively, ANOVA p=0.07). CF subjects with the AA genotype had a higher baseline exhaled Cl-, FEV1, and FEFmax than those in the AA group (Cl=0.125±0.038,0.0 27±0.007, and 0.033±0.02 mmol/L ; FEV1=71±5, 68±11, and 40±22L; FEFmax=86±4, 72±7, and 44±24L/sec; for AA, AT, and TT respectively, ANOVA p<0.05, Tukey [AA vs. TT] p<0.05) while exhaled Na+ and FVC were similar between genotypes. Conclusions: Our results suggest that CF subjects with the AA genotype of the alpha subunit of the ENaC have a higher baseline exhaled Cl- and a resulting increase in pulmonary function when compared to the overactive TT groupCF patients with the TT αENaC genotype are likely candidates for early identification and treatment with inhaled ENaC inhibitors or other modulators of this pathway in order to improve survival.

Dans cette thèse, nous étudions les fonctions de corrélation à deux points ou plus de la théorie N = 4 super Yang-Mills (SYM) et ses déformations. On pense que cette théorie et ses déformations sont intégrables dans la limite planaire pour toutes les valeurs de la constante de couplage, ce qui nous permet de calculer plus efficacement diverses grandeurs physiques. Nous commençons par passer en revue les bases de la théorie N = 4 SYM et les outils d’intégrabilité nécessaires. Dans la partie II, nous présentons le formalisme en hexagone pour le calcul des constantes de structure de la théorie N = 4 SYM. En utilisant l’ansatz de Bethe imbriqué, nous étudions la constante de structure de deux opérateurs BPS et d’un opérateur non BPS du sous-secteur su (1, 1 | 2) dans les limites de couplage faible et forte. Le résultat de couplage faible est appliqué à l’étude du développement en ondes partielles conformes des fonctions asymptotiques à quatre points. A fort couplage, le résultat est appliqué pour le calcul des constantes de structure Heavy-Heavy-Light. Dans la partie III, nous nous concentrons sur une limite de double échelle particulière de la théorie N = 4 SYM, la limite de fishnet. Le lagrangien est obtenu à partir du processus de déformation et nous en présentons les théorèmes de non-renormalisation. En outre, nous donnons la première preuve de la symétrie du Yangien pour une grande classe de diagrammes de fishnet, en utilisant la fameuse relation RTT de l’intégrabilité. En utilisant les techniques d'intégrabilité héritées de la théorie N = 4 SYM, nous trouvons également que la limite du continuum des diagrammes de Feynman en fishnet peut être décrit comme un modèle sigma AdS
In this thesis, we study two- and higher-point correlation functions of the N = 4 super Yang-Mills theory (SYM) and its deformations. This theory and its deformations are believed to be integrable in the planar limit for any values of the coupling constant, which allow us to compute various physical quantities more efficiently. The basics of N = 4 SYM and the necessary integrability tools are reviewed in the very beginning. In part II, we review the hexagon formalism for computing structure constant in N = 4 SYM. Using the nested Bethe ansatz techniques, we investigate the structure constant of two BPS operators and one non-BPS operator in the su(1, 1|2) subsector in both weak and strong coupling limit. The weak coupling result is applied to the study of the conformal partial wave expansion of asymptotic four-point functions. At strong coupling, the result is applied to the Heavy-Heavy-Light structure constants. In part III, we focus on a particular double scaling limit of N = 4 SYM, namely the fishnet limit. The Lagrangian is obtained from the deformation process, and we present the non-renormalization theorems about it. Besides, we give the first principle proof of the Yangian symmetry for a large class of fishnet Feynman diagrams, using the famous RTT-formulation of integrability. Using the integrability techniques inherited from N = 4 SYM, we also find that the continuum limit of fishnet Feynman diagrams admit an AdS sigma model description

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.

Conformal field theories (CFT) represent a framework of fruitful interplay between some of the most advanced topics in theoretical physics and algebraic geometry. In particular, the investigation of the CFT partition functions is closely related to the analysis of the correspondence between analytic and algebraic properties of closed Riemann surfaces. In the present thesis, some new aspects of this correspondence, in particular the ones arising in the CFTs associated to string and superstring theories, are considered. More precisely, the algebraic parameters, determining the canonical curve associated to a non-hyperelliptic Riemann surface, are explicitly computed in terms of Riemann theta functions, evaluated at generic points of the curve. Moreover, the techniques here introduced are applied to the analysis of the singular locus of the theta function, also considered with respect to the Andreotty-Mayer approach to the Schottky problem, and to the restriction of the Siegel's measure to the moduli space of canonical curves.

KCNE-Hilfsuntereinheiten assoziieren mit Spannungs-abhängigen K+-Kanälen und verändern dadurch deren subzelluläre Lokalisation, Regulation sowie deren biophysikalische Eigenschaften. Bei heterologer Expression interagiert KCNE3 mit mehreren Poren-bildenden K+-Kanal-Hauptuntereinheiten, deren Ströme dadurch stark modifiziert werden. Aufgrund dieser in vitro-Experimente wurden verschiedenste Funktionen von KCNE3 in den verschiedenen Geweben, wie Gehirn, Herz, Muskel, Kolon und Niere, vermutet. Außerdem wurden Variationen im kcne3-Gen mit menschlichen Skelettmuskelpathologien in Verbindung gesetzt (Abbott et al. 2001). In der gegenwärtigen Literatur wird die physiologische Funktion von KCNE3 eher als komplex und heterogen dargestellt. Auch die direkte Beteiligung von KCNE3 an Krankheiten ist immer noch spekulativ. Zur Untersuchung der physiologischen Funktion von KCNE3 in vivo sowie der potentiellen Rolle bei Krankheiten generierten wir ein kcne3-/- Mausmodell. Die vorliegende Arbeit unterstützt die kritische Rolle der KCNQ1/KCNE3-Kanäle beim Salz- und Flüssigkeitstransport über intestinale und respiratorische Epithelien. Insbesondere fanden wir für die KCNQ1/KCNE3-Heteromere eine basolaterale Lokalisation in Darm- und Trachea-Epithelzellen, wo sie die transepitheliale Cl--Sekretion über basolaterales Recycling von K+-Ionen sowie über Erhöhung der elektrochemischen Triebkraft für apikalen Cl--Austritt fördern. Da weder Veränderungen in der KCNQ1-Expressionsmenge noch in dessen subzellulärer Lokalisation festgestellt wurden, ist die durch KCNE3 verursachte Modifikation der KCNQ1-Kanaleigenschaften essenziell für die hier beschriebene physiologische Rolle im Intestinal- und Trachealtransport. Ferner wird von unserer Arbeit die postulierte Funktion von KCNE3-Heteromeren im Skelettmuskel, Herz und zentralen Nervensystem nicht unterstützt und erweckt somit erhebliche Zweifel über den Beitrag von KCNE3 zu menschlichen Krankheiten, die mit diesen Organen in Verbindung stehen.
When overexpressed in heterologous systems, KCNE3 is able to interact with several pore-forming K+ channel alpha subunits greatly modifying their currents. Based on these in vitro evidences, KCNE3 has been proposed to serve different roles in multiple tissues, including brain, heart, muscle, colon and kidney. Additional reports have also linked sequence variations in the KCNE3 gene to cardiac and skeletal muscle pathologies in human. Based on the literature, the overall picture of KCNE3 physiological function is rather complex and heterogeneous, and its direct involvement in pathologies is still speculative and far from being conclusively proven. In order to study the physiological role of KCNE3 in vivo and to address its potential pathological implications, we generated kcne3-/- mice. The present analysis of kcne3-/- mice strongly supports a crucial role of KCNQ1/KCNE3 channels in salt- and fluid secretion across intestinal and airway epithelia. In particular, we found that KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they facilitate transepithelial Cl- secretion through basolateral recycling of K+ ions and by increasing the electrochemical driving force for apical Cl- exit. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3-/- mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. In addition, our work does not support the postulated role of KCNE3 heteromers in skeletal muscle, heart and CNS physiology, and raises considerable doubts concerning its implication in human pathologies which affect these tissues.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.

Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.

S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients.

Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.

Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

The discovery of integrability in planar N=4 super Yang-Mills and ABJM has enabled a precise study of AdS/CFT. In the past decade integrability has been successfully applied to the spectrum of anomalous dimensions, which can now be obtained at any value of the coupling. However, in order to solve conformal field theories one also needs to understand their structure constants. Recently, there has been great progress in this direction with the all-loop proposal of Basso, Komatsu and Vieira. But there is still much to understand, as it is not yet possible to use that formalism to find structure constants of short operators at strong coupling. It is important to study wrapping corrections and resum them as the TBA did for the spectrum. It is also crucial to obtain perturbative data that can be used to check if the all-loop proposal is correct or if there are new structures that need to be unveiled. In this thesis we compute several structure constants of short operators at strong coupling, including the structure constant of Konishi with half-BPS operators. Still at strong coupling, we find a relation between the building blocks of superstring amplitudes and the tensor structures allowed by conformal symmetry. We also consider the case of extremal correlation functions and the relation of their poles to mixing with double-trace operators. We also study three-point functions at weak coupling. We take the OPE limit in a four-point function of half-BPS operators in order to shed some light on the structure of five-loop wrapping corrections of the Hexagon form factors. Finally, we take the first steps in the generalization of the Hexagon programme to other theories. We find the non-extremal setup in ABJM and the residual symmetry that it preserves, which we use to fix the two-particle form factor and constrain the four-particle hexagon. Finally, we find that the Watson equations hint at a dressing phase that needs to be further investigated.

Dans cette thèse, on décrit une procédure permettant de représenter les équations intégrales de l’Ansatz de Bethe sous la forme du problème de Riemann-Hilbert. Cette procédure nous permet de simplifier l’étude des chaînes de spins intégrables apparaissant dans la limite thermodynamique. A partir de ces équations fonctionnelles, nous avons explicité la méthode qui permet de trouver l’ordre sous-dominant de la solution de diverses équations intégrales, ces équations étant résolues par la technique de Wiener-Hopf à l’ordre dominant. Ces équations intégrales ont été étudiées dans le contexte de la correspondance AdS/CFT où leur solution permet de vérifier la conjecture d’intégrabilité jusqu’à l’ordre de deux boucles du développement à fort couplage. Dans le contexte des modèles sigma bidimensionnels, on analyse le comportement d’ordre élevé du développement asymptotique perturbatif. L’expérience obtenue grâce à l’étude des représentations fonctionnelles des équations intégrales nous a permis de résoudre explicitement les équations de crossing qui apparaissent dans le problème spectral d’AdS/CFT
In this thesis is given a general procedure to represent the integral Bethe Ansatz equations in the form of the Reimann-Hilbert problem. This allows us to study in simple way integrable spin chains in the thermodynamic limit. Based on the functional equations we give the procedure that allows finding the subleading orders in the solution of various integral equations solved to the leading order by the Wiener-Hopf techniques. The integral equations are studied in the context of the AdS/CFT correspondence, where their solution allows verification of the integrability conjecture up to two loops of the strong coupling expansion. In the context of the two-dimensional sigma models we analyze the large-order behavior of the asymptotic perturbative expansion. Obtained experience with the functional representation of the integral equations allowed us also to solve explicitly the crossing equations that appear in the AdS/CFT spectral problem

The aim of this study was to investigate the development of release from proactive interference (RPI) and its relations with executive working memory functions. 101 primary school children (aged 6-12 years) and 20 young adults (aged 22-30 years) participated in the study. The main task, the Categorical Free Recall Test, comprised 12 items from 3 different categories (animals, fruits, clothes). The purpose of the main task was to examine both the development of the RPI pattern and the categorization ability during childhood. As our results showed, the categorization ability and the RPI pattern were already present in the 1st graders. Although overall memory span increased with age, there was no significant development for the categorization and the RPI effect. For the additional tasks, the Word Span Test (WST, to measure the phonological WM capacity), the Wisconsin Card Sorting Test (WCST, to measure both the categorization ability and executive WM functions), and the Listening Span Test (LST, to examine executive and complex WM functions), the results indicated that children also improved with age. Overall memory capacity in the main task was best predicted by the WST
however, memory of serial position was best predicted by the LST. These findings are in accordance with the view that the WST measures the phonological working memory span, whereas the LST measures complex working memory and executive functions. The comparisons between the adult and the child sample revealed that except for the RPI pattern adults were better on all tasks than the children. The lack of a consistent RPI pattern for the adults may be due to the relatively short stimulus list.

Complexos Convectivos de Mesoescala (CCM) são eventos meteorológicos de difícil previsão, que resultam em tempestades severas e desastres. O objetivo deste trabalho é indicar as características em grande escala do ambiente atmosférico favorável para a formação de CCM no Sul do Brasil, entre 1998 e 2007. Fez-se uso da base de dados de CCM de Durkee e Mote (2009), assim como das variáveis de Potencial de Energia Convectiva Disponível (CAPE), ponto de orvalho, temperatura, altura geopotencial, componentes de vento u e v e umidade relativa da reanálise do National Center for Environmental Prediction (NCEP) Climate Forecast System Reanalysis (CFSR), coletadas entre 2,5 e 5,5 horas antes do desenvolvimento dos CCM. Com o método de Análise das Componentes Principais (ACP), geraram-se as composições do ambiente atmosférico médio favorável ao desenvolvimento dos CCM, para comparar o grupo dos que ocorreram no Sul do Brasil ao dos que atuaram em outras regiões da AS. Usando como dado de entrada as variáveis de altura geopotencial e temperatura (em 850 hPa), foram encontradas quatro componentes principais para cada um dos grupos de CCM. Com base nas componentes principais, nas variáveis atmosféricas e nas cartas sinóticas, foram reconstruídos os ambientes atmosféricos médios para identificar o comportamento das características atmosféricas prévias aos CCM para cada conjunto de eventos. Os resultados identificaram 303 CCM, 96 no Sul do Brasil, 168 em outras regiões da AS e 39 oceânicos. O ambiente atmosférico médio dos 168 CCM não apresentou características homogêneas, pois 75% das componentes não possuíam jatos de baixos níveis (JBN) dentro dos critérios adotados, mas a presença de um escoamento meridional. Esse fluxo, ao encontrar com a região de divergência dos jatos de altos níveis (JAN), foi um dos fatores favoráveis para a convecção, já que seus valores de CAPE (≥ 450 J kg-1) eram menores que a média esperada para formação de tempestades e só uma das componentes teve frentes frias associadas. Por outro lado, o grupo dos 96 CCM que atuaram no Sul do Brasil mostrou-se cerca de 50.000 km² maior em extensão que os das outras regiões da AS e dos EUA e com duração de pelo menos 3 h a mais. Além disso, as características atmosféricas do grupo de CCM do Sul do Brasil mostraram padrões homogêneos, podendo indicar a formação de CCM nessa região quando: o campo de ventos médios em 850 e 200 hPa, se encontrarem em posição ortogonal, indicando acoplamento entre os jatos de baixos e altos níveis; os valores de CAPE forem ≥ 600 J kg-1 e o cisalhamento vertical estiver entre 7 e 12 m s-1; houver atuação das frentes frias no sul da AS; a umidade relativa disponível estiver concentrada próxima à região Sul do Brasil, com valores maiores que 80%; a altura geopotencial (850 hPa) apresentar um cavado na região gênese dos CCM e a temperatura (850 hPa) estiver mais elevada próxima e ao norte da região de formação.
Mesoscale Convective Complexes (MCCs) are meteorological events difficult to forecast, which result in severe storms and other natural hazards. This study’s objective is to indicate the large-scale atmospheric environment favorable to develop MCCs in Southern Brazil during the 1998–2007 period. The MCCs database used was from Durkee and Mote (2009) and the variables selected include CAPE (Convective Available Potential Energy), dewpoint temperature, temperature, geopotential height, and relative humidity from National Center for Environmental Prediction (NCEP) Climate Forecast System Reanalysis (CFSR), collected from 2,5 to 5,5 hours before the MCCs’ development. Principal component analysis (PCA) method was used to construct the average atmospheric environments of MCCs group that occurred in Southern Brazil to compare with MCCs that occurred in other regions of South America. Temperature and geopotential height were the variables used for the PCA, resulting in four principal components to each MCCs group. Based on these principal components, meteorological variables and synoptic charts, average atmospheric environments were built to understand the atmospheric parameters that indicate the development of MCCs in each group. Results show 303 MMCs, 96 were located in Southern Brazil, 168 in South America and 39 in the South Atlantic Ocean. The average atmospheric environment from the group of 168 MCCs did not indicate homogeneous characteristics, as 75% of its principal components cannot be characterized as having a low-level jet (LLJ) in the wind field, instead only a meridional flux of humid and warm air at 850 hPa. This air coupled with the upperlevel jet (ULJ) was found to be responsible for convection developing MCCs, as CAPE (≥ 450 J kg-1) was below the average to produce storms and only one component was associated with a cold front. On the other hand, the MCCs’ group of Southern Brazil is on the order of 50.000 km² larger and 3 hours longer than MCCs from other regions of South America and from the United States. Furthermore, the atmospheric characteristics of the Southern Brazil MCCs’ group revealed homogenous patterns, which suggest that the development of MCCs in this region starts when: the mean wind field indicate a coupled LLJ (jet streak between 10 and 12 m s-1) and ULJ (jet streak ≥ 32 m s-1), CAPE value is ≥ 600 J kg-1 and the vertical wind shear is from 7 to 12 m s-1, cold fronts are active in Southern South America, the relative humidity is concentrated in Southern Brazil and above 80%, the geopotential height (850 hPa) indicate a trough in the genesis region of MCCs and the temperature (850 hPa) is higher near and northern the genesis region.

Cette thèse est dédiée à l'étude de la fonction à trois points dans la théorie de jauge super-symétrique (SYM) N=4, dans la limite du grand nombre de couleurs, à l'aide de l'intégrabilité. La théorie de jauge N=4 SYM est invariante conforme au niveau quantique est on pense qu'elle est résoluble exactement. Par la correspondance AdS/CFT, elle est duale à la théorie des cordes de type IIB dans l'espace courbe AdS5× S5. Les fonctions à trois points sont des quantités qui contiennent de l'information essentielle sur la dynamique de la théorie.Nous passons en revue les méthodes déjà existantes et outils de l'intégrabilité qui sont nécessaires pour le calcul de la fonction à trois points. Nous présentons le calcul de la fonction à trois points dans le secteur SU(3), de rang supérieur à un, nous avons utilisé une représentation sous forme de déterminant, qui nous permets de prendre la limite semi-classique. En exploitant la relation entre des chaines de spin à langue portée et la chaine de Heisenberg inhomogène, nous avons développé une nouvelle pur calculer la fonction à trois points dans le secteur SU(2) à l'ordre d'une boucle qui nous permets d'obtenir le résultat dans une forme très compacte. Dans la limite de Frolov-Tseytlin ce résultat est en accord avec celui qu'on obtient au couplage fort.Nous avons exploré des nouvelles formulations de la fonction à trois points. En nous inspirant de la formulation de la théorie des champs des cordes dans la jauge du cone de lumière nous avons construit un vertex de spin, qui est la version de couplage faible du vertex des cordes, pour tous les secteurs à l'ordre des arbres. Cette approche peut être reliée au programme des facteurs de forme pour les théories de champs bi-dimensionnelles intégrables, dont nous rappelons ici les bases. Nous étudions la dépendance dans la taille du système pour une classe spéciale de fonction à trois points qui correspond aux facteurs de forme diagonaux
This thesis is devoted to the study of three-point functions of N=4 Super-Yang-Mills (SYM) theory in the planar limit by using integrability. N=4 SYM theory is conformal invariant at quantum level and is believed to be completely solvable. By the AdS/CFT correspondence, it is dual to the type IIB superstring theory on the curved background AdS5×S5. The three-point functions are important quantities which contain essential dynamic information of the theory.The necessary tools in integrability and the existing methods of computing three-point functions are reviewed. We compute the three-point functions in the higher rank SU(3) sector and obtain a determinant representation for one special configuration, which allows us to take the semi-classical limit. By exploring the relation between long-range interacting spin chain and inhomogeneous XXX spin chain, we develop a new approach to compute three-point functions in the SU(2) sector at one-loop and obtain a compact result. In the Frolov-Tseytlin limit, this result matches the result at strong coupling.We also explore new formulations of the three-point functions. In one formulation inspired by the light-cone string field theory, we constructed the spin vertex, which is the weak coupling counterpart of the string vertex for all sectors at tree level. Another formulation which is related to the form factor boostrap program in integrable field theory is reviewed. At weak coupling, we study the finite volume dependence of a special type of three-point functions which are related to the diagonal form factors

La correspondance AdS/CFT est la première réalisation précise de la dualité jauge/gravité. Jusqu’à maintenant la correspondance AdS/CFT reste une conjecture. La dualité de N = 4 SYM et la théorie des cordes est un exemple le plus notable de correspondance AdS/CFT. Un des obstacles principaux à l’explorer est le fait que le régime de couplage faible pour la théorie de jauge est le régime de couplage fort pour la théorie des cordes et vice versa. Par conséquent, aussi longtemps que les méthodes perturbatives sont appliquées, on ne peut pas comparer les observables de deux cotés de la correspondance directement en dehors de quelques cas particuliers. A ce stade, l’énorme symétrie de N = 4 SYM joue un rôle important en permettant le calcul exact des observables de la théorie au moins dans la limite planaire. Cette thèse est consacrée au calcul des fonctions à trois, l’un des principaux observables de N = 4 SYM, et est composée de deux parties. Dans la première partie nous considérons l’approche générale pour le calcul des fonctions à trois points sur la base de soi-disant vertex de spin, qui est inspiré de la théorie de champs des cordes. Dans la deuxième partie, nous considérons un type spécifique de fonctions à trois points appelés lourd-lourd-léger, qui sont caractérisés par la propriété que la longueur de l’un des opérateurs est beaucoup plus petite des longueurs de deux autres. Il s’avère que ces fonctions de corrélations peuvent être identifiées à des facteurs de forme diagonaux et ainsi on peut appliquer les résultats concernant les facteurs de forme
N=4 SYM theory has been drawing the attention of a lot of physicists during two last decades mainly due to the two aspects: AdS/CFT correspondence and integrability. AdS/CFT correspondence is the first precise realization of the gauge/string duality whose history starts in the 60's, when a string theory was considered as a candidate for describing the strong interactions. In 1997 Maldacena made a proposal about the duality between certain conformal field theories (CFT) and string theories defined on the product of AdS space and some compact manifold, which implies a one to one map between the observables of the gauge and string counterparts. Up to now AdS/CFT correspondence still remains a conjecture. The duality of N=4 SYM and the appropriate string counterpart is the most notable example of the AdS/CFT correspondence. One of the main obstructions to exploring it is the fact that weak coupling regime for the gauge theory is the strong coupling regime for the string theory and vice versa. Therefore as long as perturbative methods are applied, one can not compare the observables of dual counterparts directly apart from some specific cases. At this point the huge symmetry of N=4 SYM plays an important role allowing exact computation of the theory observables at least in the planar limit. This property of the theory is called integrability. The observables of the N=4 SYM are Wilson loops and correlation functions built out of gauge invariant operators. The space-time dependence of the two- and three-point correlators is fixed by the conformal symmetry up to some parameters: dimensions of the operators in the case of two-point functions and dimensions of the operators and structure constants in the case of three-point functions. It's commonly accepted to refer to the problem of finding the dimensions of the operators as the spectral problem. On the classical level the operator dimension is equal to the sum of the dimensions of the fundamental fields out of which the operator is composed. When the interaction is turned on, the conformal dimension gets quantum correction. In order to compute three-point functions, apart from the conformal dimensions of corresponding operators one needs to compute the structure constants. In CFT computation of the higher-point correlators eventually can be reduced to computation of two- and three-point functions by means of the operator product expansion. Therefore two- and three-point functions appear to be building blocks of any correlator of the theory. This thesis is devoted to computation of three-point functions and consists of two parts. In the first part we consider the general approach for computing three-point functions based on the so-called spin vertex, which is inspired from the string field theory. In the second part we consider a specific kind of three-point functions called heavy-heavy-light, which are characterized by the property that the length of one of the operators is much smaller the lengthes of other two. It happens that this kind of correlators can be considered as diagonal form factors which supposes that in this case one can apply the results obtained in the form factor theory

The main aim of using radars in industrial safety system is to detect the presence of target accurately. The conventional methods of radar target detection algorithm such as the Cell averaging constant false alarm rate method (CA-CFAR), Greatest of constant false alarm method (GO-CFAR) and the Smallest of constant false alarm rate method (SO-CFAR) has their own disadvantage when it comes to precise target detection which is a key factor for a safety system. This thesis investigates the above mentioned conventional CFAR algorithms for its pros and cons in target detection and proposes a new and improved method called Cell Evaluation target detection method. The proposed method is shown to mitigate the limitations present and the assumptions made in the conventional target detection method. Further more angular estimation is performed to determine the precise location of the target and the artifacts due to the angular estimation is eliminated by aggregating the detected points from multiple radar modules by linear translation. This gives a better visualization of the target.
Radarteknik kan användas inom maskinsäkerhet (MS) för att detektera skyddsvärda objekt, typiskt människor i arbete nära maskiner. Konventionella metoder för detektering med given frekvens falsk alarm (eng. Constant False Alarm Rate (CFAR)) som baseras på medelvärden, har dock betydande brister. Främst beträffande precision och tillförlitlighet, vilket är centralt för MS. Exempel som studerats i detta examensarbete är “Cell-averaging CFAR” (CA-CFAR), “Greatest of CFAR” (GO-CFAR) samt “Smallest of CFAR” (SO-CFAR). Med målet att förbättra detektionen föreslås även en ny CFAR-metod, vilken benämns ”Cell Evaluation target detection”. I detta arbete visas denna metod undertrycka begränsningar med konventionella tekniker. Den undviker även en del antaganden som inte alltid stämmer i praktiken. Studien inkluderar även skattning av riktning. Det visas hur visualisering av skyddsobjekt kan förbättras, genom att felaktigheter elimineras efter sammanläggning av detektioner från flera radarmoduler efter koordinattransformation.

Diese Arbeit wird durch die AdS/CFT Korrespondenz, sowie durch die Dualität zwischen lichtartigen, polygonalen Wilsonschleifen und Gluonenstreuamplituden in N=4 Super-Yang-Mills-Theorie motiviert. Bei starker Kopplung haben lichtartige, polygonale Wilsonschleifen und Gluonenstreuamplituden eine Beschreibung über raumartige Minimalflächen in AdS5. Wir benutzen eine Pohlmeyerreduktion, um eine Klassifikation aller raumartigen Minimalflächen in AdS3xS3 mit flachen Projektionen herzuleiten. Diese Klassifikation enthält neun verschiedene Klassen von Flächen. Dabei treten raumartige, zeitartige und degenerierte AdS3-Projektionen auf. Bei denjenigen Lösungen, die einen geschlossenen, polygonalen und lichtartigen Rand besitzen, berechnen wir den regularisierten Flächeninhalt. Bei schwacher Kopplung erfüllen lichtartige, polygonale Wilsonschleifen und Gluonenstreuamplituden den um eine Remainderfunktion korrigierten BDS-Ansatz. Wir präsentieren eine Technik, die auf einer Renormierungsgruppengleichung für selbstschneidende Wilsonschleifen beruht, mit der wir die Divergenzen der Remainderfunktion in diesem Limes berechnen können. Mittels dieser Technik analysieren wir zwei Arten des Selbstschnittes. Im Falle des Selbstschnittes zwischen zwei Ecken berechnen wir die führenden Divergenzen bis zur vierten Schleifenordnung. Beim Selbstschnitt zwischen zwei Kanten berechnen wir die führenden und nächstfolgenden Divergenzen bis zur vierten Schleifenordnung und präsentieren eine analytische Fortsetzung in die Region der Euklidischen Wilsonschleifen und sagen bestimmte Terme vorher, die in dem unbekannten analytischen Ausdruck für die Remainderfunktion enthalten sein müssen.
This thesis is motivated by the AdS/CFT correspondence and the duality between gluon scattering amplitudes and light-like polygonal Wilson loops in N=4 super Yang-Mills theory. At strong coupling light-like polygonal Wilson loops and gluon scattering amplitudes have a description in terms of space-like minimal surfaces in AdS5. We use a Pohlmeyer reduction to derive a classification of all space-like minimal surfaces in AdS3xS3 that have flat projections. The classification consists of nine different classes and contains space-like, time-like and degenerated AdS3 projections. For solutions that admit a closed light-like polygonal boundary we calculate the regularized area. At weak coupling light-like polygonal Wilson loops and gluon scattering amplitudes obey the BDS Ansatz corrected by a remainder function. We present a renormalisation group equation technique using self-crossing Wilson loops to extract the divergences of the remainder function in this limit. Using this technique we analyse two different types of self-crossing. We present the leading and sub-leading divergences up to four loops for a crossing between two edges and the leading divergences for a crossing between two vertices. For a crossing between two edges we present an analytic continuation to the euclidean regime to predict certain terms that have to occur in the unknown analytic expression of the remainder function.

In dieser Dissertation werden Aspekte von superkonformen Quantenfeldtheorien untersucht, die für die sogenannte AdS/CFT Korrespondenz relevant sind. Die AdS/CFT Korrespondenz beschreibt eine Dualität zwischen Stringtheorien im Anti-de Sitter Raum und superkonformen Quantenfeldtheorien im Minkowskiraum. In diesem Kontext wurde die sog. Wilsonschleifen / Amplituden Dualität entdeckt, die die Übereinstimmung von n-Gluon MHV Amplituden und n-seitigen polygonalen Wilsonschleifen in der N=4 supersymmetrischen Yang-Mills (SYM) Theorie beschreibt. Im ersten Teil dieser Dissertation wird die Wilsonschleifenseite einer solchen möglichen Dualität in der N=6 superkonformen Chern-Simons (ABJM) Theorie untersucht. Das Hauptergebnis dieser Untersuchungen ist, dass der Erwartungswert der n-seitigen polygonalen Wilsonschleifen auf Einschleifenebene verschwindet, während er auf Zweischleifenebene in seiner funktionalen Form identisch zu der analogen Wilsonschleife in N=4 SYM auf Einschleifenniveau ist. Außerdem wird eine anomale konforme Wardidentität für Wilsonschleifen in Chern-Simons Theorie berechnet. Zudem werden die damit im Zusammenhang stehenden Entwicklungen für Amplituden und Korrelatoren in der ABJM Theorie diskutiert. Im zweiten Teil dieser Dissertation werden Dreipunktfunktionen von zwei geschützten Operatoren und einem Twist-Zwei Operator mit beleibigem Spin j in der N=4 SYM Theorie berechnet. Dafür werden die Indizes des Spin j Operators auf den Lichtkegel projiziert und der Korrelator wird in einem Grenzfall untersucht in dem der Impuls der bei dem Spin j Operator einfließt verschwindet. Dieser Grenzfall vereinfacht die perturbative Berechnung erheblich, da alle Dreipunktdiagramme effektiv auf Zweipunktdiagramme reduziert werden und die Abhängigkeit der Mischungsmatrix auf Einschleifenebene herausfällt. Das Ergebnis stimmt mit der Analyse der Operatorproduktentwicklung von Vierpunktfunktionen geschützter Operatoren von Dolan und Osborn aus dem Jahre 2004 überein.
In this thesis aspects of superconformal field theories that are of interest in the so-called AdS/CFT correspondence are investivated. The AdS/CFT correspondence states a duality between string theories living on Anti-de Sitter space and superconformal quantum field theories in Minkowski space. In the context of the AdS/CFT correspondence the so-called Wilson loop / amplitude duality was discovered, stating the equality of the finite parts of n-gluon MHV amplitudes and n-sided lightlike polygonal Wilson loops in N=4 supersymmetric Yang-Mills (SYM) theory. It is the subject of the first part of this thesis to investigate the Wilson loop side of a possible similar duality in N=6 superconformal Chern-Simons matter (ABJM) theory. The main result is, that the expectation value of n-sided lightlike polygonal Wilson loops vanishes at one-loop order and at two-loop order is identical in its functional form to the Wilson loop in N=4 SYM theory at one-loop order. Furthermore, an anomalous conformal Ward identity for Wilson loops in Chern-Simons theory is derived. Related developments and symmetries of amplitudes and correlators in ABJM theory are discussed as well. In the second part of this thesis we calculate three-point functions of two protected operators and one twist-two operator with arbitrary even spin j in N =4 SYM theory. In order to carry out the calculations, the indices of the spin j operator are projected to the light-cone and the correlator is evaluated in a soft-limit where the momentum coming in at the spin j operator becomes zero. This limit largely simplifies the perturbative calculation, since all three-point diagrams effectively reduce to two-point diagrams and the dependence on the one-loop mixing matrix drops out completely. The result is in agreement with the analysis of the operator product expansion of four-point functions of half-BPS operators by Dolan and Osborn in 2004.

Tese de doutoramento, Bioquímica (Genética Molecular), 2010, Universidade de Lisboa, Faculdade de Ciências
Disponível no documento
Fundação para a Ciência e Tecnologia do Ministério da Ciência, Tecnologia e Ensino Superior SFRH/BD/17475/2004

Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion in vivo. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia.

To identify the developmental requirements for cftr function, we generated cftr mutant zebrafish using transcription activator like effector nucleases (TALENs). In cftr mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in cftr mutants, which prevents directional fluid flow necessary for KV function. To examine cftr expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the cftr promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for cftr during KV morphogenesis and function in the zebrafish.

In the zebrafish pancreas, we found that loss of cftr function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of cftr BAC expression in the adult and larval zebrafish pancreata revealed that cftr is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult cftr mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that cftr is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and cftr mutant larvae. These results indicate that cftr-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of cftr function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.

Dissertation

Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2008
Cystic Fibrosis (CF) is the most common lethal monogenic autosomal recessive disease in the Caucasian population and is caused by dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, usually located at the apical membrane of epithelial cells. The most common disease-causing mutation, F508del, causes CFTR protein to be retained at the endoplasmic reticulum (ER) and targeted to proteasomal degradation. Despite great efforts to elucidate the mechanisms and the molecular partners involved in CFTR biogenesis, intracellular localization, trafficking and function, many processes are not fully understood. Protein kinases and phosphatises are known for long to regulate CFTR function (and possibly localization). However, the role of phosphorylation in CFTR biogenesis and trafficking remains uncertain. CFTR processes several CK2 phosphorylation sites (namely one at S511 and another at T1471) and one SYK consensus site at Y512. We produced CFTR mutants in which the consensus residues S511, Y512 and T1471 were substituted by either a neutral (alanine, A) or an acidic residue (aspartic acid, D) in both wt and F508del-CFTR backgrounds and used them to stable transfect BHK cells. Pulsechase experiments followed by CFTR immunoprecipitation and western blot were performed in the procuded cell lines. After quantification of bands B and C of CFTR, results show that whereas substitution of S511 does not affect the turnover or processing of either wt- or F508del-CFTR, mutation of T1471 completely impairs processing of wt-CFTR without affecting significatively F508del-CFTR turnover. However, treatment of cells with 20 uM TBB (tetrabromobenzotriazole , a specific inhibitor of CK2) shows a significant decrease in processing efficiency of wt-CFTR. Furthermore, mutation of putative SYK target Y512 also reduces the steady-state levels of fully processed CFTR without a major impact on F508del-CFTR. Altogether, our data indicate that CK2 and SYK may have a stabilizing role upon wt-CFTR. This effect seems to be independent on residue S511 (or on the putative charge added by aspartic acid replacement at this residue) but apparently dependent on residues Y512 and T1471. However, the effects observed for replacement of these residues suggest that the role of CK2 or SYK is not by direct phosphorylation of CFTR at these positions
Resumo alargado disponível em português

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

Cystic Fibrosis (CF) is caused by mutations in the CFTR gene. The relationship between CFTR and differentiation has been firmly documented. However, the mechanisms that regulate this relationship are still unclear. The aim of this work was to further characterize the differentiation defect in CF and to identify the underlying molecular mechanisms. In the first chapter, our results show that CF cells display increased proliferation, acquire lower levels of TEER upon polarization, display slower wound healing, and have altered differentiation. Moreover, our data suggest that partial epithelial-mesenchymal transition occurs in CF cells. Given that Krüppel-like factors (KLFs) play pivotal roles in the regulation of differentiation, in the second chapter we tested the levels of KLF2, 4 and 5 and found that only KLF4 is differentially expressed in CF vs non-CF cells. Therefore, we then assessed whether KLF4 modulation had an impact in CFTR levels and function. Our data show that KLF4 acts as a negative regulator of wt-CFTR expression and function. Noteworthy, F508del cells are relatively insensitive to KLF4 modulation. Next, we tested possible pathways by which KLF4 action can impact CFTR in the CF and non-CF contexts. Our data showed that AKT signalling acts as a modulator of CFTR in a KLF4-dependent way, whilst GSK3β was observed to be a negative regulator of CFTR independent of KLF4 in CF cells. In chapter three, our data show that KLF4 knock-out has no major impact in proliferation but modulates TEER acquisition, wound healing, and differentiation markers differentially in CF vs non-CF cells. Moreover, we characterized the levels of KLFs, CFTR and proteins of interest during epithelial differentiation and showed that maximum KLF4 expression occurs prior to maximum CFTR expression. Altogether these data contribute to mechanistic clarification of the relationship between CFTR and epithelial differentiation.

卵巢是女性生殖系統中一個重要的器官，負責為受精提供卵子，以及合成生殖過程中所必需，同時也在其他生理過程中起重要作用的各種激素。大約有30%的不育源於卵巢的問題，包括無排卵，無月經，月經週期不規律和激素水平異常等。雄激素:雌激素比例過高，卵泡發育異常，無排卵等卵巢功能障礙常見於各種疾病中，例如多囊性卵巢綜合征(PCOS)--一種影響5~10%育齡婦女的內分泌疾病，以及囊性纖維化( CF)--一種由囊性纖維化跨膜電導調節器(CFTR) 基因突變引起的遺傳疾病。然而引起這些卵巢功能障礙的確切機制並不清楚。
雌激素是在卵泡雌激素(FSH) 的調節下，在卵巢顆粒細胞中通過芳香化臨的住激素轉化而生成的。在論文第一部分的研究中，我們旨在證明CFTR 在卵巢顆粒細胞中的表達，以及它參與雌激素生成的過程。實驗結果證實了CFTR 在小鼠和人顆粒細胞中的表達，同時表明CFTR 通過一種碳酸氫根離子(HC0₃⁻) 敏感的可溶性腺苦酸環化梅(sAC) ，放大FSH 所刺激的雌激素生成過程。實驗結果顯示，在原代小鼠顆粒細胞中， HC0₃⁻能夠增強FSH 所引起的CREB 磷酸化，芳香化晦表達，以及雌激素的生成，而在抑制CFTR 的情況下，或在CFTR 敲除/DeltaF508 突變小鼠的顆粒細胞中， HCO3-的放大作用顯著降低。CFTR 和芳香化醋的表達水準在人顆粒細胞中具有正相關性，進一步支持CFTR 對雌激素生成的調節作用。在PCOS 患者的顆粒細胞和大鼠PCOS 模型的卵巢中， CFTR 和芳香化醋的表達水準顯著下調。這些結果提示， CFTR 對雌激素生成調節這一機制的缺陷可能參與了CF 和PCOS 中卵巢功能障礙的發病機理。
卵泡發育很大程度上依賴於顆粒細胞的增殖'生存和凋亡，這些過程在PCOS 中都會出現異常。論文的第二部分冒在研究顆粒細胞的CFTR 在PCOS 的卵泡發育異常中的作用。實驗結果表明， CFTR 在PCOS 大鼠的囊，性卵泡的顆粒細胞中表達降低，同時伴隨著PCNA 和Bcl-2 的下調，而Bax 和cleaved caspase-3則沒有變化，提示顆粒細胞的增殖和生存/抗凋亡能力降低。敲減或抑制顆粒細胞中的CFTR 導致細胞存活降低， PCNA 和Bcl-2 表達下調，以及細胞凋亡增加，提示CFTR 對顆粒細胞增殖和生存的調節作用。CFTR 通過HC0₃⁻/ sAC/PKA 信號通路，調節基礎及FSH 刺激引起的ERK I!2 磷酸化，及其下游的CyclinD2 和PCNA表達，從而促進顆樹圍胞的增殖。顆粒細胞CFTR 的下調可能通過抑制細胞增殖和降低細胞生存能力，參與了PCOS 中的囊性卵泡的形成過程。
綜上所述，本論文證明了CFTR 在卵巢顆粒細胞上的表達，並且參與調節顆粒細胞雌激素生成和細胞的增殖和生存。CFTR 的缺陷或表達下調可能是導致CF和PCOS 的卵巢功能障礙的發病機理。
The ovary is the female reproductive organ, which produces female gametes, oocytes for fertilization and sex hormones essential to reproduction and important to a wide range of physiological and pathological events as well. About 30% of infertility cases arise from ovarian problems, including anovulation, amenorrhea, irregular menstrual cycle and abnormal hormone levels. Ovarian disorders, such as high androgen to estrogen ratio, abnormal folliculogenesis and anovulation, are often seen in diseases, including polycystic ovarian syndrome (PCOS) and cystic fibrosis (CF). The former is an endocrine disorder affecting 5~10% women of reproductive age, and the latter is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). However, the exact mechanisms underlying the ovarian disorders seen in these diseases are not well understood.
Estrogen biosynthesis is profoundly influenced by follicle-stimulating hormone (FSH) that regulates the conversion of androgen to estrogen in ovarian granulosa cells by the rate-limiting enzyme aromatase. The first part of the study aims to investigate the expression of CFTR in granulosa cells and its involvement in regulating estrogen production. The results demonstrate the expression of CFTR in both mouse and human granulosa cells, and provide evidence demonstrating a previously unsuspected role of CFTR in amplification of FSH-stimulated ovarian estrogen biosynthesis and the involvement of a HC0₃⁻ sensor, the soluble adenylyl cyclase (sAC) in this synthesis. FSH-stimulated CREB phosphorylation, aromatae expression, as well as estradiol production are enhanced by HC0₃⁻ and sAC, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/deltaF508 mutant mice. The fact that CFTR expression is found positively correlated with aromatase expression in human granulosa cells supports its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in polycystic ovarian syndrome (PCOS) rodent models and human patients. These findings suggest that defective CFTR-dependent regulation of estrogen production may underline the ovarian disorders seen in CF and PCOS.
Folliculogenesis largely depends on the proliferation, survival and apoptosis of granulosa cells in the follicles and alteration in which has been found in PCOS. The second part of the study aims to investigate the possible involvement of granulosa cell CFTR in the impaired folliculogenesis in PCOS. The results show that downregulation of CFTR is found in the cystic follicles, which is accompanied by reduced expression of PCNA and Bcl-2, but not Bax and cleaved caspase-3, in the ovaries of PCOS rat models, indicating reduced cell proliferation and survival/anti-apoptotic ability. Knockdown or inhibition of CFTR in granulosa cell culture results in reduced cell viability, downregulation of PCNA and Bcl-2 and increase of apoptosis, supporting a role of CFTR in regulating granulosa cell proliferation and survival. CFTR exerts its effect on granuloa cell proliferation by modulating basal and FSH-stimulated ERKl/2 phosphorylation and the expression of its downstream target CyclinD2 and PCNA through the HC0₃⁻/sAC/PKA pathway. These findings suggest that downregulation of CFTR may play a role in the formation of cystic follicles by inhibiting granulosa cell proliferation and reducing cell survival ability, therefore providing a possible mechanism for the abnormal folliculogenesis in PCOS.
In conclusion, the present study has demonstrated the expression of CFTR in the ovarian granulosa cell and its role in regulation of granulosa cell proliferation, survival and estrogen production. Defect of CFTR in CF and downregulation of CFTR in PCOS may contribute to the abnonnal honnone profile and impaired folliculogenesis in both disease conditions.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Chen, Hui.
"October 2011."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 124-137).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
ABSTRACT --- p.i
摘要 --- p.iv
ACKNOWLEDGEMENT --- p.vi
LIST OF PUBLICATIONS --- p.viii
ABBREVIATIONS --- p.xiii
LIST OF FIGURES AND TABLES --- p.xvi
Chapter 1 --- CHAPTER I: Introduction --- p.1
Chapter 1.1 --- The ovary --- p.1
Chapter 1.1.1 --- Structure and function of the ovary --- p.1
Chapter 1.1.2 --- Follicle development --- p.5
Chapter 1.1.3 --- Ovulation and luteinization --- p.7
Chapter 1.1.4 --- Ovarian hormone biosynthesis --- p.10
Chapter 1.2 --- Diseases with ovarian dysfunction --- p.14
Chapter 1.2.1 --- Polycystic ovarian syndrome (PCOS) --- p.14
Chapter 1.2.1.1 --- Introduction to PCOS --- p.14
Chapter 1.2.1.2 --- Diagnostic criteria --- p.14
Chapter 1.2.1.3 --- Abnormal hormone profile in PCOS --- p.16
Chapter 1.2.1.4 --- Abnormal folliculogenesis in PCOS --- p.18
Chapter 1.2.1.5 --- Etiology --- p.22
Chapter 1.2.2 --- Cystic Fibrosis (CF) --- p.24
Chapter 1.2.2.1 --- Introduction to CF --- p.24
Chapter 1.2.2.2 --- Cause and pathogenesis of CF --- p.25
Chapter 1.2.2.3 --- Ovarian disorder in CF --- p.27
Chapter 1.3 --- CFTR in reproduction --- p.29
Chapter 1.3.1 --- Introduction to CFTR --- p.29
Chapter 1.3.2 --- Channel function --- p.30
Chapter 1.3.3 --- Protein regulator function --- p.32
Chapter 1.3.4 --- Regulation of CFTR expression --- p.34
Chapter 1.3.5 --- Role of CFTR in reproduction --- p.35
Chapter 1.3.6 --- CFTR in the ovary --- p.39
Chapter 1.4 --- General hypothesis and aims --- p.39
Chapter 1.4.1 --- General hypothesis --- p.39
Chapter 1.4.2 --- Aims of the study --- p.40
Chapter 2 --- CHAPTER II: General Methods --- p.42
Chapter 2.1 --- Meterials --- p.42
Chapter 2.1.1 --- Animals --- p.42
Chapter 2.1.2 --- Chemicals and reagents --- p.42
Chapter 2.1.3 --- Antibodies --- p.44
Chapter 2.1.4 --- Primers --- p.45
Chapter 2.2 --- Methods --- p.45
Chapter 2.2.1 --- Determination of estrous cycle --- p.45
Chapter 2.2.2 --- Granulosa cell culture --- p.46
Chapter 2.2.3 --- PCGS rat model --- p.47
Chapter 2.2.4 --- Collection of human granulosa cells --- p.47
Chapter 2.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.48
Chapter 2.2.6 --- Western blot --- p.50
Chapter 2.2.7 --- Histological studies --- p.53
Chapter 2.2.8 --- siRNA transfection --- p.55
Chapter 2.2.9 --- Intracellular pH measurement --- p.56
Chapter 2.2.10 --- Whole-cell patch clamp recording --- p.57
Chapter 2.2.11 --- Statistics --- p.57
Chapter 3 --- CHAPTER III: Result I - The Role of CFTR in FSH-stimulated Estrogen Production: Implication in Cystic Fibrosis and PCGS --- p.59
Chapter 3.1 --- Summary --- p.59
Chapter 3.2 --- Introduction --- p.60
Chapter 3.3 --- Methods --- p.63
Chapter 3.3.1 --- Intracellular cAMP assay --- p.63
Chapter 3.3.2 --- Nuclei isolation and nuclear cAMP measurement --- p.63
Chapter 3.3.3 --- CREB phosphorylation assay --- p.64
Chapter 3.3.4 --- Estradiol enzyme immunoassay --- p.64
Chapter 3.4 --- Results --- p.64
Chapter 3.4.1 --- Functional expression of CFTR in granulosa cells --- p.64
Chapter 3.4.2 --- Expression and localization of sAC in granulosa cells and its involvement in BC03f CFTR-dependent cAMP production --- p.66
Chapter 3.4.3 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated CREB phosphorylation --- p.67
Chapter 3.4.4 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated aromatase expression and estradiol production --- p.68
Chapter 3.4.5 --- Impaired CREB phosphorylation aromatase expression and estradiol production by granulosa cells from CFTR-deficient mice --- p.70
Chapter 3.4.6 --- Reduced CFTR and aromatase expression in human PCOS granulosa cells and rat PCOS ovaries --- p.71
Chapter 3.5 --- Discussion --- p.87
Chapter 4 --- CHAPTER IV: Result II - The Role of CFTR in Granulosa Cell Proliferation and survival in PCOS --- p.91
Chapter 4.1 --- Summary --- p.91
Chapter 4.2 --- Introduction --- p.92
Chapter 4.3 --- Methods --- p.95
Chapter 4.3.1 --- Cell viability assay (MTT and MTS assay) --- p.95
Chapter 4.3.2 --- ERKI/2 phosphorylation assay --- p.95
Chapter 4.4 --- Results --- p.96
Chapter 4.4.1 --- Reduced CFTR expression in PCOS rat models --- p.96
Chapter 4.4.2 --- Downregulation of genes related to proliferation and survival in PCOS --- p.96
Chapter 4.4.3 --- CFTR affect viability of granulosa cells --- p.97
Chapter 4.4.4 --- CFTR regulate cell cycle protein and promote proliferation via HC0₃⁻/sAC/PKA and ERK pathway --- p.98
Chapter 4.4.5 --- CFTR regulates apoptosis-related protein expression --- p.100
Chapter 4.5 --- Discussion --- p.114
Chapter 5 --- CHAPTER V: General Discussion --- p.119
Chapter 5.1 --- Role of CFTR in ovarian function --- p.119
Chapter 5.2 --- Role of CFTR/HC0₃⁻/sAC in modulating FSH signaling in the ovary --- p.120
Chapter 5.3 --- CFTR/HC0₃⁻/sAC as a general modulator in receptor-mediated signaling cascades --- p.122
Chapter 5.4 --- Concluding remarks --- p.123
REFERENCES --- p.124
APPENDICES --- p.138

碩士
輔仁大學
基礎醫學研究所碩士班
102
A mild cystic fibrosis transmembrane conductance regulator (CFTR)-associated condition, congenital bilateral absence of vas deferens (CBAVD), appeared among 1-2% of infertile men. However, approximately 17 of 36 (47.2%) Taiwanese infertile men with CBAVD were unable to identify any CFTR mutations. In our previous research, using the oligonucleotide array based CGH and Real-time PCR for genome-wide search of copy number variants (CNVs) in Taiwanese men with CBAVD revealed that SLC9A3 (solute carrier family 9 subfamily A member 3) was a potential CBAVD-related gene. It was indicated that SLC9A3, encoded by SLC9A3, acted as a sodium hydrogen exchanger mainly regulates sodium absorption in human intestine and renal proximal tubule. It was shown to interact with CFTR both in vitro and in the luminal membrane of mouse pancreatic duct. Previous research also showed dilated rete testis, efferent ducts and male infertility in Slc9a3 knockout mice. Thus, we aimed to investigated the genetic roles of SLC9A3 in Taiwanese CBAVD. According to RT-PCR, Slc9a3 expressed in vas deferens; further, SLC9A3 localized to the mouse apical and basolateral membrane of luminal epithelium in vas deferens by immunofluorescent staining. By DNA sequencing of SLC9A3 in 30 Taiwanese CBAVD patients, we found multiple genetic variants including c.676-44C>A, c.2031G>A, c.2068-43G>C and c.2128A>C. On the other hand, the phenotype of reproductive system showed increase weight in testis, but decrease weight in epididymis, vas deferens and seminal vesicle. By haeomatoxylin and eosin staining, the compression of germ cells layer in testis, absence of matured sperm in partial lumen of caput epididymis, absence of matured sperm in cauda epididymis and vas deferens were also observed in male mice lacking Slc9a3 alleles. In summary, our study demonstrated that multiple genetic variants of SLC9A3 may possibly attributed to Taiwanese CBAVD and Slc9a3-/- in mice induced male infertility and pathological changes in testis, epididymis and vas deferens.

Mast cells (MC) are present in nearly all tissues in the body and participate in many physiological processes including allergy, tissue remodelling, fibrosis, angiogenesis, and autoimmunity. They can be activated by many stimuli, including allergic and innate immune stimulation. When activated, MC release mediators through which they can regulate inflammatory processes. Recently, we have discovered that rat and human MC express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the gene responsible for Cystic Fibrosis (CF). We showed that CFTR had functional activity in MC and its expression was differentially regulated by IFNg. In this thesis, we compared CFTR expression between MC and epithelial cells (EC) by Western blot analysis and found that CFTR expression in MC is similar to that in EC, but there are some differences which suggest either glycosylation or post-transcriptional/translational differences between MC and EC. We also explored the role of CFTR in human MC secretion from various cellular compartments, in response to various stimuli. When we blocked CFTR using pharmacological inhibitors, there was an inhibition of cAMP-dependent Cl- flux. Our data also shows that CFTR pharmacological inhibition had no effect on IgE/anti-IgE-mediated b-hexosaminidase or eicosanoid release from MC. When we stimulated MC with either IgE/anti-IgE or the adenosine receptor agonist NECA (3 uM) for 24h in the presence of CFTR inhibitors, secretion of several mediators appeared to be dysregulated including IL-8, MIF, IL-13, IL-16, PAI-1 and CCL1. To add to these findings, we also used short hairpin RNA (shRNA) to reduce CFTR expression in MC. CFTR deficient MC were unresponsive to NECA and showed reduced constitutive IL-6 secretion. Finally, we cultured MC from CF and non-CF donor peripheral blood progenitors and compared several phenotypic and functional aspects of the cells. We saw no difference in growth, protease content and surface marker expression between CF and non-CF MC, but stimulation of the cells with IgE/anti-IgE or Pseudomonas aeruginosa appeared to differentially induce cytokine synthesis and secretion from CF and non-CF MC. These findings suggest that MC function is dysregulated in CF and that CF MC may be involved in the pathophysiology of CF.
Experimental Medicine

Cheung King Ho.
"August 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 140-158).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

CFTR基因突變或者功能缺失是否導致包括胃腸道在內的各種組織惡性腫瘤的發生風險增加目前仍然是一個充滿爭議的問題。同時，眾所周知，緊密連接分子在腫瘤發生和轉移的過程發揮了關鍵的作用。本論文首次發現了CFTR基因與一種緊密連接分子AF-6/afadin的在人類結直腸腫瘤中的表達水平呈高度相關，并研究了CFTR和AF-6/afadin之間潛在的相互作用及其在結直腸腫瘤轉移中的功能。
論文的第一部份首先用實時定量PCR和免疫組織化學的方法比較了CFTR在結直腸腫瘤和正常組織的表達情況，發現CFTR表達水平在腫瘤組織中有顯著的下降。令人感興趣的是，我們同時發現CFTR和AF-6/afadin在腫瘤組織中的表達呈高度正相關，并由此展開了後續的體外實驗，研究對CFTR與AF-6/afadin之間可能的相互聯繫。利用免疫螢光染色和免疫共沉澱的方法，我們發現了這兩種蛋白分子共表達在結直腸腫瘤細胞的接觸面，并存在相互作用。用CFTR突變蛋白的免疫共沉澱實驗進一步發現，這種相互作用需要CFTR分子在細胞膜表面的正確定位及其PDZ結構域結合位點。實驗還發現與CFTR的相互作用加強了AF-6/afadin與細胞骨架蛋白系統的結合。在結直腸腫瘤細胞中CFTR基因敲减导致了AF-6/afadin蛋白定位混亂，從細胞連接位點轉移到細胞漿內，并因此破壞了上皮細胞的緊密性。極性生長細胞的跨上皮電阻降低而滲透性增強的實驗結果證實了CFTR基因敲減導致的上皮細胞緊密性的破壞。同時，AF-6/afadin蛋白水平也隨著CFTR基因敲減而降低，但mRNA水平未發生明顯的改變。蛋白降解系統的抑製劑逆轉了CFTR基因敲減細胞中AF-6/afadin蛋白的減少，提示CFTR基因敲減增加了AF-6/afadin的蛋白降解。這些實驗結果揭示了通過與細胞連接分子AF-6/afadin的相互作用以及調節，CFTR可能在上皮細胞極性的調節以及腫瘤發展過程中起重要作用。
論文的第二部份研究了CFTR和AF-6/afadin在結直腸腫瘤細胞上皮細胞間充質化（EMT）和轉移過程中的功能及機制。我們之前的工作已經揭示抑制CFTR的功能可以誘導結直腸腫瘤LIM1863細胞的EMT過程。本研究在另外三株不同的結直腸腫瘤細胞（SW480，SW1116和HRT-18）中進一步證實了抑制CFTR誘導的EMT過程。細胞形態轉變，上皮細胞標誌物的下調，間充質細胞標誌物的上調以及受損的上皮細胞緊密性均證實了對CFTR的抑制可以在這三種細胞中成功誘導EMT的發生。我們發現在以上所有細胞EMT的過程中，AF-6/afadin的蛋白表達水平都發生了顯著的下調。在HRT-18細胞中過表達AF-6/afadin，可以逆轉由CFTR抑製劑誘導的上皮細胞標誌分子的下調和間充質標誌分子的上調，表明抑制CFTR誘導的EMT過程是由AF-6/afadin參與介導的。此外，CFTR基因敲減導致結直腸腫瘤細胞的惡性表型強化，包括減弱的細胞粘附性，增強的貼壁依賴性生長、侵襲和遷移。另外，CFTR基因敲減激活了ERK的磷酸化，過表達AF-6/afadin可以阻斷ERK途徑的激活。CFTR基因敲減而增強的細胞侵襲性也可以被外源性AF-6/afadin或者ERK途徑的抑製劑U0126完全逆轉，提示作為AF-6/afadin的下游靶信號，ERK介導了CFTR在腫瘤侵襲中的作用。更重要的是，我們分析了CFTR和AF-6/afadin的表達水平與結直腸癌病人腫瘤進展的關係，發現在嚴重TNM腫瘤分期或者有腫瘤遠處轉移的病人中CFTR的表達水平顯著低於輕型分期或未发生转移的病人中的水平，而且CFTR和/或AF-6/afadin低表達的病人的預後更差。這些實驗結果顯示CFTR的缺失可能通過抑制AF-6/afadin和激活ERK通路而與EMT和結直腸癌癥轉移的過程高度相關。
綜上所述，本研究揭示了以往未報道過的CFTR在結直腸腫瘤發病機理中的功能，提示CFTR可以用作一種新的腫瘤的潛在預後指標。
The question whether mutation or dysfunction of CFTR increases the risk of malignancies in various tissues, including the gastrointestinal tract, remains highly controversial. Meanwhile, it is well-known that adherens junctions play critical roles in the process of cancer development and metastasis. In this thesis we found for the first time a highly correlation between expression levels of CFTR and an adherens junction molecule AF-6/afadin in human colorectal tumours, and investigated the potential interaction between CFTR and AF-6/afadin and their functional roles in the metastasis of colorectal cancer.
In the first section of this thesis, we started our studies with comparing the expression of CFTR between human colorectal tumours and normal colorectal tissues. Real time quantitative PCR and immunohistochemistry results revealed a dramatically reduced CFTR level in the cancer tissues. Intriguingly, we noticed a highly positive correlation between CFTR and AF-6/afadin expression in tumours, which prompted the further in vitro investigation of possible interaction between CFTR and AF-6/afadin. Using immunofluoresent staining and co-immunoprecipitation, we found that the two proteins were colocalized at cell-cell junctions and interacted with each other in colorectal cancer cell lines. Further Co-IP experiments performed with CFTR mutations revealed that this protein interaction requires the proper localization of CFTR in cell membrane and its PDZ-interacting domain. Moreover the interaction with CFTR strengthens the binding of AF-6/afadin to the cytoskeleton system. Knockdown of CFTR in colorectal cancer cells resulted in the disorganized localization of AF-6/afadin protein from junctional sites to the cytoplasm and impaired epithelial tightness, which was confirmed by significantly reduced transepithelial resistance and increased permeability of polarized cells. Meanwhile, the protein level of AF-6/afadin was down-regulated in CFTR-knockdown cells, while no significant changes were detected at the mRNA level. Protein degradation inhibitor reversed the repression of AF-6/afadin protein in CFTR knockdown cells, suggesting the protein degradation of AF-6/afadin was increased by CFTR knockdown. These data revealed that CFTR interacts with and regulates the cell adhesion molecular AF-6/afadin in colorectal cells, which may be important in the regulation of epithelial cell polarity and cancer development.
In the second section of this thesis, we studied the functional roles and mechanisms of CFTR and AF-6/afadin in the epithelial-mesenchymal transition (EMT) and metastasis of human colorectal cancer cells. Our previous work has revealed inhibition of CFTR can induce EMT in a colorectal cancer cell line, LIM1863. This study further confirmed the induction of EMT by inhibiting CFTR in several other colorectal cancer cell lines (SW480, SW1116 and HRT-18), which was evaluated by morphological changes, down-regulation of epithelial markers or up-regulation of mesenchymal markers, and impaired epithelial cell tightness. In all these cell lines, we found that the protein levels of AF-6/afadin were significantly reduced. Over-expression of AF-6/afadin in HRT-18 cells reversed the down-regulated epithelial markers and up-regulated mesenchymal markers induced by CFTR inhibition, indicating that the CFTR inhibition-induced EMT is mediated by AF-6/afadin. Moreover, knockdown of CFTR in HRT-18 or RKO cells resulted in enhanced malignant phenotypes, including decreased cell adhesion, increased anchorage-independent cell growth, invasion, and migration. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was activated by CFTR knockdown, which was abolished by over-expression of AF-6/afadin. The enhanced invasiveness of CFTR knockdown cells was also completely inhibited by either exogenous AF-6/afadin or ERK inhibitor, U0126, suggesting that ERK, the downstream target of AF-6/afadin, is involved in mediating the effect of CFTR in cancer invasion. More importantly, we analyzed the association of CFTR and AF-6/afadin expression levels with tumour progression of patients with colorectal cancer, and revealed that CFTR expression was significantly lower in patients with more severe TNM stage or with metastasis to distant organs than those with milder stage or with no metastasis. The prognosis was poorer in patients with lower expression of CFTR and/or AF-6/afadin than those with higher expressions. These data showed that dysfunction of CFTR is highly associated with EMT and colorectal cancer metastasis, probably via repression of AF-6/afadin and activation of ERK pathways.
In summary, the present study has revealed a previously undefined role of CFTR in the pathogenesis of colorectal cancer and indicated its potential as a new prognostic indicator.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Sun, Tingting.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 113-127).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstract --- p.i
中文摘要 --- p.iv
Publications --- p.vi
Conference Abstract --- p.vii
Declaration --- p.viii
Acknowledgements --- p.x
List of Figures --- p.xi
List of Tables --- p.xiii
List of Abbreviations --- p.xiv
Chapter Chapter 1 --- General Introduction --- p.1
Chapter 1.1. --- Colorectal Cancer --- p.1
Chapter 1.1.1. --- Structure of Human Normal Colon and Rectum Epithelium --- p.1
Chapter 1.1.2. --- Staging of Colorectal Cancer --- p.3
Chapter 1.1.3. --- Metastasis of Colorectal Cancer --- p.3
Chapter 1.1.4. --- K-Ras mutation and It Downstream Pathways in Colorectal Cancer Metastasis --- p.11
Chapter 1.1.5. --- Prognosis of Colorectal Cancer --- p.14
Chapter 1.2. --- Epithelial Cell Junctional Complexes --- p.14
Chapter 1.2.1. --- Junctional Complexes and Epithelial Cell Polarity --- p.15
Chapter 1.2.2. --- Classic Cadherin-catenin Complex --- p.17
Chapter 1.2.3. --- Novel Nectin-afadin Complex --- p.19
Chapter 1.2.4. --- Cell Polarity and Cancer Progression --- p.23
Chapter 1.3. --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.24
Chapter 1.3.1. --- Structure of CFTR --- p.24
Chapter 1.3.2. --- Mutations of CFTR --- p.24
Chapter 1.3.3. --- Functions of CFTR --- p.26
Chapter 1.3.4. --- Cancer Risk of CF Patients --- p.33
Chapter 1.4. --- Hypothesis and Aims --- p.34
Chapter Chapter 2 --- Materials and Methods --- p.35
Chapter 2.1. --- Materials --- p.35
Chapter 2.1.1. --- Reagents and Chemicals --- p.35
Chapter 2.1.2. --- Antibodies --- p.35
Chapter 2.1.3. --- Primers --- p.35
Chapter 2.1.4. --- Solutions and Buffers --- p.35
Chapter 2.1.5. --- Human Specimens --- p.36
Chapter 2.2. --- Methods --- p.36
Chapter 2.2.1. --- Cell Culture --- p.36
Chapter 2.2.2. --- Transfection --- p.36
Chapter 2.2.3. --- Selection of Stable Clones --- p.40
Chapter 2.2.4. --- RNA Extraction and RT-PCR --- p.40
Chapter 2.2.5. --- Quantitative Real Time PCR --- p.41
Chapter 2.2.6. --- Protein Extraction and Western Blotting --- p.42
Chapter 2.2.7. --- Immunostaining --- p.45
Chapter 2.2.8. --- In vitro Cell Functional Assays --- p.46
Chapter 2.2.9. --- Epithelial Tightness Measurement --- p.48
Chapter 2.2.10. --- Statistical Analysis --- p.49
Chapter Chapter 3 --- Interaction of CFTR with AF-6/afadin and Its Importance in Maintaining Colorectal Epithelial Cell Polarity --- p.50
Chapter 3.1. --- Introduction --- p.50
Chapter 3.2. --- Objectives --- p.53
Chapter 3.3. --- Experimental plan --- p.54
Chapter 3.4. --- Results --- p.55
Chapter 3.4.1. --- The expression of CFTR and AF-6/afadin is decreased and positively correlated in human colorectal cancer --- p.55
Chapter 3.4.2. --- CFTR colocalizes and interacts with AF-6/afadin in human colorectal cancer cells --- p.58
Chapter 3.4.3. --- PDZ binding motif and membrane localization of CFTR are necessary for the interaction between CFTR and AF-6/afadin --- p.64
Chapter 3.4.4. --- Knockdown of CFTR interferes with cell junction formation in colorectal cancer cells --- p.66
Chapter 3.5. --- Discussion --- p.71
Chapter Chapter 4 --- CFTR as a Suppressor and Prognosis Indicator of Metastasis in Human Colorectal Cancer --- p.77
Chapter 4.1. --- Introduction --- p.77
Chapter 4.2. --- Objectives --- p.80
Chapter 4.3. --- Experimental plan --- p.81
Chapter 4.4. --- Results --- p.82
Chapter 4.4.1. --- CFTR inhibition-induced EMT in colorectal cancer cells involves AF-6/afadin --- p.82
Chapter 4.4.2. --- Knockdown of CFTR aggravates malignant phenotype of colorectal cancer cells --- p.86
Chapter 4.4.3. --- AF-6/afadin mediates the effect of CFTR on cell invasion in colon cancer through ERK --- p.91
Chapter 4.4.4. --- CFTR and AF-6/afadin expression is correlated with the prognosis of colorectal cancer --- p.97
Chapter 4.5. --- Discussion --- p.100
Chapter Chapter 5 --- General Discussion and Conclusion --- p.105
Chapter 5.1. --- The diversified roles of CFTR in epithelial cells --- p.105
Chapter 5.2. --- The unfolding relationship between CFTR and cancer development --- p.107
Chapter 5.3. --- Future studies --- p.109
Chapter 5.4. --- Conclusions --- p.112
Reference List --- p.113
Chapter Appendix A --- Reagents and Chemicals --- p.128
Chapter Appendix B --- Antibody List --- p.131
Chapter Appendix C --- Primer List --- p.132
Chapter Appendix D --- Solution Recipe --- p.133

We report on the first systematic study of correlation functions in N=4 super Yang-Mills theory using integrability techniques. In particular, we show how to compute three- and four- point functions of single-trace gauge-invariant operators at tree level in the SU(2) sector of the theory. Using the AdS/CFT correspondence, the correlation functions that we compute can be thought of as the joining or splitting of strings moving in AdS5 × S5. We show that when one (two) of the operators in the three-(four-)point function are taken to be small BPS operators, our weak coupling results match perfectly with the strong coupling results in the Frolov-Tseytlin limit. We conclude by presenting some results that will be needed to extend the methods presented in this thesis beyond the SU(2) sector of N=4 super Yang-Mills.

Tese de mestrado, Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2015
Cystic Fibrosis (CF) is the most common lethal autosomal recessive disorder in the Caucasian population, affecting 1 in 2500-6000 new-borns. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes a cAMP-regulated chloride (Cl-) and bicarbonate (HCO3-) channel expressed at the apical membrane of a variety of epithelial cells. The most common mutation in CF patients is F508del, a three base pair deletion that causes a single amino acid deletion from CFTR and disrupts its traffic and function, due to protein misfolding. Obstructive lung disease is the primary cause of morbidity in CF patients. Other symptoms of CF include elevated concentrations of sodium chloride (Cl-) in the sweat, pancreatic insufficiency, and male infertility, among others. Besides its function as a Cl- channel, CFTR also controls a number of other ion channels and transporters, being crucial to the ionic flow on epithelia. Among these other channels is the epithelial sodium (Na+) channel (ENaC) and members of the anoctamin family, some of which have been shown to function as Cl- channels. The latter constitutes a class of proteins that is only expressed in eukaryotic organisms. In mammals the family contains ten members (Ano1 to Ano10), with high sequence conservation, particularly around the putative channel pore forming region. Since the identification of Ano1 as a Ca2+-activated Cl- channel (CaCCs), in 2008, anoctamins have raised high interest as possible alternative Cl- channels to compensate for the loss of functional CFTR in CF. However, little is known about the regulation of their biogenesis, traffic or whether their channel function can be activated by other means, other than Ca2+ signalling. Such knowledge is, nevertheless, essential for their possible therapeutic use as alternative Cl- channels for CFTR. Anoctamins are involved in a variety of functions that besides ion transport, include phospholipid scrambling and regulation of other membrane proteins. Several lines of evidence suggest that anoctamins form homodimers, but the possibility of heterodimer formation has not been excluded. Also, some family members are reported to interact with each other. Despite some knowledge on this protein family, the structure and functions of all family members are not completely defined. The first two members of the family (Ano1 and Ano2) function as Ca2+-activated Cl− channels (CaCCs), while Ano6 was described as scramblase and also as a main component of the outwardly rectifying Cl- channels (ORCCs). Other members of the anoctamin family, such as Ano3, 4, 7, and 9, may work as phospholipid scramblases and/or ion channels, being their functions still poorly understood. Mutations in anoctamin genes (ANO3, ANO5, ANO6, and ANO10) cause various genetic disorders, suggesting the involvement of anoctamins in a variety of cellular functions. The core objective of this project was to identify novel regulators of Ano6 and also Ano9 and 10 and their interaction with other anoctamin family members and CFTR. To accomplish this goal three main tasks were proposed, namely, i) To generate novel cell lines stably expressing double-tagged Ano6, Ano9 and Ano10 constructs; ii) To use these constructs to develop robust cell-based traffic assays to be used in fluorescence high-throughput microscopy; iii) To use siRNA microscopy screens to identify novel genes involved in regulating the traffic of Ano6, Ano9 and Ano10 as potential drug targets for CF. The first task was completed for Ano6 and Ano9, while the second one was only accomplished for Ano6, and the third just started also for this protein. In summary, in this MSc project, double-tagged constructs of Ano6, 9 and 10 were created. The constructs of Ano6 and Ano9 were cloned into lentiviral vectors, and several cell lines expressing the double-tagged anoctamins under an inducible (Tet-On) promoter were generated. CFBE Ano6/Ano9 3-HA GFP cells were characterized by immunostaining (with polarized and non-polarized cells) and Western Blot techniques. Additionally, functional analysis of these proteins was assessed by both the iodide efflux technique and analysis in perfused micro-Ussing chamber on open circuit mode. Finally, a pilot screen of a small siRNA library targeting 231 genes (previously tested for CFTR traffic) was performed for CFBE Ano6 3-HA GFP cells. Overall the results showed that: The double-tagged construct of Ano6 is functional as a channel when overexpressed either in HEK 293T cells or in CFBE cells; The double-tagged construct of Ano9 seems to increase I- efflux in HEK 293T but shows no effect on ion currents on CFBE cells; Overexpressed Ano6 is located at the plasma membrane (and also at the cytoplasm); Overexpressed Ano9 is mostly intracellularly located but when at the cell membrane possibly forms dimers; CFBE cells stably expressing the inducible (Tet-On) Ano6 3-HA GFP construct constitute a robust cellular model for siRNA screens by automated high-content microscopy (HCM); Results from a pilot siRNA screen suggest that Ano6 traffic to the plasma membrane is affected by genes involved in several biological processes like G-protein coupled receptor signalling, ion transport, protein phosphorylation, and regulation of apoptosis, among others. Additional studies and further validation of these results could potentially constitute a new attractive approach for CF therapy involving alternative Cl- channels. Indeed, the regulation of anoctamins’ traffic is largely unknown and its better understanding will allow the exploration of this pathway to compensate for CFTR deficiency, making possible the development of new therapies for CF.
A Fibrose Quística (FQ) é a doença autossómica recessiva letal mais comum na população caucasiana, com uma prevalência de 1 em cada 2500-6000 nascimentos. Esta doença é causada por mutações no gene Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Este gene, localizado no braço longo do cromossoma 7, codifica para uma glicoproteína com 1480 resíduos de aminoácidos localizada na membrana apical de uma grande variedade de células epiteliais, onde transporta cloreto (Cl-) e bicarbonato (HCO3-). Entre as mais de 2000 mutações conhecidas no gene CFTR, a mais comum consiste na deleção de três pares de bases, que resulta na remoção de uma fenilalanina na posição 508 (F508del) da proteína. Esta mutação está presente em aproximadamente 85% dos pacientes com FQ e afeta o processamento da proteína que, devido a um folding incorreto, fica retida intracelularmente ao nível do retículo endoplasmático (RE). Neste organelo é rapidamente enviada para degradação proteassomal, não chegando assim à membrana plasmática. Para além do seu papel como canal de aniões, a CFTR também funciona como reguladora de outros canais, tendo um efeito inibitório crítico na absorção de sódio (Na+) pela proteína ENaC (epithelial Na+ channel). Assim, os pacientes com FQ apresentam uma secreção de aniões diminuída, acompanhada por um aumento na absorção de Na+, o que leva à desidratação do líquido que reveste as vias respiratórias, airway surface liquid (ASL), e ao aumento da viscosidade e espessura do muco. Estas características causam uma eliminação ineficiente de agentes patogénicos pulmonares, resultando em infecções bacterianas recorrentes e, consequentemente, em inflamação crónica. Esta inflamação, dominada por neutrófilos, exacerba os processos de remodelação do tecido pulmonar e acaba por culminar na fibrose dos tecidos e perda da sua função. A doença pulmonar obstrutiva é a causa de morte mais comum em pacientes com FQ, sendo responsável por aproximadamente 80% da mortalidade. Para além deste fenótipo, outras manifestações da doença incluem a elevada concentração de NaCl no suor (sendo a base dos testes de diagnóstico mais comuns), insuficiência pancreática, infertilidade masculina, e ainda outras manifestações menos comuns como diabetes, ileus meconial, e obstrução intestinal e hepática. Como referido anteriormente, a CFTR tem um papel importante na regulação de outros canais e transportadores, sendo fundamental no transporte iónico no epitélio. Para além da ENaC já evidenciada, outros canais que possivelmente interatuam com a CFTR são os canais de cloreto ativados por cálcio (CaCC) e os canais de cloreto outwardly rectifying (ORCC), entre outros. Os CaCCs funcionam como transportadores iónicos transepiteliais em células secretoras e são caracterizados por apresentarem uma ativação dependente do aumento dos níveis de Ca2+ intracelular. Por sua vez, os ORCC demonstram uma relação I/V outwardly rectifying e são ativados por despolarização celular e via PKA e UTP extracelular. Vários estudos mostram que a CFTR inibe a produção endógena de corrente pelos CaCCs, estando esta aumentada nas vias respiratórias de pacientes com FQ. No entanto, a ativação dos ORCC pela PKA encontra-se diminuída no epitélio pulmonar de pacientes. As anoctaminas, proteínas que pertencem a uma família de 10 membros (Ano1 a Ano10) com elevada semelhança estrutural, foram associadas aos canais já referidos como regulados pela CFTR. A anoctamina 1 (Ano1) foi identificada como CaCC, sendo a sua função inibida com a activação da CFTR, enquanto a função de transporte por parte da CFTR é diminuída com a sobreexpressão da Ano1. Para além disso, a Ano6 foi identificada como componente principal dos ORCC. Assim, na presença de CFTR não mutada, é observada uma ativação paralela da CFTR e da Ano6, enquanto a Ano1 é inibida. Pelo contrário, em células com a CFTR mutada, a função da Ano6 é atenuada e a função da Ano1 aparenta estar aumentada. O interesse no estudo das anoctaminas aumentou fortemente desde a identificação da Ano1 como CaCC em 2008. Tal interesse deve-se maioritariamente ao facto destas poderem ser consideradas possíveis canais de Cl- alternativos para compensar a falta de CFTR funcional na FQ. Não obstante, o conhecimento sobre a sua biogénese, tráfego e ativação, é ainda muito reduzido. Tal conhecimento é, no entanto, essencial para o seu possível uso terapêutico como canais de Cl- alternativos para a CFTR. As anoctaminas estão envolvidas numa variedade de funções que, para além da função como transportadoras de iões já referida, ainda incluem scrambling de fosfolípidos e regulação de outras proteínas membranares. Vários estudos sugerem que as anoctaminas formam homodímeros, sendo ainda possível a formação de heterodímeros. Estes estudos são corroborados pela descoberta de interação entre alguns membros da família. Entre estes encontram-se a Ano1 e a Ano9, uma vez que estudos mostram que a sobreexpressão da Ano9 origina uma forte inibição das correntes produzidas pela Ano1. Apesar das características já conhecidas das anoctaminas, a estrutura e as funções de todos os membros da família ainda não estão completamente definidas. Enquanto os dois primeiros membros da família (Ano1 e Ano2) funcionam como CaCCs, a Ano6 foi descrita como principal componente dos ORCC e como scramblase. Outros membros da família, tais como Ano3, 4, 7 e 9, podem ter funções de scramblases e/ou canais de iões, estando ainda as suas funções concretas por desvendar. Mutações nos genes das anoctaminas (nomeadamente ANO3, ANO5, ANO6 e ANO10) causam diferentes doenças genéticas, sugerindo o envolvimento destas proteínas numa variedade de funções celulares. O objetivo do presente projeto foi a identificação de novos reguladores da Ano6, e ainda possivelmente da Ano9 e 10, e a interação das mesmas com outros membros da família de proteínas e com a CFTR. Para atingir este objetivo foram propostas três tarefas principais, nomeadamente, i) Criar linhas celulares que sobreexpressam de forma estável os construtos da Ano6, Ano9 e Ano10 com dois tags, ii) Usar estes construtos para desenvolver ensaios de tráfego robustos para serem utilizados em microscopia de fluorescência high-throughput, iii) Usar screens com bibliotecas de siRNAs para identificar novos genes envolvidos na regulação do tráfego da Ano6, Ano9 e Ano10, que possam ainda vir a ser utilizados como potenciais fármacos para a FQ. O primeiro passo foi concluído, tendo o segundo sido apenas realizado para a Ano6 e o último começado para a mesma. Resumindo, neste projecto de mestrado foram criados construtos da Ano6, Ano9 e Ano10 com dois tags. Os construtos da Ano6 e Ano9 foram clonados em vetores lentivirais, e foram criadas várias linhas celulares que expressam estas anoctaminas sob o efeito de um promotor indutível Tet-On. As células CFBE Ano6/Ano9 3-HA GFP foram caraterizadas por imunofluorescência (tanto em células polarizadas como não polarizadas) e por Western Blot. Adicionalmente a função destas proteínas foi testada em ensaios funcionais (efluxo de iodeto e micro-Ussing chamber). Por fim, para as células CFBE Ano6 3-HA GFP, foi feito um screen preliminar de uma pequena biblioteca de siRNAs que afetam 231 genes (previamente testados para o tráfego da CFTR). Os resultados obtidos ao longo deste trabalho sugerem que: O construto com dois tags da Ano6 é funcional como canal quando sobreexpresso tanto em células HEK 293T como em CFBE; O construto com dois tags da Ano9 parece aumentar o efluxo de iodeto em células HEK 293T mas não apresenta nenhum efeito no transporte de iões quando sobreexpresso em células CFBE; A Ano6 sobreexpressa está localizada na membrana plasmática (e também no citoplasma); A Ano9 sobreexpressa está principalmente localizada no citoplasma, mas quando se localiza na membrana plasmática forma possivelmente dímeros; As células CFBE que expressam de forma estável o contruto Ano6 3-HA GFP constituem um modelo celular robusto para realizar screens com siRNAs por Microscopia Automatizada (Análise de Alto Conteúdo); Os resultados de um screen de siRNA preliminar sugerem que o tráfego da Ano6 para a membrana plasmática é afetado por genes envolvidos em vários processos biológicos, tais como na sinalização de recetores acoplados a proteínas G, transporte de iões, fosforilação de proteínas e regulação da apoptose, entre outros. Estudos adicionais e posterior validação destes resultados podem potencialmente resultar numa nova estratégia apelativa para desenvolver terapias para a FQ envolvendo canais de Cl- alternativos. A regulação do tráfego das anoctaminas é ainda desconhecida e o seu estudo aprofundado irá permitir a exploração destas vias para compensar o defeito da CFTR, tornando possível o desenvolvimento de novas terapias para a FQ.

Thesis (Ph.D.)--University of Alberta, 2009.
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on November 1, 2009). Includes bibliographical references.