A carregar...
Projeto de investigação
iNOVA4Health - Programme in Translational Medicine (iBET, CEDOC/FCM, IPOLFG e ITQB)
Financiador
Autores
Publicações
Rational genetic engineering of insect cells to improve rAAVs and influenza VLPs production yield utilizing the baculovirus expression vector system
Publication . Graça, Miguel Ângelo Matos; Roldão, António; Planels, Jose
The insect cells-baculovirus expression vector system (IC-BEVS) is one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, allowing for high expression levels in short time frames. Despite its benefits, IC-BEVS is a transient system due to the baculovirus lytic nature, which causes the productivity and/or quality of products generated to be impacted. Improvements to the expression platform, and in particular to the cell line, is thus essential in order to bring IC-BEVS products faster to market. However, this has been limited by the lack of well-established tools for genetic engineering in insect cells.
This thesis aimed at overcoming these bottlenecks by establishing a gene editing pipeline based on the CRISPR-Cas9 system for simple and fast gene(s) knockout in Sf9 cells towards improved phenotypes. To establish the pipeline, different transfection methods were explored as to their potential to deliver the ribonucleoprotein complex comprising the Cas9 enzyme and a guide RNA that targeted the fused lobes (fdl) gene expressing the β-N-acetylglucosaminidase enzyme. Lipofection was identified as the best method with up to 85 % of gene editing efficiency achieved in a single transfection step. To validate the system, Sf-caspase-1, a gene associated to apoptosis (pathway demonstrated by in-house data to be up-regulated upon infection and linked to increased product titers), was used as a model knockout target. Editing efficiency of Sf-caspase-1 was optimized, obtaining a highly edited population. Single-cell cloning by limited dilution was implemented and Sf-caspase-1 knockout clones identified. Isolated clones were evaluated for cell growth, showing similar kinetics to non-edited Sf9 cells. In addition, clones showed improved resistance to apoptosis and generated slightly improved titers of recombinant adeno-associated viruses and influenza virus-like particles when compared to non-edited Sf9 cells.
Overall, an efficient CRISPR-Cas9 gene editing pipeline in insect cells was successfully established, which can assist rational engineering of insect cell lines towards improved production titers.
Advancing liver stage malaria in vitro models for drug discovery
Publication . Arez, Francisca; Alves, Paula M.; Prudêncio, Miguel
"The malaria drug pipeline has been highly affected by the fastdeveloping
rate of Plasmodium resistance to the currently available
drugs. There are two promising and complementary approaches being
explored to mitigate drug resistance. One is focused on the drugs, and
includes the development of drugs exploiting novel targets and the
combination of drug partners presenting the same pharmacokinetic
properties but distinct modes of action. The other approach is to explore
the life cycle of the parasite and target the hepatic stage of the
Plasmodium life cycle.(...)"
Characterization of cellular organelles and the endo-lysosomal pathway in choroideremia patient cells
Publication . Coelho, Rita Alexandre; Lopes-da-Silva, Mafalda
Choroideremia (CHM) is a form of retinal degeneration, with an X-linked pattern of inheritance caused by mutations in the CHM gene that codifies the Rab Escort Protein 1 (REP1). Given its genetic component and the closed system of the eye, CHM is an excellent target for gene therapy. However, this type of treatment still presents its challenges, thus it is important to uncover new molecular mechanisms of the disease, with the hopes of finding new therapeutic targets. The retinal pigment epithelium (RPE) consists of a monolayer of cells, localized between the choroid and the photoreceptors. The RPE is functionally diverse, performing tasks such as the daily phagocytosis and degradation of photoreceptor outer segments (POS), secretion of growth factors and transport of nutrients and metabolites. The REP1 protein is responsible for the geranylgeranylation of Rab GTPases, a post-translational modification that facilitates the binding of these small GTPases to membranes and therefore, exert their role in the regulation of membrane traffic. CHM patients have been shown to have RPE dysfunction; accumulation of unprocessed POS accompanied by a reduced degradation capacity and a decrease in phagosomal acidity.
With this project we intended to make a full characterization of cellular organelles and pathways, to dissect new uncharacterized cellular defects, with a focus on the endo-lysosomal pathway, as well as the impact of unprocessed POS accumulation in CHM RPE cells. To carry out our objective we used two different in vitro RPE cell models: differentiated RPE from human induced pluripotent stem cells from both healthy and CHM patient donors, hiPSc-RPE, and a commonly used immortalized RPE cell line, ARPE-19 where we used CRISPR/Cas9 to generate a CHM KO line. Characterization of the endo-lysosomal pathway revealed an increase in the number of LAMP1+ and CD63+ vesicles in both hiPSc-RPE and ARPE-19 CHM cell models, suggesting an increase in the number of both lysosomes and multivesicular bodies (MVBs), respectively. Western blot analysis of CHM hiPSc-RPE whole cell lysates revealed an increase in (inactive) procathepsin D and L, suggesting an impairment in the delivery of the immature form of these antibodies cathepsin forms to their proper cellular compartments, pointing to a targeting dysfunction within the endo-lysosomal pathway. Additionally, in ARPE-19 CHM cells, after a single pulse of POS, a significant accumulation of autofluorescent granules (AFGs) was observed, verified at both 24h- and 72h- post feeding resulting from incomplete digestion of POS. Furthermore, hiPSc-RPE CHM cells revealed an increase in cell size as well as a decrease in polarization, as analysed by a decrease in the transepithelial electrical resistance (TER), suggesting a dysfunction in the establishment of a tight monolayer, this evidence should be further analysed. Overall, this work provides novel evidence of cellular dysfunction in CHM cells, specifically on the endo-lysosomal pathway, as well as POS incorporation and degradation. We also show key differences between two cellular models of RPE, describing advantages and disadvantages of each model for the use of different assays and technical approaches. In the future, we aim to achieve optimized protocols for hiPSc-RPE to overcome technical challenges and make hiPSc-RPE an even better model of human RPE to investigate the molecular and cellular mechanisms of choroideremia as well as other diseases of the RPE.
Berry-enriched diet suppress neuroinflammation by modulating microglia
Publication . Louro, Diogo Ramos de Oliveira Moura; Santos, Cláudia Nunes dos
Abstract
Pilling evidence in the recent years shows an increasing incidence of
neurodegenerative diseases in a constantly aging population, bringing to the
forefront one of the most impactful socio-economic problems future generations
may come across. Having no explicit cure, only treatment focused on managing
the symptoms, these diseases are considered permanent hindrances in the lives of
patients. Given this, the possibility of modulating the brain’s well-being through
our diet stands as an innovative and ground-breaking approach to this urgent
health problem. Fruit and plant-based diets rich in (poly)phenols, a trademark to
the Mediterranean diet, have shown potential in modulating several hallmarks of
neurodegenerative pathologies, particularly the neuroinflammation associated
with disease progression, highlighting the value of these types of compounds.
As such, the following novel study was conducted, where the anti-inflammatory
potential of a berry-enriched diet, rich in (poly)phenols, was evaluated, on an LPSinduced
mouse model of neuroinflammation. The LPS-induced
neuroinflammation resulted in classical signs of sickness-like behavior with
hypophagia and weight loss, that were attenuated by the berry-enriched diet.
Behavioral tests on exploratory habits and memory function were performed, and
they showed the capability of the berry-enriched diet to prevent LPS-induced
memory impairments, particularly in the Y-maze and novel object recognition
(NOR).
In the brain, LPS-induced neuroinflammation resulted in microgliosis, astrogliosis,
and peripheral macrophage infiltration, especially in the memory-related region of
the hippocampus. These effects were all modulated by the berry-enriched diet.
In conclusion, our study demonstrated the potential of a berry-enriched diet to
attenuate LPS-induced neuroinflammation and its associated phenotypic and
biochemical changes. This research emphasizes the emerging potential of dietary
interventions, particularly (poly)phenol-rich diets, in preventing and modifying the
progression of neurodegenerative diseases.
A Study on Biofouling and Cleaning of Anion Exchange Membranes for Reverse Electrodialysis
Publication . Tiago, Gonçalo; Cristóvão, Maria Beatriz; Marques, Ana Paula; Huertas, Rosa; Merino-Garcia, Ivan; Pereira, Vanessa Jorge; Crespo, João Goulão; Velizarov, Svetlozar; LAQV@REQUIMTE; DQ - Departamento de Química; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); MDPI AG
This study covers the modification, (bio)fouling characterization, use, and cleaning of commercial heterogeneous anion exchange membranes (AEMs) to evaluate their feasibility for reverse electrodialysis (RED) applications. A surface modification with poly (acrylic) acid resulted in an improved monovalent perm-selectivity (decreased sulfate membrane transport rate). Moreover, we evaluated the (bio)fouling potential of the membrane using sodium dodecyl sulfate (SDS), sodium dodecyl benzenesulfonate (SDBS), and Aeromonas hydrophila as model organic foulants and a bio-foulant, respectively. A detailed characterization of the AEMs (water contact angle, ion exchange capacity (IEC), scanning electron microscopy (SEM), cyclic voltammetry (CV), and Fourier Transform Infrared (FTIR) spectra) was carried out, verifying that the presence of such foulants reduces IEC and the maximum current obtained by CV. However, only SDS and SDBS affected the contact angle values. Cleaning of the biofouled membranes using a sodium hypochlorite aqueous solution allows for (partially) recovering their initial properties. Furthermore, this work includes a fouling characterization using real surface and sea water matrixes, confirming the presence of several types of fouling microorganisms in natural streams. A lower adhesion of microorganisms (measured in terms of total bacteria counts) was observed for the modified membranes compared to the unmodified ones. Finally, we propose a cleaning strategy to mitigate biofouling in AEMs that could be easily applied in RED systems for an enhanced long-term process performance.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UIDB/04462/2020