ITQB: PCB - MA Dissertations
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- Microalgae for the Production of High-Value Compounds: Evaluation of the Effect of Different Nitrogen and Carbon Sources on the Production of Fucoxanthin in Phaeodactylum tricornutumPublication . Bragança, Ana Rita; Abranches, Rita"Phaeodactylum tricornutum is a model diatom used in studies of growth and production of photosynthetic pigments, with the particularity of its great capacity to produce fucoxanthin. Fucoxanthin is a high-value pigment with applications in the food, cosmetic and pharmaceutical industries, revealing in pre-clinical studies, antioxidant, anti-obesity, and anti-cancer activity. This xanthophyllous pigment plays an important role in microalgae, as its production occurs in response to abiotic stresses, allowing the cell to survive under less favourable conditions. Thus, it is possible to optimize the production of fucoxanthin by changing the growth conditions, in particular the composition of the culture medium. In this thesis, changes in the culture medium composition were studied, through the modification of nitrogen and carbon sources in various concentrations. The results indicated that the conditions that most favoured the growth and production of fucoxanthin were achieved by increasing sodium nitrate by a factor of 10 to the f/2 medium supplemented with silica, which is used for maintenance of the diatoms growth. The increase in the concentration of sodium nitrate allowed to double the fucoxanthin production, in just 7 days of growth. Cell viability and behaviour under different light conditions were also investigated. Additionally, genetic expression studies were performed to evaluate the possible influence of N sources on the expression of target genes of the fucoxanthin synthesis pathway.(...)"
- SARS-CoV-2 Spike and Receptor Binding Domain Glycoproteins Produced in Tobacco BY-2 and Medicago Cell Suspension CulturesPublication . Ferreira, Ana Clara; Abranches, Rita Sobral"The rapid worldwide spread of SARS-CoV-2 has motivated the scientific community to develop efficient and specific diagnostics and therapies in response to the COVID-19 pandemic. A variety of approaches have been considered, including the production of COVID-related proteins, with the Spike and its RBD as the major targets for expression in various systems. RBD and Spike protein production have previously been achieved in eukaryotes, namely animal cell cultures and via transient expression in plants. Spike protein production in microbial systems has been unsuccessful until now. In this dissertation, an alternative eukaryotic system was used for the production of SARS-CoV-2 Spike and RBD. The effective production of these viral proteins was successfully achieved in two plant cell suspension cultures: Tobacco BY-2 and Medicago truncatula A17 cell lines.(...)"
- Transformation of grey poplar to introduce resistance to Phytophthora sp.Publication . Marques, Rita M; Fevereiro, Pedro"Phytophthora cinnamomi is a widespread, aggressive and destructive plant pathogen with a wide variety of economically-relevant host species. One such species is the chestnut tree, Castanea sativa, which has provided an important European food and wood source since the medieval times. A previous transcriptomics and genomics study identified differentially expressed genes on resistant and susceptible chestnut varieties. One of the most promising genes was the GINKBILOBIN2-like (GNK2-like), whose protein, secreted by the roots of Castanea crenata, has a known antifungal domain. This work aimed to transform grey poplar (Populus tremula x alba), a model for trees, with an overexpressed Cast_GNK2-like gene in order to validate the anti-Phytophthora activity of this protein through a heterologous system.(...)"
- Culturas Celulares de Tabaco BY2 para a Produção de Biofármacos: O Papel da Metilação do ADN na Expressão do TransgenePublication . Melo, Leonor Brás de Oliveira Bandeira de; Abranches, Rita"Genetic engineering strategies such as molecular farming, essential for the production of recombinant proteins and other secondary metabolites in plants, have the potential to provide virtually unlimited amounts of recombinant proteins for use as diagnostic and therapeutic tools in health care and life sciences. Tobacco cell suspension cultures such as BY2 (Bright Yellow 2) can be easily transformed and a number of approaches have been developed to increase production, including medium optimization or the use of improved expression vectors. The aim of this work was to try to optimize the production, by increasing the transcription levels of the transgene encoding the protein of interest lipocalin-type Prostaglandin D synthase (L-PGDS), through epigenetic modulation using hypomethylation agents. Treatment with 5-azacytidine in plant and animal cells results in demethylation of DNA both directly and indirectly. We attempted to establish the optimum concentration of 5- azacytidine in the cell lines by applying different concentrations of the compound, and subsequently performed the immunodetection of methylated cytosines in BY2 nuclei, as well as methylation studies using Dot-Blot methods and conversion by bisulfite.(...)
- Otimização da produção de proteínas recombinantes em culturas de células vegetais: edição de genoma de células de tabaco BY-2 através de CRISPR-Cas9Publication . Nogueira, Ana Rita de Jesus; Abranches, Rita"The use of transgenic plants for large-scale production of recombinant proteins with commercial and pharmaceutical value has emerged as an alternative to conventional systems due to its several advantages. Particularly, plant cell suspension cultures offer the advantages of production in whole plant (such as reduced costs and safety) and production in cell culture systems. Although they have numerous advantages, the yields obtained are not yet competitive enough when compared to animal cell culture systems.(...)"