Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Ciências Médicas, 2018. / Introdução: As doenças cardiovasculares são uma das mais importantes causas de morbidade e mortalidade no mundo inteiro, sendo a dislipidemia um dos fatores de risco para o desenvolvimento da aterosclerose, que é o substrato dessas doenças. O colesterol não-HDL é a fração do colesterol que inclui todo o colesterol das partículas potencialmente aterogênicas e seu controle passou a fazer parte das medidas de prevenção primária e secundária das doenças cardiovasculares. Objetivo: Correlacionar os vários padrões de alteração do colesterol com a espessura mediointimal das carótidas, como forma de estratificação não invasiva do risco cardiovascular, analisando se os pacientes com elevação isolada de colesterol não-HDL têm comportamento semelhante aos demais pacientes com fatores de risco já consagrados. Metodologia: Foram selecionados para o estudo militares submetidos a inspeção de saúde no Hospital de Força Aérea de Brasília no período de maio de 2016 a fevereiro de 2017, separando-os em três grupos: um grupo com elevação isolada de colesterol não-HDL, outro, com níveis de colesterol normal, que foi considerado o grupo controle deste estudo e um grupo com elevação de colesterol LDL ou HDL baixo. Posteriormente, foram analisados os exames de ultrassonografia de carótidas a que esses pacientes foram submetidos e coletadas informações sobre a espessura mediointimal das carótidas, sendo realizada estratificação do risco cardiovascular, conforme tabela do estudo CAPS para determinar o risco de cada grupo desenvolver doenças cardiovasculares. Resultados: Foram selecionados para esse estudo 427 pacientes divididos nos três grupos estudados. Os valores de colesterol foram em média de 118,1 mg/dl para o LDL colesterol; de 49,4 mg/dl para o HDL colesterol e de 141,7 mg/dl para o não- HDL colesterol. A espessura mediointimal (EMI) das carótidas foi de 0,71 mm em média e uma pequena porcentagem dos pacientes apresentou placas ateroscleróticas diagnosticadas pelo ultrassom. Os pacientes do grupo normal foram em sua maioria estratificados como de risco cardiovascular intermediário (41,3%), sendo esse valor maior que o dos pacientes do grupo LDL alto/HDL baixo (35,8%); enquanto que os que foram estratificados como de alto risco no grupo não-HDL elevado (46%), aproximaram-se do grupo LDL alto/HDL baixo (37,1%). A idade foi o único fator independente na alteração da espessura mediointimal das carótidas e houve uma tendência do grupo não-HDL, quando comparado ao grupo normal, de apresentar aumento dos valores da EMI das carótidas. Conclusão: Os grupos estudados não apresentaram maior risco independente. O grupo não-HDL elevado apresentou uma tendência maior de ocorrência de eventos em relação ao grupo com níveis de colesterol normais e comportamento semelhante ao grupo LDL alto/HDL baixo. A idade foi o único fator de risco independente de eventos. / Introduction: Cardiovascular diseases are one of the most important causes of morbidity and mortality worldwide, with dyslipidemia being one of the risk factors for the development of atherosclerosis, which is the substrate of these diseases. Non- HDL cholesterol is the fraction of cholesterol that includes all cholesterol from potentially atherogenic particles and its control has become part of the primary and secondary prevention measures of cardiovascular diseases. Objective: To correlate the different patterns of cholesterol change with the medianintimal thickness of the carotid arteries as a non-invasive stratification of cardiovascular risk, analyzing whether patients with isolated elevation of non-HDL cholesterol had a similar behavior to the other patients with risk factors already established. Methods: We selected for the study military personnel undergoing health inspection at the Brasília Air Force Hospital from May 2016 to February 2017, separating them into three groups: one group with isolated elevation of non-HDL cholesterol, another group with normal cholesterol levels, which was considered the control group of this study and a group with elevated LDL or low HDL cholesterol. Afterwards, the carotid ultrasound examinations were analyzed and the information was collected on the average thickness of the carotid arteries. The cardiovascular risk stratification was performed according to the CAPS study table to determine the risk of each group developing cardiovascular diseases. Results: For this study 427 patients were divided into three groups. Cholesterol values were on average 118.1 mg / dl for LDL cholesterol; of 49.4 mg / dl for HDL cholesterol and 141.7 mg / dl for non-HDL cholesterol. The medianintimal thickness (EMI) of the carotid arteries was 0.71 mm on average and a small percentage of the patients had atherosclerotic plaques diagnosed by ultrasound. The patients in the normal group were mostly stratified as intermediate cardiovascular risk (41.3%), which was higher than that of the patients in the high LDL / low HDL group (35.8%); while those who were stratified as high-risk in the high non-HDL group (46%), were close to the low HDL / HDL group (37.1%). Age was the only independent factor in the change in the medianintimal thickness of the carotid arteries and there was a tendency of the non-HDL group, when compared to the normal group, to present an increase in carotid EMI values. Conclusion: The groups studied did not present a higher independent risk. The high non-HDL group showed a greater tendency to occur in relation to the group with normal cholesterol levels and similar behavior to the high LDL / low HDL group. Age was the only independent risk factor for events.

Tese de mestrado integrado. Engenharia Electrotécnica e de Computadores (Major Telecomunicações). Faculdade de Engenharia. Universidade do Porto. 2009

Modern high-performance digital signal processing (DSP) applications face constantly increasing performance requirements and are becoming increasingly challenging to develop and work with. In DSP paradigm, many researchers see potential in achieving algorithm speed-up by employing Field Programmable Gate Arrays (FPGAs) – reconfigurable hardware with parallelism feature. However, developing applications for FPGAs incur particular challenges on the development flow. This work proposes a scalable hybrid DSP system for performing high-performance signal processing applications. The system employs hybrid CPU + FPGA architecture of commercially available, off-the-shelf (COTS) FPGAs and central processing units (CPU) of personal computers. In this work an example implementation of a multi-channel cross-correlator is investigated and delivered using a new development paradigm. The correlator is implemented on the XD1000 development system using a high-level FPGA programming tool – Impulse CoDeveloper. Analysis of DSP application development in a hybrid CPU+FPGA system employing the high-level programming tool Impulse C is presented. Potential of the selected tool to deliver algorithm speed-ups is investigated using reference multi-channel correlator software. Particular attention is devoted to input/output (I/O) implementation, which is considered one of the most challenging problems in FPGA design development. This work delivers an I/O framework based on PCI Express interface for the proposed high-performance scalable DSP system. Using Stratix II GX PCI Express Development Board from Altera Corporation, a scalable and flexible communication approach for the multi-channel correlator is delivered. This framework can be adapted to perform other high-performance streaming DSP applications. The outcomes of this work are a multi-channel correlator developed in a reconfigurable environment with new design methodology and I/O framework with software control application. The outcomes are used to demonstrate the potential of implementing DSP applications in hybrid CPU + FPGA architecture and to discuss existing challenges and suggest possible solutions.

Ce travail de thèse est consacré au développement de biocapteurs électrochimiques en utilisant des argiles ou des hydroxydes doubles lamellaires (HDL), riches en fer, comme matrices d'immobilisation de l'hémoglobine (Hb). Le but était de mettre en évidence la contribution des propriétés redox de ces matériaux dans l'amélioration des performances des biocapteurs par un phénomène de catalyse redox. L'hémoglobine est une métalloprotéine qui contient des porphyrines au fer (hème) comme sous-unité prosthétique. Une orientation privilégiée de cette biomolécule à la surface d'une électrode permet le transfert direct d'électrons entre le site actif de la protéine et l'électrode. Les propriétés électro-catalytiques de l'Hb immobilisée ont été étudiées pour la réduction du peroxyde d'hydrogène et ont permis la réalisation de différents biocapteurs ampérométriques. Nous avons immobilisé l'Hb dans plusieurs argiles cationiques contenant du fer : la nontronite de Garfield, des montmorillonites ferrifères synthétiques ou naturelles, ainsi qu'une montmorillonite synthétique servant de référence. Les propriétés électrochimiques de ces argiles ont été évaluées en voltammétrie cyclique et en impédancemétrie, en relation avec leurs propriétés structurales. Les isothermes d'adsorption de l'Hb dans ces argiles ont été établis montrant une forte affinité de l'Hb pour la nontronite. Nous avons montré que les argiles riches en fer octaédrique, notamment la nontronite, améliorent le transfert direct d'électrons entre l'Hb et l'électrode. L'immobilisation de l'Hb dans des HDL, de compositions différentes (MgAl et ZnAl) a été réalisée par adsorption et coprécipitation. Il faut noter que le fer situé en site octaédrique dans les HDL (MgFe) n'est pas électroactif et ne peut donc pas intervenir dans le processus électro-enzymatique. Les caractérisations physico-chimiques et morphologiques des biohybrides Hb-HDL ont été faites par plusieurs techniques, comme la DRX, IR, UV, MEB et MET, montrant une dénaturation partielle de la structure tertiaire de la protéine par la formation de liaisons hydrogènes entre la biomolécule et les feuillets hydroxylés des HDL; ce qui limite l'accessibilité de l'hème au transfert électronique direct. Outre l'immobilisation de l'Hb dans les HDL, nous avons également intercalé une métalloporphyrine chargée négativement, la FeTSPP, dans trois HDL de compositions différentes : Zn2Al, Mg2Al et Zn2Cr et nous avons réalisé la première étude électrochimique avec ces matériaux.

A proteomic approach was applied to examine the protein composition of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in humans. LDL and HDL were isolated by density gradient ultracentrifugation, and proteins were separated with twodimensional gel electrophoresis (2-DE) and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and with amino acid sequencing using electrospray ionization tandem mass spectrometry. To improve the identification of low abundant proteins in silver stained 2-DE gels, 2,5-dihydroxybenzoic acid was used instead of α-cyano-4-hydroxycinnamic acid as matrix in the peptide mass fingerprinting procedure; this was demonstrated to give more matching peptide peaks, higher sequence coverage, and higher signal to noise ratio. Altogether 18 different proteins were demonstrated in LDL and/or HDL: three of these (calgranulin A, lysozyme C and transthyretin) have not been identified in LDL before. Apo C-II, apo C-III, apo E, apo A-I, apo A-IV, apo J, apo M, serum amyloid A-IV and α1-antitrypsin were found in both LDL and HDL, while apo B-100 (clone), calgranulin A, lysozyme C and transthyretin were found only in LDL, and apo A-II, apo C-I, and serum amyloid A only in HDL. Salivary α-amylase wass identified only in HDL2, and apo L and glycosylated apo A-II only in HDL3. Many of the proteins occurred in a number of isoforms: in all, 47 different isoform identities were demonstrated. A 2-DE mobility shift assay and deglycosylation experiments were used to demonstrate, for the first time, that apo M in LDL and HDL occurs in five isoforms; three that are both N-glycosylated and sialylated, one that is N-glycosylated but not sialylated and one that is neither N-glycosylated nor sialylated. LDL from obese subjects was found to contain more apo J, apo C-II, apo M, α1-antitrypsin and serum amyloid A-IV than LDL from controls,, and also more of an acidic isoform (pI/Mr; 5.2 / 23 100) of apo A-I. In addition, the new LDLassociated protein transthyretin, was found to be significantly more abundant in LDL from obese subjects. On the other hand, the amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3 / 23 100) were significantly less. Altogether, these findings (i) illustrate the power of 2-DE and mass spectrometry for detailed mapping of the proteins and their isoforms in human lipoproteins; (ii) demonstrate the presence of a number of new proteins in LDL (calgranulin A, lysozyme C and transthyretin); (iii) give precise biochemical clues to the polymorphism of apo M in LDL and HDL, and; (iv) indicate that obesity is associated with significant changes in the protein profile of LDL. It is concluded that new information on lipoproteins can easily be obtained through a proteomic approach, thus facilitating the development of a new proteomic field: lipoproteomics. Much further investigation in this field is warranted, particularly because newly discovered LDL and HDL proteins may play hitherto unknown role(s) in inflammatory reactions of the arterial wall and evolve as useful biomarkers in cardiovascular disease.

No description available.

Las proteínas de la familia de las anexinas se caracterizan su unión a los fosfolípidos de la bicapa lipídica de forma dependiente de calcio, a través de los motivos "repetición anexina" presentes en su secuencia. Esta capacidad ha sido descrita mediante ensayos in-vitro, aunque poco se conoce de la funcionalidad de este comportamiento en los sistemas biológicos. Además del calcio, otros componentes celulares, como el citoesqueleto de actina, podrían modular in-vivo la unión de anexinas a las membranas biológicas.En trabajos previos nuestro grupo determinó in-vivo como la anexina A6 era capaz de reclutarse hacia el compartimento endocítico en paralelo a la internalización de las lipoproteínas de baja densidad (LDL), uno de cuyos componentes mayoritarios es el colesterol. En el presente trabajo describimos como el colesterol por si sólo es capaz de inducir este reclutamiento, de forma independiente de calcio. En primer lugar, se determinó que existía una población de anexina A6, asociada a membranas (totales o endosomales) aisladas en ausencia de calcio, y que ésta era liberada al solubilizar el colesterol presente en la bicapa (con digitonina). Además, la acumulación de colesterol en el compartimento endocítico tardío (mediante el tratamiento de las células con la droga U18666A) inducía el reclutamiento in-vivo de anexina A6 hacia éstas membranas, proceso observado mediante fraccionamiento celular (aislamiento de endosomas e immunodetección por western blot) e immunocitoquímica (co-localizando, en estos endosomas a la anexina con el lípido no esterificado -marcado con filipina-). El incremento de la asociación colesterol-dependiente se observó asimismo para la anexina A6 añadida de forma exógena (a membranas aisladas carentes de anexina endógena) descartando la afectación del tráfico intracelular como causa del efecto observado. A continuación se determinó la funcionalidad derivada de la interacción de otro tipo de lipoproteínas involucradas en el metabolismo de colesterol, las HDL (de alta densidad), con la superficie de células CHO. Describimos la activación de Ras de forma subsiguiente a la unión de la partícula a la membrana. involucrando a uno de sus receptores más importantes, el Scavenger Receptor Type B-I (SR-BI). Al estudiar el efecto de la sobreexpresión de anexina A6 sobre esta señalización a partir de las HDL y su receptor SR-BI, en células CHO, se observó una disminución de la activación de Ras y Raf (pero no del resto de MAPKs). Por otro lado, la observación in-vivo (videomicroscopía) de la distribución celular de anexina A6-GFP sobreexpresada en células CHO, muestra como incrementos del calcio intracelular ejercen un reclutamiento masivo y muy rápido de la anexina A6 hacia la membrana plasmática (o dominios subyacentes a ésta), siguiendo un patrón similar al observado para algunas GAPs (GTPase activating proteins). Esta observación, así como la interacción ya descrita in-vitro entre la anexina A6 y p120GAP, sugiere la hipótesis que la translocación calcio-dependiente de la anexina hacia la membrana modularía la concentración de p120GAP, colaborando así en la desactivación de Ras. En este sentido, observamos como la sobreexpresión de anexina A6 induce, en células CHO, un incremento de la asociación de p120 GAP a fracciones de membrana enriquecidas en Ras.La caracterización de estos dominios de concentración de anexina A6 en la membrana indica que se trata de lipid rafts (dominios enriquecidos en colesterol) con presencia de citoesqueleto de actina. Postulamos que, en la célula viva, el calcio, el colesterol y el citoesqueleto de actina colaboran determinando la localización de la anexina en la membrana, desde donde actuaría como un modulador de la señalización a través de Ras. / The annexin family of proteins is characterized by the presence of a tandemly repeated calcium dependant - phospholipid binding domain, called "annexin repeat". However, other cell constituents, like actin cytoskeleton or cholesterol, have been proposed to play a role in annexin binding to biological membranes. In this sense, here we demonstrate in-vivo that cholesterol itself is able to modulate the intracellular distribution of membrane-associated annexin A6, in a calcium - independent manner. Concretely, cholesterol accumulation in the late endocytic compartment, upon treatment of the cells with "U18666A", is accompanied by annexin VI recruitment to this structures. Also, treatment of crude membrane extracts from early or late endosomes with digitonin, a cholesterol-sequestering agent, is able to displace a significant membrane-bound population of annexin VI, associated in a calcium independent manner. Finally, we demonstrate that this redistribution correlates with an increased binding of recombinant GST-annexin VI to cholesterol-enriched endocytic membranes in-vitro.After determining the role of cholesterol in mediating annexin VI recruitment to membranes, we aimed to characterize the signalling pathway activated upon HDL (one of the key lipoproteins in cholesterol metabolism) binding to the cell surface of CHO cells. Here we describe that HDL activates MAP kinases through the small GTPase Ras. We involve in this activation one of the most physiologically important receptors for HDL, the "Scavenger Receptor Type B-I" (SR-BI). We then analyzed this activation pathway in an annexin VI-overexpressing derived cell line, finding that annexin VI partially inhibits the signalling through the small GTPase Ras and blocks the subsequent activation of Raf. This diminished Ras activation is completely dependant on the presence of the enzyme PKC, and does not ultimately lead to a deficient MAPK activation. We also observed that annexin VI overexpression promoted the association of p120 GAP (one of the Ras inactivators and also an annexin VI-interacting protein) with Ras - enriched membrane fractions, suggesting an increased Ras - p120 GAP complex formation in these cells. In summary, we suggest that annexin VI is able to modulate signalling activation from the membranes and that, in this process, both calcium- and cholesterol-mediated membrane recruitment of annexin VI are involved.

Density gradient ultracentrifugation (DGU) is a powerful method for analyzing lipoprotein particles in great detail. It yields considerable amounts of information regarding the density distribution of these particles when coupled with fluorometric analysis and is an invaluable tool in determining their relative abundance. This union allows relationships between subclasses of lipoproteins to be established that gives researchers a more focused path to aid them in developing methods to predict the early onset of coronary artery disease (CAD). The research presented here focuses on the pairing of DGU with post-separatory techniques including matrix-assisted laser desorption mass spectrometry (MALDI-MS), liquid chromatography mass spectrometry (LC-MS), capillary electrophoresis (CE), isoelectric focusing (IEF) and apoptosis studies involving cell cultures. It is becoming clearer that cholesterol concentrations themselves do not provide sufficient data to assess the quality of cardiovascular health. As a result, research is becoming more focused on identifying better markers that may be indicative of development of CAD in a patient. Of specific interest is group of particles known as high density lipoproteins (HDL). Classically, this molecule is considered the “good cholesterol”, but literature from the last decade suggests that there may be atherogenic variants to this group. By utilizing DGU as a preparatory method for secondary analyses, new dimensions can be added to the density distribution analysis to allow a better determination of markers of cardiovascular health. The aim of this work is to utilize the principles involved with these various techniques to develop a comprehensive set of methods to aid in the detection of potential risk markers. In this study, the properties of metal ion complexes of EDTA as solute systems for analysis of lipoproteins by DGU are analyzed. We show that by varying the complexing ion and counter-ion of these metal-ion complexes, we gain the ability to control the separation of lipoprotein subclasses for subsequent analyses. Qualitative and quantitative data is presented that describes the analysis of different density regions of HDL for apolipoprotein content. Trends between control and atherogenic samples are also described and a clinical link between the biological activity of these regions and the chemical analysis is discussed.

Modern high-performance digital signal processing (DSP) applications face constantly increasing performance requirements and are becoming increasingly challenging to develop and work with. In DSP paradigm, many researchers see potential in achieving algorithm speed-up by employing Field Programmable Gate Arrays (FPGAs) – reconfigurable hardware with parallelism feature. However, developing applications for FPGAs incur particular challenges on the development flow. This work proposes a scalable hybrid DSP system for performing high-performance signal processing applications. The system employs hybrid CPU + FPGA architecture of commercially available, off-the-shelf (COTS) FPGAs and central processing units (CPU) of personal computers. In this work an example implementation of a multi-channel cross-correlator is investigated and delivered using a new development paradigm. The correlator is implemented on the XD1000 development system using a high-level FPGA programming tool – Impulse CoDeveloper. Analysis of DSP application development in a hybrid CPU+FPGA system employing the high-level programming tool Impulse C is presented. Potential of the selected tool to deliver algorithm speed-ups is investigated using reference multi-channel correlator software. Particular attention is devoted to input/output (I/O) implementation, which is considered one of the most challenging problems in FPGA design development. This work delivers an I/O framework based on PCI Express interface for the proposed high-performance scalable DSP system. Using Stratix II GX PCI Express Development Board from Altera Corporation, a scalable and flexible communication approach for the multi-channel correlator is delivered. This framework can be adapted to perform other high-performance streaming DSP applications. The outcomes of this work are a multi-channel correlator developed in a reconfigurable environment with new design methodology and I/O framework with software control application. The outcomes are used to demonstrate the potential of implementing DSP applications in hybrid CPU + FPGA architecture and to discuss existing challenges and suggest possible solutions.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 5 uppsatser.