Browsing by Author "Fernandes, Rafael"
Now showing 1 - 10 of 24
Results Per Page
Sort Options
- Characterization of Two Variants at Met 1 of the Human LDLR Gene Encoding the Same Amino Acid but Causing Different Functional PhenotypesPublication . Graça, Rafael; Fernandes, Rafael; Alves, Ana catarina; Menezes, Juliane; Romão, Luísa; Bourbon, MafaldaFamilial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, characterized by increased levels of total and LDL plasma cholesterol, which leads to premature atherosclerosis and coronary heart disease. FH phenotype has considerable genetic heterogeneity and phenotypic variability, depending on LDL receptor activity and lifestyle. To improve diagnosis and patient management, here, we characterized two single nucleotide missense substitutions at Methionine 1 of the human LDLR gene (c.1A>T/p.(Met1Leu) and c.1A>C/p.(Met1Leu)). We used a combination of Western blot, flow cytometry, and luciferase assays to determine the effects of both variants on the expression, activity, and synthesis of LDLR. Our data show that both variants can mediate translation initiation, although the expression of variant c.1A>T is very low. Both variants are in the translation initiation codon and codify for the same amino acid p.(Met1Leu), yet they lead to different levels of impairment on LDLR expression and activity, corroborating different efficiencies of the translation initiation at these non-canonical initiation codons. The functional data of these variants allowed for an improved American College of Medical Genetics (ACMG) classification for both variants, which can allow a more personalized choice of the lipid-lowering treatment and dyslipidemia management, ultimately improving patients' prognosis.
- Functional characterization of variants in the 5’ UTR and promoter of LDLR genePublication . Alves, Ana Catarina; Menezes, Juliane; Fernandes, Rafael; Romão, Luísa; Bourbon, MafaldaBackground and Aims: Familia lhypercholesterolemia (FH) is the most common genetic disorder conferring an increased cardiovascular risk due to cholesterol accumulation since birth. The majority of patients with FH phenotype have mutations in LDLR, APOB or PCSK9 genes. In about 50% of patients a variant causing disease has not been possible to find. The 5'and 3’untranslated regions (UTRs) and promoter of these genes is poorly studied. Consequently, few variants were detected in these locations and functional validation is lacking for the ones described. The aim of this project is to perform an in vitro characterization of variants in 5’UTR and promoter of LDLR gene.
- Functional characterization of variants in the 5’UTR and promoter of PCSK9 genePublication . Alves, Ana Catarina; Fernandes, Rafael; Menezes, Juliane; Romão, Luísa; Bourbon, MafaldaFamilial hypercholesterolemia (FH) is the most common genetic disorder conferring an increased cardiovascular risk due to cholesterol accumulation since birth. The majority of patients with FH phenotype have mutations in LDLR, APOB or PCSK9 genes. In about 50% of patients a variant causing disease has not been possible to find. The 5' and 3’ untranslated regions (UTRs) and promoter of these genes is poorly studied. Consequently, few variants were detected in these locations and functional validation is lacking for the ones described. The aim of this project is to perform an in vitro characterization of variants in 5’ UTR and promoter of PCSK9 gene.
- Gene expression regulation by upstream open reading frames in rare diseasesPublication . Silva, Joana; Fernandes, Rafael; Romão, LuísaUpstream open reading frames (uORFs) constitute a class of cis-acting elements that regulate translation initiation. Mutations or polymorphisms that alter, create or disrupt a uORF have been widely associated with several human disorders, including rare diseases. In this mini-review, we intend to highlight the mechanisms associated with the uORF-mediated translational regulation and describe recent examples of their deregulation in the etiology of human rare diseases. Additionally, we discuss new insights arising from ribosome profiling studies and reporter assays regarding uORF features and their intrinsic role in translational regulation. This type of knowledge is of most importance to design and implement new or improved diagnostic and/or treatment strategies for uORF-related human disorders.
- Genetics of personalized medicine: cancer and rare diseasesPublication . Siefers Alves, Inês; Condinho, Manuel; Custódio, Sónia; Pereira, Bruna; Fernandes, Rafael; Gonçalves, Vânia; da Costa, Paulo J.; Lacerda, Rafaela; Marques, Ana Rita; Martins-Dias, Patrícia; Nogueira, Gonçalo; Neves, Ana Rita; Pinho, Patrícia; Rodrigues, Raquel; Rolo, Eva; Silva, Joana; Travessa, André; Pinto-Leite, Rosário; Sousa, Ana; Romão, LuísaThe 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling.
- Modulation of protein translation mediated by upstream open reading frames (uORFs) in PERK mRNAPublication . Fernandes, Rafael; Romão, LuísaObjective: PERK is one of the three sensor proteins from the ER that are responsible for inducing the Unfolded Protein Response (UPR) when stress occurs (4). It is also the kinase responsible for phosphorylating eIF2α in these conditions (4). In this work we intended to determine if PERK is regulated at the translational level by uORFs in normal and ER stress conditions, and understand the impact of this regulation for the cell-stress response.
- Nonsense-mediated mRNA decay (NMD): a bifunctional mechanism in human physiology and diseasePublication . Fernandes, Rafael; Romão, LuísaProteins are the working-units of the cell, which are encoded by thousands of genes that pass their information into an intermediate molecule, the messenger RNA (mRNA). For this flow of information – from genes to RNA (transcription), and then into proteins (translation) – to occur properly, the cell has developed several check points and mechanisms that limit the occurrence and transmission of errors. Some of these mechanisms act at the mRNA level to ensure that the information transcribed from a gene will yield a proper functional protein after mRNA translation. Nonsense-mediated mRNA decay (NMD) is a translation-dependent surveillance mechanism that rapidly degrades transcripts with premature stop codons (PTCs), like the ones bearing nonsense mutations.
- Nonsense-Mediated mRNA decay in development, stress and cancerPublication . Fernandes, Rafael; Nogueira, Gonçalo; da Costa, Paulo J.; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a well characterized eukaryotic mRNA degradation pathway, responsible for the identification and degradation of transcripts harboring translation termination codons in premature contexts. Transcriptome-wide studies revealed that NMD is not only an mRNA surveillance pathway as initially thought, but is also a post-transcriptional regulatory mechanism of gene expression, as it fine-tunes the transcript levels of many wild-type genes. Hence, NMD contributes to the regulation of many essential biological processes, including pathophysiological mechanisms. In this chapter we discuss the importance of NMD and of its regulation to organism development and its link to the cellular stress responses, like the unfolded protein response (UPR) and the integrated stress response (ISR). Additionally, we describe how tumor cells have explored both NMD functions to promote tumorigenesis. Using published data and databases, we have also performed a network-based approach that further supports the link between NMD and these (patho) physiological processes.
- Nonsense-mediated RNA decay and its bipolar function in cancerPublication . Nogueira, Gonçalo; Fernandes, Rafael; García-Moreno, Juan F; Romão, LuísaNonsense-mediated decay (NMD) was first described as a quality-control mechanism that targets and rapidly degrades aberrant mRNAs carrying premature termination codons (PTCs). However, it was found that NMD also degrades a significant number of normal transcripts, thus arising as a mechanism of gene expression regulation. Based on these important functions, NMD regulates several biological processes and is involved in the pathophysiology of a plethora of human genetic diseases, including cancer. The present review aims to discuss the paradoxical, pro- and anti-tumorigenic roles of NMD, and how cancer cells have exploited both functions to potentiate the disease. Considering recent genetic and bioinformatic studies, we also provide a comprehensive overview of the present knowledge of the advantages and disadvantages of different NMD modulation-based approaches in cancer therapy, reflecting on the challenges imposed by the complexity of this disease. Furthermore, we discuss significant advances in the recent years providing new perspectives on the implications of aberrant NMD-escaping frameshifted transcripts in personalized immunotherapy design and predictive biomarker optimization. A better understanding of how NMD differentially impacts tumor cells according to their own genetic identity will certainly allow for the application of novel and more effective personalized treatments in the near future.
- Regulation of nonsense-mediated mRNA decay (NMD) and the transcriptome: implications for physiology and myocardial infarctionPublication . Fernandes, Rafael; Bourbon, Mafalda; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation termination codons (PTCs) that would otherwise lead to the production of potentially harmful truncated proteins (1). Recent studies demonstrated that NMD also targets physiologic mRNAs transcribed from a large subset of wild-type genes, being responsible for the regulation of up to 10% of the mammalian transcriptome (2,3). This raises the possibility that NMD itself is under regulatory control. Indeed, recent studies have shown that NMD activity is modulated in specific cell types and that key components of the NMD pathway are regulated by several pathways, including NMD itself (4). Cellular stress, such as endoplasmic reticulum (ER) stress, hypoxia, reactive oxygen species, and nutrient deprivation also modulates the magnitude of NMD by mechanisms that are beginning to be understood (5). For example, the activation of kinases, as part of the cell-stress corrective pathways, induces the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), reducing protein translation and thus impairing NMD activity (6,7). There is currently great interest in decoding the mechanisms that couple stress signaling to human pathology. Only recently has ER stress been considered a potential contributor to cardiac and vascular diseases (8). Myocardial infarction is a pathological state that occurs during ischemia, where there is nutrient and oxygen deprivation in the heart, causing aggregation of proteins in the ER. This aggregation triggers ER stress and the three arms of the unfolded protein response (UPR), to mitigate or eliminate the stress (8). Given that NMD can respond to ER stress (6), here we aim to study 1) how NMD is regulated in cardiomyocytes under stress conditions, and 2) what is the influence of NMD on the transcriptome of cardiomyocytes and how it is involved in the cell-stress corrective strategies. So far, we have built a database that contains around 149 transcripts which are natural NMD-targets and are dysregulated under stress conditions, based on data from transcriptomic and UPF1-silencing studies. Using bioinformatics and gene ontology analysis we have classified the transcripts by biological function, and we intend to choose a few molecular targets for further studies in cardiomyocytes cultured cells in order to accomplish the objectives proposed.
- «
- 1 (current)
- 2
- 3
- »