Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica Estrutural

Organocatálise assimétrica: estudo mecanístico de reacções intramoleculares de aldol

Dissertação apresentada para obtenção do Grau de Doutor em Química, especialidade de Química Orgânica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Fitting the yield curve

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

Characterization of sol-gel matrices with entrapped cutinase

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

The fitting of the German yield curve: A dynamic approach using latent, macroeconomic and stock market variables

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

Three essays on the term structure of interest rates

A PhD Dissertation, presented as part of the requirements for the Degree of Doctor of Philosophy from the NOVA - School of Business and Economics

Structural characterization of the urea-unfolded state of Colicin Immunity Protein Im7 and Im9

Dissertação para obtenção do Grau de Mestre em Bioquímica Estrutural e Funcional

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

Redes de Nova Geração: Uma Proposta para Cadastro em Sistemas de Informação Geográfica de Redes Fixas de Telecomunicações em Fibra Óptica

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Gestão do Território variante de Sistemas de Informação Geográfica e Detecção Remota

RingDings market analysis

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

On the physical-chemical properties of ceramide C16

Dissertation presented to obtain the Ph.D degree in Chemistry.

Estudo de interações proteína-carbohidrato em sistemas bacterianos

Bacillus subtilis tem a capacidade de utilizar arabinoligossacáridos presentes na parede das células vegetais, através de um consórcios de enzimas envolvidas na hidrólise enzimática dos mesmos. A captação destes carbohidratos por parte do Bacillus subtilis depende de dois transportadores membranares AraE e AraNPQ-MsmX. Após a captação de L-arabinose e arabinoligossacáridos, estes substratos serão metabolizados pelos produtos enzimáticos codificados por genes pertencentes ao operão responsável pela metabolização de arabinose araABDLMNPQabfA. Neste operão, para além dos genes responsáveis pelo transportador AraNPQ, o gene araL determina a síntese de uma enzima com atividade de fosfatase, AraL, que tem putativamente como principal função a destoxificação de intermediários metabólicos fosforilados, em situações particulares. O objectivo deste trabalho consistiu em descobrir quais os determinantes moleculares envolvidos em ambos os processos de reconhecimento molecular, ora o reconhecimento de arabinoligossacáridos por parte da AraN, do ponto de vista dos carbohidratos ora de carbohidratos fosforilados do ponto de vista da AraL. Para elucidar estes processos de reconhecimento molecular proteína-carbohidrato foi utilizada a técnica de RMN, modelação computacional e mutagénese dirigida. No primeiro capitulo são introduzidos conceitos fundamentais para a percepção da ação de ambas as proteínas (AraN e AraL), no organismo Bacillus subtilis. O segundo capitulo, refere-se ao estudo dos mecanismos envolvidos no reconhecimento proteína-carbohidrato através da técnica de STD-RMN para estudar interações do ponto de vista do carbohidrato, bem como abordagens bioinformáticas tais como alinhamentos de sequencia primária e dockings moleculares, para identificar resíduos do ponto de vista da proteína passiveis de se encontrarem envolvidos no reconhecimento molecular, que numa última instância são mutados para se confirmar a sua relevância no processo de reconhecimento molecular do ponto de vista da proteína. Neste capitulo é demonstrado que a AraN é responsável pelo reconhecimento de arabinoligossacáridos e celoligossacáridos. No que diz respeito aos primeiros, a AraN reconhece preferencialmente arabinoligossacáridos com três subunidades, verificando-se a perda de saturação na subunidade não redutora da arabinotetraose. O processo de reconhecimento molecular apresenta uma constante de dissociação com uma ordem de grandeza na ordem dos μM. Do ponto de vista da proteína são identificados resíduos (W253, Y254 e Y54) putativos de se encontrarem também eles envolvidos no reconhecimento molecular do ponto de vista da AraN através de interações CHstacking. Finalmente no terceiro capitulo, é apresentada a optimização da sobrexpressão da AraL, dado que numa primeira fase a sua produção ocorria sob a forma de corpos de inclusão. Para tal são descritas metodologias de solubilização e renaturação da mesmas, com recurso a agentes caotrópicos (ureia e GmdCl) e detergentes (SDS).

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.

How do current term structure model behave beyond the last liquid point?: A comparison of the DNS and Smith-Wilson methods

A Work Project, presented as part of the requirements for the Award of a Master’s Double Degree in Finance from Maastricht University and NOVA – School of Business and Economics