ITQB: ECAL - PhD Theses
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- In vitro tools to explore cardiac fibrosis target spacePublication . Moita, Maria Raquel; Barbas, Ana; Simão, Daniel; Hoet, RenéCardiac fibrosis is driven by activation of cardiac fibroblasts. To date, there are no effective therapies for this disease, which affects many lives, especially patients with cardiovascular disease. This lack of therapeutic options is due to the fact that there are few selective markers for activated cardiac fibroblasts. The aim of this work is to expand the number of selective markers and discover new ligands for the activated state of cardiac fibroblasts. We have studied the transcriptomic and proteomic changes on cardiac fibroblasts upon activation, investigated the impact of using different cell systems in whole-cell phage display selections and performed phage display selections directly in an in vitro model of cardiac fibroblasts.
- Engineering stable insect cell expression systems using site-specific integration technology for production of complex proteinsPublication . Dias, Mafalda M; Roldão, António; Teixeira, Ana P."Recombinant protein production plays a pivotal role in the industrial manufacturing of therapeutics, vaccines and diagnostics. A variety of expression systems are available, whereby insect High FiveTM (Hi5) cells are one of the main platforms largely due to the development of the baculovirus expression vector system (BEVS). While there are continuous efforts to improve the BEVS, key limitations remain, including the baculovirus-mediated cell lysis and the lack of homogeneous humanized glycosylation. To address these limitations, there is a driving force towards the adoption of virus-free gene expression approaches, notably the use of random or site-specific integration systems (the most widely used technique for stable cell expression from a previously identified and characterized locus)(...)"
- Streamlining Upstream Processing of Complex BiopharmaceuticalsPublication . Sousa, Marcos; Roldão, António; Carrondo, Manuel"The market demand for new biopharmaceuticals is increasing with the aging of the global population and associated (chronic) diseases, rising interest for targeted (cell and gene) therapy, and the advent of new infectious diseases (e.g. Covid-19 pandemics). It becomes evident the need to accelerate the development of commercial processes for the manufacturing of such biopharmaceuticals to meet such demand. This thesis targets such needs creating solid knowledge on the upstream processing of animal cell cultures for the production of complex biopharmaceuticals using bioreactors and microcarriers technology, perfusion and integrated biomanufacturing as optimization tools. The work developed attempted to cover some latitude of challenges based on differences in product characteristics: (i) recombinant protein for drug discovery (human recombinant Bone Marrow Tyrosine kinase on the chromosome X – hrBMX, Chapter 2), (ii) virus as vaccine candidate (Peste des Petites ruminants virus – PPRV – vaccine, Chapter 3), (iii) virus for cancer therapy (oncolytic adenovirus type 5 – OV-Ad5, Chapter 4), and (iv) stem cells for cell therapy (human Mesenchymal Stem Cells – hMSC, Chapter 4). System’s complexity was embraced using bioreactors scale-down models and bioengineering correlations defining optimal process operation and scale-up.(...)"
- Metabolic Systems Biology for Stem Cell BioprocessesPublication . Sá, João Alberto Pacheco Marques de Vasconcelos; Carrondo, Manuel; Isidro, Inês"The application of stem cell-derived products for human health is in its infancy. Different applications of stem cell bioprocesses – cell therapy, discovery of new targets for regenerative medicine, disease modeling, drug and toxicity testing – have common hurdles. The most relevant hurdle is the lack of quality of cell products due to inefficient and ineffective stem cell differentiation procedures. Differentiated cells in vitro do not behave similarly to mature cells in vivo despite presenting similar surface and intracellular protein biomarkers. Our current weak understanding of cell “behavior” contributes to lack of cell functionality of produced cells and make these inadequate for cell therapy, disease modeling, drug screening and toxicity. Cells communicate with the environment and with neighboring cells, usually through secreted factors and through metabolites. This communication is guaranteed by an adjustable and controlled internal system in which metabolism is very important. (...)"
- Downstream strategies for VLPs as candidate universal vaccine for InfluenzaPublication . Baptista De Carvalho, Sofia; Carrondo, Manuel J.T.; Peixoto, CristinaInfluenza virus seasonal epidemics in a global public health concern. (...)
- The role of the miR-200 family in the epithelial-to-mesenchymal transition (EMT)Publication . Henriques, Ricardo Perdigão; Carrondo, ManuelOver the last decade, microRNAs (miRNAs) have earned a lot of attention due to their critical roles in several biological processes and human diseases. However, progress in this field has been limited by the difficulty of discovering miRNA target genes, as each miRNA can potentially bind to hundreds of different mRNAs.(...)
- Improving downstream processing for viral vectors and viral vaccinesPublication . Nestola, Piergiuseppe; Carrondo, ManuelViral vectors are playing an increasingly important role in the vaccine and gene therapy elds. The broad spectrum of potential applications, together with expanding medical markets, drives the e orts to improve the production processes for viral vaccines and viral vectors. Developing countries, in particular, are becoming the main vaccine market. It is thus critical to decrease the cost per dose, which is only achievable by improving the production process. In particular advances in the upstream processing have substantially increased bioreactor yields, shifting the bioprocess bottlenecks towards the downstream processing. The work presented in this thesis aimed to develop new processes for adenoviruses puri cation. The use of state-of-the-art technology combined with innovative continuous processes contributed to build robust and cost-e ective strategies for puri cation of complex biopharmaceuticals.(...)
- Downstream processing development of enveloped viruses for clinical applications: innovative tools for rational process optimizationPublication . Vicente, Tiago; Carrondo, Manuel J. T.; Alves, Paula M.Viral vectors and virus-like particles hold a tremendous potential in various clinical applications in the areas of gene therapy and/or vaccination, drawing the attention of biotechnology and pharmaceutical companies. The majority of these products are manufactured in animal cell cultures, inherently making the process costly. A great deal of effort is taking place to generate optimized biological and engineering strategies to find scalable and cost-effective processes, easily transferable to cGMP facilities. However, the implementation of robust downstream processes generating this type of biopharmaceuticals in the amounts required for pre-clinical and clinical trials is still lacking and lagging. By including a labile lipid membrane layer harboring glycoproteins (often critical for infection) over the viral capsid, enveloped viruses bring extra challenges in terms of their bioprocessing particularly downstream. The work developed during this thesis aimed at improving the state-of-the-art purification processes for these types of viral particles. The rationale was to integrate process understanding with product characterization, still scarce in such biological systems.(...)
- Production optimization of rotavirus-like particles: a system biology approachPublication . Roldão, António Manuel Missionário; Oliveira, Rui M. F.; Carrondo, Manuel J. T.Rotavirus-like particles (RLPs), a vaccine candidate against rotavirus disease, were produced by infecting Spodoptera frugiperda Sf-9 cells with genetically engineered recombinant baculoviruses. RLPs are spherically shaped particles composed by three viral proteins (vp) of rotavirus, vp2, vp6 and vp7, arranged in a triple layered structure. A diversity of protein structures, other than the correctly assembled RLP, are observed at the end of a typical production run suggesting that the protein assembly process is rather inefficient. Contaminants such as trimers of vp6 and vp7, vp6 tube-like structures, single-layered vp2 particles, double layered particles of vp2 and vp6 or RLPs lacking one or more subunits represent almost 88% of the total mass of proteins expressed. Thus, optimal control of protein expression concomitant with efficient particle assembly are critical factors for economical RLP production in the baculovirus/insect cells system.
- Gammaretroviral and lentiviral vectors for gene therapy: stability and inactivation mechanismsPublication . Carmo, Marlene; Carrondo, Manuel J. T.; Cruz, Pedro E.