STRUCTURE-FUNCTION ANALYSIS OF MULTI-COPPER OXIDASES

Dissertation presented to obtain the Ph.D degree in Biochemistry, Structural Biochemistry

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.

Ageing on commercial automotive catalysts, a structure reactivity study

Increasingly stringed regulations for diesel engine emissions have a significant impact on the required efficiency of DOC. Lowered DOC oxidation efficiency due to thermal aging effects influences the efficiency of the exhaust aftertreatment systems downstream of the DOC. In this work carried out in the Jean Le Rond d’Alembert Institute the effect of hydrothermal aging on the reactivity and structure of a commercial DOC was investigated. The characterization of the catalytic performance was carried out on a synthetic gas bench using carrots catalyst under conditions close to the realistic conditions i.e. using a synthetic gas mixture, representative of the exhaust gases from diesel engines. Different structural characterization techniques were performed: textural and morphological proprieties were analyzed by BET and TEM, the characterization of the presented crystallographic phases was performed by DRX and the determination of the number of reducible species was possible by TPR. TEM results shown, an increase of the metal particle size with the aging caused by the agglomeration of metal particles, revealing the presence of metal sintering. DRX results also suggest the presence of support sintering. Furthermore, DRX and BET results unexpectedly reveal that the most drastic aging conditions used actually activated the catalyst surface. As expected, the aging affected negatively the catalyst performance on the oxidation of methane and CO, however an improvement of the NO oxidation performance with the aging was observed. Nevertheless, for the aging conditions used, catalytic activity results show that the influence of aging in DOC performance was not significant, and therefore, more drastic aging conditions must be used.

Follow the red road of triheme cytochromes in Geobacter sulfurreducens

The bacterium Geobacter sulfurreducens (Gs) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membrane in a process designated as extracellular electron transfer. The Gs genome was fully sequenced and a family composed by five periplasmic triheme cytochromes c7 (designated PpcA-E) was identified. These cytochromes play an important role in the reduction of extracellular acceptors. They contain approximately 70 amino acids, three heme groups with bis-histidinyl axial coordination, and share between 57 and 77% sequence identity. The triheme cytochrome PpcA is highly abundant in Gs and is most likely the reservoir of electrons destined for outer surface. In addition to its role in electron transfer pathways this protein can perform e-/H+ energy transduction, a process that is disrupted when the strictly conserved aromatic residue phenylalanine 15 is replaced by a leucine (PpcAF15L). This Thesis focuses on the expression, purification and characterization of these proteins using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The orientations of the heme axial histidine ring planes and the orientation of the heme magnetic axis were determined for each Gs triheme cytochrome. The comparison of the orientations obtained in solution with the crystal structures available showed significant differences. The results obtained provide the paramagnetic constraints to include in the future refinement of the solution structure in the oxidized state. In this work was also determined the solution structure and the pH-dependent conformational changes of the PpcAF15L allowing infer the structural origin for e-/H+ energy transduction mechanism as shown by PpcA. Finally, the backbone and side chain NH signals of PpcA were used to map interactions between this protein and the putative redox partner 9,10-anthraquinone-2,6-disulfonate (AQDS). In this work a molecular interaction was identified for the first time between PpcA and AQDS, constituting the first step toward the rationalization of the Gs respiratory chain.

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

UAV - based imagery processing using structure from motion and remote sensing technology

With the recent advances in technology and miniaturization of devices such as GPS or IMU, Unmanned Aerial Vehicles became a feasible platform for a Remote Sensing applications. The use of UAVs compared to the conventional aerial platforms provides a set of advantages such as higher spatial resolution of the derived products. UAV - based imagery obtained by a user grade cameras introduces a set of problems which have to be solved, e. g. rotational or angular differences or unknown or insufficiently precise IO and EO camera parameters. In this work, UAV - based imagery of RGB and CIR type was processed using two different workflows based on PhotoScan and VisualSfM software solutions resulting in the DSM and orthophoto products. Feature detection and matching parameters influence on the result quality as well as a processing time was examined and the optimal parameter setup was presented. Products of the both workflows were compared in terms of a quality and a spatial accuracy. Both workflows were compared by presenting the processing times and quality of the results. Finally, the obtained products were used in order to demonstrate vegetation classification. Contribution of the IHS transformations was examined with respect to the classification accuracy.

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

Design and characterization of novel biopolymeric structure using biocompatible ionic liquids

The main objective of this thesis was the development of polymeric structures from the dissolution of FucoPol, a bacterial exopolysaccharide (EPS), in a biocompatible ionic liquid, choline acetate. The FucoPol was produced by the bacteria Enterobacter A47 using glycerol as carbon source at controlled temperature and pH (30ºC and 7, respectively). At the end of 3 days it was produced 7 g/L of FucoPol. The net yield of Fucopol in glycerol (YP/S) was 0.22 g/g and the maximum productivity 2.37 g/L.d This polymer was characterized about its composition in sugars and acyl groups (by High-Performance Liquid Chromatography - HPLC), containing fucose (35 % mol), galactose (21 % mol), glucose (29 % mol), rhamnose (3% mol) and glucuronic acid (12% mol) as well as acetate (14.28 % mol), pyruvate (2.15 % mol) and succinate (1.80 % mol). Its content of water and ash was 15% p/p and 2% p/p, respectively, and the chemical bonds (determined by Infrared Spectroscopy - FT-IR) are consistent to the literature reports. However, due to limitations in Differential Scanning Calorimetry (DSC) equipment it was not possible to determine the glass transition temperature. In turn, the ionic liquid showed the typical behavior of a Newtonian fluid, glass transition temperature (determined by DSC) -98.03ºC and density 1.1031 g/cm3. The study of chemical bonds by FT-IR showed that amount of water (8.80%) influenced the visualization of the bands predicted to in view of their chemical structure. After the dissolution of the FucoPol in the ionic liquid at different temperatures (50, 60, 80 and 100 ° C) it was promoted the removal of this by the phase inversion method using deionized water as a solvent, followed by drying in an oven at 70 ° C. The mixtures before and after the phase inversion method were characterized through the studies mentioned above. In order to explore possible application field’s biocompatibility assays and collage on balsa wood tests were performed. It was found that the process of washing with water by the phase inversion method was not totally effective in removing the biocompatible ionic liquid, since all FucoPol – IL mixtures still contained ionic liquid in their composition as can be seen by the DSC results and FT-IR. In addition, washing the mixtures with water significantly altered the composition of FucoPol. However, these mixtures, that developed a viscous behavior typical of a non-Newtonian fluid (shear-thinning), have the potential to be applied in the biomedical field as well as biological glues.

Does ownership structure matter? Three essays in finance

Management

Technical optimization of the synthesis of flavours

The goal of this thesis is the investigation and optimization of the synthesis of potential fragrances. This work is projected as collaboration between the University of Applied Sciences in Merseburg and the company Miltitz Aromatics GmbH in Bitterfeld‐Wolfen (Germany). Flavoured compounds can be synthesized in different ways and by various methods. In this work, methods like the phase transfer catalysis and the Cope‐rearrangement were investigated and applied, for getting a high yield and quantity of the desired substances and without any by‐products or side reactions. This involved the study of syntheses with different process parameters such as temperature, solvent, pressure and reaction time. The main focus was on Cope‐rearrangement, which is a common method in the synthesis of new potential fragrance compounds. The substances synthesized in this work have a hepta‐1,5‐diene‐structure and that is why they can easily undergo this [3,3]‐sigma tropic rearrangement. The lead compound of all research was 2,5‐dimethyl‐2‐vinyl‐4‐hexenenitrile (Neronil). Neronil is synthesized by an alkylation of 2‐methyl‐3‐butenenitrile with prenylchloride under basic conditions in a phase‐transfer system. In this work the yield of isolated Neronil is improved from about 35% to 46% by according to the execution conditions of the reaction. Additionally the amount of side product was decreased. This synthesized hexenenitrile involved not only the aforementioned 1,5‐diene‐structure, but also a cyano group, that makes this structure a suitable base for the synthesis of new potential fragrance compounds. It was observed that Neronil can be transferred into 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid by a hydrolysis under basic conditions. After five hours the acid can be obtained with a yield of 96%. The following esterification is realized with isobutanol to produce 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid isobutyl ester with quantitative conversion. It was observed that the Neronil and the corresponding ester can be converted into the corresponding Cope‐product, with a conversion of 30 % and 80%. Implementing the Cope‐rearrangement, the acid was heated and an unexpected decarboxylated product is formed. To achieve the best verification of reaction development and structure, scrupulous analyses were done using GC‐MS, 1H‐NMR and 13C‐ NMR.

The Multidimensional and Centred Structure of Our Interested Perception in Hierocles

O objectivo desta dissertação é identificar a estrutura da percepção tal como ela é desenhada por Hiérocles nos <,(#= >1!(?$%&'(). Para alcançar este objectivo focar-me-ei na análise que o filósofo estóico leva a cabo em torno da percepção. O foco começa por incindir sobre a distinção entre percepção de si e percepção do exterior, com todas as suas subtilezas. Tal análise implicará então a consideração da importância da não-indiferença nessa estrutura de percepção. O objectivo é compreender como a percepção é sempre relacional e interessada. . Centrar-me-ei então na noção de !"#$%&'(), tentanto explorar a complexidade e multiplicidade do fenómeno em causa. Será também estabalecida a relação com o fragmento de Estobeu (6.671), que é, a par da obra, a fonte mais importante do pensamento de Hiérocles. Através desta relação introduzir-se-á a estrutura circular e concêntrica de uma percepção relacional e interessada. Toda esta dissertação será levada a cabo focando-se principalmente nos textos do próprio Hiérocles.

Inovcity- Structure of trials evaluation

In 2007, the UK government commissioned the Energy Demand Research Project to conduct a large scale experiment of smart metering technologies to test the impacts from many different forms of feedback to residential consumers. A full evaluation of the results was completed in 2011. In Portugal, EDP is also conducting smart meter trials in a project called InovCity in the city of Évora whose results will be evaluated during 2012. In this work, the case of Great Britain is studied as a reference on how an evaluation of trial results should be conducted. I also discuss potential limitations of the experiments, implications for national roll-out decisions, and finally draw some lessons that can be applied to the Portuguese case.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.

Impact of chromossomal structure on the evolution of Schizosaccharomyces pombe undergoing Mutation Accumulation

Large chromosomal rearrangements are common in natural populations and thought to be involved in speciation events. In this project, we used experimental evolution to determine how the speed of evolution and the type of accumulated mutations depend on the ancestral chromosomal structure and genotype. We utilized two Wild Type strains and a set of genetically engineered Schizosaccharomyces pombe strains, different solely in the presence of a certain type of chromosomal variant (inversions or translocations), along with respective controls. Previous research has shown that these chromosomal variants have different fitness levels in several environments, probably due to changes in the gene expression along the genome. These strains were propagated in the laboratory at very low population sizes, in which we expect natural selection to be less efficient at purging deleterious mutations. We then measured these strains’ changes in fitness throughout this accumulation of deleterious mutations, comparing the evolutionary trajectories in the different rearrangements to understand if the chromosomal structure affected the speed of evolution. We also tested these mutations for possible epistatic effects and estimated their parameters: the number of arising deleterious mutations per generation (Ud) and each one’s mean effect (sd).