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.

Development of process control strategies exploiting knowledge from systems biology: application to MDCK suspension cells

Madine Darby Canine Kidney (MDCK) cell lines have been extensively evaluated for their potential as host cells for influenza vaccine production. Recent studies allowed the cultivation of these cells in a fully defined medium and in suspension. However, reaching high cell densities in animal cell cultures still remains a challenge. To address this shortcoming, a combined methodology allied with knowledge from systems biology was reported to study the impact of the cell environment on the flux distribution. An optimization of the medium composition was proposed for both a batch and a continuous system in order to reach higher cell densities. To obtain insight into the metabolic activity of these cells, a detailed metabolic model previously developed by Wahl A. et. al was used. The experimental data of four cultivations of MDCK suspension cells, grown under different conditions and used in this work came from the Max Planck Institute, Magdeburg, Germany. Classical metabolic flux analysis (MFA) was used to estimate the intracellular flux distribution of each cultivation and then combined with partial least squares (PLS) method to establish a link between the estimated metabolic state and the cell environment. The validation of the MFA model was made and its consistency checked. The resulted PLS model explained almost 70% of the variance present in the flux distribution. The medium optimization for the continuous system and for the batch system resulted in higher biomass growth rates than the ones obtained experimentally, 0.034 h-1 and 0.030 h-1, respectively, thus reducing in almost 10 hours the duplication time. Additionally, the optimal medium obtained for the continuous system almost did not consider pyruvate. Overall the proposed methodology seems to be effective and both proposed medium optimizations seem to be promising to reach high cell densities.

José de Figueiredo, 1871-1937. Acção e contributos no panorama historiográfico, museológico e patrimonialista em Portugal

Estruturado sob a influência dos modelos de “vida e obra” próprios da narrativa biográfica, este trabalho assume uma abordagem metodológica de compromisso entre a fixação da cronologia do percurso de José de Figueiredo e o desenvolvimento de uma reflexão sobre o seu papel nas áreas em que se destacou. Numa primeira parte são contextualizados e analisados as suas origens familiares, o seu percurso formativo em Coimbra, os anos de formação artística informal que teve em Paris no final de oitocentos e o seu processo de integração na sociedade erudita lisboeta do início do século XX. De seguida é apresentada uma análise das várias áreas da sua atuação no panorama cultural português: a sua integração na Academia Real de Belas- Artes de Lisboa, num período de afirmação do seu nome enquanto especialista em “assuntos de arte”; o seu papel na campanha de estudo, restauro e divulgação dos painéis de S. Vicente; as suas ideias e contribuições na definição da legislação artística e patrimonial portuguesa, nos diversos contextos político-sociais que integrou; a sua atividade como crítico e historiador de arte, num período marcado pelas narrativas nacionalistas e pelo desenvolvimento da História da Arte enquanto área disciplinar autónoma; o seu papel na divulgação da arte portuguesa, dentro e fora do país; e a sua ação no âmbito da museologia da arte em Portugal, destacando-se a identificação das suas ideias e influências no contexto europeu e a leitura descritiva e crítica da atividade desenvolvida ao longo dos 26 anos em que dirigiu o Museu Nacional de Arte Antiga. Propõe-se assim um balanço crítico das ações e contribuições desta personalidade no panorama cultural português, bem como do seu enquadramento na cultura Europeia. É ainda apresentada uma reflexão sobre a criação do “mito José de Figueiredo”, que se verifica ser fruto de três fatores que se interrelacionam: a sua ambição pessoal, marcada por uma enérgica vontade de singrar e de deixar uma marca na cultura portuguesa; o seu forte carisma, alimentado estrategicamente através da gestão eficaz da sua imagem pública; e os contextos que o acolheram e que simultaneamente estimularam o seu trabalho, proporcionando-lhe recursos materiais e humanos que geriu com sucesso, dentro e fora das instituições a que pertenceu.

Von der Idee: Kultur

Im Werk Portugal Hoje. O Medo de Existir - Portugal heute. Die Angst des Existierens, zitiert F. Gil ein Lissabonner Graffitti. Não há drama, tudo é intriga." [[Kein Drama. Es ist alles Intrige], um daraus ein Motto für seine Analyse der gegenwãrtigen portugiesischen Gesellschaft zu entnechmen. Unfãhig, ihre herkõmmlichen Formen von Solidaritãt und gegenseitiger Hilfe wieder in Kraft zu stzen, sei die portugiesische Gesellschaft im chaotischen Ozean der Konkurrenz, der Arbeitslosigkeit und des Ausschlusses so tief gesunken, dass sie sich wegen der ihr gestellten Herausforderungen bedroht fühle. Der portugiesischen Gesellschaft würde es nicht gelingen, die Angst zu überwinden, die sie direkt aus der ehemaligen autoritãren und diktatorialen Gesellschaft als Erbe bekommen hatte, deren strukturelle Hauptstütze die Angst war - wie auch ein System, dessen Hauptziel es war, die Fõrderung der Kreativitãt , der schõpferischen und unternehmerischen Krãfte des sozialen, wirtschaftlichen Lebens zu unterdrücken; unter der Diktatur Salazars lebte man schon unter dem Einfluss der Gehorsamkeit, der Korruption, des Nicht-Teilnehmens, des Nicht-Einwerbens, des Nicht-Ereignens.