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.

Risk of employing an evolvable production system

Nowadays manufacturing companies are facing a more challenging environment due to the unpredictability of the markets in order to survive. Enterprises need to keep innovating and deliver products with new internal or external characteristics. There are strategies and solutions, to different organisational level from strategic to operational, when technology is growing faster in operational level, more specifically in manufacturing system. This means that companies have to deal with the changes of the emergent manufacturing systems while it can be expensive and not easy to be implement. An agile manufacturing system can help to cope with the markets changeability. Evolvable Production Systems (EPS) is an emergent paradigm which aims to bring new solutions to deal with changeability. The proposed paradigm is characterised by modularity and intends to introduce high flexibility and dynamism at shop floor level through the use of the evolution of new computational devices and technology. This new approach brings to enterprises the ability to plug and unplug new devices and allowing fast reformulation of the production line without reprogramming. There is no doubt about the advantages and benefits of this emerging technology but the feasibility and applicability is still under questioned. Most researches in this area are focused on technical side, explaining the advantages of those systems while there are no sufficient works discussing the implementation risks from different perspective, including business owner. The main objective of this work is to propose a methodology and model to identify, classify and measure potential risk associated with an implementation of this emergent paradigm. To quantify the proposed comprehensive risk model, an Intelligent Decision system is developed employing Fuzzy Inference System to deal with the knowledge of experts, as there are no historical data and sufficient research on this area. The result can be the vulnerability assessment of implementing EPS technology in manufacturing companies when the focus is more on SMEs. The present dissertation used the experts’ knowledge and experiences, who were involved in FP7 project IDEAS, which is one of the leading projects in this area.

An agent based architecture to support monitoring in plug and produce manufacturing systems using knowledge extraction

In recent years a set of production paradigms were proposed in order to capacitate manufacturers to meet the new market requirements, such as the shift in demand for highly customized products resulting in a shorter product life cycle, rather than the traditional mass production standardized consumables. These new paradigms advocate solutions capable of facing these requirements, empowering manufacturing systems with a high capacity to adapt along with elevated flexibility and robustness in order to deal with disturbances, like unexpected orders or malfunctions. Evolvable Production Systems propose a solution based on the usage of modularity and self-organization with a fine granularity level, supporting pluggability and in this way allowing companies to add and/or remove components during execution without any extra re-programming effort. However, current monitoring software was not designed to fully support these characteristics, being commonly based on centralized SCADA systems, incapable of re-adapting during execution to the unexpected plugging/unplugging of devices nor changes in the entire system’s topology. Considering these aspects, the work developed for this thesis encompasses a fully distributed agent-based architecture, capable of performing knowledge extraction at different levels of abstraction without sacrificing the capacity to add and/or remove monitoring entities, responsible for data extraction and analysis, during runtime.

Relatório de estágio no departamento central de investigação e acção penal

The means of obtaining evidence, the amount of evidence obtained, the number of defendants related to each criminal case and the gravity of the crimes for which the magistrates of the Department are holders of penal action, define its real importance to the Rule of Law. I have deeply studied the subject of the institution of hierarchical intervention required by the assistant and the application of an opening statement by the defendant, starting from a hypothetical case, provided when the query of an investigation with the subject of the crime of active corruption, where this institution was called as a reaction to the archiving dispatch delivered by the Public Ministry. I have study about the implementation of the institution of provisional suspension of the process, specifically in the scope of fiscal criminality, analyzing the effective satisfaction of the purposes of the sentences in two slopes: general prevention and special prevention. I went for my first time to a Central Court of Criminal Instruction, where I attended the measures of inquiry and instructive debate of a process that culminated with the prosecution and pronunciation of the defendants. In addition to this criminal experience, I have deepened and consolidated the academic knowledge with the study of various criminal cases from various fields in the scope of criminality investigated by the Department. I could therefore check the basis of procedural delays, regarding to our legal system, especially in this type of crime, raising issues that I analyzed and discussed, always in a critical and academic way. I had the opportunity to attend and witness a seminar in the Lisbon Directorate of Finance as well of entering the Centre for Judicial Studies to attend a conference on the International Anti-Corruption Day. Focus on the investigatory importance of the international judicial cooperation, through the various organs, with special interest to EUROJUST. I comprehended the organization and functioning of these communitarian organs and means of communication of procedural acts, in particular, the rogatory letters and european arrest warrants. This involvement is motivated by the moratorium factor of the investigations where rogatory letters are necessary for the acquisition of evidence or information relevant to the good continuation of the process. For this reason the judicial cooperation through the relevant communitarian organs, translates a streamlined response between the competent judicial authorities of the Member States, through the National Member that integrates EUROJUST. This report aims to highlight some of the difficulties and procedural issues that Public Prosecutors of DCIAP and criminal police bodies that assist them, face in combating violent and organized crime, of national and transnational nature, of particular complexity, according to the specifics of criminal types.

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.

Towards a knowledge society

Economies are moving towards competitive stadiums based on knowledge and innovation. The changing environment and the level of globalization demand important efforts in order to sustain competitive advantages. Portugal has experienced a remarkable evolution since its adhesion to the European Union in several fields: economic development, Research & Development (R&D) consolidation, health parameters and social cohesion. As other developed economies, Portugal started its journey towards a knowledge-based economy and has been consolidating an innovation system during the last 35 years. The following report aims to analyze the evolution of a system since its creation to its last transformation within a globalized context. Challenges such as the lack of maturity of the system, the economic crisis, the European paradox, and closing the gap with other European countries are addressed in the next chapters. Likewise, recommendations on these points are provided by the end of the report as potential solutions.