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

Economic analysis in health care regulation : three essays

ABSTRACT - It is the purpose of the present thesis to emphasize, through a series of examples, the need and value of appropriate pre-analysis of the impact of health care regulation. Specifically, the thesis presents three papers on the theme of regulation in different aspects of health care provision and financing. The first two consist of economic analyses of the impact of health care regulation and the third comprises the creation of an instrument for supporting economic analysis of health care regulation, namely in the field of evaluation of health care programs. The first paper develops a model of health plan competition and pricing in order to understand the dynamics of health plan entry and exit in the presence of switching costs and alternative health premium payment systems. We build an explicit model of death spirals, in which profitmaximizing competing health plans find it optimal to adopt a pattern of increasing relative prices culminating in health plan exit. We find the steady-state numerical solution for the price sequence and the plan’s optimal length of life through simulation and do some comparative statics. This allows us to show that using risk adjusted premiums and imposing price floors are effective at reducing death spirals and switching costs, while having employees pay a fixed share of the premium enhances death spirals and increases switching costs. Price regulation of pharmaceuticals is one of the cost control measures adopted by the Portuguese government, as in many European countries. When such regulation decreases the products’ real price over time, it may create an incentive for product turnover. Using panel data for the period of 1997 through 2003 on drug packages sold in Portuguese pharmacies, the second paper addresses the question of whether price control policies create an incentive for product withdrawal. Our work builds the product survival literature by accounting for unobservable product characteristics and heterogeneity among consumers when constructing quality, price control and competition indexes. These indexes are then used as covariates in a Cox proportional hazard model. We find that, indeed, price control measures increase the probability of exit, and that such effect is not verified in OTC market where no such price regulation measures exist. We also find quality to have a significant positive impact on product survival. In the third paper, we develop a microsimulation discrete events model (MSDEM) for costeffectiveness analysis of Human Immunodeficiency Virus treatment, simulating individual paths from antiretroviral therapy (ART) initiation to death. Four driving forces determine the course of events: CD4+ cell count, viral load resistance and adherence. A novel feature of the model with respect to the previous MSDEMs is that distributions of time to event depend on individuals’ characteristics and past history. Time to event was modeled using parametric survival analysis. Events modeled include: viral suppression, regimen switch due virological failure, regimen switch due to other reasons, resistance development, hospitalization, AIDS events, and death. Disease progression is structured according to therapy lines and the model is parameterized with cohort Portuguese observational data. An application of the model is presented comparing the cost-effectiveness ART initiation with two nucleoside analogue reverse transcriptase inhibitors (NRTI) plus one non-nucleoside reverse transcriptase inhibitor(NNRTI) to two NRTI plus boosted protease inhibitor (PI/r) in HIV- 1 infected individuals. We find 2NRTI+NNRTI to be a dominant strategy. Results predicted by the model reproduce those of the data used for parameterization and are in line with those published in the literature.

Regulation and disclosure about financial instruments: Evidence on financial reporting in the Portuguese banking sector

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics

Basel III: The impact of an increase in regulation in a recession framework

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics

EDP - Energias de Portugal: Electric utilities: Equity Investment Report

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics

EDP - Energias de Portugal: Electric utilities: Equity Investment Report

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics

The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus

Dissertation for the Master’s Degree in Structural and Functional Biochemistry

The tight grip of a debt based global economy: A study on banking regulation and the implementation of the Basel II Accord in Portugal

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Regulation of the rice gene OsRMC under salt stress: identification and functional characterization of novel transcription factors

Dissertation presented to obtain the Ph.D degree in Biology

New roles of Rab GTPases on eye diseases: targeting VEGF secretion

RESUMO: A retina é composta, entre outras estruturas, pelo epitélio pigmentar da retina (EPR)e pela coróide. A região central da retina denomina-se mácula, e é a zona mais afetada na degenerescência macular relacionada com a idade, a forma mais comum de degenerescência da retina. Nesta doença, a secreção de fatores de crescimento pelo EPR é afetada, nomeadamente a do fator de crescimento vascular endotelial (VEGF), e pouco se sabe ainda sobre os mecanismos moleculares conducentes a esta condição. A família de proteínas Rab GTPases está envolvida nas vias intracelulares de sinalização e tráfego membranares, essenciais na transdução de sinais extracelulares em respostas biológicas. A sua crucial importância nestes mecanismos levou-nos a considerar o seu potencial envolvimento nas vias de secreção do VEGF, e a questionar-nos se teriam algum papel regulador sobre as mesmas. O principal objetivo deste trabalho é identificar Rab GTPases importantes para as vias de secreção e endocitose do VEGF no EPR. Essa identificação ajudará a esclarecer a patogénese da degenerescência macular da retina, e poderá servir para uma procura mais direcionada de novos agentes terapêuticos. A caracterização de dois modelos in vitro do EPR, células primárias isoladas de murganho e a linha celular B6-RPE07,levou-nos a concluir que são ambos semelhantes. Contudo, a linha celular foi escolhida como protótipo do EPR por permitir o acesso a um número ilimitado de células. No decurso deste trabalho, desenvolvemos e caracterizámos uma biblioteca de ferramentas moleculares que nos permitiram reduzir os níveis proteicos das proteínas Rab GTPases, com base na tecnologia de ácido ribonucleico (ARN) de interferência. O papel das proteínas Rab GTPases na secreção do VEGF no EPR foi estudado com base no silenciamento de apenas uma proteína, ou combinando várias, segundo a sua localização e funções intracelulares descritas. Este trabalho permitiu-nos concluir que as proteínas Rab GTPases são importantes intervenientes no processo de secreção de VEGF pelo EPR, e confirmar dados anteriores que relatam o envolvimento de algumas Rab GTPases endocíticas no processo. Propomos ainda um novo modelo para a interação destas proteínas no EPR, e sugerimos que a Rab10 e a Rab14 atuam negativamente sobre a Rab8, controlando o seu funcionamento. Os nossos resultados evidenciam a importância das proteínas Rab GTPases na secreção do VEGF pelas células do EPR, e servem de base a futuros estudos que melhor procurem compreender este mecanismo e de que modo a sua alteração se relaciona com a degenerescência da retina.--------ABSTRACT: Retinal pigment epithelium (RPE) and choroid are components of the mammalian retina, of which the central region is called macula. The most common form of retinaldegeneration, age-related macular degeneration (AMD), involves primarily deregulation of growth factors secretion by the RPE. Very little is known about the molecular mechanisms that lead to impairment of RPE’s homeostatic intracellular processes, namely the secretion of vascular endothelial growth factor (VEGF). Rab GTPases’ family regulates membrane targeting and traffic, being essential in the transduction of signal pathways. Given Rab proteins’ role in intracellular trafficking, we propose to identify key regulatory Rab proteins involved in either the secretory or the recycling pathways of VEGF in RPE. Understanding how Rab proteins’ function disruption could lead to retinal and choroidal pathology would ultimately contribute to find new therapeutic agents. Here, we characterized two mouse RPE in vitro cell models, primary cells and B6-RPE07 cell line, and concluded that both display important epithelial features as the RPE presents in vivo. Considering unlimited cell number and results reproducibility, we chose B6-RPE07 cells to further study Rab proteins’ function. To scrutinize the consequences of Rab proteins’ absence or diminished levels, we have developed novel molecular tools to achieve silencing of these key proteins using miRNA technology. We further addressed the effect of Rab proteins’ absence on VEGF secretion by performing an extensive screening where different Rab proteins were silenced, both individually and in multiple combinations considering their cellular/ compartment location. We conclude that Rab GTPases are important intervenients in VEGF secretion by RPE cells, confirming endocytic Rab proteins’ role in regulation of VEGF biology. We also propose a novel model for Rab proteins’ interaction in RPE. Our results suggest that Rab10 and Rab14 might influence Rab8 in a negative feedback mechanism, important for controlling VEGF secretion. Our achievements’ unravel Rab proteins’ role in VEGF secretion by RPE cells and are the basis for future studies to better understand RPE molecular secretory machinery.

Centriole elimination in Drosophila melanogaster oogenesis

Dissertation presented to obtain the Ph.D degree in Cellular Biology

The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium

Dissertation presented to obtain the Ph.D degree in Biology

Investigation of the regulation mechanisms for bioplastics production from industrial residues

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Disclosing Carbon Monoxide Protection in Cerebral Ischemia: Insights into the cellular mechanisms

Dissertation presented to obtain the PhD degree in Biochemistry, Neurosciences

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology