Autor(es): Olivença, Daniel Vigário, 1976-
Data: 2018
Identificador Persistente: http://hdl.handle.net/10451/35920
Origem: Repositório da Universidade de Lisboa
Assunto(s): Teses de doutoramento - 2018; Domínio/Área Científica::Ciências Naturais::Ciências Biológicas
Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018
Cystic fibrosis is a condition caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride and bicarbonate channel. The epithelial sodium channel (ENaC) may also be affected. The defective function of these ion channels is thought to reduce the airway surface liquid (ASL) and lead to the accumulation of mucus in the airways that characterizes the disease and causes the recurrent pulmonary infections and inflammation that will ultimately destroy the lungs of the affected subjects. Phosphoinositides are rare signaling lipids that constitute a complex network regulating many cellular processes. One of phosphoinositides’ many functions is as cell membrane protein regulators, and several studies implicate phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) in ENaC regulation. Diacylglycerol kinase (DGK), an enzyme of the phosphoinositide pathway that catalyses the phosphorylation of diacylglycerol (DAG) into phosphatidic acid (PA). When DGK is inhibited, it will cause the moderation of ENaC function, and this could be exploited as a therapeutic in cystic fibrosis. But the mechanism of ENaC regulation by DGK is not completely understood. The usually accepted hypothesis is that DGK influences PI(4,5)P2 production by halting the phosphoinositide recycling. In Chapter 2 we present a model of the phosphoinositide pathway that simulates one square micrometer of the inner layer of the membrane. The objective of this project was to create a model that could simulate the phosphoinositide pathway and be used to study how perturbations to the pathway impact the levels of pertinent lipids, especially the ones known to affect ENaC. The model replicates the steady-state of the phosphoinositide pathway as recorded in the literature and replicates most known dynamic phenomena. Furthermore, sensitivity analysis demonstrates that the model is robust to moderate perturbations to the parameters. The model suggests that the main source of material to the PI(4,5)P2 pool is a flux representing the direct transformation of phosphatidylinositol (PI) into PI(4,5)P2 that defies the traditional view that the main source is the sequential phosphorylation of phosphoinositol into phosphatidylinositol 4-phosphate (PI(4)P) by the enzyme phosphoinositol 4-kinase (PI4K) followed by the transformation to PI(4,5)P2 by phosphoinositide 4-phosphate 5-kinase I (PIP5KI). The model also suggests that phosphatidylinositol 5-phosphate (PI(5)P) could be a significant source for PI(4,5)P2 production. We compared the model results to data from a siRNA screens, where the expression of several enzymes in the pathway were knocked down and the activity of ENaC was monitored. Our model suggests control strategies where the activity of the enzyme PIP5KI or the PI4 +PIP5K +DVL protein complex are decreased and cause an efficacious reduction in PI(4,5)P2 levels while avoiding undesirable alterations in other phosphoinositide pools. In Chapter 3 we present a model that enables the study of the interplay between ENaC, CFTR, airway surface liquid (ASL), PI(4,5)P2 and the protein SPLUNC1 (short palate, lung, and nasal epithelial clone). It presents a good fit to experimental observations, and the available data can constrain the model’s parameters without ambiguities. The model analysis shows that ASL at the steady state is sensitive to small changes in PI(4,5)P2 abundance, particularly in cystic fibrosis conditions, which suggests that manipulation of phosphoinositide metabolism may promote therapeutic benefits for cystic fibrosis patients. Finally, in Chapter 4, we bring the phophoinositide pathway and ENaC/ASL model together. These models enabled us to study DGK and ENaC and strongly suggest that, contrary to the usually accepted hypothesis, this regulation is effected by the control of PI(4,5)P2 production by the PIP5KI that in turn is controlled by PA, the product of DGK. In this work we also use a model of the phosphoinositide cycle to test the hypothesis that DGK influence PI(4,5)P2 production by halting the phosphoinositide recycling. This model is unable to replicate the available data if the activation of PIP5KI by PA is not implemented, which strengthens our belief that ENaC regulation by phosphoinositides is accomplished through PA and PIP5KI.
Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52486/2014; Programa doutoral - BioSys em Sistemas Biológicos, Genómica Funcional & Integrativa, (FCT/PD/00065/2012)
