Dissertations / Theses on the topic 'Plasmid Profile Analysis'

Neste estudo 60 cepas de Aeromonas, 15 A. hydrophila, 15 A. caviae, 15 A. sobria e 15 A. jandaei, isoladas de 5 diferentes pontos do reservatório de Guarapiranga, São Paulo, e previamente testada quanto à produção de fatores de virulência, acúmulo de fluído em alça ligada e hemólise em ágar sangue, foram submetidas a ribotipagem e a análise do perfil plasmidial. Cada cepa apresentou um perfil de ribotipagem diferente tendo-se observado para as espécies A. hydrophila e A. caviae a diferenciação em 3 agrupamentos, e A. sobria e A. jandaei dois agrupamentos cada. A análise do perfil plasmidial demonstrou que 13,4 por cento das A. hydrophila apresentaram um ou no máximo 2 plasmídios, enquanto 33,3 por cento das A. sobria e 53,3 por cento das A. jandaei apresentaram de 1 a 6 plasmídios para cada espécie; A. caviae não apresentou cepas contendo plasmídios. Não foi observada correlação entre a presença de plasmídios e a produção de fatores de virulência pelas cepas estudadas. A ribotipagem demonstrou haver um polimorfismo genômico dentro de uma mesma espécie de Aeromollas e, ainda, diferenciou cepas isoladas de um mesmo ponto de amostragem. Estas metodologias, ribotipagem e análise do perfil plasmidial, apresentam em geral características que são complementares, demonstrando ser ferramentas importantes a serem empregadas, tanto em estudos epidemiológicos como ecológicos.
In this work 60 Aeromonas strains, 15 A. hydrophila, 15 A. caviae, 15 A. sobria and 15 A. jandaei isolated from 5 different points of Guarapiranga Dam, São Paulo, and previously tested for virulence factors production (ileal loop assay and hemolysis on blood agar) were submitted to ribotyping and plasmidial profiles analysis. Each strain showed a different ribopattern and there were observed that for A. hydrophila and A. caviae each specie were grouped in 3 ribotypes, A. sobria and A. jandaei in 2 ribotype each. Plasmidial profiles analysis demonstrated that 13,4 per cent af A. hydrophila had at least one but no more than 2 plasmids, 33,3 per cent of A. sobria and 53,3 per cent af A. jandaei had from one to 6 plasmids each, and A. caviae didn\'t show to have any plasmids. There were not observed correlation between presence of plasmids and virulence factor production. Ribotyping showed that there are genomic polymorphism within the same Aeromonas specie and differentiate strains that were isolated from the same sample point, indicating that those methodologies have in general characteristics that are complementary and are important tools to be used either in epidemiological or ecological studies.

Dans ce travail, nous avons développé un système expert qui intègre les fonctions suivantes :i) l'identification des profils à partir des mesures sur les plasmas de tokamak, par les méthodes bayésiennes; ii) La prédiction des quantités reconstruites, selon les modèles choisis et; iii) une comparaison intelligente des résultats provenant des deux premières étapes. Ce système comprend une vérification systématique de la cohérence interne des quantités reconstruites, permet la validation automatique des modèles. Avec un modèle validé, il peut aider à la détection de nouveaux effets physique dans une expérience. Trois applications de ce système sont présentées dans ce travail. Le système expert peut détecter automatiquement les défauts dans la reconstruction des profils et fournir (lorsque la reconstruction est valide) des statistiques sur l'accord des modèles avec les données expérimentales, c'est à dire des informations sur la validité du modèle
In this work we developed an expert system that carries out in an integrated and fully automated way i) a reconstruction of plasma profiles from the measurements, using Bayesian analysis ii) a prediction of the reconstructed quantities, according to some models and iii) an intelligent comparison of the first two steps. This system includes systematic checking of the internal consistency of the reconstructed quantities, enables automated model validation and, if a well-validated model is used, can be applied to help detecting interesting new physics in an experiment. The work shows three applications of this quite general system. The expert system can successfully detect failures in the automated plasma reconstruction and provide (on successful reconstruction cases) statistics of agreement of the models with the experimental data, i.e. information on the model validity

Human disease due to food-borne pathogens remains a medical problem worldwide. Campylobacter jejuni is the most common cause of bacterial food-borne diarrhoeal disease, but the mechanisms by which it causes disease remain unclear. This bacterium is also the most common antecedent to the peripheral neuropathies Guillain-Barre syndrome (GBS) and Miller-Fisher syndrome (MFS). The main aim of this project was to compare cytotoxicity of isolates of C. jejuni towards mammalian cells in culture, to purify any cytotoxic activity and to compare strains by plasmid and proteomic analysis. Using the API Campy identification kit, 37 of the 39 strains were identified as C. jejuni jejuni serotype 1 or 2 with identification (ID) scores > 98.3%; and two strains were identified as C. jejuni jejuni 1 with ID scores of 88.3%. GOT (Gamma Glutamyl Transferase) test was the most important test to differentiate between C. jejuni jejuni 1 and 2 which showed negative and positive results, respectively. The cytotoxic activity against Vero and Caco-2 cells of some of the typed strains of C. jejuni was tested. Cytotoxic activity was clearly demonstrated using Vero and Caco-2 cells and these activities peaked after incubation of the cell culture with sample for 16h. Comparison of wild-type strains 81-176 and 11168 with mutants of these strains lacking cytolethal distending toxin (CDT) or putative haemolysin or phospholipase toxins indicated that the toxicity observed was not due to any of these proteins, as cytotoxicity was unaffected by their absence. Some isolates (e.g. C0L12) showed low cytotoxicity while others, especially C. jejuni 81-176, consistently produced high cytotoxin activity which was heat-labile and lethal to tissue culture cells. Cytotoxicity comparisons of inner and outer membrane preparations with a cytoplasmic fraction of C. jejuni strain 81-176 and C0L12 indicated that greatest activity was found in the inner membrane fraction. Proteins in the membrane preparation could not be easily fractionated and cell-free extracts of strains with low (COL12) and high (81-176) cytotoxic effects were chosen for further purification and characterisation studies. Although some differences were detected after DEAE- Sepharose fractionation and two-dimensional electrophoretic (2-DE) separation of proteins, no individual proteins could be clearly related to cytotoxicity. However, proteomic analysis did reveal some interesting properties of the C. jejuni strains. Of the proteins characterised by mass spectrometry after 2-DE, oxidoreductases were the most frequently identified in the virulent strain 81-176, but not in the type strain 11168. There was no apparent correlation between toxin production and clinical symptoms in patients from whom the strains were isolated. The study indicated that not all C. jejuni strains tested produced cytotoxin (s), but that cytotoxin producers seemed to be predominantly human strains. The presence of virB11 and tetO genes carried by plasmids was examined by PCR in the 39 C. jejuni isolates together with strains 81-176 and NCTC 11168. Detection rates for the virB11 and tetO genes in the clinical isolates were 28.2% and 12.82%, respectively.

Analysis of the shapes of diffraction peak profiles (DPPA) is a widely used method for characterising the microstructure of crystalline materials. The DPPA method can be used to determine details about a sample that include, the micro-strain, crystal size or dislocation cell size, dislocation density and arrangement, quantity of planar faults and dislocation slip system population.The main aim of this thesis is to evaluate the use of DPPA in studying the deformation of metals. The alloys studied are uni-axially deformed samples of nickel alloy, nickel-200, 304 and 316 stainless steel alloys and titanium alloys, Ti-6Al-4V and grade 2 CP-titanium.A number of DPPA methods were applied to these metals: a full-width method; a method that attributes size and strain broadening to the Lorentzian and Gaussian integral breadth of a Voigt; different forms of the variance method; the Williamson-Hall method; the alternative method; and variations of the Warren-Averbach method. It is found that in general the parameters calculated using the different methods qualitatively agree with the expectations and differences in the deformation of the different metals. For example, the dislocation density values found for all metals, are approximately the same as would be expected from TEM results on similar alloys. However, the meaning of the results are ambiguous, which makes it difficult to use them to characterise a metal. The most useful value that can be used to describe the state of a metal is the full-width. For a more detailed analysis the Warren-Averbach method in a particular form, the log format fitted to individual Fourier coefficients, is the most useful method.It was found that the shape of different diffraction peaks change in different texture components. These changes were found to be different for the different metals. A method to calculate the shape of diffraction peaks, in different texture components, using a polycrystal plasticity models was investigated. It was found that for FCC metals, the use of a Taylor model was able to qualitatively predict the changes in the shape of diffraction peaks, measured in different texture components. Whereas, for titanium alloys, a model which used the Schmid factor was able to qualitatively explain the changes. The differences in the FCC alloys was attributed to being due to differences in the stacking fault energy of the alloys. For nickel, which develops a heterogeneous cell structure, an additional term describing changes in the crystal size in different orientations is required. The differences between the titanium alloys were shown to be due the presence of twinning in CP-titanium and not in Ti-6Al-4V. This difference was thought to cause an additional broadening due to variations in intergranular strains in twinned and non-twinned regions. The use of polycrystal plasticity models, to explain the shape of diffraction peaks, raises questions as to the validity of some of the fundamental assumptions made in the use of most DPPA methods.

As the bottled water trend continues to rise across the nation, drinking water utilities have become more concerned with ensuring consumer satisfaction of their product. Although public water supplies are safeguarded by regulations, aesthetically unappealing taste-and-odor problems have led consumers to search for alternative water sources, such as bottled water or tap water processed by point-of-use filters. Consequently, taste-and-odor monitoring has become important to the drinking water industry. Because many utilities use chlorine to disinfect the water, chlorine odor often masks other more subtle odors that may eventually cause consumer complaints. As treated water travels from the water treatment plant to the consumer, chlorine residual diminishes and may reveal a water's naturally less-pleasing odors. Consequently, odor monitoring at the water treatment plant, where chlorine concentrations are at a peak, may not identify potential displeasing smells. Proper evaluation of these odor-causing substances requires that the chlorine odor first be eliminated before evaluating any remaining odors. Dechlorinating agents can remove chlorine, but some will produce other unwanted odors or even remove certain odorous compounds. This research describes the efficiency of several of these agents (ascorbic acid, hydrogen peroxide, oxalic acid, sodium nitrite, and sodium thiosulfate) in dechlorinating chlorinated solutions of the earthy-smelling compound geosmin and musty-smelling MIB. Interfering odors in reusable containers pose another problem in drinking water odor analysis. The most common odor-analysis methods (TON and FPA) involve the use of glass flasks, which often either develop chalky odors or have persistent lingering odors from previous evaluations. Furthermore the glass flasks break easily and are difficult to clean. This research also evaluates the suitability of four types of disposable plastic containers for odor analyses.
Master of Science

Temperature profiles of and combined thermal-mechanical induced strains for Carbon Fibre Reinforced Plastic (CFRP)-Aluminium multi-fastener double lap joints in a wingbox structure are examined. Two dimensional (2D) FE analyses for cases of full, empty, and half-full fuel tank scenarios are used to develop temperature profiles. The influence of conduction, convection, and radiation on temperature profiles is examined. Results show that the empty tank scenario produces the highest temperatures, with the joint region having the peak temperatures, and that convection and radiation must both be modelled in order to accurately estimate wingbox temperatures for the empty, and halffull tank scenarios. Analytical temperature prediction models, both at and away from the joint region, are developed for combined convection and radiation boundary conditions at both external surfaces of this unique finite geometry. For transient analyses, single and multiple layer models are designed using integral transforms and separation of variables, respectively. To show the joint region is critical in terms of induced strains, sequentially coupled thermal-stress analysis is performed using the resulting temperature profiles. Based on these results, and on the results of the temperature profiling, an experimental model is designed to study the effects of thermal and mechanical 1 oading on a threefastener double lap joint with CFRP'skin and aluminium laps. To fully explore the joint region, three dimensional (3D) FE results are compared with experimental data. Mechanical tensile stress in the elastic range is applied at room temperature (295K) and at an elevated temperature (373K). Increasing temperature alters the strain patterns among the fasteners and generally decreases' the peak radial strains at individual fasteners, but increases tangential strains. The effect of torque on the strain distribution in these multi-fastener double lap joints is examined by comparing finger-tight and operationally-tight (35Nm) torques at both temperatures. Increasing torque significantly reduces peak strains on individual fasteners and evens the strain distribution across the joint

Inspirée des techniques radars de sondage de l'ionosphère par réflexion d'onde, la réflectométrie est aujourd'hui fréquemment utilisée pour la mesure du profil de densité dans les plasmas de fusion. Ce travail s'intéresse à la réflectométrie de pulse, qui consiste à sonder le plasma à l'aide de signaux très courts présentant une largeur spectrale finie. Le profil de densité est alors déduit des mesures du temps d'aller-retour (ou temps de vol) de l'onde pour différentes fréquences sondes. Une meilleure compréhension de la propagation de tels signaux dans un plasma est nécessaire afin d'améliorer la détermination du profil de densité. Cela a motivé l'écriture d'un code numérique résolvant l'équation d'onde dépendante du temps. Une étude qualitative et quantitative du rôle des effets dispersifs sur la forme du pulse réfléchi est tout d'abord réalisée. Il est montré qu'une information supplémentaire sur la forme du profil de densité peut être déduite de la mesure de l'élargissement du pulse. En conséquence, les méthodes existantes de reconstruction de profil peuvent être améliorées. Des tests d'applicabilité à des cas réalistes rencontrés sur textor, ou la dispersion dans les guides d'onde est prise en compte, et sur tore supra, ou des données expérimentales sont traitées par une méthode de compression de pulse, sont effectues. Afin de comprendre l'origine des perturbations des mesures, l'effet de fluctuations de densité cohérentes sur la forme du signal réfléchi est étudié. Les simulations réalisées montrent que ces perturbations sont prédominantes pour des fluctuations au voisinage de la couche de coupure et donnent des variations de temps de vol comparables à celles observées expérimentalement. Ces résultats confirment que la présence de fluctuations de densité dans les plasmas de tokamak est la principale cause de perturbation des mesures de réflectométrie.

Le déplacement des raies de l'hélium hydrogenoïde émises dans les plasmas denses a été calculé en considérant l'effet des électrons libres et celui des ions perturbateurs sur l'émetteur. Le premier, déterminé par une méthode de potentiel auto-consistant, induit un déplacement vers le rouge alors que le second, calcule par perturbation, décale les raies vers le bleu. Deux méthodes de calcul de cette grandeur ont été développées : l'une permet d'évaluer le déplacement comme moment spectral du profil complet; l'autre, plus simple, séparé les deux contributions et peut s'appliquer à des états très excités. Les résultats théoriques obtenus sont en accord quantitatif avec les expériences les plus récentes.

Le profilage métabolomique global de matrices biologiques dans de larges séries d'échantillon est un enjeu majeur. Dans ce contexte, notre travail vise à développer des approches LC-HRMS et outils bioinformatiques pour les analyses métabolomique et lipidomique de larges cohortes. Dans un premier temps, nous avons développé puis évalué la pertinence de 4 méthodes LC-HRMS dans l'annotation du métabolome/lipidome sérique humain. Ainsi, une base de données spectrales a été implémentée à l'aide de spectres MS, MS/MS et les temps de rétention de composés de référence afin d'assurer l'annotation de jeux de données. La combinaison de méthodes RP, HILIC et PFPP-HRMS a permis l'identification de 266 métabolites et 706 espèces lipidiques sériques répartis sur 20 et 24 classes chimiques respectivement dont 27% d'espèces isomères. Ces outils ont été appliqués, dans un second temps, à la stratification de 78 patients diabétiques. Outre le syndrome métabolique marqué (perturbation du métabolisme énergétique), nos analyses ont montré l'impact délétère de facteurs physiologiques confondants -âge et IMC-. Nous en avons évalué l'influence sur une cohorte de 227 salariés du CEA. Les empreintes lipidomiques sont robustes, néanmoins l'impact de l'IMC est marqué pour les lipides neutres. L'effet du genre démontre un catabolisme masculin important. L'effet de l'âge se manifeste par des activités enzymatiques altérées. Ces études combinent une analyse globale métabolomique et lipidomique des mêmes échantillons humains. Elles visent à construire une base de données relationnelle incluant données spectrales et biologiques servant à la caractérisation de biomarqueurs dans le cas d'études cliniques
Global metabolomic profiling of biological media in large sample sets is a major challenge. In this context, our work aims to develop LC-HRMS approaches and data mining tools for metabolomics and lipidomics analysis of large cohorts. We have first developed and evaluated the reliability of four LC-HRMS methods in the annotation of human serum metabolome and lipidome. Thus, spectral database was implemented using MS spectra, MS/MS and retention times of reference compounds to further ensure datasets annotation. The combination of RP, PFPP and HILIC-HRMS methods allowed identification of 266 metabolites and 706 lipid species in human serum over 20 to 24 chemical classes respectively including 27% of isomeric species. These analytical tools were then applied for the stratification of 78 diabetic patients. Unsurprisingly, we highlighted a metabolic syndrome (energy metabolism disruption), moreover our analyses have shown the deleterious impact of confounding physiological factors on diabetes biomarker discovery –age and BMI-. We finally evaluated their influence on a cohort of 227 CEA employees. Lipidomic fingerprints are robust, however BMI impact is marked for neutral lipids. Gender effect shows significant male catabolism and age altered enzyme activities. These studies combine an overall metabolomics and lipidomics analyses of the same human samples. They aim to build up a relational database including spectral and biological data for biomarker characterization in clinical studies

Les récents travaux du laboratoire de Thèse ont montré une pluralité et une diversité de Ca2+ATPases dont les expressions sont modulées au cours de la mégacaryocytopoïèse, suggérant que ces protéines puissent être des nouveaux marqueurs de la maturation plaquettaire. De fait un défaut d'expression de ces Ca²+ATPases vient d'être montré dans les plaquettes de patients atteints d'une thrombocytopénie sévère. A la lumière de ces résultats, nous avons étudié l'expression de ces Ca²+ATPases dans les plaquettes pathologiques de patients atteints de Diabète (type I & II) ou d'Hypertension et ainsi confirmé un défaut de maturation plaquettaire chez ces patients. Ensuite, nous avons étudié les effets de certaines Ca²+ATPases recombinantes transfectées dans les cellules HEK-293, afin de comprendre comment les Ca²+ATPases pouvaient intervenir dans la mégacaryocytopoïèse. Ces travaux ont ainsi pu montrer pour la première fois que SERCA3 peut induire l'activation des voies du stress du Réticulum Endoplasmique (RE) aboutissant à l'activation du facteur de transcription XBP-1 et les caspases-3 & 9, et l'augmentation de l'expression de la protéine chaperonne GRP78 (BIP). Ces résultats suggèrent donc une relation étroite entre la surexpression de SERCA3 et l'activation du stress du RE conduisant à l'activation des voies proapoptotiques observées au cours de la phase précoce de la maturation plaquettaire. En conclusion, la réalisation de l'ensemble de ce programme de recherches, apportera des réponses concernant la nature précise du rôle des Ca²+ATPases durant la mégacaryocytopoïèse normale et pathologique. Ce projet devrait ainsi permettre d'identifier de nouvelles cibles anti-thrombotiques
Megakaryocytopoiesis gives rise to circulating platelets through proliferation and differentiation of the megakaryocyte cells (MKs). Calcium signalling plays a key role in this process. Among the structures involved in Ca²+ signalling are Ca²+ATPases able to deplete free Ca2+ ions from the cytosol. Two families of such proteins are defined depending on the membranes they are inserted in: the Plasma Membrane Ca²+ATPases (PMCAs) and the Sarco/Endoplasmic Reticulum Ca²+ATPases (SERCAs). Previous works showed that expression of SERCA3 increased, while PMCA4b decreased in platelets compared to different human MK cell lines (MEG 01, UT7). Ca²+ATPases can sign an abnormality in platelet maturation in patients with type I (T1D) diabetes, type II (T2D) diabetes and hypertension. Pathological platelets showed slight modulation of the expression of SERCA3 proteins coupled to a net up-regulation of PMCA4b. Finally, we postulated for a functional role of SERCAS in apoptosis induction during early stage leading to platelet formation. In this purpose, we used the overexpression model of SERCAS in HEK-293 cell lines. Further analysis indicated that SERCAS overexpression resulted in the activation of both caspase and ER stress signalling pathways (including an increased GRP78 expression and processing of XBP-1 mRNA). Taken together, our results suggest that platelet imbalance between SERCAS and PMCA-type Ca²+ATPases is a new marker of abnormal magakaryocytopoiesis in patients with Diabetes and Hypertension. In addition, up-regulation of SERCAS expression is correlated with ER stress induction during early phase of platelet maturation

Doutoramento em Química
This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.
A presente tese reporta a aplicação da metabolómica ao estudo de tecidos e biofluidos humanos (plasma sanguíneo e urina), com o intuito de caracterizar a assinatura metabólica do cancro pulmonar primário. No Capítulo 1, apresenta-se uma breve introdução sobre a epidemiologia e a patogénese deste tipo de cancro, bem como um sumário das principais alterações metabólicas tipicamente associadas ao cancro em geral. Descreve-se ainda a abordagem metabolómica, nomeadamente os métodos analíticos e estatísticos utilizados, assim como o estado da arte da sua aplicação em estudos clínicos do cancro do pulmão. No Capítulo 2, apresentam-se os detalhes experimentais deste trabalho, no que diz respeito ao grupo de indivíduos envolvidos, à colheita e análise das amostras e ao posterior tratamento dos dados. O Capítulo 3 descreve a caracterização metabólica de tecidos do pulmão (de 56 doentes) por espetroscopia de Ressonância Magnética Nuclear (RMN) de alta resolução com rotação no ângulo mágico. Após a otimização cuidada das condições de aquisição e a identificação detalhada dos sinais espetrais (mais de 50 metabolitos identificados), os perfis metabólicos dos tumores e dos tecidos adjacentes não envolvidos (controlos) foram comparados por análise multivariada, tendo sido discriminados com uma exatidão de 97%. Os metabolitos que mais significativamente contribuíram para esta diferenciação foram: glucose e acetato (diminuídos nos tumores), lactato, alanina, glutamato, GSH, taurina, creatina, fosfocolina, glicerofosfocolina, fosfoetanolamina, nucleótidos de uracilo e péptidos (aumentados nos tumores). Algumas destas variações corroboraram alterações típicas do metabolismo do cancro (e.g., glicólise e glutaminólise aumentadas), enquanto outras sugeriram novas pistas sobre a possível relevância de processos como a proteção antioxidante e a degradação proteica. Um outro resultado novo e importante descrito neste capítulo foi a dependência da assinatura metabólica em relação ao tipo histológico do tumor. Enquanto as principais alterações observadas nos adenocarcinomas (AdC) se relacionaram com o metabolismo fosfolipídico e proteico, os carcinomas de células escamosas (SqCC) apresentaram perfis glicolíticos e glutaminolíticos mais pronunciados, sendo possível construir um modelo válido para a discriminação destes subtipos. No Capítulo 4, apresenta-se o estudo metabolómico por RMN de plasma sanguíneo de mais de 100 doentes e quase 100 controlos saudáveis, do qual resultou um modelo multivariado com uma taxa de classificação de 87%. A distinção entre os grupos foi feita essencialmente com base nos níveis de lactato, piruvato, acetoacetato, lipoproteínas LDL+VLDL e glicoproteínas (aumentados nos doentes), juntamente com os níveis de glutamina, histidina, valina, metanol, lipoproteínas HDL e dois compostos não identificados (diminuídos nos doentes). Estas variações foram detetadas desde os estádios iniciais da doença e a magnitude de algumas delas dependeu do tipo histológico, embora não permitindo discriminar AdC de SqCC. Para além disso, mostra-se neste capítulo que o desequilíbrio dos grupos controlo e cancro em termos da idade dos indivíduos poderá ter alguma influência nos resultados, e apresenta-se uma tentativa exploratória de validação externa, que resultou numa taxa de classificação de 85%. O estudo por RMN do perfil metabólico da urina dos doentes com cancro do pulmão e dos controlos é apresentado no Capítulo 5. Comparativamente ao plasma, o modelo construído com os perfis urinários apresentou uma taxa de classificação superior (97%). Após uma avaliação cuidada da possível influência do género, idade e hábitos tabágicos, um conjunto de 19 metabolitos foi proposto como estando relacionado com a doença (incluindo 3 compostos desconhecidos e 6 parcialmente identificados como metabolitos N-acetilados). Tal como no caso do plasma, estas variações foram detetadas em doentes no estádio inicial e mostraram alguma dependência em relação ao tipo histológico, obtendo-se um modelo válido para a discriminação AdC vs. SqCC, ainda que com um poder preditivo modesto. Para além disso, o teste preliminar de validação externa revelou 100% de sensibilidade e 90% de especificidade, o que é um resultado bastante promissor em termos da potencial utilização dos perfis urinários em aplicações clínicas futuras. No Capitulo 6, descreve-se a caracterização dos perfis metabólicos da urina (de um subgrupo de indivíduos) por cromatografia líquida de ultra-eficiência acoplada a espetrometria de massa (UPLC-MS). Embora não avançando muito na identificação estrutural de possíveis marcadores, este estudo reforçou o valor diagnóstico da urina, já que os modelos multivariados resultantes apresentaram taxa de classificação e poder preditivo elevados. Finalmente, no Capítulo 7, apresentam-se as principais conclusões deste trabalho, realçando o contributo da metabolómica integrada de tecidos e biofluidos para a compreensão do metabolismo alterado do cancro do pulmão e para a deteção de novos perfis marcadores com valor diagnóstico.

Les monnaies anciennes à base d'argent présentent fréquemment des variations de composition en fonction de la profondeur qui apparaissent principalement suite à leur enfouissement. Une analyse juste de ces échantillons impose de s'affranchir de cette couche superficielle perturbée pour caractériser l'alliage qui n'a pas été affecté par ces modifications. L'analyse par spectrométrie de masse couplée à un plasma inductif avec prélèvement par ablation laser (LA-ICP-MS) permet une approche en profils de concentration qui peut rendre compte des variations de teneur selon la profondeur. Un protocole analytique par LA-ICP-MS a été développé spécifiquement pour les monnaies d'argent, qui permet de visualiser la couche de surface perturbée pour caractériser l'alliage sain lors de l'analyse des éléments majeurs. Les caractéristiques intrinsèques de la méthode LA-ICP-MS ont bénéficié au dosage des éléments mineurs et traces : le nombre d'éléments dosés et les limites de détection très basses permettent de caractériser l'ensemble des constituants de ces alliages. Des méthodes d'analyse complémentaires ont été mises en œuvre pour valider le protocole analytique proposé.
Les monnaies d'argent des premiers souverains carolingiens sont les premières à avoir bénéficié des avancées analytiques permises par l'application de la méthode LA-ICP-MS aux échantillons de ce type. L'évolution du titre d'argent de pièces frappées entre 751 et 864 a été étudiée, et corrélée aux données numismatiques et historiques. Des spécificités régionales ou locales du point de vue de la pureté des alliages monétaires ont été observées. L'examen des teneurs en éléments mineurs et traces caractéristiques, susceptibles de témoigner de mélanges ou de mouvements de stocks de métaux précieux, a mis en évidence des particularités de l'argent frappé par certains ateliers monétaires, en particulier celui de Venise.

博士
國立中興大學
食品科學系
93
Salmonella enterica serovar Choleraesuis is one of the major bacterial pathogens for swine. The Salmonella serovar has been often isolated from diarrheagenic and septicemia swine in Taiwan. In this study, pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis were used for the subtyping of 95 S. Choleraesuis strains isolated from swine with diarrhea and systemic infection during 2000~2002. The swine were grown in the swine fields in southern Taiwan. For PFGE, XbaI, AvrⅡ, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 95 Salmonella strains, 86 PFGE patterns combinations were found. Of them, pattern X1A1S1N1, was the major subtype since 11 strains belong to this pattern. Strains of this pattern may be the most epidemic strains. On the other hand, RAPD analysis generated 74 patterns for these 95 isolated strains. Comparison of the PFGE and RAPD patterns, RAPD could subdivide strains within a PFGE type. Thus, RAPD was a better discriminative tool for subtyping of S. Choleraesuis, if suitable primer was selected for typing. Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Since conventional method for the detection of this Salmonella serovar may take 3-5 days, in this study, PCR method was developed. By comparison of the sequence of phase-1 flagellin (fliC) gene of S. Choleraesuis with those of other Salmonella serovars and the sequences of other bacteria spp. available in GenBank, two PCR primers, i.e, flinC-F and flinC-R, were designed. Using these primers, all the 97 S. Choleraesuis strains assayed generated expected PCR products with molecular weight equal to 963 bp. Except of S. Paratyphi C, Salmonella isolates other than S. Choleraesuis, and non-Salmonella isolates including strains of Enterobacteriaceae, all generated negative PCR results. S. Paratyphi C could be differentiated from S. Choleraesuis through the use of primers designed from viaB gene. When S. Choleraesuis in swine stool, pork, liver, feed, and human whole-blood samples were assayed, with a pre-enrichment step, as low as 100 CFU per g or ml of the sample could be detected. Antimicrobial susceptibility test was also preformed for the 95 S. Choleraesuis strains collected from the swine field in Southern Taiwan. A total of 18 antibiotics were tested. Results showed that 46 antibiograms were obtained. All these strains were multidrug resistant strains. PCR was used to screen the gene encoding the resistance to ampicillin (TEM, and PSE group), streptomycin-spectinomycin (aadA), kanamycin (aphA), sulfadiazine (sulI, and sul3), trimethoprim (dfrA12), tetracycline (tetA), streptomycin (strA, and strB), chloramphenicol (cmlA), gentamycin (aac(3)Ⅳ), fluoroquinolone (gyrA, parC, and parE), and that class I integron. Results showed that 32.6% of the 95 strains of Salmonella Choleraesuis are with the class I integron. The presense of antibiotic resistance genes on integron cassette was further analyzed by PCR mapping. Four types of patterns were detected. They were dfrA12-aadA2, aadA5-aadA2, dfrA12-aad2-su1l, and dfrA12-aadA5. Our data demonstrated that the problem of antibiotic resistance in Salmonella Choleraesuis need strains to be paid with attention. Certain Salmonella serovars have virulence plasmid, including serovars Typhimurium, Choleraesuis, Dublin, and Enteritidis. In this study, plasmid profile analysis of 95 swine S. Choleraesuis isolates showed that S. Choleraesuis isolates harbored 1~3 large virulence plasmids. These plasmids sizes ranging from 40 kb to 190 kb, a total of 14 types plasmid profiles were obtained. Forty-two strains of S. Choleraesuis belong to a major type which containing 50 kb and 90 kb plasmids. When using spvR gene specific primers were used for PCR, all the 95 swine isolates generated the expected PCR product. The results indicted that spvR gene may be present in plasmid with size large than 50 kb. The variation of plasmids sizes may be caused by high frequency gene exchange. Since microarray method has become an important technology for detection of mass genes. This method was rapid, highly specific, and sensitive. To develop a microarray method for detecting different antibiotic resistant genes, identification of bacterial strains and their virulence genes, in this study, a total of 32 gene specific PCR products were printed on the microarray, including the products from 22 antibiotic resistant genes, 3 virulence genes, 5 specific genes for Salmonella, and the 16S rDNA gene for positive control. Genomic DNAs from swine isolates were fluorescently labeled for array hybridization. Three different multi-drug resistant S. Choleraesuis isolates SCV28, SCV62, and SCV27a were tested. The results showed that all tested strains were detected by the microarray as expected. Our results suggested that our microarray detected the antibiotic resistant genes, virulence genes, and specific genes and may be used as a platform for S. Choleraesuis isolates. Furthermore, this microarray might also be useful for the detection of antibiotic resistant genes for other serovar Salmonella or closely related bacteria. Salmonella enterica serovar Choleraesuis was one of the highly invasive serotypes to animal and human. In this study, we attempt to elucidate the relationship between their molecular subtypes and their invasiveness to different epithelial celltypes. Meanwhile, the invasiveness for different serotypes of Salmonella were also compared. Twenty-three strains representing randomly selected PFGE subtypes were studied for their invasiveness into Intestine 407 cells. Four strains were tested of the invasiveness to different celltypes. On the other hand, serovar Typhimurium, Derby, Enteritidis, Paratyphi C, and Choleraesuis strains were compared for their invasiveness into Intestine 407 cells and LLC-PK1 cells. The results showed that there were no significant differences in invasiveness between different molecular subtypes. The results also showed that Intestine 407 cells, HEp-2 cells, and LLC-PK1 cells were variable in susceptibility to Salmonella invasion. Comparison with different serotypes of Salmonella for invasion, S. Choleraesuis strain showed higher invasion rate to Intestine 407 cell, but low invasion rate to LLC-PK1 cells.

碩士
大葉大學
生物產業科技學系
93
ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens infected in community hospitals, and the incidence of MRSA strains is increasing and becomes a common problem among the hospitals in Taiwan recently. In this study, the polymer chain reaction (PCR) was used to detect the mecA resistant gene, and the Sma I restriction enzyme was used to digest the chromosomal DNA of MRSA. In addition, the plasmid profile and the pulsed field gel electrophoresis (PFGE) analysis were used to investigate the distribution of the toxin types for MRSA strains from Veterinary General Hospital (VGH) in Taichung and Center for Disease Control, Taiwan, ROC. (TCDC), and trace the MRSA infection in the hospitals. The PCR results showed that 45 (59.21 %) clinical strains from VGH and 23 (16.3 %) human S. aureus strains from food-poisoning cases provided by TCDC possessed mecA resistant gene. The ratio of MRSA strains from hospitals was higher than that from food-poisoning cases. As for the distribution for enterotoxin types, it was found that the major enterotoxin types for the strains from hospitals and food-poisoning cases were the enterotoxin A and B, respectively. From the results of the plasmid profiles for MRSA strains, totally 29 distinct plasmid types were found. Of them, no co-shared types were found in the isolates from hospitals and food-poisoning cases. The PFGE results showed that 18 PFGE types were found in the 68 MRSA strains from hospitals and food-poisoning cases in 2003, and the major types for the strains from hospitals were different from those for the strains from food-poisoning cases. In addition, the clinical strains isolated between 1998 and 2003 were found high similarity in PFGE type. The results of this study reveal that the MRSA with same PFGE type still prevail in hospitals.

by Ho, Wai Fun.
Thesis (M.Sc.)--Chinese University of Hong Kong, 1994.
Includes bibliographical references (leaves 79-87).
LIST OF TABLES --- p.iv
LIST OF FIGURES --- p.vi
ACKNOWLEDGEMENTS --- p.vii
ABSTRACT --- p.viii
Chapter 1. --- INTRODUCTION --- p.1
Chapter 1.1 --- METABOLIC DERANGEMENTS AND CACHEXIA IN CANCER --- p.1
Chapter 1.2 --- PROTEIN METABOLISM IN MALIGNANCY --- p.4
Chapter 1.3 --- REVIEW OF REPORTS ON AMINO ACID DISTURBANCES IN MALIGNANCY --- p.5
Chapter 1.4 --- AMINO ACID ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY --- p.10
Chapter 1.4.1 --- Amino Acid Analysis by Ion-Exchange HPLC --- p.11
Chapter 1.4.2 --- Amino Acid Analysis by Reversed-Phase HPLC --- p.13
Chapter 1.4.3 --- Derivatizing Agents --- p.15
Chapter 1.5 --- CHOICE OF CANCER PATIENTS AND METHODOLOGY FOR THIS STUDY --- p.19
Chapter 1.5.1 --- Choice of Cancer Patients --- p.19
Chapter 1.5.2 --- Methodology Chosen and Its Principle --- p.20
Chapter 2. --- OBJECTIVES --- p.23
Chapter 3. --- MATERIALS AND METHODS --- p.24
Chapter 3.1 --- STUDY SUBJECTS --- p.24
Chapter 3.1.1 --- Patients --- p.24
Chapter 3.1.2 --- Control Subjects --- p.25
Chapter 3.2 --- CLINICAL FEATURES --- p.25
Chapter 3.3 --- BLOOD COLLECTION --- p.25
Chapter 3.4 --- GENERAL BIOCHEMICAL TESTS --- p.26
Chapter 3.5 --- PLASMA AMINO ACID ANALYSIS BY HPLC --- p.26
Chapter 3.5.1 --- Apparatus --- p.26
Chapter 3.5.2 --- Reagents --- p.27
Chapter 3.5.3 --- Reagent Preparation --- p.28
Chapter 3.5.3.1 --- Mobile phase --- p.28
Chapter 3.5.3.2 --- Derivatizing reagent --- p.29
Chapter 3.5.4 --- Standard Preparation --- p.29
Chapter 3.5.4.1 --- Internal standard solution --- p.29
Chapter 3.5.4.2 --- Composite standard solution --- p.30
Chapter 3.5.4.3 --- Composite standard-internal standard mixture --- p.32
Chapter 3.5.5 --- Sample Preparation --- p.32
Chapter 3.5.5.1 --- Protein removal --- p.32
Chapter 3.5.5.2 --- Addition of internal standard --- p.32
Chapter 3.5.6 --- Preparation of Samples for the WISP Sample Processor --- p.33
Chapter 3.5.7 --- Sample Queue for the WISP Sample Processor --- p.33
Chapter 3.5.8 --- Automated Derivatization Procedure --- p.36
Chapter 3.5.9 --- Chromatographic Conditions --- p.36
Chapter 3.6 --- STATISTICAL STUDIES --- p.38
Chapter 4. --- RESULTS --- p.40
Chapter 4.1 --- ANALYTICAL PERFORMANCES --- p.40
Chapter 4.1.1 --- Chromatograms --- p.40
Chapter 4.1.2 --- Precision --- p.47
Chapter 4.1.3 --- Linearity --- p.47
Chapter 4.1.4 --- Analytical Recovery --- p.51
Chapter 4.2 --- DATA DISTRIBUTION STUDIES --- p.53
Chapter 4.3 --- PATIENTS' ANTHROPOMETRIC DATA AND BLOOD BIOCHEMISTRY --- p.55
Chapter 4.4 --- FREE PLASMA AMINO ACID CONCENTRATIONS IN NORMAL CONTROLS --- p.59
Chapter 4.5 --- FREE PLASMA AMINO ACID CONCENTRATIONS IN CANCER PATIENTS --- p.59
Chapter 5. --- DISCUSSION --- p.68
Chapter 5.1 --- METHOD ESTABLISHMENT --- p.68
Chapter 5.2 --- NORMAL CONTROLS --- p.68
Chapter 5.3 --- CANCER PATIENTS --- p.71
Chapter 5.3.1 --- Nasopharyngeal Cancer --- p.71
Chapter 5.3.2 --- Lung Cancer --- p.71
Chapter 5.3.3 --- Breast Cancer --- p.72
Chapter 5.3.4 --- Colorectal Cancer --- p.74
Chapter 5.4 --- SUMMARY OF THE PLASMA AMINO ACID PROFILES IN CANCER --- p.75
Chapter 5.5 --- FURTHER STUDIES --- p.76
Chapter 6. --- CONCLUSION --- p.78
Chapter 7. --- REFERENCES --- p.79

碩士
國立高雄應用科技大學
機械與精密工程研究所
103
This paper mainly discuss the impact of gear profile shifted design and material temperature coefficient for the load-bearing capacity of plastic gears and plastic gears deformation. Using finite element analysis software MSC.Marc, to simulate tooth engagement for different amounts of profile shifted. Discuss stress and strain of gears in several different temperature environment, and summarized results of the analysis to predict the plastic gear damage scenarios. Nonlinear plastic material used in the simulation is produced by the Ticona company's Celcon-M90.During the study, the first to use MatLab to calculate Litvin toothed algorithms with a number of different transfer coefficient, and then simulate and discuss the dedendum stress and deformation and interference problems for plastic gear in -40 ℃, 23 ℃, 40 ℃, 60 ℃, 80 ℃, 100 ℃, 120 ℃ operating temperature environment. The simulation results show that the use of multiple teeth or complete gear pair module for contact analysis can more accurately calculate the interference situation due to deformation caused by plastic gears. Increased profile shifted can effectively increase the ability to withstand the load of gear. When the gear to withstand the same load situation, because of the increased profile shifted changed tooth, so reducing the amount of deformation of gear and reduce interference. And the change in temperature will affect the strength of the plastic material, but do not have much effect on the interference position. On this simulation of plastic materials, increase profile shifted that can non-linear enhance the ability to withstand load. Before most of plastic gear case reaching the yield or cracks, that occur deformation interference. And the 0.7 profile shifted gear case cracks appeared before it interferes.

Phenylketonuria (PKU) is the most prevalent innate error in amino acid metabolism. PKU is characterized by the deficiency of a phenylalanine (Phe) metabolism enzyme, phenylalanine hydroxylase, which is responsible for the conversion of Phe into tyrosine (Tyr). Deficiency of this enzyme causes the accumulation of Phe, which may cause severe cognitive impairment. Early diagnosis through neonatal screening and a rapid therapeutic implementation are essential to prevent irreversible sequelae. Therapeutic approach for PKU is based on strict control of Phe intake through a lifelong diet restricted in proteinrich food. High dietary restrictions can lead to imbalances in other nutrients, notably lipids. Previous plasma/serum and red blood cells studies of PKU patients with Phe-restricted diet revealed lipid changes, namely in lipoprotein’s components and fatty acid profile. Despite the changes already reported, further studies are needed to understand in detail the changes in the lipid profile of PKU patients, particularly at the level of phospholipids, which also have important signalling and regulatory functions. Thus, the main aim of this work was to study the phenylketonuric phospholipidome in order to identify the occurrence of changes in lipid profile. To this end, the plasma phospholipidenriched extracts obtained from PKU and healthy children (control group, CT) were analysed by HILIC-MS/MS and GC-MS. Using this approach, 187 lipid species belonging to 9 different phospholipid classes and 3 ceramides were identified. Principal component analysis of lipid species dataset showed a distinction between PKU and CT groups. Univariate analysis revealed that 146 phospholipid species were significantly different between groups. The species with major variation included phosphatidylcholines (PC), bearing polyunsaturated fatty acids (PUFA), which were more abundant in the PKU group. For patients with diet supplemented with PUFA, the higher level of PUFA-containing lipid species may be related with such supplementation. This study was the first report comparing the plasma polar lipidome of PKU and healthy children, highlighting that the phospholipidome of PKU children is significantly altered when compared with healthy children. In conclusion, the lipidomic approach used in this work allowed the analysis and identification of the phospholipid profile of PKU and healthy children. However, further studies with larger cohorts are needed to clarify if the changes identified are specific to the phenylketonuric children, namely those with PUFA supplementation.
A fenilcetonúria (PKU) é o erro inato mais prevalente no metabolismo de aminoácidos. A PKU é caracterizada pela deficiência de uma enzima do metabolismo da fenilalanina (Phe), a fenilalanina hidroxilase, a qual é responsável pela conversão de Phe em tirosina (Tyr). A deficiência desta enzima causa a acumulação de Phe, podendo causar um comprometimento cognitivo grave. Assim, o diagnóstico precoce através do rastreio neonatal e uma rápida implementação do tratamento são essenciais para evitar sequelas irreversíveis. A abordagem terapêutica usada na PKU baseia-se no controlo rigoroso do aporte de Phe através uma dieta restrita em alimentos ricos em proteínas que deve ser mantida durante toda a vida. As elevadas restrições na dieta podem levar a desequilíbrios noutros nutrientes, nomeadamente em lípidos. Estudos anteriores com plasma/soro e glóbulos vermelhos de pacientes com PKU com dieta restrita em Phe revelaram alterações lipídicas, nomeadamente nos componentes de lipoproteínas e no perfil de ácidos gordos. Apesar das alterações já reportadas, são necessários mais estudos para compreender em detalhe as alterações no perfil lipídico de pacientes com PKU, nomeadamente a nível dos fosfolípidos, os quais também têm importantes funções de sinalização e regulação. Assim, o principal objetivo deste trabalho foi estudar o fosfolipidoma de fenilcetonúricos, a fim de identificar a ocorrência de alterações no perfil lipídico. Para tal, extratos plasmáticos enriquecidos em fosfolípidos obtidos a partir de crianças com PKU e de crianças saudáveis (grupo controlo, CT) foram analisados por HILICMS/MS e GC-MS. Deste modo, foram identificadas 187 espécies lipídicas pertencentes a 9 classes diferentes de fosfolípidos e 3 ceramidas. A análise de componentes principais do conjunto de dados das espécies lipídicas mostrou uma distinção entre os grupos PKU e CT. A análise univariada revelou que 146 espécies de fosfolípidos foram significativamente diferentes entre os dois grupos. As espécies com maior variação incluíram principalmente fosfatidilcolinas, contendo ácidos gordos polinsaturados (PUFA), estando estas mais abundantes no grupo PKU. Para doentes com uma dieta suplementada com PUFA, os níveis elevados de espécies contendo PUFA podem estar relacionados com a suplementação. Este estudo foi o primeiro a comparar o lipidoma polar plasmático de crianças com PKU e de crianças saudáveis, realçando que o fosfolipidoma de crianças com PKU está significativamente alterado quando comparado com crianças saudáveis. Em suma, a abordagem lipidómica usada neste estudo permitiu a análise e identificação do perfil fosfolipídico de crianças com PKU e de crianças saudáveis. Contudo, mais estudos são necessários para esclarecer se alterações identificadas são específicas de crianças fenilcetonúricas, nomeadamente daquelas que fazem suplementação com PUFA.
Mestrado em Bioquímica

This paper intends to study the acceptance of sneakers made out of recycled plastic in the Portuguese market, through the assessment of main drivers and barriers to consumption, as well as of potential consumer profiles. For that purpose, an analysis of the impact of perceived product benefits, risks, brand name, and individual characteristics on purchase intention and willingness to pay was conducted. An online questionnaire with an experiment section allowed the collection of 200 valid answers for further research and hypotheses testing. The main findings showed that anticipated conscience, environmental benefits, and brand name positively impacted the consumption of sneakers manufactured with recycled plastic. While the risk of contamination and low quality negatively influenced consumption decisions. Moreover, well-established brands seemed to be able to reduce perceived risks, such as low quality and performance, to a higher extent than non-well-established eco-brands. Finally, individuals with high levels of environmental concern and for whom it is important to signal green consumption, were more prone to purchase sneakers made out of recycled plastic than others. The present research adds to the literature regarding green consumption behaviors and recycled plastic products, in specific. Moreover, besides studying an innovative solution to tackle the plastic environmental issue, it provides relevant insights to incentivize green consumption and for businesses to establish a source of competitive advantage.
Este estudo tem como objetivo avaliar a aceitação de ténis fabricados com plástico reciclado no mercado português, através da análise dos principais impulsionadores e barreiras ao consumo, bem como de potenciais perfis de consumidores. Para este propósito, foi estudado o impacto dos benefícios e dos riscos do produto, marca, e características individuais na intenção de compra, bem como a quantia que o comprador se encontra disposto a pagar. A aplicação de um questionário online permitiu a recolha de 200 respostas válidas para futura investigação. Os resultados demonstraram que a consciência antecipada, os benefícios ambientais, e a marca tiveram uma influência positiva no consumo deste tipo de ténis. Enquanto que os riscos de contaminação e a fraca qualidade influenciaram negativamente as decisões de consumo. Adicionalmente, marcas bem estabelecidas foram capazes de reduzir a perceção de riscos, tais como o de fraca qualidade e performance de forma mais explícita do que marcas não estabelecidas de carácter ambiental. Por fim, indivíduos com elevada preocupação ambiental e para os quais sinalizar o consumo verde é importante, demonstraram maior intenção de compra do que os restantes. Este estudo complementa a literatura sobre comportamentos de consumo verde e de produtos de plástico reciclado, em específico. Para além de estudar uma solução inovadora, que pretende lidar com o plástico enquanto problema ambiental, disponibiliza conhecimentos relevantes para o incentivo do consumo verde e para o estabelecimento de uma vantagem competitiva.

Dissertação de Mestrado Integrado em Engenharia Mecânica apresentada à Faculdade de Ciências e Tecnologia
A eficácia da simulação numérica de um processo de estampagem está intrinsecamente ligada ao modo como o contacto com atrito é modelado. Nos últimos dois milénios, têm sido desenvolvidos muitos estudos para perceber as causas do aparecimento de atrito e como o reduzir. No entanto, a simulação numérica ainda recorre à lei de Coulomb, expressando as forças de atrito apenas por um parâmetro obtido experimentalmente.O principal objectivo deste trabalho é estudar numericamente o contacto entre superfícies rugosas. Para concretizar esta análise, construíram-se dois modelos de elementos finitos de uma asperidade, um em duas dimensões e outro em três dimensões, sendo as simulações realizadas com recurso ao programa de elementos finitos académico DD3IMP. Vários materiais foram testados, comparando a influência dos vários parâmetros (modulo de Young, tensão de cedência e coeficiente de Poisson) assim como a geometria (altura) da asperidade. Este procedimento permite estudar uma vasta gama de situações de contacto e perceber qual a importância de cada parâmetro na deformação da asperidade. Em ambos os modelos estudados verificou-se que a transição do regime elástico para o regime plástico ocorre mais cedo quando a ponta da asperidade é mais alta. Com base no parâmetro de carga e no índice de plasticidade , é possível prever a resposta do material no modelo 2D. Em relação ao modelo 3D, à excepção do coeficiente de Poisson, todos os parâmetros do material apresentam uma forte influência no raio de contacto aquando da transição para o regime plástico. A razão entre a área de contacto real e a aparente revela-se bastante pequena e pode ser descrita por uma função linear quando representada em função da área real de contacto. Para além disso, o rácio é mais elevado para o modelo 2D do que para o modelo 3D.
The effectiveness of the sheet metal forming simulation is strongly affected by the accuracy of the friction model used. Studies about the cause and mitigation of friction have been developed over the last two millenniums. Despite the all information reported, numerical simulation still relies on Coulomb’ law, which considers only a single parameter obtained by experimental tests.The main objective of this work is to study numerically the contact between roughness surfaces. In order to develop this analysis, two- and three-dimension finite element models of an asperity were developed and the simulations were carried out by the in-house finite element code DD3IMP. Several materials are compared by changing the material parameters (Young’s modulus, yield stress and Poisson’s ratio) and different geometrical parameters (height of the asperity’s tip) are studied. This allows to cover a wide range of contact situations, highlighting the role of each parameter on the asperity deformation. In both models studied, for an asperity with larger tip, the material reaches the yield inception at a lower interference and consequently the contact area is smaller. Based on the regarding load parameter and the plasticity index , it is possible to predict the response of the 2D model. Regarding the 3D model, as the exception of Poisson’s ratio, all the material parameters considered present a great influence at the value of contact radius and the yield inception. The ratio between the effective contact area and the apparent contact area, which is very low, presents a linear increase as a function of the real contact area. Besides, this ratio is higher for the 2D model than in the 3D model.