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Sustained gene expression in the retina by improved episomal vectors

Publication . Calado, Sofia; Oliveira, Ana; Machado, Susana; Haase, Rudolf; Silva, Gabriela

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.

2014-10Journal article

Open Access

Effects of dietary curcumin in growth performance, oxidative status and gut morphometry and function of gilthead seabream postlarvae

Publication . Xavier, Maria João; Navarro-Guillén, Carmen; Lopes, André; Colen, R.; Teodósio, Rita; Mendes, Rodrigo; Oliveira, Beatriz; Valente, Luisa M.P.; Conceição, Luís E.C.; Engrola, Sofia

An imbalance in the production and detoxification of reactive oxygen species and other oxidants can disrupt a l l types of cellular compounds, and lead to a state of oxidative stress. Preventing this state is essential to improve growth and health in animal production. Therefore, the aim of this work was to assess i f the dieta r y supple-mentation of curcumin cou l d improve the antioxidant status and intestine structu r e and functionalit y of gilthead seabream postlarvae, with the global objective of improving fish growth performance and robustness. Two experimental diets supplemented with different doses of curcumin (LOW and HIGH diets), and a commercial diet were fed to quadruplicate groups of postlarvae, for 20 days. At the end of the feeding trial fish fed the supple-mented diets significantly improved their antioxidant status compared to CTRL fed fish. LOW and HIGH fed fish presented lower protein oxidative damage (P < 0.05) and higher total antioxidant capacity (P < 0.05). Moreover, postlarvae fed curcumin supplemented diets also presented an upregulation of nuclear factor erythroid 2 - related factor 2 (nrf2) and glutathione-disulfide reductase (gr) in HIGH (P < 0.05) and heat shock protein 70 (hsp70) in LOW treatments (P < 0.05). No differences were observed in growth performance, intestine morphome t r y , and digestive enzymes activities among treatments (P > 0.05). In conclusion, dieta r y curcumin supplementation was able to enhance gilthead seabream postlarvae robustness through a modulation of the oxidative status, increasing total antioxidant capacity and decreasing protein oxidative damage. This data pro-vide evidence that curcumin can be a suitable feed additive to promote heath status and robustness of fish at early stages of development, therefore contributing for the development and sustainabilit y of marine fish hatchery production.

2022Journal article

Open Access

Modality effects in implicit artificial grammar learning: An EEG study

Publication . Silva, Susana; Folia, Vasiliki; Inácio, Filomena; Castro, Sao Luis; Petersson, Karl Magnus

Recently, it has been proposed that sequence learning engages a combination of modality-specific operating networks and modality-independent computational principles. In the present study, we compared the behavioural and EEG outcomes of implicit artificial grammar learning in the visual vs. auditory modality. We controlled for the influence of surface characteristics of sequences (Associative Chunk Strength), thus focusing on the strictly structural aspects of sequence learning, and we adapted the paradigms to compensate for known frailties of the visual modality compared to audition (temporal presentation, fast presentation rate). The behavioural outcomes were similar across modalities. Favouring the idea of modality-specificity, ERPs in response to grammar violations differed in topography and latency (earlier and more anterior component in the visual modality), and ERPs in response to surface features emerged only in the auditory modality. In favour of modality-independence, we observed three common functional properties in the late ERPs of the two grammars: both were free of interactions between structural and surface influences, both were more extended in a grammaticality classification test than in a preference classification test, and both correlated positively and strongly with theta event-related-synchronization during baseline testing. Our findings support the idea of modality-specificity combined with modality-independence, and suggest that memory for visual vs. auditory sequences may largely contribute to cross-modal differences. (C) 2018 Elsevier B.V. All rights reserved.

2018-05Journal article

Open Access