Percorrer por autor "Moreira, Luciana"
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- Assessing the in vitro toxicity of airborne (nano)particles to the human respiratory system: from basic to advanced modelsPublication . Bessa, Maria João; Brandão, Fátima; Rosário, Fernanda; Moreira, Luciana; Reis, Ana Teresa; Valdiglesias, Vanessa; Laffon, Blanca; Fraga, Sónia; Teixeira, João PauloSeveral studies have been conducted to address the potential adverse health risks attributed to exposure to nanoscale materials. While in vivo studies are fundamental for identifying the relation-ship between dose and occurrence of adverse effects, in vitro model systems provide important information regarding the mechanism(s) of action at the molecular level. With a special focus on exposure to inhaled (nano)particulate material toxicity assessment, this review provides an over-view of the available human respiratory models and exposure systems for in vitro testing, advan-tages, limitations, and existing investigations using models of different complexity. A brief overview of the human respiratory system, pathway and fate of inhaled (nano)particles is also presented.
- Can Cell-type specific variability be involved in a rare variant of Unverricht-Lundborg? Investigation with iPSC generated modelsPublication . Duarte, Ana Joana; Ribeiro, Diogo; Moreira, Luciana; Amaral, OlgaHomozygosity for a private synonymous mutation in the cystatin-B gene (CSTB, MIM:601145; c.66G>A; p.Q22Q) was detected in a Portuguese patient with a rare, atypical form of Unverricht-Lundborg disease (ULD, MIM #254800). This apparently silent mutation leads to mis-splicing of CSTB pre-mRNA where a normal and an abnormal transcript were detected. Using iPSCs as a source of different cell types, we intend to clarify if the observed abnormal RNA splicing is cell-type specific, and to characterise the subsequent protein mislocalization. In conclusion, we hope to be able to contribute to the understanding of cell-type specific implications in the pathogenesis of ULD.
- Comparative Analysis of the Toxicity Profile of Eleven Consumer-Relevant Nanomaterials in Human Intestinal and Placental Barrier CellsPublication . Pires, Joana; Moreira, Luciana; Teixeira, João Paulo; Fraga, SóniaBackground: The growing number of items incorporating nanomaterials (NM) has prompted considerable concerns about human health and safety [1]. Metal nanoparticles, inorganic non-metallic, and carbon-based NM are among the types with the highest market volume [2]. Objective: The purpose of this study was to determine the effect of chemical composition [Ag, Au, TiO2, SiO2, and graphene oxide (nano_GO)], primary size (10, 30 and 60 nm AgNP and AuNP), crystal structure (TiO2NP rutile/anatase and anatase), and surface coating (citrate and PEGylated AuNP) on potential toxicity to human intestinal (Caco-2) and placental (BeWo b30) epithelial cells. Methods: Changes in cell morphology, metabolic activity, plasma membrane integrity, intracellular ROS and ATP levels, and DNA integrity were assessed to investigate their potential toxicity at 24 h after exposure. Results: In both barrier models, the toxicity profile was similar, however placentalwere more sensitive than intestinal epithelial cells. Overall, NM may be ranked for cytotoxicity as AgNP > nano_GO > AuNP ~ TiO2NP ~ SiO2NP, with the effects becoming more evident at greater concentrations. The influence of size was more pronounced for AgNP than for AuNP, with the smaller nanoparticles producing higher cytotoxic effects. The cytotoxicity of AuNP was prevented by PEG capping. AgNP and nano_GO exposure markedly raised the levels of ROS, indicating that oxidative stress may play a role in their cytotoxicity. Except for 10 nm AuNP, every NM tested markedly increased intracellular ATP levels. One interesting finding was that a higher cytotoxic potential did not necessarily equate to a higher genotoxic potential, since only AgNP (classified as positive) and anatase TiO2NP (classified as equivocal) caused DNA damage. Conclusions: Our findings alert to the potential risks associated with human barriers exposure to NM, where the physicochemical properties are important determinants of their toxicity. Additional research is needed for a deeper understanding of NM impact on human barriers.
- CRISPR/Cas in iPSCs from Sphingolipidoses patientsPublication . Amaral, Olga; Duarte, Ana; Ribeiro, Diogo; Moreira, LucianaClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were found as an immune adaptive mechanism in bacteria and quickly were applied to various fields as a promising tool for gene editing. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by defects in lysosomal proteins leading to accumulation of undigested macromolecules within the cells. The lack of good in vitro models hinders research of the pathophysiologic mechanisms and the development of new therapies. Induced pluripotent stem cells (iPSCs) are patient-specific and can be differentiated in any cell type. The advantage of iPSCs is to enable targeted studies in cells with the patient’s own background leading to more straightforward results than other models. Combining CRISPR and iPSCs is, therefore, a promising strategy. We aim to use CRISPR/Cas-mediated gene editing to provide more specific cellular models of disease, to correct causal mutations in LSDs and to create mutants for functional studies. In this work, we generated and characterized iPSCs from human fibroblasts obtained from Gaucher and Fabry patients (through Gaslini Institute) and will edit them with a CRISP/Cas9 approach. Because both gene editing and iPSCs generation require manipulating the cell’s genome, we envisage multiple check points along the workflow. It will be useful to compare the “native” mutated cells with the corrected cells that modulate the “disease in a dish”. Gene editing is still recent and the methods require improvement, namely increasing transfection rates and mutagenesis efficiency with less off-targets. Nevertheless, CRISPR/Cas is a promising alternative to other therapies, and every result contributes to the enhancement of this technology, broadening the validation of CRISPR application and making it an accessible option.
- CRISPR/Cas in iPSCs from sphingolipidoses patientsPublication . Moreira, Luciana; Duarte, Ana Joana; Ribeiro, Diogo; Amaral, OlgaBackground: Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) were found as an immune adaptive mechanism in bacteria and quickly were applied to various fields as a promising tool for gene editing. Ultimately, the use of CRISPR/Cas-mediated gene editing can provide specific cellular models of disease, correct causal mutations in LSDs and create mutants for functional studies. Aim: In this work, experimental conditions were tested to validate a CRISPR/Cas9 approach to generate a KO. Conclusion: The cells were amenable to edition by CRISPR/Cas9 and the results obtained proved that editing was achieved. All results contribute to the improvement of our knowledge regarding this technology, broadening the validation of CRISPR application and making it an accessible option.
- Doenças lisossomais de sobrecarga: da epidemiologia genética ao desenvolvimento de modelos de doençaPublication . Moreira, Luciana; Coutinho, Maria Francisca; Moutinho, Maria Eduarda; Almeida, Matilde Barbosa; Gonçalves, Francisca; Carvalho, Sofia; Amaral, Olga; Duarte, Ana Joana; Encarnação, Marisa; Gaspar, Paulo; Gonçalves, Mariana; Matos, Liliana; Ribeiro, Diogo; Rocha, Hugo; Santos, Juliana Inês; Alves, Sandra; .
- Fibrinogen A alpha-chain amyloidosis: a non-negligible cause of chronic kidney disease in dialysis patientsPublication . Tavares, Isabel; Moreira, Luciana; Costa, Paulo Pinho; Lobato, LuísaBackground: Fibrinogen A alpha-chain (AFib) amyloidosis is a rare and late-onset disease, that result from amyloidogenic autosomal dominant mutations in the gene-encoding AFib (FGA). Patients invariably develop chronic kidney disease (CKD), typically progressing to end-stage renal failure within 5 years of recognition of renal involvement [1]. In Portugal, four apparently unrelated patients with AFib amyloidosis were identified in the district of Braga, Northern Portugal. They all carried the FGA p.Glu545Val mutation, three were heterozygous and one homozygous [2,3]. This observation led us to assess the prevalence of AFibE526V (p.Glu545Val) amyloidosis among Portuguese patients undergoing hemodialysis in the same district, through genetic screening for the FGA p.Glu545Val mutation.
- Gene editing as a tool for developing cell based models of a lysosomal storage disorder: preliminary resultsPublication . Duarte, Ana Joana; Moreira, Luciana; Gaspar, Paulo; Alves, Sandra; Bragança, José; Amaral, OlgaIn this work, we aimed to establish a Fabry Disease (FD, OMIM: #301500) disease model using the CRISPR/Cas 9 system by knocking out a HDFa iPSC line. We also aimed to correct a nonsense mutation (p. W 287 X) in the iPSCs derived from a patient with FD. The cell lines used were generated in our laboratory, and the FD iPSC line is registered in the Human Pluripotent Stem Cell Registry with identification "INSAi 002-A". To fully evaluate the molecular and cellular physiological changes, further studies are still required. The development of innovative cell models, particularly for rare diseases like Lysosomal Storage Disorders, is beneficial for studying the pathophysiology of the disease.
- Gene Editing in Fabry Disease: A Strategy DelineationPublication . Duarte, Ana J.; Moreira, Luciana; Ribeiro, Diogo; Amaral, OlgaThe use of iPSCs, in the last years became wide spread, even in our group at INSA, the use of iPSCs to develop models of disease is now envisaged for various Lysosomal Storage Diseases. Such cell models are being used to experiment several types of therapeutic methodologies, as well as approaches that interfere with normal pathways to provide understanding about pathologic mechanisms, and gene editing is particularly interesting among the latter strategies. Recently, a new CRISPR-based method – Prime Editing (PE) – provides all-possible base-to-base conversions, “indels”, and combinations; the human genome can be edited without the need of double-strand breaks (DSBs) or donor DNA templates. This method proved its efficacy to correct a pathogenic insertion that causes Tay-Sachs disease (HEXA 1278+TATC; OMIM 606869). In this work, our aim is to correct one of the Fabry Disease (FD) causing mutations, the p.W287X, located on the GLA gene (OMIM 300644). For this purpose, our strategy is to use a construct that uses a one-step golden gate digestion-ligation cloning that is called Prime Editing All-in-One (PEA1) plasmid, consisting in a cassette for expression of all PE3 components and a selection marker. A few years ago we developed iPSCs from skin fibroblasts of patients. The present correction approach will be tested in our FD iPSC line. At this moment, we are initiating the work but we hope to achieve positive results soon. The use of new genetic engineering tools, like PE, and its use as possible therapeutic strategy should provide further comprehension of FD and act as a potential therapy.
- Generation of Cellular Models for Fabry Disease: Unlocking the Potential of iPSCs and Gene EditingPublication . Duarte, Ana Joana; Moreira, Luciana; Ribeiro, Diogo; Alves, Sandra; Gaspar, Paulo; Bragança, José; Amaral, OlgaIntroduction: Fabry Disease (FD) is a lysosomal storage disorder caused by mutations in the GLA gene, resulting in a defective α-GAL A enzyme. This deficiency leads to the accumulation of Gb3 and lyso-Gb3 within lysosomes, resulting in a multisystem disease. Through reprogramming, we obtained induced pluripotent stem cells (iPSCs) derived from fibroblasts of a patient with FD2 and from a wild-type (WT) control. We used CRISPR/Cas9 to correct the c.860G>A mutation present in the patient’s cells, as well as to generate a WT GLA knockout (KO). The resulting cells were then differentiated into cardiomyocytes, a cell type affected by this disease. Methods: We reprogrammed the fibroblasts into iPSCs using episomal vectors or Sendai virus. For gene editing, single-guide RNAs (sgRNAs) and Cas9 were nucleofected, and the editing was confirmed by Sanger sequencing. Following colony selection, isogenic cell lines were established. The FD iPSCs, the corrected FD iPSCs, and the WT iPSCs were then differentiated into iPSC-derived cardiomyocytes (iPSC-CMs). Results: Seven new cell models were generated. Functional studies of the FD iPSCs showed the maintenance of the molecular and biochemical characteristics and a normal karyotype. The KO cell line recapitulated the biological features observed in FD patient cells, with reduced GLA expression, lower α-Galactosidase A (α-Gal A) activity (1.5 nmol/h/mg protein), and Gb3 accumulation. The corrected cell line was generated with 75.8% efficiency and 69.6% on-target efficacy. Enzyme activity increased to 579 nmol/h/mg protein (vs. 0.78 nmol/h/mg protein in FD iPSCs), accompanied by a marked reduction in Gb3 levels. We successfully generated iPSC-CM lines, which were validated by qRT-PCR and immunofluorescence. Discussion: Cell modelling is essential for studying the pathophysiology of disease mechanisms. By retaining the characteristics of the original cells, iPSCs are a valuable biological resource for generating specific differentiated cell types affected by the disease, which would otherwise be difficult to access. This study also explored the therapeutic potential of gene editing as a promising approach to altering the course of rare diseases.