DGH - Apresentações orais em encontros internacionais
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- An engineered U1 snRNA-based therapeutic approach can efficiently rescue a 5’ splice site mutation causing Mucolipidosis type IIIPublication . Peretto, L.; Gonçalves, M.; Santos, J.I.; Duarte, A.J.; Moreira, L.; Encarnação, M; Coutinho, M.F.; Pinotti, M.; Balestra, D.; Alves, S.; Matos. L.A significant number of splicing mutations have been identified in Lysosomal Storage Disorders (LSDs). Mucolipidosis III (ML III) is a LSD caused by GlcNAc-1-phosphotransferase deficiency, which impairs the trafficking of lysosomal hydrolases. 10% of the genetic defects in ML III are splicing mutations, and around 45% affect 5' splice-sites (ss) thus constituting a good target for mutation specific therapies. The use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s - ExSpeU1s) has been applied as a potential therapeutic strategy to correct 5’ss defects. Here we used engineered U1 snRNAs to correct the GNPTAB exon 17 skipping caused by the 5’ss mutation (c.3335+6T>G) found in a ML III patient. First, we performed transfection of exon-trapping minigenes expressing exon 17 surrounded by a portion of introns - pGNPTAB_WT and pGNPTAB_+6, in HEK293T cells to analyze if they reproduce the WT and mutant splicing patterns. Then, to evaluate the potential of 2 modified U1’s, 3 ExSpeU1s and 2 modified U6’s to restore mRNA splicing, these vectors were cotransfected into HEK293T cells along with the mutant +6 minigene as well as electroporated in patient’s fibroblasts. Then, cells were harvested, and RT-PCR analysis was performed. Both minigenes reproduced the control or ML III patient cDNA’s splicing patterns, thus, different concentrations of the modified U1’s and ExSpeU1s were tested together with the mutant minigene. The cDNA analysis showed almost 100% of exon 17 inclusion when one of the ExSpeU1s, was overexpressed in HEK293T cells. The combination of the 2 modified U6’s with the modified U1’s or the ExSpeU1s allowed exon 17 inclusion at some extent, but not as effectively as with the best ExSpeU1 alone. The electroporation of the 2 modified U1’s and of the 3 ExSpeU1s was done, and the cDNA analysis of patient’s fibroblasts treated with 2 ExSpeU1s (ExSpeU1 int17-1 or int17-2) showed around 35% and 15% of exon 17-including transcripts, respectively. To confirm these results, given that the lentiviral transduction is a more efficient delivery technique than electroporation, the gene cassettes of the 2 most promising ExSpeU1s were cloned in a lentivirus vector and after obtaining the viral mediums, their transduction in patient’s fibroblasts is being optimized. The cDNA analysis of preliminary experiments is still ongoing. In conclusion, we have developed an RNA therapy based on engineered U1 snRNAs for a ML III 5’ss mutation. We showed that an ExSpeU1 (binding downstream of the mutated 5´ss) can restore proper exon 17 definition in vitro, opening the opportunity for a personalized therapeutic intervention.
- Tracking Mycotoxin Exposure in Portugal: New Insights and Key DeterminantsPublication . Namorado, Sónia; Maris, Elias; Chen, A.; Pero-Gascon, Roger; de Boevre, Marthe; De Saeger, Sarah; Silva, Maria João; Alvito, Paula; .Human biomonitoring (HBM) is a vital tool for assessing exposure to environmental chemicals. Mycotoxins have been associated with various adverse health effects, including estrogenic, immunotoxic, nephrotoxic, and teratogenic outcomes. In this presentation an overview of two recent HBM studies on multiple mycotoxins exposure biomarkers conducted in Portugal will be given. The first study analyzed 37 mycotoxin biomarkers in urine samples from 94 adult participants (48.4 ± 15.2 years), as part of the National Food, Nutrition, and Physical Activity Survey (2015–2016). Six different mycotoxins -DON, ZEN, AOH, OTA, FB1, and CIT- was confirmed through the quantification of 12 urinary biomarkers in paired 24 h and first-morning urine samples. DON and its metabolites were among the most frequently detected biomarkers and AOH was identified for the first time in urine samples from a European population. Associations between urinary mycotoxin biomarkers and consumption of specific food items were also observed. More recently, a subset of 295 first-morning urine samples from adults (28–39 years) was collected between 2019-2020, as part of a cross-sectional study embedded within the Portuguese National Health Examination Survey (INSEF). These samples were analyzed using a newly optimized and validated LC-MS/MS method capable of detecting 40 mycotoxins and/or their metabolites in urine. DON and tenuazonic acid were the most frequently detected, with detection rates of 85% and 96%, respectively. Further investigations into key exposure determinants—including dietary habits, demographic factors, and geographical variations—are ongoing and will be presented. Altogether, these findings highlight the importance of continued surveillance and the integration of HBM into national food safety and public health strategies.