DGH - Posters/abstracts em congressos internacionais
URI permanente para esta coleção:
Navegar
Percorrer DGH - Posters/abstracts em congressos internacionais por Domínios Científicos e Tecnológicos (FOS) "Ciências Médicas"
A mostrar 1 - 7 de 7
Resultados por página
Opções de ordenação
- Dissecting the DIS3L2 target-specificity of transcripts committed to nonsense-mediated decay in human cellsPublication . Garcia-Moreno, Juan F.; Carvalho, Miguel P.; Lacerda, Rafaela; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates mRNAs harboring premature termination codons (PTCs) and regulates the expression of certain physiological transcripts. The 3’-to-5’ exoribonuclease DIS3L2 degrades different RNAs independently of the RNA exosome, following uridylation at the 3' end by the terminal uridylyl transferases TUT4 and TUT7. We and others have shown that DIS3L2 is involved in NMD in an uridylation-dependent manner, being its function in NMD target-specific (Kurosaki et al. 2018; da Costa et al. 2019). Now, we aim to characterize the mechanisms involved in DIS3L2/NMD-target specificity. We used our RNA-seq data already obtained and validated and compared the transcripts upregulated upon DIS3L2 knockdown (REF) with a validated NMD-target set (Colombo et al., 2017). We observed that about 7% of DIS3L2-sensitive transcripts overlap with known NMD-targets. Considering the different groups of transcripts, we then analyzed specific features that make some NMD-targets sensitive to DIS3L2 (so called DIS3L2/NMD-targets; group 1), versus the remaining NMD-targets (DIS3L2-resistant NMD-targets; group 2), the remaining DIS3L2-sensitive targets (group 3), or the remaining transcriptome (DIS3L2-resistant NMD-targets plus NMD-resistant transcriptome; group 4). We assessed the following genomic features: 5’ and 3’ untranslated region (UTR) lengths, 3’UTR GC-, AU-, G-, C-, A-, and U-contents, presence of 5’UTR upstream open reading frames (uORFs), and 3’UTR introns. Elevated G-, C-, and GC-contents in the 3’UTRs were the most consistent features distinguishing DIS3L2/NMD-targets from the group 4. Comparison between group 1 and 2, and 1 and 3 was not significant. To better characterize the importance of each transcript portion, we are also analyzing hybrid constructs combining regions of the DIS3L2/NMD-resistant human β-globin (HBB) gene and the DIS3L2/NMD-sensitive GADD45A gene expressed in DIS3L2 depleted cultured cells.
- Early diagnosis of acid sphingomyelinase deficiency (ASMD) through biomarkers analysisPublication . Neiva, Raquel; da Silva Gaspar, Paulo Jorge Miranda; Sousa e Silva, Lisbeth Elena; Gonçalves, I.; Ferreira, S.; Diogo, Luisa; Vilarinho, LauraIntroduction: Acid sphingomyelinase deficiency (ASMD), historically known as Niemann–Pick disease (NPD) types A, A/B, and B, is a rare, progressive, potentially fatal lysosomal storage disease caused by pathogenic variants in SMPD1 gene. It presents a wide spectrum of symptoms, age of onset, and degree and type of organ effected. The disease manifestations frequently involve hepatosplenomegaly with progressive organ dysfunction, interstitial lung disease, and bleeding. In this work, we will present a patient whose lysosomal biomarkers study allowed the diagnosis of ASMD. Methods: This patient had hepatosplenomegaly, elevated transaminases in which the primary clinical suspicion was an acid lipase deficiency. By the analysis of our multiplex biomarker panel by LC-MS/MS analysis, we were able to do a differential diagnosis. Results/Case report: The lysosphingomyelin (lysoSM) and lysosphingomyelin-509 (lysoSm-509) were approximately 100 a 150x than normal, suggestive of Niemann–Pick disease. The diagnosis of ASMD was confirmed by reduced acid sphingomyelinase enzyme activity measured in peripheral blood leukocytes and the presence of a pathogenic variant in both alleles in the SMPD1 gene. Conclusion: ASMD can be underestimate and the diagnostic odissey arise from an overlap in symptomology with other diseases, including primary hepatic disease, Gaucher disease, Niemann–Pick disease, and lysosomal acid lipase deficiency. The multiplex biomarker panel, with different lysolipids, allows simultaneously diagnosis of different LSDs, in a timely manner, leading to an early intervention, before the appearance of more deleterious symtpoms.
- Is Antisense Oligonucleotide-Mediated Exon Skipping a Potential Therapeutic Approach for Mucolipidosis II?Publication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Duarte, Ana Joana; Gaspar, Paulo; Santos, Juliana Inês; Coutinho Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntridution: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs lysosomal hydrolases trafficking. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) to promote targeted skipping of GNPTAB exon 19, which harbors c.3503_3504del, the most frequent disease-causing variant. Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping, successfully generating an in-frame mRNA1. Now, our aim is to determine if this in-frame transcript leads to increased GlcNAc-PT levels. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts, but activity levels were similar in ML II and control fibroblasts (treated/non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without exon 19) and a mutant (with the mutation c.3503_3504del) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Also, we measured the activity of several hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment. To further validate this therapy we also generated a novel GlcNAc-PT antibody in rabbits. Results: Our results showed that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β-subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, in the delTC construct WB no α/β precursor band was detected. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts after treatment. However, only the α-Gal values were statistically significant, but the WB analysis for this enzyme did not reveal any band in ASO-treated ML II cells. We also developed a novel antibody for GlcNAc-PT. Preliminary results showed a β-subunit band both in control and patient fibroblasts (unexpected), but in overexpression both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess its specificity. Conclusion: Our ASO-based approach effectively promotes exon 19 skipping. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.
- Olipudase alfa enzyme replacement therapy. One-year outcomes in an adult patient with acid sphingomyelinase deficiency type BPublication . Cardoso, M.; Chaves, P.C.; Pintalhão, M.; da Silva Gaspar, Paulo Jorge Miranda; Castro, R.; Bastos, J.; Silva, A.; Campos, T.; Macedo, Fatima; Rodrigues, E.; Leão Teles, ElisaIntroduction: Acid Sphingomyelinase Deficiency (ASMD) is a rare autosomal recessive lysosomal storage disorder caused by variants in the SMPD1 gene, leading to a deficiency in the activity of sphingomyelinase (ASM) that catabolizes sphingomyelin (SPM). ASMD Type B is a late-onset, severe disease characterized by progressive hepatosplenomegaly, gradual deterioration of liver and pulmonary function, osteopenia and an atherogenic lipid profile. Olipudase alfa is a recombinant human ASM enzyme replacement therapy indicated for the treatment of non-C-NS manifestations of ASMD.
- The potential function of alternative translation initiation of Argonaute 1 in cancerPublication . Vieira da Silva, Verónica; Lacerda, Rafaela; Romão, LuísaTranslation is one of the most regulated and energy-consuming cellular processes crucial for proper cell function. Translation is initiated by the canonical cap-dependent mechanism. However, under stress conditions, the initiation of canonical translation is inhibited, which allows the translation of specific proteins via alternative mechanisms. This project aims to understand the biological relevance of alternative protein synthesis mechanisms in Argonaute 1 (AGO1) expression. The AGO1 protein is involved in microRNA regulation, gene expression modulation and inhibition. AGO1 is also involved in the regulation of gene expression by RNA interference (RNAi), and its deregulation can lead to the activation of oncogenes or the suppression of tumor suppressor genes, contributing to the development and progression of cancer. Our work has shown that AGO1 mRNA can be translated through a cap-independent initiation mechanism. An upstream open reading frame (uORF) has also been identified in its 5’ untranslated region (5’UTR), which may play a role in the initiation of AGO1 translation. The results showed that the 5’UTR of human AGO1 supports a cap-independent mechanism of translation initiation, which is maintained under stress conditions. However, our analyses did not provide conclusive evidence for a regulatory role of the uORF in this initiation process. To this end, the 5’UTR of human AGO1 was cloned into a bicistronic vector containing Renilla (RLuc) and Firefly luciferase (FLuc), with FLuc positioned downstream of the 5’UTR. Luminometry assays will be used to evaluate the relative FLuc/RLuc translation efficiency under the control of the AGO1 5’UTR. The same approach will be used with monocistronistic reporter vectors, contaning only FLuc. This project aims to understand how these alternative mechanisms of mRNA translation initiation can influence AGO1 expression and help explain their potential roles in certain pathologies and cancer progression, such as colorectal cancer.
- The role of UPF1 cap-independent translation in colorectal cancerPublication . Lacerda, Rafaela; Menezes,Juliane; Elias, Adriana; Sousa, Sofia de; Romão, LuísaColorectal cancer (CRC) is one of the deadliest diseases worldwide with projections pointing towards an increase for the next two decades. Translation dysregulation of many genes contributes to CRC development, and here we are studying the role of translation dysregulation of up-frameshift 1 (UPF1) in CRC. This protein is involved in many cellular mechanisms such as nonsense-mediated mRNA decay, cell cycle progression, or telomere maintenance and homeostasis. It also works as a tumour suppressor protein in most cancers but not in CRC, in which UPF1 plays an oncogenic role. We used the Xena platform to perform in silico analyses that revealed UPF1 protein overexpressed in CRC, contrary to several other analysed cancers. Besides, UPF1 protein levels are increased in CRC compared to the counterpart normal tissues. Experimentally, we confirmed that UPF1 protein expression is maintained in different CRC cell lines under normal conditions or endoplasmic reticulum (ER) stress. To understand the mechanism underlying such maintenance, we used a bicistronic reporter construct to test whether UPF1 5’ untranslated region (UTR) can mediate alternative translation initiation and we concluded that such sequence drives cap-independent translation initiation, in both normal and stress conditions. Deletional and mutational analyses of UPF1 5’UTR showed that nucleotides 1–100 [stem-loop (SL) I] and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the cap-independent translation initiation mechanism to work properly. Using RNA antisense oligonucleotides (ASOs) targeting UPF1 SL I and III, we observed a reduced UPF1 expression in HCT116 (CRC) cells, supporting the functional role of SL I and SL III in mediating cap-independent translation. Altogether, these results highlight the importance of cap-independent translation initiation in UPF1 expression regulation, in conditions that mimic the tumour microenvironment, and this might be used as a therapeutic target.
- Upstream Open Reading Frames Regulate PERK Translation InitiationPublication . Fernandes, Rafael; Silvestre, Samuel; Ponte, João; Lacerda, Rafaela; Romão, LuísaIntroduction: Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS).