Insights into cell wall synthesis and cell division in Staphylococcus aureus

Dissertation presented to obtain the Ph.D degree in Biology

Directivas antecipadas de vida : modelo de aferição da sua aceitabilidade nas unidades de saúde em Portugal

RESUMO - As Directivas Antecipadas de Vida, são instruções escritas ou orais formuladas por uma pessoa competente relativamente à prestação ou suspensão de cuidados médicos numa eventual situação de doença geradora de incapacidade para decidir ou expressar a sua vontade (Neves et al., 2010). No presente estudo, o problema de investigação centra-se em saber de que forma a construção de um modelo de Directivas Antecipadas de Vida pode contribuir para uma melhor gestão nas unidades de saúde? Para a realização do presente projecto de investigação, foi efectuada uma pesquisa bibliográfica sobre os principais conceitos e estado actual do conhecimento, os quais contribuíram para a definição dos objectivos de investigação empírica:  Contribuir para a criação de um corpo de conhecimento no que diz respeito às Directivas Antecipadas de Vida com a elaboração de uma proposta de um modelo de aferição da sua aceitabilidade nas unidades de saúde em Portugal.  Identificar os diferentes intervenientes, e qual o seu papel na implementação do modelo proposto. No que se refere ao estudo das Directivas Antecipadas de Vida em Portugal, e em especial nas unidades de saúde, recorreu-se a uma metodologia exploratória e descritiva, para identificar as principais características e trabalhos desenvolvidos na área em investigação. A primeira conclusão reside nas características da própria sociedade, a qual não parece ainda estar suficientemente desperta para a problemática em estudo, não obstante se verificar a existência de iniciativas legislativas recentemente apresentadas. Esta constatação verifica-se igualmente ao nível das unidades prestadoras de cuidados de saúde.

Medicago truncatula as a platform for Molecular Farming: a study of the subcellular localization of a human recombinant protein!

Dissertation presented at Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa to obtain the Degree of Master in Biotecnology

Real-time labview assessment on adsorption systems

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

Smart windows with permanent memory effect

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

Role of surfactants in filtration and fouling of colloidal silica

The EM3E Master is an Education Programme supported by the European Commission, the European Membrane Society (EMS), the European Membrane House (EMH), and a large international network of industrial companies, research centres and universities

Improved polymeric medical devices for active substances delivery

The work presented in this thesis was developed in collaboration with a Portuguese company, BeyonDevices, devoted to pharmaceutical packaging, medical technology and device industry. Specifically, the composition impact and surface modification of two polymeric medical devices from the company were studied: inhalers and vaginal applicators. The polyethylene-based vaginal applicator was modified using supercritical fluid technology to acquire self-cleaning properties and prevent the transport of bacteria and yeasts to vaginal flora. For that, in-situ polymerization of 2-substituted oxazolines was performed within the polyethylene matrix using supercritical carbon dioxide. The cationic ring-opening polymerization process was followed by end-capping with N,N-dimethyldodecylamine. Furthermore, for the same propose, the polyethylene matrix was impregnated with lavender oil in supercritical medium. The obtained materials were characterized physical and morphologically and the antimicrobial activity against bacteria and yeasts was accessed. Materials modified using 2-substituted oxazolines showed an effective killing ability for all the tested microorganisms, while the materials modified with lavender oil did not show antimicrobial activity. Only materials modified with oligo(2-ethyl-2-oxazoline) maintain the activity during the long term stability. Furthermore, the cytotoxicity of the materials was tested, confirming their biocompatibilty. Regarding the inhaler, its surface was modified in order to improve powder flowability and consequently, to reduce powder retention in the inhaler´s nozzle. New dry powder inhalers (DPIs), with different needle’s diameters, were evaluated in terms of internal resistance and uniformity of the emitted dose. It was observed that they present a mean resistance of 0.06 cmH2O0.5/(L/min) and the maximum emitted dose obtained was 68.9% for the inhaler with higher needle´s diameter (2 mm). Thus, this inhaler was used as a test and modified by the coating with a commonly-used force control agent, magnesium stearate, dried with supercritical carbon dioxide (scCO2) and the uniformity of delivered dose tests were repeated. The modified inhaler showed an increase in emitted dose from 68.9% to 71.3% for lactose and from 30.0% to 33.7% for Foradil.

Green synthesis of 2-Oxazoline-based polymers with antimicrobial activity using scCO2

Using a green methodology, 17 different poly(2-oxazolines) were synthesized starting from four different oxazoline monomers. The polymerization reactions were conducted in supercritical carbon dioxide under a cationic ring-opening polymerization (CROP) mechanism using boron trifluoride diethyl etherate as the catalyst. The obtained living polymers were then end-capped with different types of amines, in order to confer them antimicrobial activity. For comparison, four polyoxazolines were end-capped with water, and by their hydrolysis the linear poly(ethyleneimine) (LPEI) was also produced. After functionalization the obtained polymers were isolated, purified and characterized by standard techniques (FT-IR, NMR, MALDI-TOF and GPC). The synthesized poly(2-oxazolines) revealed an unusual intrinsic blue photoluminescence. High concentration of carbonyl groups in the polymer backbone is appointed as a key structural factor for the presence of fluorescence and enlarges polyoxazolines’ potential applications. Microbiological assays were also performed in order to evaluate their antimicrobial profile against gram-positive Staphylococcus aureus NCTC8325-4 and gram-negative Escherichia coli AB1157 strains, two well known and difficult to control pathogens. The minimum inhibitory concentrations (MIC)s and killing rates of three synthesized polymers against both strains were determined. The end-capping with N,N-dimethyldodecylamine of living poly(2- methyl-2-oxazoline) and poly(bisoxazoline) led to materials with higher MIC values but fast killing rates (less than 5 minutes to achieve 100% killing for both bacterial species) than LPEI, a polymer which had a lower MIC value, but took a longer time to kill both E.coli and S.aureus cells. LPEI achieved 100% killing after 45 minutes in contact with E. coli and after 4 hours in contact with S.aureus. Such huge differences in the biocidal behavior of the different polymers can possibly underlie different mechanisms of action. In the future, studies to elucidate the obtained data will be performed to better understand the killing mechanisms of the polymers through the use of microbial cell biology techniques.

Aqueous biphasic system based on cholinium ionic liquids: extraction of biologically active phenolic acids

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Due to their biological and pharmacological properties, they have been playing an important role in phytotherapy and consequently techniques for their separation and purification are in need. This thesis aims at exploring new sustainable separation processes based on ionic liquids (ILs) in the extraction of biologically active phenolic acids. For that purpose, three phenolic acids with similar chemical structures were selected: cinnamic acid, p-coumaric acid and caffeic acid. In the last years, it has been shown that ionic liquids-based aqueous biphasic systems (ABSs) are valid alternatives for the extraction, recovery and purification of biomolecules when compared to conventional ABS or extractions carried out with organic solvents. In particular, cholinium-based ILs represent a clear step towards a greener chemistry, while providing means for the implementation of efficient techniques for the separation and purification of biomolecules. In this work, ABSs were implemented using cholinium carboxylate ILs using either high charge density inorganic salt (K3PO4) or polyethylene glycol (PEG) to promote the phase separation of aqueous solutions containing three different phenolic acids. These systems allow for the evaluation of effect of chemical structure of the anion on the extraction efficiency. Only one imidazolium-based IL was used in order to establish the effect of the cation chemical structure. The selective extraction of one single acid was also researched. Overall, it was observed that phenolic acids display very complex behaviours in aqueous solutions, from dimerization to polymerization and also hetero-association are quite frequent phenomena, depending on the pH conditions. These phenomena greatly hinder the correct quantification of these acids in solution.

A harmonização fiscal europeia

With this dissertation we aim to analyze the most relevant aspects of the excise duties harmonized regime, considering Community origins, but having also in attention all legal specifications of its implementation in Portugal. The legal regime of excise duties is presented as an ambitious theme, considering the challenge of different branches of law that influence this subject, such as Tax, Economic and Community laws, the inescapable influence of customs procedures, or regarding environmental objectives. In the European context, the harmonization of excise duties was seen as a condition for the implementation of the internal market, contributing to undo secular tax barriers between Member States and, since so, ensure fair competition and free movement of services and goods. Along with VAT, the excise duties harmonization process could represent a potential European tax system, essential for a full and integrated single market. In this context, it is essential to pay special attention to specific characteristics of excise duties regime, such as ‘duty suspension arrangement’ applicable during the production phase, storage and movement in certain conditions. The growing importance of excise duties, as for revenue or extra-fiscal purposes, recommends new academic studies on this subject, seeking new opportunities and challenges.

Contratos de prestação de serviços de comunicações electrónicas

The object of this dissertation is the analysis of the legal framework applicable to contracts for provision of electronic communications services, while trying to offer solutions to some of the issues regarding this matter. The main focus of this study will be the rules concerning service’s suspension, which have been recently amended. The technological development and the establishment of these services as information transmitters and work tools were noteworthy for its growing importance at the present time. These services include cable television, telephone (landline and mobile) and internet and they are regulated by Law nr 23/96, July 26th, along with other essential public services. Said law sets a group of principles and duties, such as good faith (article 3), continuity and quality of the service (article 7) and the duty to rightfully inform the user (article 4), in order to protect the users. For the analysis of legal framework applicable to these particular contracts it is also fundamental to mention Law nr 5/2004, February 10th, known as Electronic Communications Law. The provisions regarding the service’s suspension are currently prescribed in articles 52.º and 52.º-A of the law. Given the amendments introduced by Law nr 10/2013, January 28th, consumers are subjected to a regulation different from the one applicable to the other users, established in the new article 52.º-A. From our analysis, we have concluded that the main change from past provisions has to do with the automatic termination of the contract as consequence of the consumer’s failure to pay the price or to conclude a written payment arrangement after service’s suspension.

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.

O contrato de prestação de serviços de fornecimento de água

The theme of thid thesis is the water supply services contract. The texto starts with an analysis of the service, aimed at clarifying what is the system organization and the principles applicable to this essential public service. Then the water supply services contract is analyzed according to the law on essential public services. Subsequently, connections are established with other applicable laws and the differing doctrinal and jurisprudential perspectives are presented. The thesis ends with an outlook on the dispute resolution mechanisms at the users’ disposal.

Production and characterization of electrospun composite fibers: confinement of thermosensitive microgels

Materials engineering focuses on the assembly of materials´ properties to design new products with the best performance. By using sub-micrometer size materials in the production of composites, it is possible to obtain objects with properties that none of their compounds show individually. Once three-dimensional materials can be easily customized to obtain desired properties, much interest has been paid to nanostructured poly-mers in order to build biocompatible devices. Over the past years, the thermosensitive microgels have become more common in the framework of bio-materials with potential applicability in therapy and/or diagnostics. In addition, high aspect ratio biopolymers fibers have been produced using the cost-effective method called electrospinning. Taking advantage of both microgels and electrospun fibers, surfaces with enhanced functionalities can be obtained and, therefore employed in a wide range of applications. This dissertation reports on the confinement of stimuli-responsive microgels through the colloidal electro-spinning process. The process mainly depends on the composition, properties and patterning of the precur-sor materials within the polymer jet. Microgels as well as the electrospun non-woven mats were investigated to correlate the starting materials with the final morphology of the composite fibers. PNIPAAm and PNIPAAm/Chitosan thermosensitive microgels with different compositions were obtained via surfactant free emulsion polymerization (SFEP) and characterized in terms of chemical structure, morphology, thermal sta-bility, swelling properties and thermosensitivity. Finally, the colloidal electrospinning method was carried out from spinning solutions composed of the stable microgel dispersions (up to a concentration of about 35 wt. % microgels) and a polymer solution of PEO/water/ethanol mixture acting as fiber template solution. The confinement of microgels was confirmed by Scanning Electron Microscopy (SEM). The electrospinning process was statistically analysed providing the optimum set of parameters aimed to minimize the fiber diameter, which give rise to electrospun nanofibers of PNIPAAm microgels/PEO with a mean fiber diameter of 63 ± 25 nm.

As conversas informais na reconstituição

The theme of thid thesis is the water supply services contract. The texto starts with an analysis of the service, aimed at clarifying what is the system organization and the principles applicable to this essential public service. Then the water supply services contract is analyzed according to the law on essential public services. Subsequently, connections are established with other applicable laws and the differing doctrinal and jurisprudential perspectives are presented. The thesis ends with an outlook on the dispute resolution mechanisms at the users’ disposal.