Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

Enrichment of a PHA producing microbial community in a continuous bioreactor setup

Polyhydroxyalkanoates (PHAs) are biosynthetic polyesters, biodegradable and biocompatible making them of great interest for industrial purposes. The use of low value substrates with mixed microbial communities (MMC) is a strategy currently used to decrease the elevated PHA production costs. PHA production process requires an important step for selection and enrichment of PHA-storing microorganisms which is usually carried out in a Sequencing Batch Reactor (SBR). The aim of this study was to optimize the PHA accumulating culture selection stage using a 2-stage Continuous Stirrer Tank Reactor (CSTR) system. The system was composed by two separate feast and famine bioreactors operated continuously, mimicking the feast and famine phases in a SBR system. Acetate was used as carbon source and biomass seed was highly enriched in Plasticicumulans acidivorans obtained from activated sludge. The system was operated under two different sets of conditions (setup 1 and 2), maintaining a system total retention time of 12 hours and an OLR of 2.25 Cmmol/L.h-1. An average PHB-content of 3.3 % wt was obtained in setup 1 and 4.8% wt in setup 2. Several other experiments were performed in order to better understand the continuous system behaviour, using biomass from the continuous system. With the fed-batch experiment a maximum of 8.1% PHB was stored and the maximum substrate uptake and specific growth rates obtained in the growth experiment (1.15 Cmol Cmol-1.h-1 and 0.53 Cmol Cmol-1.h-1) were close to the ones from continuous system (1.12 Cmol Cmol-1.h-1 and 0.59 Cmol Cmol-1.h-1). The microbial community was characterized trough microscopic visualization, Denaturing Gradient Gel Electrophoresis (DGGE) analysis and Fluorescent in situ hybridization (FISH). The last studied performed mimicked the continuous system by building up a SBR system with all the same operational conditions while adding an extra acetate dosage during the 12 h cycle, simulating the substrate passing from the feast to the famine reactors under continuous operation. It was shown that possibly the continuous system was not able to efficiently select for PHB storing organisms under the operational conditions imposed, although the selected culture was capable of consuming the substrate and grow fast. This main conclusion might have resulted from two major factors affecting the system performance: the ammonium concentration in the Feast reactor and the amount of substrate leaching from the Feast to the Famine reactor.

Transport mechanism in ionic liquid membranes and the study of thiomersal biodegradation

The initial goal of this work was the development of a supported liquid membrane (SLM) bioreactor for the remediation of vaccine production effluents contaminated with a highly toxic organomercurial – thiomersal. Therefore, two main aspects were focused on: 1) the development of a stable supported liquid membrane – using room temperature ionic liquids (RTILs) – for the selective transport of thiomersal from the wastewater to a biological compartment, 2) study of the biodegradation kinetics of thiomersal to metallic mercury by a Pseudomonas putida strain. The first part of the work focused on the evaluation of the physicochemical properties of ionic liquids and on the SLMs’ operational stability. The results obtained showed that, although it is possible to obtain a SLM with a high stability, water possesses nonnegligible solubility in the RTILs studied. The formation of water clusters inside the hydrophobic ionic liquid was identified and found to regulate the transport of water and small ions. In practical terms, this meant that, although it was possible to transport thiomersal from the vaccine effluent to the biological compartment, complete isolation of the microbial culture could not be guaranteed and the membrane might ultimately be permeable to other species present in the aqueous vaccine wastewater. It was therefore decided not to operate the initially targeted integrated system but, instead, the biological system by itself. Additionally, attention was given to the development of a thorough understanding of the transport mechanisms involved in the solubilisation and transport of water through supported liquid membranes with RTILs as well as to the evaluation of the effect of water uptake by the SLM in the transport mechanisms of water-soluble solutes and its effect on SLM performance. The results obtained highlighted the determinant role played by water – solubilised inside the ionic liquids – on the transport mechanism. It became clear that the transport mechanism of water and water-soluble solutes through SLMs with [CnMIM][PF6] RTILs was regulated by the dynamics of water clusters inside the RTIL, rather than by molecular diffusion through the bulk of the ionic liquid. Although the stability tests vi performed showed that there were no significant losses of organic phase from the membrane pores, the formation of water clusters inside the ionic liquid, which constitute new, non-selective environments for solute transport, leads to a clear deterioration of SLM performance and selectivity. Nevertheless, electrical impedance spectroscopy characterisation of the SLMs showed that the formation of water clusters did not seem to have a detrimental effect on the SLMs’ electrical characteristics and highlighted the potential of using this type of membranes in electrochemical applications with low resistance requirements. The second part of the work studied the kinetics of thiomersal degradation by a pure culture of P. putida spi3 strain, in batch culture and using a synthe tic wastewater. A continuous ly stirred tank reactor fed with the synthetic wastewater was also operated and the bioreactor’s performance and robustness, when exposed to thiomersal shock loads, were evaluated. Finally, a bioreactor for the biological treatment of a real va ccine production effluent was set up and operated at different dilution rates. Thus it was possible to treat a real thiomersal-contaminated effluent, lowering the outlet mercury concentration to values below the European limit for mercury effluent discharges.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Modelling and optimization of the ion exchange membrane bioreactor for removal of anionic pollutants from drinking water streams

Dissertação para obtenção do Grau de Doutor em Engenharia Química, especialidade de Engenharia Bioquímica

Fate of nitrogen in bioreactor landfills: lab-scale in situ aeration in well decomposed MSW

Dissertação para obtenção do Grau de Mestre em Engenharia do ambiente, perfil de engenharia sanitária

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.

Development of biocompatible and “smart” porous structures using CO2-assisted processes

Dissertação apresentada para a obtenção do grau de Doutor em Engenharia Química, especialidade Engenharia da Reacção Química, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

The influence of sludge characteristics from fullscale MBR in membrane filterability

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para a obtenção do grau de Mestre em Engenharia do Ambiente,perfil Sanitária

Novas linhas celulares humanas para produção de adenovírus com aplicação em terapia génica

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

Estudo do desenvolvimento e da produtividade lipídica da levedura Rhodotorula glutinis NCYC 921 em culturas descontínuas monitorizadas por citometria de fluxo

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Process engineering of liver cells for drug testing applications

Dissertação para obtenção do Grau de Doutor em Bioengenharia

Development of novel human cellular models for neurotoxicity studies

Dissertation to obtain master degree in Genética Molecular e Biomedicina

A systems biology framework for pathway level culture media engineering: pplication to Pichia pastoris cultures

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Food waste valorization through the production of polyhydroxyalkanoates by mixed microbial cultures

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica