1,721,045 research outputs found
Morphological Modifications of the Early Secretory Pathway in Differentiating Skeletal Muscle Cells
No full textSkeletal muscle (SKM) cells present a regular and striking arrangement of organelles and membrane systems that is essential for their function. It has been largely investigated on the structural events occurring during the organization of transverse (T) tubules, sarcoplasmic reticulum (SR), and Calcium Release Units (CRUs), and it has been proposed that during SKM differentiation, membrane systems follow a highly coordinated plan.
In addition, SKM cell differentiation and remodeling requires synthesis and transport of an enormous amount of proteins addressed to build up the contractile apparatus, the SR and the sarcolemma. This process most probably requires an important involvement of endoplasmic reticulum (ER), ER exit sites (ERES), Golgi Complex (GC), or early secretory pathway components, and vesicular traffic in general.
In terminally differentiated skeletal muscle cells early secretory pathway components are regularly arranged; the GC is organized in a unique fashion, fiber-type dependent and, formed by very small GC elements, which are localized around the nucleus and in all the fiber. GC elements in mature muscle fibers are very small compared to other cells, but still formed by cisternae. It has been shown that GC undergoes a dramatic reorganization during SKM cell differentiation resulting in the formation of small elements, and interestingly, in mature myotubes GC is found associated to ERES. Whether GC reorganization consists in fragmentation or ex-novo biogenesis is not known, and whether its co-localization with ERES is the result of structural hindrance by the myofibrils and membrane systems, or depends on interaction mechanisms has not been explained.
The present work is aimed at a better understanding how GC and ERES achieve their mature structure, what is their role during SKM differentiation, and whether morphological modifications are followed by alterations of membrane traffic efficiency. Therefore we investigated the processes underlying the morphological transformation of the secretory pathway structures as CG and ER exit sites (ERES) during SKM differentiation. The project envisaged the analysis of the localization of selected markers of secretory pathway components in C2C12 differentiating myoblasts and regenerating fibers by applying imaging techniques in order to follow the differentiation of the early secretory pathway compartments.
We report preliminary data on the morphological modifications occurring to GC and ERES in differentiated C2C12 cells
Functional genomics in familial dyslipidaemia
No full textDyslipidaemia is defined as an abnormal deviation of plasma lipid levels, being a significant risk factor for cardiovascular disease, which can be due to both genetic and environmental factors. There are several familial dyslipidaemias, and their occurrence is regulated by multiple genes. Familial hypercholesterolaemia (FH) is one of those dyslipidaemias, being the most common inherited life-threatening disorder of the lipid metabolism, affecting around 1:300 individuals. This disorder is characterised by increased levels of total and LDL plasma cholesterol, which is associated with premature atherosclerosis and coronary heart disease. The FH phenotype has a considerable genetic heterogeneity and phenotypic variability, depending on both the LDL receptor activity and lifestyle habits. So far, about 3,500 unique LDLR variants have been described, most of them lacking functional evidence proving their effect on the LDLR function. The high number of LDLR variants described as variants of uncertain significance (VUS) is the largest obstacle to achieve a definitive FH diagnosis, being early diagnosis and treatment the key to reduce the cumulative life-long cardiovascular burden of patients with FH.
Being a correct diagnosis one of the most important steps for FH patients’ identification this work concentrated in achieving a better diagnosis for FH patients by improving variants functional characterisation. Another aim was to unravel the genetic background of phenotype-positive genotype-negative patients in order to provide a more personalised medicine for these patients.
In the initial stage of this work, the combined efforts for the functional characterisation of 28 rare LDLR variants found in the Portuguese population (and other countries worldwide) allowed the classification reassignment of 15 variants, from VUS to likely benign (1), benign (3), likely pathogenic (10) or pathogenic (1). The variants were analysed by flow cytometry to assess their individual function and the results showed a large spectrum of functional levels, indicating that the impact of variants is not uniform across the LDLR gene. This methodology is deemed as the gold standard for functional characterisation and recommended by the FH Variant Curation Expert Panel from the Clinical Genome Resource. These results allowed 82 Portuguese patients to receive a definitive FH diagnosis, as well as many other FH patients with the same variants scattered across the 21 different countries where the same variants have been described.
Following the first stage of this work, the efforts were focused on the development of a high-throughput cell-based methodology to identify disruptive variants in the LDLR gene, with the objective of providing a fast and reliable method for variant characterisation. This assay showed to be a time- and cost-effective method of profiling rare variants, helping to differentiate between rare variants that are clinically significant and those that are benign. Two sets of variants were characterised: 1 – control set, consisting of 22 previously characterised variants as well as variants that achieve a pathogenic and benign classification without functional studies (n=5), where no variants were misclassified; 2 – test set, with 19 newly functionally characterised variants: 11 displaying abnormal function, five displaying normal function, and three remaining unclassified. This work generated a valuable resource for systematic functional characterisation of LDLR variants, helping to solve one of the major issues to achieve a definitive FH diagnosis. Large scale up of this method is now possible.
This project also assessed the impact of rare variants from an array of 112 genes on a FH-negative cohort gathered from the Portuguese FH study. From the targeted sequencing of 185 variant-negative individuals and subsequent cascade family screening, it was possible to determine a definitive or possible genetic aetiology of the dyslipidaemia for 14 patients. The obtained data uncovered a high prevalence of rare variants in genes associated with sitosterolaemia and hypertriglyceridaemia, namely ABCG5, ABCG8 and GPD1 genes. Heterozygous variants in these genes are linked to increases of LDL cholesterol levels of up to 71.38, 76.11 and 65.96 mg/dL, respectively, compared to normal controls. From the analysis of this cohort, it was described the second case of sitosterolaemia in Portugal. Finally, in three unrelated families it was identified a variant that largely co-segregates with the phenotype in each family, in a new potential FH gene. However, further studies and supportive evidence are still required to establish a direct association to the FH phenotype and pathogenesis.
Overall, this work makes a significant contribution to the field of dyslipidaemias and FH, by introducing new data and fresh perspectives to various domains within it. It contributed for the functional characterisation of over 40 rare LDLR variants, which can allow a more personalised choice of the lipid-lowering treatment and dyslipidaemia management, ultimately improving patients’ prognosis. Moreover, a new high-throughput cell-based methodology that generates a useful resource for systematic functional characterisation of rare LDLR variants has been developed. This method has the potential to quickly profile a significant number of variants of uncertain significance, addressing a major gap in the diagnosis of FH. Additionally, this research shed light on the genetic dynamics of FH-positive and FH-negative individuals, highlighting the importance of accurate identification of dyslipidaemia's aetiology and introduces a potential candidate FH gene, providing a new avenue for future investigation
Functional genomics in familial dyslipidaemia
No full textDyslipidaemia is defined as an abnormal deviation of plasma lipid levels, being a significant risk factor for cardiovascular disease, which can be due to both genetic and environmental factors. There are several familial dyslipidaemias, and their occurrence is regulated by multiple genes. Familial hypercholesterolaemia (FH) is one of those dyslipidaemias, being the most common inherited life-threatening disorder of the lipid metabolism, affecting around 1:300 individuals. This disorder is characterised by increased levels of total and LDL plasma cholesterol, which is associated with premature atherosclerosis and coronary heart disease. The FH phenotype has a considerable genetic heterogeneity and phenotypic variability, depending on both the LDL receptor activity and lifestyle habits. So far, about 3,500 unique LDLR variants have been described, most of them lacking functional evidence proving their effect on the LDLR function. The high number of LDLR variants described as variants of uncertain significance (VUS) is the largest obstacle to achieve a definitive FH diagnosis, being early diagnosis and treatment the key to reduce the cumulative life-long cardiovascular burden of patients with FH.
Being a correct diagnosis one of the most important steps for FH patients’ identification this work concentrated in achieving a better diagnosis for FH patients by improving variants functional characterisation. Another aim was to unravel the genetic background of phenotype-positive genotype-negative patients in order to provide a more personalised medicine for these patients.
In the initial stage of this work, the combined efforts for the functional characterisation of 28 rare LDLR variants found in the Portuguese population (and other countries worldwide) allowed the classification reassignment of 15 variants, from VUS to likely benign (1), benign (3), likely pathogenic (10) or pathogenic (1). The variants were analysed by flow cytometry to assess their individual function and the results showed a large spectrum of functional levels, indicating that the impact of variants is not uniform across the LDLR gene. This methodology is deemed as the gold standard for functional characterisation and recommended by the FH Variant Curation Expert Panel from the Clinical Genome Resource. These results allowed 82 Portuguese patients to receive a definitive FH diagnosis, as well as many other FH patients with the same variants scattered across the 21 different countries where the same variants have been described.
Following the first stage of this work, the efforts were focused on the development of a high-throughput cell-based methodology to identify disruptive variants in the LDLR gene, with the objective of providing a fast and reliable method for variant characterisation. This assay showed to be a time- and cost-effective method of profiling rare variants, helping to differentiate between rare variants that are clinically significant and those that are benign. Two sets of variants were characterised: 1 – control set, consisting of 22 previously characterised variants as well as variants that achieve a pathogenic and benign classification without functional studies (n=5), where no variants were misclassified; 2 – test set, with 19 newly functionally characterised variants: 11 displaying abnormal function, five displaying normal function, and three remaining unclassified. This work generated a valuable resource for systematic functional characterisation of LDLR variants, helping to solve one of the major issues to achieve a definitive FH diagnosis. Large scale up of this method is now possible.
This project also assessed the impact of rare variants from an array of 112 genes on a FH-negative cohort gathered from the Portuguese FH study. From the targeted sequencing of 185 variant-negative individuals and subsequent cascade family screening, it was possible to determine a definitive or possible genetic aetiology of the dyslipidaemia for 14 patients. The obtained data uncovered a high prevalence of rare variants in genes associated with sitosterolaemia and hypertriglyceridaemia, namely ABCG5, ABCG8 and GPD1 genes. Heterozygous variants in these genes are linked to increases of LDL cholesterol levels of up to 71.38, 76.11 and 65.96 mg/dL, respectively, compared to normal controls. From the analysis of this cohort, it was described the second case of sitosterolaemia in Portugal. Finally, in three unrelated families it was identified a variant that largely co-segregates with the phenotype in each family, in a new potential FH gene. However, further studies and supportive evidence are still required to establish a direct association to the FH phenotype and pathogenesis.
Overall, this work makes a significant contribution to the field of dyslipidaemias and FH, by introducing new data and fresh perspectives to various domains within it. It contributed for the functional characterisation of over 40 rare LDLR variants, which can allow a more personalised choice of the lipid-lowering treatment and dyslipidaemia management, ultimately improving patients’ prognosis. Moreover, a new high-throughput cell-based methodology that generates a useful resource for systematic functional characterisation of rare LDLR variants has been developed. This method has the potential to quickly profile a significant number of variants of uncertain significance, addressing a major gap in the diagnosis of FH. Additionally, this research shed light on the genetic dynamics of FH-positive and FH-negative individuals, highlighting the importance of accurate identification of dyslipidaemia's aetiology and introduces a potential candidate FH gene, providing a new avenue for future investigation
Inhibition of intracellular cholesterol transport alters presenilin localization and amyloid precursor protein processing in neuronal cells
No full textGeneration of amyloid-beta (Abeta) from the amyloid precursor protein (APP) requires proteolytic cleavage by two proteases, beta- and gamma-secretase. Several lines of evidence suggest a role for cholesterol on secretase activities, although the responsible cellular mechanisms remain unclear. Here we show that alterations in cholesterol transport from late endocytic organelles to the endoplasmic reticulum have important consequences for both APP processing and the localization of gamma-secretase-associated presenilins (PS). Exposure of neuronal cells to cholesterol transport-inhibiting agents resulted in a marked decrease in beta-cleavage of full-length APP. In contrast, gamma-secretase activity on APP C-terminal fragments was enhanced, increasing the production of both Abeta40 and Abeta42. Remarkably, retention of cholesterol in endosomal/lysosomal compartments induced PS1 and PS2 to accumulate in Rab7-positive vesicular organelles implicated in cholesterol sorting. Accumulation of PS in vesicular compartments was prominent in both Chinese hamster ovary cells deficient in Niemann-Pick C1 protein as well as in neuronal cells exposed to the cholesterol transport-inhibiting agent U18666A. Because Abeta42 also localized to PS1-containing vesicular compartments, organelles involved in cholesterol transport might represent an important site for gamma-secretase activity. Our results suggest that the subcellular distribution of cholesterol may be an important factor in how cholesterol alters Abeta production and the risk of Alzheimer's disease
Search for new modulators of Phe508del-CFTR retention at the plasma membrane
No full textTese de doutoramento, Biologia (Biologia de Sistemas), apresentada à Faculdade de Ciências da Universidade de Lisboa, 2018Cystic fibrosis (CF) is a complex inherited disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Around 2000 disease causing mutations are known for this gene, which encodes a Chloride (Cl−) channel expressed at the plasma membrane (PM) of epithelial cells. The most frequent CFTR mutation, the deletion of phenylalanine 508 (Phe508del), causes the protein to misfold and be prematurely degraded. Low temperature or pharmacological “correctors” can partly rescue Phe508del-CFTR processing defect and enhance the channel traffic to the cell surface. Nevertheless, the rescued channels show partial channel function and a highly decreased PM half-life, due to accelerated endocytosis and fast turnover. Given this accelerated endocytic rate, new strategies aiming to retain rescued Phe508del-CFTR at the cell surface could be relevant as to enhance the efficacy of currently available pharmacological correctors. For that reason, the major objective of this dissertation is to identify novel cellular pathways or key interactors for the modulation of CFTR surface retention. Previous results from the host laboratory had showed that stimulation of endogenous RAC1 by Hepatocyte Growth Factor (HGF) signaling potentiated the retention of rescued Phe508del-CFTR at the PM by promoting an interaction between the actin-binding adaptor ezrin (EZR) and the Na+/H+ exchange regulatory factor-1 (NHERF1), enhancing CFTR anchoring to the actin cytoskeleton. In chapter 2 we showed that the mechanism behind this stabilization lies on a conformational change in NHERF1, triggered by EZR activation upon RAC1 signaling, which is then able to bind and stabilize misfolded CFTR at the PM. However, HGF/RAC1 signaling pathway is known to have proliferative and pleiotropic biological functions, which limit its application for therapeutic intervention. Therefore, in chapter 3, we investigated the effect of HGF treatment in epithelium-like cellular models, in combination with the most common administrated drugs. Contrary to what would be commonly assumed, we found that prolonged co-administration of HGF actually prevented previously unrecognized epithelial dedifferentiation effects of prolonged exposure to the FDA-approved Phe508del-CFTR corrector VX-809. It also significantly increased the Phe508del-CFTR functional rescue by the FDA- and EMA-approved VX-809/VX-770 drug combination, preventing the destabilization of the PM rescued channels by prolonged exposure to the VX-770 potentiator drug. These results suggest that HGF co-administration could indeed be beneficial for CF patients and should be further clinically explored. Lastly, since we showed that the type of protein interactions that wt- and rescued Phe508del-CFTR establish at the cell surface can be major determinants of their different PM stabilities, in chapter 4 we identified, for the first time, the core components of the macromolecular complexes assembled around wt- and rescued Phe508del-CFTR proteins at the PM. By identifying exclusive PM interactions between rescued Phe508del-CFTR, NHERF1 and EZR, we were able to recognize Calpain 1 as a key contributor for the decreased surface stability of pharmacologically rescued Phe508del-CFTR, probably acting through the disruption of the EZR-actin cytoskeleton binding.A Fibrose Quística (FQ) é uma doença hereditária, letal, cujo nome deriva do aspeto quístico
e fibroso do pâncreas dos portadores da doença. É uma das doenças genéticas mais comuns,
afetando aproximadamente 70.000 pessoas em todo o mundo, com maior expressão na população
caucasiana. Em Portugal estima-se que nasçam cerca de 30 a 40 crianças com FQ por ano. A doença
é causada por alterações que ocorrem num único gene – o gene CFTR (do inglês, Cystic Fibrosis
Transmembrane Conductance Regulator). Este gene codifica um canal de cloreto (Cl-
), expresso
maioritariamente na membrana apical das células epiteliais. As mutações neste canal levam a uma
secreção deficiente de Cl- no epitélio, causando um desequilíbrio eletrolítico que provoca várias
manifestações sistémicas. Os sistemas mais afetados são o pulmonar e gastrointestinal, onde muco
extremamente viscoso é acumulado, causando infeções e inflamações pulmonares crónicas e
obstrução pancreática com consequente malnutrição. A principal causa de morte por FQ é a falência
pulmonar, sendo a esperança média de vida atual entre os 35 e os 40 anos de idade.
À data existem mais de 2000 mutações descritas para o gene CFTR. A mutação mais frequente
é uma deleção de um resíduo de fenilalanina na posição 508, chamada Phe508del, estando presente
em 85% dos doentes em pelo menos num alelo. Em Portugal os números indicam que esta está
presente em cerca de 85% dos doentes com FQ. Esta mutação provoca, em primeira instância,
defeitos no processamento (folding) da CFTR, impedindo que esta atinja a membrana plasmática
(MP). Adicionalmente, as poucas moléculas CFTR-Phe508del que conseguem chegar à superfície das
células apresentam defeitos de função, ao nível da abertura do canal, bem como um tempo de semivida na membrana (estabilidade na MP) bastante reduzido, quando comparado com a CFTR normal.
Atualmente existem compostos capazes de promover o resgate parcial do folding da CFTR-Phe508del
e o seu tráfego para a MP (denominados “corretores”, como o VX-809) e a abertura do canal na MP
(denominados “potenciadores”, como o VX-770). No entanto, não existe de momento qualquer
composto que aumente o tempo de semi-vida e a estabilidade do canal na MP. Nesse sentido, o
principal objetivo desta dissertação é identificar novos modeladores ou vias celulares que aumentem
a retenção na MP da CFTR-Phe508del resgatada farmacologicamente.
É sabido que o canal CFTR está associado a várias outras proteínas, formando um grande
complexo proteico à superfície das células. Estas interações entre proteínas são determinantes para
a função e regulação do canal na MP. De acordo com este conhecimento, o laboratório de
acolhimento mostrou, anteriormente, que a ativação da via RAC1 regula a ancoragem da CFTRPhe508del à MP e ao citoesqueleto de actina mediante a sua interação com duas proteínas
adaptadoras, a ezrina (EZR) e o NHERF1 (do inglês Na/H exchanger regulatory factor 1). Este aumento
da ancoragem ao citoesqueleto de actina deriva da ativação da proteína EZR, que após estimulada
pelo RAC1 adquire uma conformação aberta e ativa, o que promove uma maior afinidade tanto para
a ligação à actina como para a ligação ao complexo NHERF1-CFTR, prevenindo a endocitose do canal
CFTR mutante. Usando o fator de crescimento de hepatócitos (HGF), um conhecido ativador do RAC1
endógeno, foi possível observar um aumento de estabilidade três vezes maior na retenção da CFTRPhe508del resgatada farmacologicamente em células epiteliais tratadas com HGF, em comparação
com células não tratadas com o fator. Assim sendo, no capítulo 2 desta dissertação, foi aprofundada a interação entre as proteínas CFTR-Phe508del, EZR e NHERF1, com o objetivo de compreender o
mecanismo molecular que leva ao aumento da estabilidade do canal após a ativação da via
HGF/RAC1. Demonstrámos que a ligação da EZR ativa à proteína NHERF1 promove uma mudança de
conformação na mesma, levando a uma mudança de ligação da CFTR do domínio NHERF1-PDZ1 para
o NHERF1-PDZ2. Esta mudança na estrutura do complexo proteico CFTR-NHERF1, induzida pelo
RAC1, antecipa e previne a deteção pelos mecanismos de controlo periférico de proteínas anómalas
(PPQC, do inglês Peripheral Protein Quality Control), levando a uma menor taxa de endocitose e,
consequentemente, a um aumento da estabilidade da CFTR-Phe508del resgatada.
Contudo, embora tenhamos mostrado e caracterizado que a via HGF/RAC1 promove a
estabilidade da CFTR mutada à superfície das células, devido à descrita pleiotropia funcional do HGF
e a sua associação ao desenvolvimento de certas neoplasias, a aplicabilidade deste fator em terapias
é limitada. Assim sendo, no capítulo 3 desta dissertação procedeu-se ao estudo do efeito do
tratamento, tanto agudo como prolongado, do HGF em modelos celulares de epitélio polarizado, em
combinação com o tratamento com VX-809 e/ou VX-770. Tanto o tratamento de HGF agudo como o
prolongado aumentaram os níveis de CFTR-Phe508del funcional à superfície das células, quando
combinado com os fármacos VX-809 e VX-770, aprovados para a tratamento de doentes com a
mutação Phe508del pelas entidades reguladoras FDA e EMA. Curiosamente, tendo já sido descrita
uma destabilização por parte do potenciador VX-770 na CFTR-Phe508del resgatada por VX-809, o
triplo co-tratamento com HGF teve um efeito protetor na estabilidade da CFTR-Phe508del. Mais
importante, ao invés de promover a desdiferenciação e proliferação das células epiteliais polarizadas,
o tratamento prolongado com HGF teve o efeito contrário, prevenindo o aumento dos marcadores
Ki-67 e CK8 que, surpreendentemente, aumentaram com a exposição prolongada de VX-809 e VX770. Em conjunto, estes resultados sugerem que a co-administração de HFG com fármacos corretores
e potenciadores poderá ser benéfica para pacientes com FQ.
Tendo-se anteriormente demonstrado que as interações entre a CFTR e as proteínas que
compõem o seu complexo macromolecular à superfície das células são importantes para a
estabilidade do canal, no capítulo 4 propusemos estudar em detalhe os seus componentes, com o
objetivo de identificar novos modeladores da retenção da CFTR na MP. Para isso, isolámos e
identificámos as proteínas que compõem os complexos macromoleculares associados à CFTR em
CFTR-wt e em CFTR-Phe508del resgatada pela baixa temperatura ou o fármaco VX-809, usando
ferramentas bioinformáticas para identificar diferenças entre os mesmos. Tendo como base as
interações exclusivas entre as proteínas EZR, NHERF1 e a CFTR-Phe508del resgatada identificámos a
protease Calpain 1 como um modelador da estabilidade do canal na membrana. Ao diminuirmos a
quantidade e função desta proteína observámos um aumento substancial na abundância, função e
retenção na MP da CFTR-Phe508del resgatada. Sabendo que a EZR é um substrato conhecido da
protease Calpain 1 (Calcium-Activated Neutral Proteinase 1), mostrámos que, à superfície das células,
a EZR aumenta quando diminuímos a quantidade de Calpain 1 disponível, não sendo observadas
quaisquer diferenças na quantidade de NHERF1. Assim sendo, propusemos que a Calpain 1 é um
regulador negativo da estabilidade da CFTR-Phe508del na MP, atuando através da proteólise da
proteína EZR, destabilizando o seu complexo macromolecular. Como a Calpain 1 foi identificada como
sendo uma proteína exequível de ser farmacologicamente modelada pela base de dados chEMBL, o seu estudo pode vir a ser importante para o desenvolvimento de novas terapias que promovam a
estabilidade na MP da CFTR.
Em resumo, esta dissertação cumpriu os objetivos propostos, dado que identificou e
caracterizou vários modeladores da retenção da CFTR-Phe508del resgatada farmacologicamente
para a superfície das células. Estes resultados abrem portas para o estudo e desenvolvimento de
novas terapias combinadas, pois, como anteriormente referido, neste momento não existe qualquer
composto disponível que aumente o tempo de semi-vida e a estabilidade do canal na MP. Algumas
das implicações deste estudo não são exclusivas apenas para a mutação Phe508del, podendo
também ser aplicados a outras mutações que diminuam a estabilidade da proteína CFTR, como a
mutação c.120del23 e Q1412x.Fundação para a Ciência e a Tecnologia (PD/BD/52490/2014)info:eu-repo/semantics/draf
Using a systems approach to identify the mechanism of action of correctors
No full textTese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel expressed at the apical membrane of epithelial cells. High-throughput screens (HTS) identified several novel molecules potentially targeting the underlying CFTR defect but only for some patients: potentiator VX-770 (Ivacaftor/Kalydeco), for subjects bearing G551D and other gating mutations, the combination corrector/potentiator VX-809 (Lumacaftor)/VX-770 (Orkambi) for F508del-homozygous patients and another similar combination VX-661 (Tezacaftor)/VX-770 is under approval. The main objective of this PhD work was to study new compounds that correct the basic CF defect, by rescuing CFTR protein traffic and function, focusing both on individual responses of CF patients with different CFTR mutations to these new drugs, and their mechanism of action. Chapter 1 focusses on the measurement of functional responses on human bronchial epithelial cells (HBE’s) derived from CF lung explants bearing different CFTR mutations to VX-809 namely: A561E, N1303K, G542X and Y1092X. Our data showed a positive response of A561E/A561E to VX-809 and F508del/Y1092X but not F508del/G542X. In Chapter 2, we evaluated the efficacy of CFTR modulators (correctors/potentiators) in physiologically relevant tissues, namely rectal biopsies, intestinal organoids, (HBE’s) and human nasal epithelial cells (HNE’s), from CF patients with rare CFTR mutations. Data obtained here showed that neither R560S nor H1079P-could not be rescued by any of the CFTR modulators, but 3849+10kbC>T and R334W and c.120del23-CFTR were rescued by VX-770 alone or with VX-809. In Chapter 3 we evaluated the efficacy of two novel CFTR correctors (B9, E12) in primary HBE cells, and three novel compounds E-act mimics (C2, C5, and C7) as enhancers of alternative Cl- channel TMEM16A in human intestinal organoids. In Chapter 4 (final) we assessed the effect of CFTR modulators and their possible additivity with F508del-CFTR genetic revertants 4RK, R1070W, and G550E to understand the mechanism of action of small molecule correctors and another variant diacidic ER exit code DD/AA in CFBE mCherry cells expressing these varinats by Ussing chamber analysis with or without CFTR modulators. Our data show that C18 and VX-661 and low temperature (But not VX-809) rescued DD/AA to the cell surface and genetic revertants restore the channel function without any CFTR modulator. Altogether, results from this work bring new insights into how the CFTR genotype may influence CFTR function and response to CFTR modulators and how each patient should be assessed individually for the responsiveness to the CFTR modulators towards personalized therapeutics
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Using a systems approach to identify the mechanism of action of correctors
No full textTese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel expressed at the apical membrane of epithelial cells. High-throughput screens (HTS) identified several novel molecules potentially targeting the underlying CFTR defect but only for some patients: potentiator VX-770 (Ivacaftor/Kalydeco), for subjects bearing G551D and other gating mutations, the combination corrector/potentiator VX-809 (Lumacaftor)/VX-770 (Orkambi) for F508del-homozygous patients and another similar combination VX-661 (Tezacaftor)/VX-770 is under approval. The main objective of this PhD work was to study new compounds that correct the basic CF defect, by rescuing CFTR protein traffic and function, focusing both on individual responses of CF patients with different CFTR mutations to these new drugs, and their mechanism of action. Chapter 1 focusses on the measurement of functional responses on human bronchial epithelial cells (HBE’s) derived from CF lung explants bearing different CFTR mutations to VX-809 namely: A561E, N1303K, G542X and Y1092X. Our data showed a positive response of A561E/A561E to VX-809 and F508del/Y1092X but not F508del/G542X. In Chapter 2, we evaluated the efficacy of CFTR modulators (correctors/potentiators) in physiologically relevant tissues, namely rectal biopsies, intestinal organoids, (HBE’s) and human nasal epithelial cells (HNE’s), from CF patients with rare CFTR mutations. Data obtained here showed that neither R560S nor H1079P-could not be rescued by any of the CFTR modulators, but 3849+10kbC>T and R334W and c.120del23-CFTR were rescued by VX-770 alone or with VX-809. In Chapter 3 we evaluated the efficacy of two novel CFTR correctors (B9, E12) in primary HBE cells, and three novel compounds E-act mimics (C2, C5, and C7) as enhancers of alternative Cl- channel TMEM16A in human intestinal organoids. In Chapter 4 (final) we assessed the effect of CFTR modulators and their possible additivity with F508del-CFTR genetic revertants 4RK, R1070W, and G550E to understand the mechanism of action of small molecule correctors and another variant diacidic ER exit code DD/AA in CFBE mCherry cells expressing these varinats by Ussing chamber analysis with or without CFTR modulators. Our data show that C18 and VX-661 and low temperature (But not VX-809) rescued DD/AA to the cell surface and genetic revertants restore the channel function without any CFTR modulator. Altogether, results from this work bring new insights into how the CFTR genotype may influence CFTR function and response to CFTR modulators and how each patient should be assessed individually for the responsiveness to the CFTR modulators towards personalized therapeutics
Using a systems approach to identify the mechanism of action of correctors
No full textTese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel expressed at the apical membrane of epithelial cells. High-throughput screens (HTS) identified several novel molecules potentially targeting the underlying CFTR defect but only for some patients: potentiator VX-770 (Ivacaftor/Kalydeco), for subjects bearing G551D and other gating mutations, the combination corrector/potentiator VX-809 (Lumacaftor)/VX-770 (Orkambi) for F508del-homozygous patients and another similar combination VX-661 (Tezacaftor)/VX-770 is under approval. The main objective of this PhD work was to study new compounds that correct the basic CF defect, by rescuing CFTR protein traffic and function, focusing both on individual responses of CF patients with different CFTR mutations to these new drugs, and their mechanism of action. Chapter 1 focusses on the measurement of functional responses on human bronchial epithelial cells (HBE’s) derived from CF lung explants bearing different CFTR mutations to VX-809 namely: A561E, N1303K, G542X and Y1092X. Our data showed a positive response of A561E/A561E to VX-809 and F508del/Y1092X but not F508del/G542X. In Chapter 2, we evaluated the efficacy of CFTR modulators (correctors/potentiators) in physiologically relevant tissues, namely rectal biopsies, intestinal organoids, (HBE’s) and human nasal epithelial cells (HNE’s), from CF patients with rare CFTR mutations. Data obtained here showed that neither R560S nor H1079P-could not be rescued by any of the CFTR modulators, but 3849+10kbC>T and R334W and c.120del23-CFTR were rescued by VX-770 alone or with VX-809. In Chapter 3 we evaluated the efficacy of two novel CFTR correctors (B9, E12) in primary HBE cells, and three novel compounds E-act mimics (C2, C5, and C7) as enhancers of alternative Cl- channel TMEM16A in human intestinal organoids. In Chapter 4 (final) we assessed the effect of CFTR modulators and their possible additivity with F508del-CFTR genetic revertants 4RK, R1070W, and G550E to understand the mechanism of action of small molecule correctors and another variant diacidic ER exit code DD/AA in CFBE mCherry cells expressing these varinats by Ussing chamber analysis with or without CFTR modulators. Our data show that C18 and VX-661 and low temperature (But not VX-809) rescued DD/AA to the cell surface and genetic revertants restore the channel function without any CFTR modulator. Altogether, results from this work bring new insights into how the CFTR genotype may influence CFTR function and response to CFTR modulators and how each patient should be assessed individually for the responsiveness to the CFTR modulators towards personalized therapeutics
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