Analysis of metabolic flux distributions in relation to the extracellular environment in Avian cells

Continuous cell lines that proliferate in chemically defined and simple media have been highly regarded as suitable alternatives for vaccine production. One such cell line is the AG1.CR.pIX avian cell line developed by PROBIOGEN. This cell line can be cultivated in a fully scalable suspension culture and adapted to grow in chemically defined, calf serum free, medium [1]–[5]. The medium composition and cultivation strategy are important factors for reaching high virus titers. In this project, a series of computational methods was used to simulate the cell’s response to different environments. The study is based on the metabolic model of the central metabolism proposed in [1]. In a first step, Metabolic Flux Analysis (MFA) was used along with measured uptake and secretion fluxes to estimate intracellular flux values. The network and data were found to be consistent. In a second step, Flux Balance Analysis (FBA) was performed to access the cell’s biological objective. The objective that resulted in the best predicted results fit to the experimental data was the minimization of oxidative phosphorylation. Employing this objective, in the next step Flux Variability Analysis (FVA) was used to characterize the flux solution space. Furthermore, various scenarios, where a reaction deletion (elimination of the compound from the media) was simulated, were performed and the flux solution space for each scenario was calculated. Growth restrictions caused by essential and non-essential amino acids were accurately predicted. Fluxes related to the essential amino acids uptake and catabolism, the lipid synthesis and ATP production via TCA were found to be essential to exponential growth. Finally, the data gathered during the previous steps were analyzed using principal component analysis (PCA), in order to assess potential changes in the physiological state of the cell. Three metabolic states were found, which correspond to zero, partial and maximum biomass growth rate. Elimination of non-essential amino acids or pyruvate from the media showed no impact on the cell’s assumed normal metabolic state.

Engineering bio-based polymers using alternative solvents and processes

The work presented in this thesis explores novel routes for the processing of bio-based polymers, developing a sustainable approach based on the use of alternative solvents such as supercritical carbon dioxide (scCO2), ionic liquids (ILs) and deep eutectic solvents (DES). The feasibility to produce polymeric foams via supercritical fluid (SCF) foaming, combined with these solvents was assessed, in order to replace conventional foaming techniques that use toxic and harmful solvents. A polymer processing methodology is presented, based on SCF foaming and using scCO2 as a foaming agent. The SCF foaming of different starch based polymeric blends was performed, namely starch/poly(lactic acid) (SPLA) and starch/poly(ε-caprolactone) (SPCL). The foaming process is based on the fact that CO2 molecules can dissolve in the polymer, changing their mechanical properties and after suitable depressurization, are able to create a foamed (porous) material. In these polymer blends, CO2 presents limited solubility and in order to enhance the foaming effect, two different imidazolium based ILs (IBILs) were combined with this process, by doping the blends with IL. The use of ILs proved useful and improved the foaming effect in these starch-based polymer blends. Infrared spectroscopy (FTIR-ATR) proved the existence of interactions between the polymer blend SPLA and ILs, which in turn diminish the forces that hold the polymeric structure. This is directly related with the ability of ILs to dissolve more CO2. This is also clear from the sorption experiments results, where the obtained apparent sorption coefficients in presence of IL are higher compared to the ones of the blend SPLA without IL. The doping of SPCL with ILs was also performed. The foaming of the blend was achieved and resulted in porous materials with conductivity values close to the ones of pure ILs. This can open doors to applications as self-supported conductive materials. A different type of solvents were also used in the previously presented processing method. If different applications of the bio-based polymers are envisaged, replacing ILs must be considered, especially due to the poor sustainability of some ILs and the fact that there is not a well-established toxicity profile. In this work natural DES – NADES – were the solvents of choice. They present some advantages relatively to ILs since they are easy to produce, cheaper, biodegradable and often biocompatible, mainly due to the fact that they are composed of primary metabolites such as sugars, carboxylic acids and amino-acids. NADES were prepared and their physicochemical properties were assessed, namely the thermal behavior, conductivity, density, viscosity and polarity. With this study, it became clear that these properties can vary with the composition of NADES, as well as with their initial water content. The use of NADES in the SCF foaming of SPCL, acting as foaming agent, was also performed and proved successful. The SPCL structure obtained after SCF foaming presented enhanced characteristics (such as porosity) when compared with the ones obtained using ILs as foaming enhancers. DES constituted by therapeutic compounds (THEDES) were also prepared. The combination of choline chloride-mandelic acid, and menthol-ibuprofen, resulted in THEDES with thermal behavior very distinct from the one of their components. The foaming of SPCL with THEDES was successful, and the impregnation of THEDES in SPCL matrices via SCF foaming was successful, and a controlled release system was obtained in the case of menthol-ibuprofen THEDES.