Publicação
New in vitro models and synthetic biology tools for research and drug discovery against hepatotropic pathogens
| datacite.subject.fos | Departamento de Biologia Vegetal | pt_PT |
| dc.contributor.advisor | Rodrigues, Ana Filipa | |
| dc.contributor.advisor | Leão, Raquel Sá | |
| dc.contributor.author | Fonseca, Maria Marques da Silva Mega da | |
| dc.date.accessioned | 2020-02-07T14:30:08Z | |
| dc.date.available | 2022-12-01T01:32:08Z | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019 | |
| dc.description | Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019 | pt_PT |
| dc.description.abstract | The lack of competent in vitro hepatic cell models has hindered the study of hepatotropic pathogens such as Hepatitis C virus (HCV) and consequently the development of new therapies. The main limitation is the shortage of a primary-like phenotype and functionality as well as limited permissiveness to infection. This work aimed at developing highly competent hepatic cell lines through lentiviral vector mediated immortalization of primary human hepatocytes (PHH). To this end, we started by developing a strategy to sustain lentiviral vector transduction of PHH by improving lentiviral vector design and optimizing transduction conditions. A dualcolor VCN PCR method was also implemented to titrate the immortalizing vectors with increased throughput. The data obtained and the tools established allowed the construction of immortalization libraries capable of driving appropriate expression. The functionality of one of these libraries was confirmed by qPCR and notable changes in morphology of PHH after one week of transduction. More, transduced PHH generated a heterogeneous population and cell clones. A total of 39 colonies was obtained by limiting dilution isolation. Proliferative cells that survived to expansion have demonstrated distinct morphologies confirming the ability of this strategy to generate biologically diverse cells. This high diversity will enable to dissect the determinants leading to the generation of cells permissive to infection and the identification of key factors underlying the hepatic primarylike phenotype. This work contributed to the creation of a platform for PHH immortalization by establishing a high throughput lentiviral vector production and titration methods compatible with use in PHH and by optimizing transduction conditions. A functional immortalization library was also constructed which led to the expansion of a heterogeneous population and 2 proliferating clones. With further optimization this platform will contribute to the establishment of new highly permissive and competent cells to support HCV entry and infection progression that ultimately can assist the efficient drug or vaccine development against HCV. | pt_PT |
| dc.identifier.tid | 202380840 | |
| dc.identifier.uri | http://hdl.handle.net/10451/41699 | |
| dc.language.iso | eng | pt_PT |
| dc.subject | Modelos hepáticos | pt_PT |
| dc.subject | Imortalização | pt_PT |
| dc.subject | Vetores lentivirais | pt_PT |
| dc.subject | VHC | pt_PT |
| dc.subject | Teses de mestrado - 2019 | pt_PT |
| dc.title | New in vitro models and synthetic biology tools for research and drug discovery against hepatotropic pathogens | pt_PT |
| dc.type | master thesis | |
| dspace.entity.type | Publication | |
| rcaap.rights | openAccess | pt_PT |
| rcaap.type | masterThesis | pt_PT |
| thesis.degree.name | Mestrado em Biologia Molecular e Genética | pt_PT |