Tesis sobre el tema "L-pgd"

Ces travaux de thèse sont consacrés au développement d'un algorithme de résolution de problèmes non-linéaires pour lesquels il existe une variabilité sur certains paramètres du modèle ou du chargement définis par leur intervalle de définition. Le cadre d'étude est le projet SINAPS@, qui a pour but d'évaluer les incertitudes dans les structures de génie civil, et de quantifier leur influence sur la réponse mécanique globale d’une structure sujette à un aléa sismique. Contrairement aux approches statistiques ou probabilistes classiques, une résolution déterministique est privilégiée dans notre étude. Cependant, afin de réduire le coût de calcul de cette famille de problèmes, une approche de type réduction de modèle PGD est mise en place, pour laquelle les paramètres incertains sont considérés comme des variables supplémentaires du problème. Cette méthode est mise en place au sein de l'algorithme LATIN, qui utilise une approche itérative pour résoudre le caractère non-linéaire des équations rencontrées lors de la résolution du problème mécanique. Ces travaux présentent donc l'extension de l'algorithme classique temps-espace LATIN-PGD à des problèmes paramétriques, pour lesquels les paramètres sont considérés comme des variables additionnelles dans la définition des quantités d’intérêt, ainsi que l'application de cette méthode à un modèle endommageant avec refermeture de fissure, présentant une variabilité à la fois sur des paramètres matériaux et sur l'amplitude du chargement. La faisabilité de ce couplage est illustrée par des exemples numériques sur des structures en béton armé pour divers types de chargement cycliques (traction—compression, flexion)
This thesis is dedicated to the development of an algorithm for the resolution of nonlinear problems for which there is a variability on some of the model parameters or on the loading conditions, which are only described by their intervals of variation. This study is part of the SINAPS@ project, which aims at evaluating the uncertainties in civil engineering structures and to quantify their influence on the global mechanical response of a structure to a seismic hazard. Unlike statistical or probabilistic approaches, we rely here on a deterministic approach. However, in order to reduce the computation cost of such problems, a PGD-based reduced-order modeling approach is implemented, for which the uncertain parameters are considered as additional variables of the problem. This method was implemented into the LATIN algorithm, which uses an iterative approach to solve the nonlinear aspect of the equations of the mechanical problem. This work present the extension of the classical time-space LATIN—PGD algorithm to parametric problems for which the parameters are considered as additional variables in the definition of the quantities of interest, as well as the application of such method to a damage model with unilateral effect, highlighting a variability on both material parameters and the loading amplitude. The feasibility of such coupling is illustrated on numerical examples for reinforced concrete structures subjected to different types of cyclic loading conditions (tension—compression, bending)

Les prostaglandines (PGs) sont des médiateurs lipidiques impliqués dans une multitude de processus physiologiques, tels que le sommeil et l'équilibre osseux, mais également dans des phénomènes pathologiques comme l'inflammation chronique et le cancer. Les PGs sont produites à partir de l'acide arachidonique par l'action des cyclooxygénases (COXs) et des PGs synthétases. La régulation de ces dernières est très mal comprise par la communauté scientifique. La L-PGDS est la principale synthétase qui produit la PGD 2 , une PG impliquée entre autres dans la nociception, l'asthme, l'athérosclérose et les allergies. Un criblage par double hybride effectué avec l'arrestine-3 (Arr3) a permis d'identifier la L-PGDS comme partenaire d'interaction. Les arrestines non visuelles sont connues pour leurs rôles dans la désensibilisation et l'endocytose des récepteurs couplés aux protéines G (RCPGs). Cependant, au fil des ans, plusieurs partenaires d'interaction de ces protéines ont été identifiés et ont permis de leur découvrir de nouvelles fonctions. Nous en sommes donc venus à penser que ces protéines multifonctionnelles pourraient réguler la L-PGDS en interagissant avec celle-ci. L'interaction a été confirmée par des essais GST-Pulldown, et de co-immunoprécipitation, dans des systèmes transfecté et endogène dans la lignée ostéoblastique MG-63. La microscopie confocale suggère que la modulation de l'activité enzymatique de la L-PGDS semble modifier la localisation cellulaire des protéines Arr3 et L-PGDS. Des dosages de PGD 2 indiquent que la présence d'Arr3 dans des essais de production de PGD2 in vitro augmente la production de PGD2 , tandis que les fibroblastes embryonnaires de souris (MEFs) déficientes pour les arrestines produisent moins de PGD 2 que les MEFs de type sauvage suite à une stimulation à la PGH2 , ainsi qu'avec l'IL-1?. Cette diminution de production est renversée par la transfection d'arrestines. Un peptide comprenant la région en acides aminés 86 à 100 sur l'Arr3 est suffisant pour augmenter les niveaux de PGD 2 observés in vitro et in cellulo dans les MG-63. Dès lors, nos études identifient pour la première fois un partenaire d'interaction pour la L-PGDS, l'Arr3, qui régule la production de PGD2 . De plus, un peptide comprenant la région d'interaction de l'Arr3 sur la L-PGDS est suffisant pour augmenter la production de PGD2 . Cette nouvelle approche pourrait être utilisée afin de synthétiser des peptides mimétiques spécifiques à la PGD2 dans un traitement anti-inflammatoire alternatif aux inhibiteurs des COXs

L'objet de ce projet de recherche est de prédire la durée de vie d'éléments mécaniques qui sont soumis à des phénomènes de fatigue cyclique. L'idée est de développer un schéma numérique novateur pour prédire la rupture de structures sous de tels chargements. Le modèle est basé sur la mécanique des milieux continus qui introduit des variables internes pour décrire l'évolution de l'endommagement. Le défi repose dans le traitement des cycles de chargement pour la prédiction de la durée de vie, particulièrement pour la prédiction de la durée de vie résiduelle de structures existantes. Les approches traditionnelles de l'analyse de la fatigue sont basées sur des méthodes phénoménologiques utilisant des relations empiriques. De telles méthodes considèrent des approximations simplificatrices et sont incapables de prendre en compte aisément des géométries ou des charges complexes associées à des problèmes d'ingénierie réels. Une approche basée sur la description de l'évolution thermodynamique d'un milieu continu est donc utilisée pour modéliser le comportement en fatigue. Cela permet de considérer efficacement des problèmes d'ingénierie complexe et la détérioration des propriétés du matériau due à la fatigue peut être quantifiée à l'aide de variables internes. Cependant, cette approche peut être numériquement coûteuse et, par conséquent, des approches numériques sophistiquées doivent être utilisées.La stratégie numérique sur laquelle ce projet est basé est singulière par rapport aux schémas incrémentaux en temps usuellement utilisés pour résoudre des problèmes élasto-(visco)plastique avec endommagement dans le cadre de la mécanique des milieux continus. Cette stratégie numérique appelée méthode LATIN (Large Time Increment method) est une méthode non-incrémentale qui recherche la solution de manière itérative sur l'ensemble du domaine spacio-temporel. Une importante innovation de la méthode LATIN est d'incorporer une stratégie de réduction de modèle adaptative pour réduire de manière très importante le coût numérique. La Décomposition Propre Généralisée (PGD) est une stratégie de réduction de modèle a priori qui sépare les quantités d'intérêt spacio-temporelles en deux composantes indépendantes, l'une dépendant du temps, l'autre de l'espace, et estime itérativement les approximations de ces deux composantes. L'utilisation de l'approche LATIN-PGD a montré son efficacité depuis des années pour résoudre des problèmes élasto-(visco)plastiques. La première partie de ce projet vise à étendre cette approche aux modèles incorporant de l'endommagement.Bien que l'utilisation de la PGD réduise les coûts numériques, le gain n'est pas suffisant pour permettre de résoudre des problèmes considérant un grand nombre de cycles de chargement, le temps de calcul peut être très conséquent, rendant les simulations de problèmes de fatigue intraitables même en utilisant les techniques LATIN-PGD. Cette limite peut être dépassée en introduisant une approche multi-échelle en temps, qui prend en compte l'évolution rapide des quantités d'intérêt lors d'un cycle et leur évolution lente au cours de l'ensemble des cycles. Une description type « éléments finis » en temps est proposée, où l'ensemble du domaine temporel est discrétisé en éléments temporels, et seulement les cycles nodaux, qui forment les limites des éléments, sont calculés en utilisant la technique LATIN-PGD. Puis, des fonctions de forme classiques sont utilisées pour interpoler les quantités d'intérêt à l'intérieur des éléments temporels. Cette stratégie LATIN-PGD à deux échelles permet de réduire le coût numérique de manière significative, et peut être utilisée pour simuler l'évolution de l'endommagement dans une structure soumise à un chargement de fatigue comportant un très grand nombre de cycles
The motivation of the research project is to predict the life time of mechanical components that are subjected to cyclic fatigue phenomena. The idea herein is to develop an innovative numerical scheme to predict failure of structures under such loading. The model is based on classical continuum damage mechanics introducing internal variables which describe the damage evolution. The challenge lies in the treatment of large number of load cycles for the life time prediction, particularly the residual life time for existing structures.Traditional approaches for fatigue analysis are based on phenomenological methods and deal with the usage of empirical relations. Such methods consider simplistic approximations and are unable to take into account complex geometries, and complicated loadings which occur in real-life engineering problems. A thermodynamically consistent continuum-based approach is therefore used for modelling the fatigue behaviour. This allows to consider complicated geometries and loads quite efficiently and the deterioration of the material properties due to fatigue can be quantified using internal variables. However, this approach can be computationally expensive and hence sophisticated numerical frameworks should be used.The numerical strategy used in this project is different when compared to regular time incremental schemes used for solving elasto-(visco)plastic-damage problems in continuum framework. This numerical strategy is called Large Time Increment (LATIN) method, which is a non-incremental method and builds the solution iteratively for the complete space-time domain. An important feature of the LATIN method is to incorporate an on-the-fly model reduction strategy to reduce drastically the numerical cost. Proper generalised decomposition (PGD), being a priori a model reduction strategy, separates the quantities of interest with respect to space and time, and computes iteratively the spatial and temporal approximations. LATIN-PGD framework has been effectively used over the years to solve elasto-(visco)plastic problems. Herein, the first effort is to solve continuum damage problems using LATIN-PGD techniques. Although, usage of PGD reduces the numerical cost, the benefit is not enough to solve problems involving large number of load cycles and computational time can be severely high, making simulations of fatigue problems infeasible. This can be overcome by using a multi-time scale approach, that takes into account the rapid evolution of the quantities of interest within a load cycle and their slow evolution along the load cycles. A finite element like description with respect to time is proposed, where the whole time domain is discretised into time elements, and only the nodal cycles, which form the boundary of the time elements, are calculated using LATIN-PGD technique. Thereby, classical shape functions are used to interpolate within the time element. This two-scale LATIN-PGD strategy enables the reduction of the computational cost remarkably, and can be used to simulate damage evolution in a structure under fatigue loading for a very large number of cycles

In this study, three subspecies of Apis mellifera L. (A. m. caucasica, A. m. carnica, and A. m. syriaca) from different climatic regions were evaluated electrophoretically at ontogenetic level by means of four enzymes, namely Phosphoglucomutase (PGM), Hexokinase (HK), Phosphoglucose isomerase (PGI) and Glukose-6-phosphate dehydrogenase (G6PD). It is determined that only Pgm and Hk loci were polymorphic. Allele and genotype frequencies at Pgm locus changes seasonally whereas Hk locus does not exhibit seasonal variation. Within the scope of this study we investigated at which developmental stage shifting to heterozygotes prior to winter occurs. It is found that there is a seasonal fluctuation throughout the year in Pgm genotype frequencies at each developmental stage studied and correlated with enzyme activity and glycogen content. As the studied enzymes have crucial v roles in insect energy metabolism, results of this study provided further information about the relationship between carbohydrate metabolism and enzyme polymorphism of honey bees.

Phosphoglucomutase (PGM) is one of the central enzymes in energy metabolism at a branch point at the head of the metabolic pathway leading into glycogen metabolism, pentose shunt and the main glycolytic cycle, catalyzing the reversible interconversion of glucose-1-phosphate to glucose-6-phosphate. Whole year, month to month analysis of pattern of allozyme variation at Pgm and Hk loci in Apis mellifera L. from three provinces
Kirklareli, Artvin, and Hatay revealed that there is significant seasonal variation of allozyme frequencies at Pgm locus (P<
0.001). The difference in genotype frequencies between summer and winter samples is apparent in Pgm, whereas at Hk locus, which is analyzed as a control there is seasonal variation in genotype frequencies. Biochemical measurements of the enzyme activities and glycogen content of different Pgm genotypes were performed to determine the effect of different Pgm genotypes on the physiological performance of the honeybees and it was observed that both enzyme activity and glycogen amount is higher in heterozygote individuals which are in high frequency during winter months (P<
0.0001). Furthermore, PGM enzyme activity and glycogen content was found to be significantly correlated. These findings clearly demonstrate that biochemical differences between different Pgm genotypes have functional correlates that lead to significant variations in glycogen content of the honeybees and may have adaptive consequences.

In an earlier project completed in our laboratory a seasonal fluctuation in Phosphoglucomutase (PGM) phenotype frequencies was found, so that the winter bees were almost all heterozygotes and long lived than the summer bees among which homozygotes were significantly at high frequencies at Pgm locus. Same results were obtained in populations of three subspecies, A. m. meda, A. m. caucasica, and A. m. carnica from different climatic regions. In the current study environmental cues related with seasonal change in PGM phenotype frequency was examined along with the correlation between PGM heterozygosity and overwintering success. Cessation of food influx was found to be effective by itself as an environmental cue that causes a sudden and sharp increase in PGM heterozygosity. In addition to that, PGM heterozygosity of the colonies with greater overwintering success was found to be significantly higher than the ones with intermediate or low overwintering success. Benefiting from the previous studies and the results of current study, ethyl oleate was suggested as a chemical signal that functions in the regulation of PGM heterozygosity.

Ce que l’on appelle l’« implémentation » des progiciels de gestion intégrés (PGI), conduit généralement à une refonte du système d’information de gestion, et à une remise à plat des procédures de gestion en place, lesquelles peuvent être par la suite réorganisées suivant les orientations stratégiques ciblées par l’entreprise. Les principaux problèmes commencent lorsqu’il s’agit d’intégrer les fonctionnalités spécifiques à chaque entreprise dans le PGI. Entre la standardisation des processus de gestion et la personnalisation du PGI, les entreprises semblent être souvent en quête d’un équilibre qui reflète la cohérence de leurs choix et de leurs décisions avec leurs orientations stratégiques. C’est sur ce thème de la recherche d’équilibre que notre thèse se positionne. Elle vise dans ce cadre à explorer les différents choix stratégiques effectués par les entreprises pour intégrer un PGI. C’est sur la base de ces choix que nous avons tenté de décrypter les différentes modalités adoptées pour intégrer ces progiciels, à partir d’une enquête qualitative par entretiens semi-directifs auprès de 16 entreprises industrielles. Il en est sorti trois configurations de base construites au niveau local des entités concernées par l’intégration, et une quatrième configuration dite "mixte" qualifiant la synergie de l’ensemble des configurations adoptées à l’échelle globale de l’entreprise. L’analyse de l’évolution de ces modalités dans le temps est une question qui s’est révélée indispensable, dans la mesure où elle permet de prendre en compte le caractère ouvert et dynamique des processus d’intégration organisationnelle des PGI
What is called « implementation » of Enterprise Resource Planning (ERP) usually leads to a rebuilding of management information systems, and to a revisit of management procedures that can be thereafter reorganized according to the company’s strategic orientations. The main problems start when one wants to integrate specific functionalities of a particular company into the ERP. Between the standardization of management procedures and the personalization of ERP, companies often seem to look for a balance that reflects the coherence of their choices and decisions according to their strategic orientations. It’s this topic of balance which constitutes the heart of this dissertation. This research aims to explore the various strategic choices done by companies to integrate ERP. It’s on the basis of these choices that we have tried to decrypt the different modalities adopted to integrate these softwares. We have found three basic configurations built on local levels of the entities concerned by the integration, and a fourth mixed configuration qualifying the synergy of the overall configurations adopted to the wider level of the company. The analysis of the longitudinal evolution of these modalities is a question that appeared to be directly related to the basic problem, because it allows to take into consideration the opened and dynamic behavior of integration processes

Dissertation presented at Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa to obtain the Degree of Master in Biotecnology
The use of transgenic plants for the production of recombinant proteins with commercial and pharmaceutical value offers several advantages when compared to the standard systems. Whole plant systems have potential for studies of the recombinant protein trafficking and suspension cell cultures combine the advantages of plants with the benefits of protein production by cell cultures. In this master thesis work, Medicago truncatula plants expressing a lipocalin-type human prostaglandin D2 synthase (L-PGDS) were used and the protein trafficking was studied. This study was the first analysis of the production of this protein in this specific plant and the first study of the subcellular trafficking of this protein in plants. Two production systems were analyzed: whole-plant systems and suspension cell cultures. The presence of the L-PGDS was studied in three different plant organs (leaf, root and seed) and in the cells and cell medium, by Western Blotting and fluorescence and electron microscopy. The L-PGDS protein appeared to accumulate in different places and patterns depending on which type of cell it is being produced. Recent work has shown that functional specialization of plant cells in storage organs can influence the subcellular trafficking of recombinant proteins, so the protein subcellular fate could be different between seeds and leaves of the same transformed plant. It has also been demonstrated that the plant species where the recombinant protein is produced, can alter the trafficking parameters. Medicago truncatula is, thus, a promising system for the production of recombinant proteins. The initial results obtained in this study could contribute to future studies and development in Molecular Farming.

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A busca por novos antibióticos de amplo espectro de ação tem aumentado nas últimas décadas devido ao número crescente de bactérias resistentes aos antibióticos convencionais. O peptídeo recombinante Pg-AMP1, expresso em um sistema heterólogo em Escherichia coli, apresentou atividade antibacteriana contra linhagens Gram-positivas e Gram-negativas. A partir da seqüência de aminoácidos do peptídeo isolado de sementes de goiaba (Psidium guajava L.) o gene correspondente ao peptídeo foi modificado utilizando-se o códon preferencial para expressão em E. coli e construído um vetor de expressão. Uma região codificadora para cauda de histidina foi fusionada ao gene pg-amp1 permitindo a purificação por cromatografia de afinidade com íons de níquel imobilizados em coluna de sefarose. Utilizando SDS-PAGE e análise in sílico identificamos a massa molecular do PgAMP1 recombinante de 7,368 kDa e pI 8.93. O peptídeo recombinante foi expresso principalmente na forma insolúvel, agregando-se em corpos de inclusão que foram tratados com agentes desnaturantes obtendo o peptídeo solúvel. Após a purificação pela coluna de níquel obtivemos um rendimento de 13 mg por litro de meio de cultura. O peptídeo Pg-AMP1 recombinante apresentou atividade contra as bactérias Gram-negativas Escherichia coli e Pseudomonas aeruginosa e contra as Grampositivas Staphylococcus aureus e Staphylococcus epidermides. O peptídeo recombinante Pg-AMP1 não mostrou atividade contra os fungos fitopatogênicos testados. Devido a sua ação contra estas cepas de bactérias patogênicas humanas, o Pg-AMP1 recombinante torna-se um promissor antimicrobiano a ser utilizado no desenvolvimento de novos antibióticos contra cepas resistentes aos fármacos comumente usados.
The search for new antibiotics of broad spectrum of activity has increased in recent decades due to the increasing number of bacteria resistant to conventional antibiotics. The Pg-AMP1 recombinant peptide expressed in a heterologous system in Escherichia coli, strains showed antibacterial activity against Gram-positive and Gram-negative. From the sequence of amino acid peptide isolated from the seeds of guava (Psidium guajava L.) peptide corresponding to the gene was modified using the preferred codon for expression in E. coli and constructed a vector for expression. A region coding for the histidine tail was merged the gene-pg amp1 allowing purification by affinity chromatography with nickel ions immobilized on the column sepharose. Using SDS-PAGE and in silico analysis identified the molecular weight of Pg-AMP1 recombinant with 7.368 kDa and pI 8.93. The recombinant peptide was expressed mostly as insoluble form, adding up in inclusion bodies, which were treated with denaturants agents to solubilize the peptide. The purification of peptide by nickel sepharose column yielded 13 mg per liter of culture medium. The Pg-AMP1 recombinant peptide showed activity against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and against gram-positive Staphylococcus aureus and Staphylococcus epidermidis. The recombinant peptide Pg-AMP1 showed no activity against the phytopathogenic fungi tested. Due to its action against these strains of human pathogenic bacteria, the recombinant Pg-AMP1 is a promising antimicrobial for use in developing new antibiotics against strains resistant to commonly used drugs.

Soil fertility is an important component of forest ecosystem, yet evaluating soil fertility remains one of the least understood aspects of forest science. Phytocentric and geocenctric approaches were used to assess soil fertility in loblolly pine plantations throughout their geographic range in the United States. The model to assess soil fertility using a phytocentric approach was constructed using the relationship between site index and aboveground productivity. Geocentric models used physical and chemical properties of the A-horizon. Soil geocentric models were constructed using two modeling approaches. In the first approach, ordinary least squares methods of multiple regression were used to derive soil fertility estimated from site index using soil physical and chemical properties from the A-horizon. Ordinary least squares methods were found unsuitable due to multicollinearity among the soil variables. In the second approach, a multivariate modeling approach, partial least squares regression, was used to mitigate multicollinearity effects. The best model to quantify soil fertility using soil physical and chemical properties included N, Ca, Mg, C, and sand percentage as the significant predictors. The 3-PG process-based model was evaluated for simulating the response of loblolly pine to changes in soil fertility. Fertility rating (FR) is a parameter in 3-PG that scales soil fertility in the range of 0 to 1. FR values estimated from phytocentric and geocentric approaches were tested against observed production. The 3-PG model prediction of aboveground productivity described 89% percent of the variation in observed aboveground productivity using FR derived from site index and 84% percent of the vari- ation in observed aboveground productivity using FR derived from physical and chemical properties of the A-horizon. A response function to model dynamics of FR (∆FR) due to one time midrotatoin fertilization of N and P was developed using the Weibull function. The magnitude of ∆FR varied with intensity of N and time since application of fertilizer. The hypothesis that repeated fertilization with N and P eliminate major nutrient deficiency in the southeastern US was tested and a relationship between baseline fertility rating and fertilizer response was developed. An inverse relationship was observed between fertilizer response and baseline FR.
Ph. D.

Forests are a valuable resource for humans providing a range of products and services such as construction timber, paper and fuel wood, recreation, as well as living quarters for indigenous populations and habitats for many animal and bird species. Most recent international political agreements such as the Kyoto Protocol emphasise the role of forests as a major sink for atmospheric carbon dioxide mitigation. However, forest areas are rapidly decreasing world wide. Thus, it is vital that efficient strategies and tools are developed to encourage sustainable ecosystem management. These tools must be based on known ecological principles (such as tree physiological and soil nutrient cycle processes), capable of supplying fast and accurate temporal and spatial predictions of the effects of management on both timber production and carbon sequestration. This thesis had two main objectives. The first was to investigate the environmental factors affecting growth and carbon sequestration of Scots pine (Pinus sylvestris L.) across Scotland, by developing a knowledge base through a statistical analysis of old and novel field datasets. Furthermore, the process-based ecosystem model 3-PGN was developed, by coupling the existing models 3-PG and ICBM. 3-PGN calibrated using a Bayesian approach based on Monte Carlo Markov Chain simulations and it was validated for plantation stands. Sensitivity and uncertainty analyses provided an understanding of the internal feedbacks of the model. Further simulations gave a detailed eco-physiological interpretation of the environmental factors affecting Scots pine growth and it provided an assessment of carbon sequestration under the scenario of sustainable, normal production and its effects from the environment. Finally, the study investigated the spatial and temporal patterns of timber production and carbon sequestration by using the spatial version of the model and applying advanced spatial analyses techniques. The second objective was to help close the gap between environmental research and forest management, by setting a strategic framework for a process-based tool for sustainable ecosystem management. The thesis demonstrated the procedures for a site classification scheme based on modelling results and a yield table validation procedure, which can provide a way forward in supporting policies for forest management and ensuring their continued existence in the face of the present and future challenges.

In this thesis, porous graphitic carbon (PGC) has been used as packing material in packed capillary liquid chromatography. The unique chromatographic properties of PGC has been studied in some detail and applied to different analytical challenges using both electrospray ionization-mass spectrometry (ESI-MS) and ultra violet (UV) absorbance detection.

The crucial importance of disengaging the conductive PGC chromatographic separation media from the high voltage mass spectrometric interface has been shown. In the absence of a grounded point between the column and ESI emitter, a current through the column was present, and changed retention behaviors for 3-O-methyl-DOPA and tyrosine were observed. An alteration of the chromatographic properties was also seen when PGC was chemically oxidized with permanganate, possibly due to an oxidation of the few surface groups present on the PGC material.

The dynamic adsorption of the chiral selector lasalocid onto the PGC support resulted in a useful and stable chiral stationary phase. Extraordinary enantioselectivity was observed for 1-(1-naphthyl)ethylamine, and enantioseparation was also achieved for other amines, amino acids, acids and alcohols.

Finally, a new strategy for separation of small biologically active compounds in plasma and brain tissue has been developed. With PGC as stationary phase it was possible to utilize a mobile phase of high content of organic modifier, without the addition of ion-pairing agents, and still selectively separate the analytes.

L’obiettivo di questo lavoro è guidare il docente attraverso la letteratura esistente, gli strumenti già in uso e si propone di fornire una modellizzazione per la pratica didattica delle lingue straniere ad alunni sordi. Nel primo e nel secondo capitolo si raccolgono, analizzano e definiscono le basi teoriche di riferimento per la didattica delle lingue a soggetti sordi. Nello specifico, si definiscono il quadro della glottodidattica speciale, i principi di bisogno speciale e specifico e le linee guida per la didattica in contesti di disabilità, procedendo con l’inclusione della sordità tra le esigenze a cui riferirsi con tali strumenti. Il terzo e il quarto capitolo rappresentano l’applicazione concreta dei modelli esposti con l’applicazione del modello SOMA al mondo della sordità e la costruzione del Profilo Glottomatetico Funzionale dell’alunno sordo. Nell’ultimo capitolo si presenta la proposta operativa di didattica dell’inglese ai sordi, partendo dall’analisi e la scelta dei materiali più accessibili, passando per la progettazione fino ad arrivare alla strutturazione specifica del lavoro di classe.
This work aims to guide the foreign language teacher trough the literature on special education needs and deafness, the available tools, and the modeling for everyday practice to encourage the inclusion of deaf students until now considered not teachable. In the first and second chapters, the theoretical approaches to deaf students learning are presented in the contexts of special language teaching, special education, and disabilities. The third and fourth chapters represent the application of those principles to include deaf students in the language classroom activities following the operational models usually used for different learning disabilities. The last chapter offers the concrete model that may guide teachers from their deaf students' needs analysis and the correct material choice for them, passing through the learning project management to get to the specific class lessons, in collaboration with support staff members.

L’obiettivo di questo lavoro è guidare il docente attraverso la letteratura esistente, gli strumenti già in uso e si propone di fornire una modellizzazione per la pratica didattica delle lingue straniere ad alunni sordi. Nel primo e nel secondo capitolo si raccolgono, analizzano e definiscono le basi teoriche di riferimento per la didattica delle lingue a soggetti sordi. Nello specifico, si definiscono il quadro della glottodidattica speciale, i principi di bisogno speciale e specifico e le linee guida per la didattica in contesti di disabilità, procedendo con l’inclusione della sordità tra le esigenze a cui riferirsi con tali strumenti. Il terzo e il quarto capitolo rappresentano l’applicazione concreta dei modelli esposti con l’applicazione del modello SOMA al mondo della sordità e la costruzione del Profilo Glottomatetico Funzionale dell’alunno sordo. Nell’ultimo capitolo si presenta la proposta operativa di didattica dell’inglese ai sordi, partendo dall’analisi e la scelta dei materiali più accessibili, passando per la progettazione fino ad arrivare alla strutturazione specifica del lavoro di classe.
This work aims to guide the foreign language teacher trough the literature on special education needs and deafness, the available tools, and the modeling for everyday practice to encourage the inclusion of deaf students until now considered not teachable. In the first and second chapters, the theoretical approaches to deaf students learning are presented in the contexts of special language teaching, special education, and disabilities. The third and fourth chapters represent the application of those principles to include deaf students in the language classroom activities following the operational models usually used for different learning disabilities. The last chapter offers the concrete model that may guide teachers from their deaf students' needs analysis and the correct material choice for them, passing through the learning project management to get to the specific class lessons, in collaboration with support staff members.

La prostaglandina D sintasi di tipo lipocalinico (L-PGDS) catalizza, in presenza di composti sulfidrilici, l’isomerizzazione del gruppo 9,11 endoperossido della PGH2 (Prostaglandina H2) nei gruppi 9-idrossi e 11-cheto della PGD2 (Prostaglandina D2). La PGH2 è il precursore comune di tutti i prostanoidi, i quali comprendono trombossani, prostacicline e prostaglandine. La PGD2 è sintetizzata sia nel sistema nervoso centrale che nei tessuti periferici dove è coinvolta nel mantenimento della temperatura corporea, nella regolazione del corretto funzionamento delle cellule nervose e del ciclo sonno-veglia. E’ inoltre implicata nella sensibilità al dolore, nell’asma allergica, nell’inibizione dell’aggregazione delle piastrine e nel reclutamento chemotattile delle cellule del sistema immunitario. La L-PGDS appartiene alla famiglia delle lipocaline ed è in grado di trasportare piccole molecole idrofobiche; è inoltre la prima proteina di questa famiglia ad essere stata identificata come enzima. Recentemente si è scoperto che la L-PGDS era già conosciuta con il nome di proteina β-trace, la quale nel fluido cerebro spinale rappresenta la seconda proteina per abbondanza. La presenza della L-PGDS è stata individuata anche nel cervello, nei testicoli e prostata umani, nelle cellule della placenta, nel tessuto cardiaco e anche in macrofagi infiltrati in placche aterosclerotiche. In questi tessuti la L-PGDS è coinvolta sia in numerose attività fisiologiche che in risposta a patologie come diabete, lesioni cardiovascolari, sclerosi multipla, malattia di Alzheimer e varie neoplasie. Attualmente i maggiori studi biochimici e strutturali presenti in letteratura riguardano la L-PGDS ricombinante di ratto. Lo scopo di questo lavoro riguarda l’espressione, la purificazione e la cristallizzazione della L-PGDS ricombinante umana allo scopo di risolverne la struttura tridimensionale utilizzando la diffrazione di raggi X. La L-PGDS umana WT e tre mutanti (C65A; C65A/K59A; C89/186A) sono stati espressi in cellule di E. coli e successivamente purificati mediante cromatografia di affinità, con resina di chitina, seguita da gel filtrazione e cromatografia ad interazione idrofobica. Il protocollo di purificazione è stato ottimizzato al fine di ottenere una proteina estremamente pura e di conseguenza adatta ad esperimenti di cristallizzazione. Le soluzioni di cristallizzazione sono state migliorate per ottenere cristalli di ordine e dimensioni tali da poter essere utilizzati in esperimenti di diffrazione di raggi X mediante sorgente ad anodo rotante. La struttura tridimensionale è stata risolta mediante la sostituzione isomorfa multipla e i derivati di atomi pesanti sono stati ottenuti mediante la tecnica del soaking. All’interno della cavità della L- PGDS-C65A si è notata una densità elettronica residua che sembrava interagire con la lisina 59. In quel momento, con i dati a disposizione, non è stato possibile caratterizzare la molecola sconosciuta sebbene siano stati provati numerosi protocolli, compreso l’uso di ligandi fisiologici. La cavità della L-PGDS-C65A/K59A risulta completamente libera. Si è notato come i cristalli della L-PGDS-C65A/K59A crescano in assenza di PEG come precipitante, il quale è, al contrario, necessario per la crescita dei cristalli della L-PGDS-C65A. Questo fatto indica come proprio il PEG potrebbe essere la molecola presente all’interno della cavità. Una parziale conferma a questa ipotesi è arrivata dall’esperimento di seeding nel quale la L-PGDS-C65A/K59A, fatta cristallizzare con semi e nelle condizioni della L-PGDS-C65A, presenta la medesima densità residua all’interno della cavità. Si è potuta definitivamente confermare la nostra ipotesi grazie ad una raccolta ad alta risoluzione della L-PGDS-C65A nella quale la densità residua risulta meglio definita e conforme ad una molecola di PEG. Un altro importante risultato è stato ottenuto dalla parziale risoluzione della struttura tridimensionale del complesso tra la L-PGDS-C65A/K59A e il peptide β amiloide (1-40), infatti è la prima volta che si raggiunge la prova strutturale di questa interazione. La L-PGDS wild type e la L-PGDS-C89/186A, da noi purificate, si sono rivelate non omogenee e non è stato ottenuto nessun cristallo in tutte le prove effettuate.
Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precurson of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central nervous system and the peripheral tissues where it is involved in the maintenance of the body temperature, regulation of nerve cell function, regulation of the sleep wake cycle, tactile pain sensitivity, allergic asthma, inhibition of platelet aggregation and chemotactic recruitment of inflammatory cells. L-PGDS belongs to the lipocalin family and it is able to bind and transport small hydrophobic molecules; it is also the first member of this important family to be recognized as an enzyme. Recently L-PGDS was identified to be already known as the β-trace protein, which is the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, human testis and prostate, endothelial cells, placenta cells, heart tissue and even in macrophages infiltrated to atherosclerotic plaques. In those tissues L-PGDS is involved in many physiological activities as well as in the response to diseases such as diabetes, cardiovascular lesions, multiple sclerosis, Alzheimer’s disease and tumors. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. The aim of this work was to express, purify and crystallize recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction experiments. Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion chromatography and hydrophobic interaction chromatography. The purification method was improved to obtain highly homogeneous protein suitable for preliminary crystallization trials. Crystallization conditions were optimized to obtain large and highly ordered crystals that were tested by X-ray diffraction using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement technique was used to solve the phase problem and heavy atom derivatives were obtained by soaking. An unidentified electron density was observed that seemed to interact with lysine 59 inside the L-PGDS-C65A cavity. It was not possible, at that moment, to characterize this residual molecule although different protocols were tested, including the use of physiological ligands. The L-PGDS-C65A/K59A structure showed a completely free cavity. It was noticed that L-PGDS-C65A/K59A crystals grew without PEG as precipitant, which instead was necessary for the L-PGDS-C65A crystals, suggesting that PEG could be the foreign molecule. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. A high resolution data set of a L-PGDS-C65A gave us the possibility to well define the boundaries of the unknown electron density showing that the foreign molecule was probably PEG. An important crystal structure was obtained by mixing L-PGDS-C65A/K59A with the amyloid β peptide (1-40). Although structure refinement is work in progress, it was the first structural evidence of the interaction of L-PGDS with the amyloid β peptide (1-40). Wild type L-PGDS and L-PGDS C89/186A were purified but they were not homogeneous and no crystals grew in any of the crystallization trials.

L'arthrose (OA) est la maladie dégénérative la plus fréquente (ou) et la principale cause d'incapacité physique avec un coût socioéconomique important. Les manifestations cliniques de l'arthrose peuvent inclure des douleurs, des raideurs et des mouvements articulaires réduits. Pathologiquement, l'OA se caractérise par une dégénérescence progressive du cartilage articulaire, une augmentation de l'expression des médiateurs inflammatoires et cataboliques et le remodelage osseux sous-chondral. Il a été démontré que la protéine prostaglandine D2 (PGD2) est synthétisée par différents types cellulaires et possède des propriétés pro et anti-inflammatoires, selon le récepteur activé. Plusieurs stimuli pro-inflammatoires ont été discernés et étudiés, mais par contre, les voies anti-inflammatoires restent toujours un univers inexploré. Le récepteur DP1 de la PGD2, ainsi que l’enzyme de synthèse L-PGDS, jouent des rôles importants dans l'inflammation et le métabolisme du cartilage. Cependant, leurs rôles dans la pathogenèse de l'arthrose (OA) restent inconnus. Nous avons entrepris l’étude (de quoi) d’une part, pour explorer les rôles de la L- PGDS et de DP1 dans le développement d'OA, et d’autre part pour évaluer l'efficacité d'un agoniste sélectif de DP1 et d’un virus d'AAV2 / 5 codant pour L-PGDS dans le traitement de l'OA. En premier, nos travaux par histologie ont démontré que la dégradation du cartilage est plus prononcée chez les souris Knock-out L-PGDS et DP1, comparativement au souris sauvages Wild-Type (WT). Ensuite une augmentation de l’expression des médiateurs cataboliques (ADAMTS5 et MMP-13), chez les souris L-PGDS -/- et DP1 -/- par rapport au WT a été démontré. Après, la stimulation des explants de cartilage des souris L-PGDS -/- et de DP1 -/- avec l’IL-1a, ont montré une dégradation élevée en protéoglycanes. En outre ces souris ont développer aussi des modifications osseuses sous- chondral. Enfin, nos résultats suggèrent qu’à la suite d'injection intrapéritonéale de l’agoniste spécifique de DP1, le BW245C a atténué la gravité de la dégradation du cartilage induite par une déstabilisation du ménisque médiale (DMM) et des modifications osseuses chez les souris WT. Pareillement, l'injection intra-auriculaire d'AAV2 / 5 codant pour L- PGDS a atténué aussi la dégradation du cartilage induite par DMM et l'expression de ADAMTS-5 et MMP-13 chez des souris L-PGDS -/-. En conclusion, l’ensemble de nos résultats suggèrentque le récepteur DP1 et l’enzyme L-PGDS au niveau du cartilage articulaire arthrosique joue un rôle très important. . Elle pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et aussi dans le traitement d’autres pathologies musculo-squelettiques.
Osteoarthritis (OA) is the most common degenerative disease (or) and the leading cause of physical disability with significant socioeconomic costs. Clinical manifestations of osteoarthritis can include pain, stiffness, and reduced joint movement. Pathologically, OA is characterized by progressive degeneration of articular cartilage, increased expression of inflammatory and catabolic mediators, and subchondral bone remodeling. It has been shown that prostaglandin D2 protein (PGD2) is synthesized by different cell types and has pro and anti-inflammatory properties, depending on the activated receptor. Several pro-inflammatory stimuli have been discerned and studied, but the anti- inflammatory pathways remain an unexplored universe. The DP1 receptor of PGD2, as well as the synthetic enzyme L-PGDS, play important roles in inflammation and cartilage metabolism. However, their roles in the pathogenesis of osteoarthritis (OA) remain unknown. We undertook the study (of what) on the one hand, to explore the roles of L-PGDS and DP1 in the development of OA, and on the other hand to evaluate the efficacy of a selective agonist DP1 and an AAV2 / 5 virus encoding L-PGDS in the treatment of OA. First, our histology work demonstrated that cartilage degradation is more pronounced in L- PGDS Knock-out and DP1 mice compared to Wild-Type (WT) wild-type mice. Then an increase in the expression of catabolic mediators (ADAMTS5 and MMP-13), in L-PGDS - / - mice and DP1 - / - compared to WT was demonstrated. Subsequently, stimulation of cartilage explants with IL-α from L-PGDS - / - and DP1 - / - mice showed high degradation in proteoglycans. In addition, these mice also develop subchondral bone changes. Finally, our results suggest that following intraperitoneal injection of the DP1-specific agonist, BW245C attenuated the severity of cartilage degradation induced by medial meniscus destabilization (DMM) and bone changes in mice. WT. Similarly, the intra-atrial injection of AAV2 / 5 encoding L-PGDS also attenuated DMM-induced cartilage degradation and the expression of ADAMTS-5 and MMP-13 in L-PGDS - / - mice. In conclusion, all our results suggest that the recepteur DP1 and the L-PGDS enzyme in osteorthritis cartilage plays a very important role. It may be a potential therapeutic avenue in the treatment of OA and also in the treatment of other musculoskeletal conditions.

L’arthrose (OA) est la maladie articulaire la plus répandue dans le monde faisant l’objet de nombreux travaux de recherche en raison de son lourd impact socioéconomique. Plusieurs travaux dans ce domaine ont pour objectif de déterminer les mécanismes moléculaires impliqués dans sa physiopathologie. Plusieurs travaux ont appuyés l’implication de la prostaglandine (E2) PGE2 dans sa physiopathologie, contrairement à la prostaglandine (D2) (PGD2) dont le rôle reste à déterminer. C’est pourquoi, nous nous sommes penchés dans cette thèse à l’étude de cette dernière molécule. Dans la première partie de nos travaux, nous avons montré que la PGD2 diminue au niveau du cartilage articulaire et au niveau niveau des explants de cartilage humains, la production des métalloprotéases-1(MMP-1) et MMP-13 induites par (Interleukine-1β) l’IL-1β. Cette diminution de la production protéique est accompagnée d’une diminution de l’expression au niveau de l’ARNm, et d’une diminution de l’activité du promoteur de MMP-1 et MMP-13. Cet effet est exercé via le récepteur D prostanoïde (DP1), bien que le Chemoattractant receptor expressed on Th2 cells (CRTH2) soit également exprimé chez les chondrocytes humains, mais ne semble pas être impliqué dans l’effet observé. Cette action inhibitrice se fait via la voie DP1/AMPc/protéine kinase A (AMPc/PKA). Dans la suite de nos travaux, nous avons montré pour la première fois l’expression des prostaglandines D-synthases responsables de la biosynthèse de la PGD2 au niveau des chondrocytes humains par immunohistochimie, avec des niveaux d’expression de l’ARNm plus élevés de la L-PGDS au niveau du cartilage OA comparativement au cartilage normal. L’IL-1β pourrait être responsable de cette augmentation via l’activation de la voie JNK et p38 MAPK, ainsi que par la voie NF-κB. L’ensemble de ces données indiquent que la modulation des niveaux de la PGD2 au niveau de l’articulation pourrait être pourvue d’un important potentiel thérapeutique. La L-PGDS pour sa part semble avoir un rôle important dans la physiopathologie de l’OA.
Osteoarthritis (OA) is the most common joint disease world wide, because of its higher socioeconomic impact it is one of the most studied joint diseases. The aims of these studies was to determine the molecular mechanisms involved in the pathophysiology of osteaarthritis. Previous studies have mainly focused on the involvement of prostaglandin (E2) PGE2 in contrast to PGD2 in the pathogenesis osteoarthritis as such the role of PGD2 remains unclear. In this thesis we examined the involvment of PGD2 in the pathogenesis of OA. In the first part of our work, we showed that in a dose dependent manner PGD2 decreased the interleukin-1β (IL-1β)–induced mettalloproteases (MMP-1) and MMP-13 expression both at protein and mRNA levels by supression of their promoter activity. The inhibitory effect was exerted via the D prostanoid receptor (DP1) and mediated through the cAMP/protein kinase A (PKA) signalling pathway. Although human chondrocytes do express the Chemoattractant Receptor Expressed on Th2 cells (CRTH2) the latter were not implicated in the inhibiton of MMP-1 and MMP-13. In the second part of our work, we showed the expression of prostaglandin D synthases (PGDS) responsible for the biosynthesis of PGD2 in human chondrocytes, with higher levels of mRNA expression of lipocaline type prostaglandin D-synthase (L-PGDS) in OA cartilage compared to normal cartilage. IL-1β may be responsible for this increase via the activation of Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK), as well as the nuclear factor-κB (NF-κB). Together, these data indicate that modulation of the levels of PGD2 at the joint may be provided with an important therapeutic potential. L-PGDS in turn seems to have an important role in the pathogenesis of OA.

碩士
國立臺灣科技大學
高分子系
94
Na+-γ-poly(glutamic acid) (PGA), a highly water-soluble, biodegradable, hygroscopic and non-cytotoxic polypeptides, was chosen to fabricate the PGA/polyvinyl alcohol (PVA) blending membranes. By mixing PGA and PVA together, a hydrogel would be formed at room temperature without any chemical treatment.It was proven to be a highly hygroscopic by mixing at different ratio of PGA/PVA (0~50%) blending membranes for testing its swelling ratio in normal saline. By glutaraldehyde to covalently bond L(+)-Ascorbic acid with PGA/PVA, the application of PGA/PVA blending membranes can be broadened in medical devices and cosmetic industries.

"The use of transgenic plants for large-scale production of recombinant proteins with commercial and pharmaceutical value has emerged as an alternative to conventional systems due to its several advantages. Particularly, plant cell suspension cultures offer the advantages of production in whole plant (such as reduced costs and safety) and production in cell culture systems. Although they have numerous advantages, the yields obtained are not yet competitive enough when compared to animal cell culture systems.(...)"

碩士
國立中正大學
化學工程研究所
100
The purpose of this study is to blend chitosan with gamma-PGA in different quantity proportion and combine with genipin (crosslinker) ,then producing bioavailable scaffold through freeze-drying and analyzing material properties of porosity, swelling raito, modulus and viability. The surface of scaffold will be modified with albumin, elastin and poly-L-lysine to provide good environment for in vitro cultivation, promote chondrocyte growth and benefit for extracellular matrix (ECM) secretion. The result of experiment reveals that when chitosan and gamma-PGA form with the ratio of 1:3 in the scaffold has the optimal porosity, pressure resistance and extensibility. It’s the most appropriate condition for cultivating bovine knee chondrocytes. Besides, when the ratio of gamma-PGA raises, the increase of swelling ratio equivalent to the enhancement of hydrophilic ability on scaffold surface. However, due to the effect of porosity and pore size, chondrocytes cell numbers, secreted glycosaminoglycans and produced type II collagen shown a descendant tendency. The experiments utilize surface-modified scaffold with a pair of components of albumin, elastin and poly-L-lysine then mixed in volume ratio. The result indicated that when the albumin and elastin mixed with the ratio of 1:3 for four weeks, the increase chondrocytes cell numbers, secretion glycosaminoglycans and production type II collagen has the optimal condition. In the apoptosis detection, elastin is the most long-lived one to appear the phenomenon of cell death. As the result of mitochondria damage revealed, chondrocytes damaged slightly when in vitro cultivation with elastin surrounded. It is concluded that utilizing chitosan/gamma-PGA scaffold can be effectively promoting the growth of bovine knee chondrocytes, secreting extracellular matrix and improving the regeneration ability of cartilage tissues.