Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

The dilution effect: The influence of expertise and abstraction on consumer's judgements of products

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

Collateral, default penalties and infinite horizon equilibrium

Pascoa and Seghir (2009) noticed that when collateralized promises become subject to utility penalties on default, Ponzi schemes may occur. However, equilibrium exists in some interesting cases. Under low penalties, equilibrium exists if the collateral does not yield utility (for example, when it is a productive asset or a security). Equilibrium exists also under more severe penalties and collateral utility gains, when the promise or the collateral are nominal assets and the margin requirements are endogenous: relative inflation rates and margin coefficients can make the income effects dominate the penalty effects. An equilibrium refinement avoids no-trade equilibria with unduly repayment beliefs. Our refinement differs from the one used by Dubey, Geanakoplos and Shubik (2005) as it does not eliminate no trade equilibria whose low delivery rates are consistent with the propensity to default of agents that are on the verge of selling.

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.

A coherent approach to non-integer order derivatives

Signal Processing, vol. 86, nº 10

Critical Velocity obtained using Simplified Models of the Railway Track: Viability and Applicability

Increased demands on the capacity of the railway network gave rise to new issues related to the dynamic response of railway tracks subjected to moving vehicles. Thus, it becomes important to evaluate the applicability of traditionally used simplified models which have a closed form solution. Regarding simplified models, transversal vibrations of a beam on a visco-elastic foundation subjected to a moving load are considered. Governing equations are obtained by Hamilton’s principle. Shear distortion, rotary inertia and effect of axial force are accounted for. The load is introduced as a time varying force moving at a constant velocity. Transversal vibrations induced by the load are solved by the normal-mode analysis. Reflected waves at the extremities of the full beam are avoided by introduction of semi-infinite elements. Firstly, the critical velocity obtained from this model is compared with results of an undamped Euler- Bernoulli formulation with zero axial force. Secondly, a finite element model in ABAQUS is examined. The new contribution lies in the introduction of semi- infinite elements and in the first step to a systematic comparison, which have not been published so fa

Transverse Vibrations in Beams Supported by a Piece-Wise Homogeneous Visco- Elastic Foundation

Transversal vibrations induced by a load moving uniformly along an infinite beam resting on a piece-wise homogeneous visco-elastic foundation are studied. Special attention is paid to the additional vibrations, conventionally referred to as transition radiations, which arise as the point load traverses the place of foundation discontinuity. The governing equations of the problem are solved by the normalmode analysis. The solution is expressed in a form of infinite sum of orthogonal natural modes multiplied by the generalized coordinate of displacement. The natural frequencies are obtained numerically exploiting the concept of the global dynamic stiffness matrix. This ensures that the frequencies obtained are exact. The methodology has restrictions neither on velocity nor on damping. The approach looks simple, though, the numerical expression of the results is not straightforward. A general procedure for numerical implementation is presented and verified. To illustrate the utility of the methodology parametric optimization is presented and influence of the load mass is studied. The results obtained have direct application in analysis of railway track vibrations induced by high-speed trains when passing regions with significantly different foundation stiffness.

La fin (l’infini) du bruit

Á... Á propos d’Erosphère propos d’Érosphère, dans un entretien pour la Revue LIEN2 en 1992, François Bayle explique que le projet d’une Suite fut déclenché par la composition de la 1ère œuvre du cycle – Tremblement de terre très doux. Il y indique : « je me suis petit à petit aperçu, en réalisant cette pièce, que j’y traitais d’un problème inhérent à l’acous- matique : celui du désir, [...] c’est-à-dire des sons appétissants, des sons voluptueux, ce qui m’a entraîné vers un projet plus vaste que j’ai appelé Érosphère, cette loi de gravitation du désir. »3 Plus tard le compositeur déclare : « Au moment d’Érosphère mon projet portait sur les mécanismes du désir, l’érotisme sonore, d’où le titre d’Éros- phère. Découvrir les archétypes érotiques des formes sonores. »4 Dans les notes qui accompagnaient la première édition en disque, Bayle présente sa définition du mot Érosphère : Ce tissu nerveux qui enveloppe notre monde habité d’un réseau d’ondes modulées à une infinité de fréquences, ce nuage de chaleur infra et supra sensorielle que diffusent des mégas milliards d’ émetteurs biologiques, cet anneau où circule la force de ce cosmos, je l’appelle l’érosphère.

Framework development for providing accessibility to qualitative spatial calculi

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Mobile persuasive interfaces for public ambient displays

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

Strategic Noise Mapping with GIS for the Universitat Jaume I Smart Campus: best methodology practices

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Study of nuclear reactions relevant for astrophysics by Micro-AMS

Dissertação para obtenção do Grau de Doutor em Física

Development of novel active pharmaceutical ionic liquids and salts based on antibiotics and anti-fungal drugs

Ionic Liquids (ILs) are class of compounds, which have become popular since the mid-1990s. Despite the fact that ILs are defined by one physical property (melting point), many of the potential applications are now related to their biological properties. The use of a drug as a liquid can avoid some problems related to polymorphism which can influence a drug´s solubility and thus its dosages. Also, the arrangement of the anion or cation with a specific drug might be relevant in order to: a) change the correspondent biopharmaceutical drug classification system; b) for the drug formulation process and c) the change the Active Pharmaceutical Ingredients’ (APIs). The main goal of this Thesis is the synthesis and study of physicochemical and biological properties of ILs as APIs from beta-lactam antibiotics (ampicillin, penicillin G and amoxicillin) and from the anti-fungal Amphotericin B. All the APIs used here were neutralized in a buffer appropriate hydroxide cations. The cation hydroxide was obtained on Amberlite resin (in the OH form) in order to exchange halides. The biological studies of these new compounds were made using techniques like the micro dilution and colorimetric methods. Overall a total of 19 new ILs were synthesised (6 ILs based on ampicillin, 4 ILs, based on amoxicillin, 6 ILs based on penicillin G and 4 ILs based on amphotericin B) and characterized by spectroscopic and analytical methods in order to confirm their structure and purity. The study of the biological properties of the synthesised ILs showed that some have antimicrobial activity against bacteria and yeast cells, even in resistant bacteria. Also this work allowed to show that ILs based on ampicillin could be used as anti-tumour agents. This proves that with a careful selection of the organic cation, it is possible to provoke important physico-chemical and biological alteration in the properties of ILs-APIs with great impact, having in mind their applications.

Optimal choice between even- and uneven-aged forestry: the case of non-industrial private forest owners

An infinite-horizon discrete time model with multiple size-class structures using a transition matrix is built to assess optimal harvesting schedules in the context of Non-Industrial Private Forest (NIPF) owners. Three model specifications accounting for forest income, financial return on an asset and amenity valuations are considered. Numerical simulations suggest uneven-aged forest management where a rational forest owner adapts her or his forest policy by influencing the regeneration of trees or adjusting consumption dynamics depending on subjective time preference and market return rate dynamics on the financial asset. Moreover she or he does not value significantly non-market benefits captured by amenity valuations relatively to forest income.

Cell functional enviromics: applications to Pichia pastoris

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.