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.

A Problemática da Erosão Costeira: O Caso Específico da Praia do Pedrógão

Ao longo das últimas décadas, tem-se verificado uma crescente ocupação populacional e urbanística, em paralelo com a concentração de diversas actividades económicas, ao longo das zonas costeiras. Neste sentido, torna-se fulcral o desenvolvimento de políticas de gestão integrada destes ecossistemas naturais, dada a sua grande vulnerabilidade, no que respeita aos usos e ocupação do solo. Como resultado de acções naturais e do Homem (na sua grande maioria), a erosão estabelece-se como o principal problema que assola as zonas costeiras, em Portugal. Neste sentido, as Tecnologias de Informação Geográfica, em especial os Sistemas de Informação Geográfica, conferem-se como importantes ferramentas, tanto na monitorização do litoral, como no apoio aos processos de tomada de decisão. A presente dissertação detém, como principal objectivo, demonstrar as mais-valias dos SIG no estudo das zonas costeiras, especialmente na área da prevenção. Tendo como área de estudo a praia do Pedrógão, em Leiria, considerando o período temporal 1947 – 2014 (1947-1983; 1983-2003; 2003-2007; 2007-2012; 2012-2014), propõe-se: estabelecer uma base cartográfica que analise, espacialmente, o comportamento da linha da costa face à gradual erosão; quantificar as taxas de erosão/acreção e, por fim, proceder à delimitação de faixas de perigo, em paralelo com a análise dos resultados previamente conseguidos, assim como da topografia do local em análise. Para o troço costeiro seleccionado, é de constatar que existe, efectivamente, um recuo da linha da costa, especialmente na praia Sul (Praia dos Campistas), onde se regista o maior recuo médio anual (-17.72m, entre 2012-2014). Na praia Norte, as v taxas anuais variam entre os -0.64m (1947-1983) e os -15.07m (2012-2014), tendo apresentado valores de acreção, com excepção para o intervalo de tempo mais recente. É certo que as recargas de areia, iniciadas em 2011, procuram minimizar os impactos da erosão, o que pode condicionar os resultados obtidos. Por outro lado, no que respeita às faixas de perigo, é certo que, com o recuar da costa, há uma tendência da frente urbana aproximar-se do nível de “perigo muito elevado”. Nesse sentido, a grande maioria do edificado situa-se na faixa de perigo moderado (211 edifícios), constatando-se que outra grande parte (193 edifícios) não se encontre ameaçado. De um total de 413 edifícios, apenas 1 encontra-se em perigo elevado. Mantendo-se esta tendência de recuo da costa e tendo por base o trabalho de campo efectuado, estima-se que os valores apresentados venham a alterar-se, isto é, tanto a população local, como o edificado tendem a aproximar-se do perigo máximo/elevado.

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.

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

8-Phase Ring oscillator for modern receivers

The evolution of receiver architectures, built in modern CMOS technologies, allows the design of high efficient receivers. A key block in modern receivers is the oscillator. The main objective of this thesis is to design a very low power and low area 8-Phase Ring Oscillator for biomedical applications (ISM and WMTS bands). Oscillators with multiphase outputs and variable duty cycles are required. In this thesis we are focused in 12.5% and 50% duty-cycles approaches. The proposed circuit uses eight inverters in a ring structure, in order to generate the output duty cycle of 50%. The duty cycle of 1/8 is achieved through the combination of the longer duty cycle signals in pairs, using, for this purpose, NAND gates. Since the general application are not only the wireless communications context, as well as industrial, scientific and medical plans, the 8-Phase Oscillator is simulated to be wideband between 100 MHz and 1 GHz, and be able to operate in the ISM bands (447 MHz-930 MHz) and WMTS (600 MHz). The circuit prototype is designed in UMC 130 nm CMOS technology. The maximum value of current drawn from a DC power source of 1.2 V, at a maximum frequency of 930 MHz achieved, is 17.54 mA. After completion of the oscillator layout studied (occupied area is 165 μm x 83 μm). Measurement results confirm the expected operating range from the simulations, and therefore, that the oscillator fulfil effectively the goals initially proposed in order to be used as Local Oscillator in RF Modern Receivers.

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.

High similarity of Trypanosoma cruzikDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

IntroductionDetermining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease.MethodsAn epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture).ResultsT. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected.ConclusionsLSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

AbstractINTRODUCTION:The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.METHODS:IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting.RESULTS:Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls.CONCLUSIONS:These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.

Quadrature generators based on ring oscillators and shift registers

Quadrature oscillators are key elements in modern radio frequency (RF) transceivers and very useful nowadays in wireless communications, since they can provide: low quadrature error, low phase-noise, and wide tuning range (useful to cover several bands). RC oscillators can be fully integrated without the need of external components (external high Q-inductors), optimizing area, cost, and power consumption. The conventional structure of ring oscillator offers poor frequency stability and phasenoise, low quality factor (Q), and besides being vulnerable to process, voltage and temperature (PVT) variations, its performance degrades as the frequency of operation increases. This thesis is devoted to quadrature oscillators and presents a detailed comparative study of ring oscillator and shift register (SR) approaches. It is shown that in SRs both phase-noise and phase error are reduced, while ring oscillators have the advantage of occupying less area and less consumption due to the reduced number of components in the circuit. Thus, although ring oscillators are more suitable for biomedical applications, SRs are more appropriate for wireless applications, especially when specification requirements are more stringent and demanding. The first architecture studied consists in a simple CMOS ring oscillator employing an odd number of static single-ended inverters as delay cells. Subsequently, the quadrature 4-stage ring oscillator concept is shown and post-layout simulations are presented. The 3 and 4-phase single-frequency local oscillator (LO) generators employing SRs are presented, the latter with 50% and 25% duty-cycles. The circuits operate at 600 MHz and 900 MHz, and were designed in a 130 nm standard CMOS technology with a voltage supply of 1.2 V.

Analysis and design of sinusoidal quadrature RC-oscillators

Modern telecommunication equipment requires components that operate in many different frequency bands and support multiple communication standards, to cope with the growing demand for higher data rate. Also, a growing number of standards are adopting the use of spectrum efficient digital modulations, such as quadrature amplitude modulation (QAM) and orthogonal frequency division multiplexing (OFDM). These modulation schemes require accurate quadrature oscillators, which makes the quadrature oscillator a key block in modern radio frequency (RF) transceivers. The wide tuning range characteristics of inductorless quadrature oscillators make them natural candidates, despite their higher phase noise, in comparison with LC-oscillators. This thesis presents a detailed study of inductorless sinusoidal quadrature oscillators. Three quadrature oscillators are investigated: the active coupling RC-oscillator, the novel capacitive coupling RCoscillator, and the two-integrator oscillator. The thesis includes a detailed analysis of the Van der Pol oscillator (VDPO). This is used as a base model oscillator for the analysis of the coupled oscillators. Hence, the three oscillators are approximated by the VDPO. From the nonlinear Van der Pol equations, the oscillators’ key parameters are obtained. It is analysed first the case without component mismatches and then the case with mismatches. The research is focused on determining the impact of the components’ mismatches on the oscillator key parameters: frequency, amplitude-, and quadrature-errors. Furthermore, the minimization of the errors by adjusting the circuit parameters is addressed. A novel quadrature RC-oscillator using capacitive coupling is proposed. The advantages of using the capacitive coupling are that it is noiseless, requires a small area, and has low power dissipation. The equations of the oscillation amplitude, frequency, quadrature-error, and amplitude mismatch are derived. The theoretical results are confirmed by simulation and by measurement of two prototypes fabricated in 130 nm standard complementary metal-oxide-semiconductor (CMOS) technology. The measurements reveal that the power increase due to the coupling is marginal, leading to a figure-of-merit of -154.8 dBc/Hz. These results are consistent with the noiseless feature of this coupling and are comparable to those of the best state-of-the-art RC-oscillators, in the GHz range, but with the lowest power consumption (about 9 mW). The results for the three oscillators show that the amplitude- and the quadrature-errors are proportional to the component mismatches and inversely proportional to the coupling strength. Thus, increasing the coupling strength decreases both the amplitude- and quadrature-errors. With proper coupling strength, a quadrature error below 1° and amplitude imbalance below 1% are obtained. Furthermore, the simulations show that increasing the coupling strength reduces the phase noise. Hence, there is no trade-off between phase noise and quadrature error. In the twointegrator oscillator study, it was found that the quadrature error can be eliminated by adjusting the transconductances to compensate the capacitance mismatch. However, to obtain outputs in perfect quadrature one must allow some amplitude error.

Medium access control design for distributed opportunistic radio networks

Existing wireless networks are characterized by a fixed spectrum assignment policy. However, the scarcity of available spectrum and its inefficient usage demands for a new communication paradigm to exploit the existing spectrum opportunistically. Future Cognitive Radio (CR) devices should be able to sense unoccupied spectrum and will allow the deployment of real opportunistic networks. Still, traditional Physical (PHY) and Medium Access Control (MAC) protocols are not suitable for this new type of networks because they are optimized to operate over fixed assigned frequency bands. Therefore, novel PHY-MAC cross-layer protocols should be developed to cope with the specific features of opportunistic networks. This thesis is mainly focused on the design and evaluation of MAC protocols for Decentralized Cognitive Radio Networks (DCRNs). It starts with a characterization of the spectrum sensing framework based on the Energy-Based Sensing (EBS) technique considering multiple scenarios. Then, guided by the sensing results obtained by the aforementioned technique, we present two novel decentralized CR MAC schemes: the first one designed to operate in single-channel scenarios and the second one to be used in multichannel scenarios. Analytical models for the network goodput, packet service time and individual transmission probability are derived and used to compute the performance of both protocols. Simulation results assess the accuracy of the analytical models as well as the benefits of the proposed CR MAC schemes.

Fatigue life assessment of endodontic instruments subjected to multi planar curvature

This present study aimed to investigate the fatigue life of unused (new) endodontic instruments made of NiTi with control memory by Coltene™ and subjected to the multi curvature of a mandibular first molar root canal. Additionally, the instrument‟s structural behaviour was analysed through non-linear finite element analysis (FEA). The fatigue life of twelve Hyflex™ CM files was assessed while were forced to adopt a stance with multiple radius of curvature, similar to the ones usually found in a mandibular first molar root canal; nine of them were subjected to Pecking motion, a relative movement of axial type. To achieve this, it was designed an experimental setup with the aim of timing the instruments until fracture while worked inside a stainless steel mandibular first molar model with relative axial motion to simulate the pecking motion. Additionally, the model‟s root canal multi-curvature was confirmed by radiography. The non-linear finite element analysis was conducted using the computer aided design software package SolidWorks™ Simulation, in order to define the imposed displacement required by the FEA, it was necessary to model an endodontic instrument with simplified geometry using SolidWorks™ and subsequently analyse the geometry of the root canal CAD model. The experimental results shown that the instruments subjected to pecking motion displayed higher fatigue life values and higher lengths of fractured tips than those with only rotational relative movement. The finite element non-linear analyses shown, for identical conditions, maximum values for the first principal stress lower than the yield strength of the material and those were located in similar positions to the instrument‟s fracture location determined by the experimental testing results.

Mapping the Pasture Steppe in Bayankhongor, Mongolia: comparison of classification methods, using Landsat-8 and geophysical data

Grasslands in semi-arid regions, like Mongolian steppes, are facing desertification and degradation processes, due to climate change. Mongolia’s main economic activity consists on an extensive livestock production and, therefore, it is a concerning matter for the decision makers. Remote sensing and Geographic Information Systems provide the tools for advanced ecosystem management and have been widely used for monitoring and management of pasture resources. This study investigates which is the higher thematic detail that is possible to achieve through remote sensing, to map the steppe vegetation, using medium resolution earth observation imagery in three districts (soums) of Mongolia: Dzag, Buutsagaan and Khureemaral. After considering different thematic levels of detail for classifying the steppe vegetation, the existent pasture types within the steppe were chosen to be mapped. In order to investigate which combination of data sets yields the best results and which classification algorithm is more suitable for incorporating these data sets, a comparison between different classification methods were tested for the study area. Sixteen classifications were performed using different combinations of estimators, Landsat-8 (spectral bands and Landsat-8 NDVI-derived) and geophysical data (elevation, mean annual precipitation and mean annual temperature) using two classification algorithms, maximum likelihood and decision tree. Results showed that the best performing model was the one that incorporated Landsat-8 bands with mean annual precipitation and mean annual temperature (Model 13), using the decision tree. For maximum likelihood, the model that incorporated Landsat-8 bands with mean annual precipitation (Model 5) and the one that incorporated Landsat-8 bands with mean annual precipitation and mean annual temperature (Model 13), achieved the higher accuracies for this algorithm. The decision tree models consistently outperformed the maximum likelihood ones.

Exploring TM image texture and its relationships with biomass estimation in Rondônia, Brazilian Amazon

Many texture measures have been developed and used for improving land-cover classification accuracy, but rarely has research examined the role of textures in improving the performance of aboveground biomass estimations. The relationship between texture and biomass is poorly understood. This paper used Landsat Thematic Mapper (TM) data to explore relationships between TM image textures and aboveground biomass in Rondônia, Brazilian Amazon. Eight grey level co-occurrence matrix (GLCM) based texture measures (i.e., mean, variance, homogeneity, contrast, dissimilarity, entropy, second moment, and correlation), associated with seven different window sizes (5x5, 7x7, 9x9, 11x11, 15x15, 19x19, and 25x25), and five TM bands (TM 2, 3, 4, 5, and 7) were analyzed. Pearson's correlation coefficient was used to analyze texture and biomass relationships. This research indicates that most textures are weakly correlated with successional vegetation biomass, but some textures are significantly correlated with mature forest biomass. In contrast, TM spectral signatures are significantly correlated with successional vegetation biomass, but weakly correlated with mature forest biomass. Our findings imply that textures may be critical in improving mature forest biomass estimation, but relatively less important for successional vegetation biomass estimation.

DOTA-based Ga(III) and Gd(III) chelates for medical imaging (PET, SPECT and MRI)

PhD Thesis in Sciences Specialization in Chemistry