Reactivity of human fetal and adult immortalized hepatocytes to potentially toxic bilirubin and bile acid species

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Calcareous nannofossil assemblages across the Pliensbachian/Toarcian boundary at the Peniche section (Ponta do Trovão, Lusitanian Basin)

The Peniche section has revealed moderately-to-well preserved calcareous nannofossil assemblages across the Pliensbachian/Toarcian boundary. This good record has allowed the proposition of a refined biostratigraphic scheme. The stage boundary, as defined by ammonites, is comprised within the NJ5b C. impontus (NW Europe; BOWN & COOPER, 1998) or the NJT5b L. sigillatus (Mediterranean Tethys; MATTIOLI & ERBA, 1999) nannofossil subzones. Since in the Lusitanian Basin a mixing of N- and S-Tethyan taxa is observed, both biozonation schemes can be applied. Some nannofossil events (mainly first occurrences) are observed earlier in Portugal than in other Tethyan settings. It is still unclear if these events are real first occurrences. A diversification phase occurred across the Pliensbachian/Toarcian boundary. This phase is well recorded at Peniche, where a change is observed passing from the Pliensbachian, when assemblages are dominated by muroliths, to the Toarcian showing assemblages where placoliths are abundant. A quantification of nannofossils per gram of rock shows that absolute abundances are the highest across the Pliensbachian/Toarcian boundary. Indeed, Peniche exhibits nannofossil abundances very high with respect to correlative levels in other Tethyan settings. The pelagic carbonate fraction (produced by nannofossils) is important in the marly hemi-couplets of Peniche. In some levels, nannofossils account for more than 50% of the total carbonate fraction.

Process engineering of liver cells for drug testing applications

Dissertação para obtenção do Grau de Doutor em Bioengenharia

Novel approaches for culturing hepatocytes for drug testing applications

Dissertation presented to obtain the Ph.D degree in Biochemisry, Biotechnology

Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes

Clin Sci (Lond). 2002 Nov;103(5):475-85

Development of 3D in vitro models for prediction of hepatic metabolism and toxicity

The development of human cell models that recapitulate hepatic functionality allows the study of metabolic pathways involved in toxicity and disease. The increased biological relevance, cost-effectiveness and high-throughput of cell models can contribute to increase the efficiency of drug development in the pharmaceutical industry. Recapitulation of liver functionality in vitro requires the development of advanced culture strategies to mimic in vivo complexity, such as 3D culture, co-cultures or biomaterials. However, complex 3D models are typically associated with poor robustness, limited scalability and compatibility with screening methods. In this work, several strategies were used to develop highly functional and reproducible spheroid-based in vitro models of human hepatocytes and HepaRG cells using stirred culture systems. In chapter 2, the isolation of human hepatocytes from resected liver tissue was implemented and a liver tissue perfusion method was optimized towards the improvement of hepatocyte isolation and aggregation efficiency, resulting in an isolation protocol compatible with 3D culture. In chapter 3, human hepatocytes were co-cultivated with mesenchymal stem cells (MSC) and the phenotype of both cell types was characterized, showing that MSC acquire a supportive stromal function and hepatocytes retain differentiated hepatic functions, stability of drug metabolism enzymes and higher viability in co-cultures. In chapter 4, a 3D alginate microencapsulation strategy for the differentiation of HepaRG cells was evaluated and compared with the standard 2D DMSO-dependent differentiation, yielding higher differentiation efficiency, comparable levels of drug metabolism activity and significantly improved biosynthetic activity. The work developed in this thesis provides novel strategies for 3D culture of human hepatic cell models, which are reproducible, scalable and compatible with screening platforms. The phenotypic and functional characterization of the in vitro systems performed contributes to the state of the art of human hepatic cell models and can be applied to the improvement of pre-clinical drug development efficiency of the process, model disease and ultimately, development of cell-based therapeutic strategies for liver failure.