Dissertations / Theses on the topic 'SbyD'

Doenças causadas por agentes infecciosos e parasitários são chamadas negligenciadas por não despertarem interesse das indústrias farmacêuticas para o desenvolvimento de novas alternativas terapêuticas. Essas doenças são responsáveis por levar milhões de pessoas à morte todos os anos e afetam principalmente os países pobres e em desenvolvimento. Dentre estas, a doença de Chagas e as leishmanioses, parasitoses causadas por parasitas flagelados pertencentes à família Trypanosomatidae, T. cruzi e Leishmaina sp., respectivamente, se apresentam como um sério problema de saúde pública mundial. Endêmicas em vários países e causando milhões de mortes anualmente, ainda hoje não existem fármacos eficientes e seguros para o tratamento dessas doenças. Este panorama torna eminente a necessidade de pesquisa e desenvolvimento de novos fármacos para essas parasitoses. A busca por agentes quimioterápicos envolve a seleção de vias metabólicas essenciais à sobrevivência dos parasitas. Dentre estas, destacamse cisteíno-proteases presentes nesses tripanossomatídeos, deste modo a cruzaína no T. cruzi, e a CPB2.8 na Leishmania mexicana, se mostram como alvos bioquímicos promissores. A disponibilidade de estruturas cristalográficas da cruzaína e do sequenciamento genômico da CPB2.8, nos permite utilizar estratégias de planejamento de fármacos baseado no receptor (SBDD) na identificação de candidatos a fármacos para essas doenças. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar promissores candidatos a novos fármacos. Assim neste trabalho, obteve-se por meio da técnica de modelagem comparativa o modelo da enzima CPB2.8 de L. mexicana, visto a indisponibilidade da estrutura cristalográfica no Protein Data Bank (PDB). De modo a refinar o modelo construído realizou-se a simulação por dinâmica molecular de 100ns, apresentando estabilização a partir de 80ns. A simulação por dinâmica molecular foi validada por meio do gráfico de Ramachandran, gráfico de raio de giro, RMSD, gráfico de superfície hidrofóbica. Foram calculados os mapas de interação molecular no programa GRID das seguintes proteínas: cruzaína, CPB2.8, catepsina B e catepsina L, e, posteriormente, foi construído um modelo farmacofórico baseado no sítio ativo das enzimas cruzaína e CPB2.8. O modelo farmacofórico da cruzaína foi validado por curva ROC apresentando valor de AUC 61%. A triagem virtual foi realizada para ambas as proteínas e foram obtidos 369 compostos para a cuzaína e 225 compostos para a CPB2.8. Foi realizado o ancoramento molecular desses compostos obtidos pela triagem virtual a fim de diminuir a quantidade de compostos a serem avaliados experimentalmente.
Neglected diseases are caused by parasites and infectious agents and affect mainly people in poor areas being prevalent in 149 countries and causing 534,000 deaths per year. Among neglected diseases we can highlight Chagas Disease and Leishmaniasis, both have a high rate of morbidity and mortality and both are addressed in this project in the search of new drugs against a NTD. Nowadays, the search for new drugs involves the selection of biological pathways essential for parasite survival, in this class of parasites we can suggest the cysteine proteases, a proteases family present in Trypanosoma cruzi and and Leishmania ssp. In order to obtain a new agent against Neglected Disease in this work was obtained the model of the enzyme CPB2.8 of L. mexicana using the comparative modeling technique, due to the unavailability of the crystallographic structure in the Protein Data Bank (PDB). In order to refine the constructed model was performed the molecular dynamics simulation of 100ns, stabilization was achieved from 80ns. Molecular dynamics simulation was validated using the Ramachandran graph, radius of rotation graph, RMSD, hydrophobic surface area graph. The molecular interaction fields were calculated in the GRID program to cruzain, CPB2.8, cathepsin B and cathepsin L. Based on molecular interaction fields generated pharmacophoric models were constructed using information about the active site of the enzymes cruzain and CPB2.8. The pharmacophoric model of cruzain was validated by ROC curve presenting AUC value of 61%. Virtual screening was performed for both proteins and 369 compounds were obtained for cuzain and 225 compounds for CPB2.8. Docking studies of these compounds was performed in order to decrease the amount of compounds to be evaluated experimentally.

Over the last twenty years, fragment-based drug discovery (FBDD) has emerged as a highly successful way to provide lead compounds for subsequent optimisation into drug candidates. Initial hits usually exhibit lower potency than those identified by more traditional techniques, such as High-Throughput Screening (HTS), but the optimisation phase of FBDD is highly efficient, thus providing superior lead-like compounds. The recent application of FBDD in a variety of protein kinase campaigns has successfully led to the identification of novel binding sites and highly efficient chemical ligands. This demonstrates the utility of the FBDD strategy against well-established kinase targets, where selectivity is otherwise challenging due to significant conservation of the ATP-binding site. Protein kinase CK2 is a ubiquitously expressed and constitutively active regulator of cell growth, proliferation and apoptosis. Elevated levels of CK2 protein and activity have historically been involved in human cancer, including lung, cervical and head and neck cancer types, and its overexpression is associated with worse prognosis. A number of CK2 inhibitors are currently available displaying activity against multiple cancers in vitro and in the clinic, however the majority of these candidates target the ATP-binding site and thus display poor selectivity in kinase panel assays. Here we explore the application of FBDD towards the development of potent and selective inhibitors of the catalytic α-subunit of CK2. This project exploits a novel, conserved binding site, named the αD pocket, for the generation of allosteric inhibitor molecules. Following structure-based optimisation of a potent inhibitor series, and characterisation of a previously unreported binding mode, a fragment linking strategy between the lead αD-site fragment and a low-affinity pseudosubstrate peptide is investigated. This work validates the utility of FBDD towards the discovery of new binding modes, presents a first in class CK2α allosteric inhibitor series and provides the first X-ray crystal structure of protein kinase CK2 in complex with a ligand binding in the substrate-binding channel.

A doença de Chagas, causada pelo parasita Trypanosoma cruzi, acomete entre 6 a 8 milhões de pessoas em todo o mundo. Conhecida como tripanossomíase americana, por ter sido considerada endêmica apenas na América Latina, esta doença, se espalhou para outros continentes devido aos movimentos migratórios se tornando um problema de sáude mundial. Estima-se que 56.000 novos casos e cerca de 12.000 mortes por complicações relacionadas à doença de Chagas anualmente. A quimioterapia disponível para o tratamento é composta apenas por dois fármacos, nifurtimox e benznidazol, no entanto são pouco eficazes na fase crônica da doença. Estes fármacos apresentarem, ainda, efeitos adversos graves e resistência por parte de algumas cepas do parasita. Diante deste panorama, é iminente a necessidade da busca de novos fármacos contra T. cruzi. Para a busca racional de novos quimiterapicos antiparasitários é fundamental a identificação e caracterização de vias metabólicas essenciais à sobrevivência dos parasitas. Assim, a enzima sirtuína 2 - Silent Information Regulator 2 (Sir2), tem importante papel para a infecção por T. cruzi, pois está totalmente envolvida no seu ciclo celular do parasita. Esta é uma enzima NAD+ dependente da classe III histona desacetilases, e se mostra como um interessante alvo bioquímico para o desenvolvimento de antichagásicos. A disponibilidade do sequenciamento genômico da Sir2 nos permite utilizar estratégias de planejamento de fármaco baseado no receptor (SBDD - Structure Based Drug Design) na identificação de candidatos a fármacos para essa doença. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar e selecionar inibidores enzimáticos potentes e seletivos para o alvo escolhido. Assim, neste trabalho, foi construído por meio da técnica de modelagem comparativa o modelo da enzima Sir2 de T. cruzi. Uma simulação por dinâmica molecular de 200ns, foi realizada para averiguar a estabilidade do modelo obtido. Diante da estabilização do modelo a partir de 100ns, o mesmo foi validado utilizando análise de clusters, RMSD (Root-mean-square Deviation) e análises de frequência de ligações de hidrogênio com o Cofator (NAD+) e os aminoácidos do sítio de catálise foram observadas, estes passos de simulação e validação foram realizados no programa DESMOND. Com o modelo robusto, os campos de interações moleculares (MIFs) foram gerados no programa GRID (Molecular Discovery v2.1) com o intuito de elucidar as regiões favoráveis a interação com a enzima em relação a propriedades físico-químicas da Sir2. A partir dos MIFs favoráveis a Sir2 de T. cruzi foi possível a construção de dois modelos farmacofóricos, o qual se baseou nas interações do Cofator (NAD+) e o sítio de catálise (Nicotinamida). O mesmo foi apliacdo como filtro para Triagem Virtual no programa UNITY da plataforma SYBYL X 2.0, utilizando os bancos de dados ZINC15 e GSK. A triagem resultou na seleção de 8 compostos candidatos a inibidores. Destes foram adquiridos 6 compostos por serem considerados mais promissores devido a complementariedade molecular. Estes foram testados contra a enzima de T. cruzi Sri2. Após o ensaio foi possível avaliar a potência de 4 compostos, sendo o composto CDMS-01 (IC50 = 39,9uM) o mais promissor que será submetido à processos de otimização molecular.
Chagas disease, caused by the parasite Trypanosoma cruzi, affects between 6 and 8 million people worldwide. Also known as American trypanosomiasis, because it is considered endemic only in Latin America, but has spread to other continents due to migratory movements. It is estimated that 56,000 new cases and about 12,000 deaths from complications related to Chagas disease annually. The chemotherapy available for treatment consists of only two drugs, nifurtimox and benznidazole, however these are poorly effective in the chronic phase. These drugs also have serious adverse effects and resistance from strains of the parasite. Faced with this scenario, the need to search for new drugs against T. cruzi is imminent. For the drug planning for new antiparasitic chemotherapics, the identification and characterization of metabolic pathways essential to the survival of parasites is fundamental. Therewith, the sirtuin 2 - Silent Information Regulator 2 (Sir2) enzyme has an important role for T. cruzi infection, since Sir2 in the parasite is totally involved in its cell cycle. This is an NAD+-dependent enzyme of class III histone deacetylases, and it shows an interesting biochemical target for the development of antichagasic. The availability of Sir2 genomic sequencing allows us to use SBDD (Structure Based Drug Design) strategies in identifying drug candidates for this disease. Among the modern techniques of SBDD used, virtual screening makes it possible to identify and select potent and selective enzyme inhibitors for the chosen target. The model of the T. cruzi Sir2 enzyme was constructed using the comparative modeling technique. A molecular dynamics simulation of 200ns was performed to ascertain the stability of the obtained model. Considering the stabilization of the model from 100ns, it was validated using cluster analysis, Root-mean-square Deviation (RMSD) and hydrogen bond frequency analyzes with Cofator (NAD+) and the amino acids of the catalysis site were observed, these simulation and validation steps were performed in the DESMOND program. With the robust model, the molecular interaction fields (MIFs) were generated in the GRID program (Molecular Discovery v2.1) in order to elucidate the regions favorable to the interaction with the enzyme in relation to the physicalchemical properties of Sir2. From the MIFs favorable to Sir2 of T. cruzi it was possible to construct two pharmacophoric models, which was based on the interactions of Cofator (NAD+) and the catalysis site (Nicotinamide). It was also applied as a Virtual screening filter in the UNITY program of the SYBYL X 2.0 platform, using the ZINC15 and GSK databases. Screening resulted in the selection of 8 inhibitor candidate compounds. Six compounds were obtained from the screening, because they were considered more promising, and were tested against T. cruzi Sri2 enzyme. After the assay it was possible to evaluate the potency of 4 compounds, the most promising compound being CDMS-01 (IC50 = 39.9 µM) that will be submitted to molecular optimization processes.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq
O Sistema Brasileiro de Coleta de Dados Ambientais (SBCDA) ? respons?vel pela coleta e transmiss?o de dados meteorol?gicos e ambientais, utilizados por dezenas de institui??es e organiza??es nacionais. O segmento espacial do sistema, composto pelos sat?lites de coleta de dados, ? uma pe?a fundamental para seu funcionamento. Para garantir a continuidade e qualidade desses servi?os, esfor?os s?o voltados para o desenvolvimento de alternativas para a constru??o de novos sat?lites. Visando reduzir o tamanho do atual transponder brasileiro, ? proposto neste trabalho a integra??o de uma interface de r?dio frequ?ncia (do ingl?s FrontEnd) a ser embarcado nos receptores da pr?xima gera??o dos sat?lites SBCDA. O circuito ? concebido respeitando os requisistos do padr?o de coleta de dados internacional, ARGOS, e do SBCDA. Este trabalho foca, portanto, na integra??o de um amplificador de baixo ru?do e dois misturadores na faixa UHF em tecnologia CMOS padr?o. As especifica??es de projeto s?o primeiramente descritas e ent?o as topologias dos circuitos s?o escolhidas. A concep??o dos circuitos ? analisada e os seus par?metros de projeto s?o derivados. Finalmente, seu layout ? concebido e os resultados finais s?o divulgados. O chip ser? fabricado utilizando a tecnologia CMOS padr?o de 130 nm da STmicroelectronics.
The Brazilian Environmental Data Collecting System (SBCDA) collects and broadcasts meteorological and environmental data, to be handled by dozens of institutions and organizations. The system space segment, composed by the data collecting satellites, plays an important role for the system operation. To ensure the continuity and quality of these services, efforts are being made to the development of new satellite architectures. Aiming a reduction of size and power consumption, the design of an integrated circuit containing a receiver front-end is proposed, to be embedded in the next SBCDA satellite generations. The circuit will also operate under the requirements of the international data collecting standard ARGOS. This work focuses on the design of an UHF low noise amplifier and mixers in a CMOS standard technology. The specifi- cations are firstly described and the circuit topologies presented. Then the circuit conception is discussed and the design variables derived. Finally, the layout is designed and the final results are commented. The chip will be fabricated in a 130 nm technology from ST Microelectronics.

The automotive field is becoming more and more complex and cars are no longer just pure mechanical artifacts. Today much more than 50 % of the functionality of a car is computerized, so, a modern car system is obviously based on mixed technologies which emphasize the need for new approaches to the design process compared to the processes of yesterday. A corresponding technology shift has been experienced in the aerospace industry starting in the late sixties and today aircraft could not fly without its computers and the pilots’ environment has turned to a so called glass cockpit with no iron-made instrumentation left. A very similar change is still going on in the automotive area.

Simulator-Based Design (SBD) refers to design, development and testing new products, systems and applications which include an operator in their operation. Simulator-Based Design has been used for decades in the aviation industry. It has been a common process in this field. SBD may be considered as a more specific application of simulation-based design, where the specific feature is a platform, the simulator itself. The simulator could consist of a generic computer environment in combination with dedicated hardware components, for instance a cockpit. This solution gives us the possibility of including the human operator in the simulation.

The name of the project is Simulator-Based Design in Practice. The purpose of this master thesis is to get a complete practice in how to use a human-in-the-loop simulator as a tool in design activities focusing on the automotive area. This application area may be seen as an example of systems where an operator is included in the operation and thus experience from the car application could be transferred to other areas like aviation or control rooms in the process industry.

During the performance of the project we have gone through the main parts of the SBD process. There are many steps to complete the whole cycle and many of them have iterative loops that connect these steps with the previous one. This process starts with a concept (product/system) and continues with a virtual prototyping stage followed by implementation, test design, human-in-the-loop simulation, data analysis, design synthesis and in the end a product/system decision. An iterative process approach makes the cycle flexible and goal oriented.

We have learnt how to use the simulator and how to perform the whole cycle of SBD. We first started getting familiar with the simulator and the ASim software and then we were trying to reduce the number of computers in the simulator and changing the network in order to find good optimization pf the computer power. The second step has been to implement a new application to the simulator. This new application is the rear mirror view and consists of a new LCD monitor and the rear view vision that must be seen in the new monitor. Finally we updated the cockpit to the new language program Action Script 3.0.

The information gathering consisted of the course Human-System interaction in the University, the introduction course to ASim software and the course of Action Script 3.0.

The synthesis of a number of alkyl esters such as methyl lactate, methyl decanoate, and ethyl benzoate via esterification in a reactive distillation is quite challenging. It is due to the complexity in the thermodynamic behaviour of the chemical species in the reaction mixture in addition to the difficulty of keeping the reactants together in the reaction section. One of the reactants (in these esterification reactions) having the lowest boiling point can separate from the other reactant as the distillation continues. This can result in a significant drop in the reaction conversion in a conventional reactive distillation whether it is a batch or a continuous column. To overcome this challenge, new different types of batch reactive distillation column configurations: (1) integrated conventional (2) semi-batch (3) integrated semi-batch (4) integrated dividing-wall batch distillation columns have been proposed here. Four esterification reaction schemes such as (a) esterification of lactic acid (b) esterification of decanoic acid (c) esterification of benzoic acid (d) esterification of acetic acid are investigated here. A detailed dynamic model based on mass, energy balances, chemical reaction, and rigorous thermodynamic (chemical and physical) properties is considered and incorporated in the optimisation framework within gPROMS (general PROcess Modelling System) software. It is found that for the methyl lactate system, the i-SBD operation outperforms the classical batch operations (CBD or SBD columns) to satisfy the product constraints. While, for the methyl decanoate system, the i-DWCBD operation outperforms all CBD, DWBD and sr-DWBD configurations by achieving the higher reaction conversion and the maximum product purity. For the ethyl benzoate system, the performance of i-CBD column is superior to the CBD process in terms of product quality, and conversion rate of acid. The CBD process is found to be a more attractive in terms of operating time saving, and annual profit improvement compared to the IBD, and MVD processes for the benzyl acetate system.
The Higher Committee for Education Development in Iraq (HCED)

A esquistossomose é uma das principais causas de morbidade em países Tropicais e Subtropicais, gerando graves consequências socioeconômicas. Atualmente, os fármacos disponíveis para o tratamento da desta doença são praziquantel e oxamniquina, porém relatos de baixa susceptibilidade do parasita a esses medicamentos sugerem a necessidade de novas estratégias terapêuticas para o tratamento da doença. Todavia, existe pouco interesse da indústria farmacêutica no desenvolvimento de fármacos contra doenças tropicais e negligenciadas, entre as quais se encontra a esquistossomose. Devido a estes fatores, o presente trabalho teve por objetivo geral utilizar ferramentas computacionais para identificar inibidores da SmDHFR candidatos a novos fármacos. Avaliou-se as características exclusivas para a proteína de S. mansoni por meio de uma análise das sequências FASTA em comparação com a DHFR de outros organismos. A fim de garantir a ação seletiva dessas moléculas frente a enzima do parasita, os campos moleculares de interação seletivos para SmDHFR foram calculados e empregados na construção do modelo farmacofórico, o qual foi utilizado na triagem virtual de inibidores de SmDHFR. Os estudos computacionais realizados nos permitiram a seleção de 20 moléculas com uma boa complementariedade com o modelo farmacofórico gerado e com potencial para serem inibidores de SmDHFR.
Schistosomiasis is one of morbidity\'s main causes in tropical and subtropical countries, which leads to serious socioeconomic consequences. Praziquantel and oxamniquina are the drugs currently available for treating this disease, but reports points that the parasite has been resistant to both drugs, which suggests the need for new therapeutic strategies for the treatment of this disease. However, there is little interest in the pharmaceutical industry in developing drugs against neglected tropical diseases, including schistosomiasis. Due to these factors, the present work has the general objective to use computational tools to identify SmDHFR inhibitors which could be good candidates for developing new drugs. Evaluation of the exclusive characteristics of the S. mansoni protein were performed by FASTA sequence analyses in comparison to DHFR from other organisms. In order to guarantee the selective action of these molecules against the parasite enzyme, the molecular interaction fields selective for SmDHFR were calculated and used in the construction of the pharmacophoric model, which was further used in the virtual screening of SmDHFR inhibitors. Computational studies were performed and those led us to 20 molecules with a good complementarity with the pharmacophoric model that was previously generated and with potential to be SmDHFR inhibitors.

The gradually increasing competition that occurs in mature markets, pushes the companies to consider new ways of competing. The more mature the market becomes the higher level of competitive fulfillment and edge is needed. E.g. for Scania the dimension high quality was until now considered a competitive advantage, but is currently evolving towards a dimension that is necessary to fulfill to even act within the market. Thus, companies such as Scania have to push their limits everywhere possible across their organization. One way to push the limits of R&D and product development (PD) is, based on this thesis and other work, implementing a simulation based product development (SBPD) process. The aim of this thesis has been, by applying a quality function deployment (QFD) model, to show the influence a SBPD process can have on increased competitiveness. By combining published literature within the field with empirical results mostly gained from interviews, nine components representing a SBPD process could be found. These components were characterized in the following categories: IT-infrastructure, design, model & test, and organizational. Depending on the level of fulfillment of these components the SBPD process is more or less implemented within the company. The two components that are considered the most important for Scania in terms of both increased competitiveness and a possible transition towards a SBPD process are Virtual representa-tion of the product and the process throughout the whole PD process and A model driven/model based approach. The work of this thesis has also shown, in correlations with previous published literature, that a SBPD process can significantly decrease the lead-time and cost of development, and at the same time increase the knowledge about the product and the process. The research has also shown that a SBPD process can considerably improve both the internal and the external collaboration. However attaining such a process where these competitive advantages can be gained affects the whole organization, the way development is performed and also the way resources are used. A fully implemented SBPD process has been shown to positively impact the following competitive dimensions; Frequency of product introductions, Lead-time of development, PD process flexibility, Degree of innovation, Customization of the offer, Collaboration with internal and external partners, Knowledge about products and processes, R&D cost, manufacturing cost, Focus on customers operational economics, Product quality, Product safety and Focus on environmental sustainability of the product & the processes. But, to get a positive impact on all these dimensions the components representing a SBPD process must be implemented and applied. Furthermore, this thesis also provides a framework for other companies to evaluate the impact a SBPD process can have on their competitiveness. The analysis model aims to guide companies that want to adopt a SBPD process. The framework can indicate how a SBPD process would impact the firm's competitive strategy. But also to provide guidance to which components that would be more important to implement in order to reach a competitive SBPD process.
Den gradvis ökande konkurrens som uppstår på mogna marknader driver företagen att finna nya sätt att konkurrera. Ju mer mogen marknaden blir desto högre blir kraven på konkurrens. För Scania har faktorn hög kvalitet tills nu betraktas som en konkurrensfördel, men utveckling sker istället mot att det kommer att vara en faktor som är nödvändig att uppfylla för att ens kunna agera på marknaden. Företag som Scania måste således utvecklas över hela organisationen. Ett sätt att tänja på gränserna för FoU och produktutveckling (PD) är, baserat på detta arbete och annan tidigare forskning, att genomföra en förändring mot en simulering baserad produktutvecklings-process (SBPD). Syftet med detta examensarbete har varit att, genom att tillämpa en quality function deployment (QFD) modell, visa effekten en SBPD-process kan ha på ökad konkurrenskraft. Genom att kombinera tidigare publicerad litteratur inom området med empiriska resultat, mestadels insamlat genom intervjuer, kunde nio komponenter som utgör en SBPD-process finnas. Dessa komponenter karakteriserades i följande kategorier: IT-infrastruktur, design, modellera och testa och organisatorisk. Beroende på graden av uppfyllnad på dessa komponenter blir den SBPD-processen mer eller mindre implementerad inom företaget. De två komponenterna som anses viktigast för Scania i form av både ökad konkurrenskraft och en eventuell övergång till en SBPD-process är Virtuell representation av produkten och processen genom hela PD-processen och Ett modellbaserat/ modelldrivet arbetssätt används. Arbetet har också visat i korrelation med tidigare publicerad litteratur, att en SBPD-process kommer att minska ledtiden och kostnaden för utveckling avsevärt och samtidigt öka kunskapen om produkten och processen. Denna studie har också visat att en SBPD-process kan förbättra både interna och externa samarbeten. Men att uppnå en sådan process där dessa konkurrens-fördelar kan vinnas kommer att påverka hela organisationen, det sätt som utveckling utförs på och också hur resurserna används. En fullt implementerad SBPD-process har visat positivt påverkan följande konkurrens faktorer; frekvens av produktlanseringar, ledtiden för utveckling, PD-processflexibilitet, innovationsgrad, anpassning av erbjudandet, samarbete med interna och externa partners, kunskap om produkter och processer, FoU kostnader, tillverkningskostnad, fokus på kundernas driftsekonomi, produktkvalitet, produktsäkerhet och miljöpåverkan av både produkten och processen. För att få en positiv inverkan på alla dessa faktorer måste alla komponenter som utgör en SBPD-process implementeras och tillämpas. Dessutom så presenteras även ett ramverk för andra företag att utvärdera effekterna en SBPD-process skulle ha på deras konkurrenskraft. Analysmodellen syftar till att vägleda företag som vill övergå till en SBPD-process. Ramverket kan ange hur en SBPD-process skulle kunna påverka företagets konkurrensstrategi men också ge vägledning om vilka komponenter som kan vara viktigare att börja implementera för att nå en konkurrenskraftig SBPD-process.

Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods typically assess the pocket as a whole, doing little to suggest which regions and interactions are the most important for binding. This thesis introduces Fragment Hotspot Maps, a grid-based method that samples atomic propensities derived from interactions in the Cambridge Structural Database (CSD) with simple molecular probes. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores, offering more precision over other binding site prediction methods. The method is validated by scoring the positions of 21 fragment and lead pairs. Fragment atoms are found in the highest scoring parts of the map corresponding to their atom type, with a median percentage rank of 98%. This is reduced to 72% for lead atoms, showing that the method can differentiate between the hotspots, and the warm spots later used during fragment elaboration. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. These calculations are easily accessible through a simple to use web application, which only requires an input PDB structure or code. High scoring specific interactions predicted by the Fragment Hotspot Maps can be used to guide existing computer aided drug discovery methods. The Hotspots Python API has been created to allow these work flows to be executed programmatically through a single Python script. Two of the functions use scores from the Fragment Hotspot Maps to guide virtual screening methods, docking and field-based ligand screening. Docking virtual screening performance is improved by using a constraint selected from the highest scoring polar interaction. The field-based ligand screener uses modified versions of the Fragment Hotspot Maps directly to predict and score the binding pose. This workflow gave comparable results to docking, and for one target, Glucocorticoid receptor (GCR), showed much better results, highlighting its potential as an orthogonal approach. Fragment Hotspot Maps can be used at multiple stages of the drug discovery process, and research into these applications is ongoing. Their utility in the following areas are currently being explored: to assess ligandability for both individual structures and across proteomes, to aid in library design, to assess pockets throughout a molecular dynamics trajectory, to prioritise crystallographic fragment hits and to guide hit-to-lead development.

Ligand-protein binding models have experienced an evolution during time: from the lock-key model to induced-fit and conformational selection, the role of protein flexibility has become more and more relevant. Understanding binding mechanism is of great importance in drug-discovery, because it could help to rationalize the activity of known binders and to optimize them. The application of computational techniques to drug-discovery has been reported since the 1980s, with the advent computer-aided drug design. During the years several techniques have been developed to address the protein flexibility issue. The present work proposes a strategy to consider protein structure variability in molecular docking, through a ligand-based/structure-based integrated approach and through the development of a fully automatic cross-docking benchmark pipeline. Moreover, a full exploration of protein flexibility during the binding process is proposed through the Supervised Molecular Dynamics. The application of a tabu-like algorithm to classical molecular dynamics accelerates the binding process from the micro-millisecond to the nanosecond timescales. In the present work, an implementation of this algorithm has been performed to study peptide-protein recognition processes.
I modelli di riconoscimento ligando-proteina si sono evoluti nel corso degli anni: dal modello chiave-serratura a quello di fit-indotto e selezione conformazionale, il ruolo della flessibilità proteica è diventato via via più importante. Capire il meccanismo di riconoscimento è di grande importanza nella progettazione di nuovi farmaci, perchè può dare la possibilità di razionalizzare l’attività di ligandi noti e di ottimizzarli. L’applicazione di tecniche computazionali alla scoperta di nuovi farmaci risale agli anni ‘80, con l’avvento del cosiddetto “Computer-Aided Drug Design”, o, tradotto, progettazione di farmaci aiutata dal computer. Negli anni sono state sviluppate molte tecniche che hanno affrontato il problema della flessibilità proteica. Questo lavoro propone una strategia per considerare la variabilità delle strutture proteiche nel docking, attraverso un approccio combinato ligand-based/structure-based e attraverso lo sviluppo di una procedura completamente automatizzata di docking incrociato. In aggiunta, viene proposta una piena esplorazione della flessibilità proteica durante il processo di legame attraverso la Dinamica Molecolare Supervisionata. L’applicazione di un algoritmo simil-tabu alla dinamica molecolare classica accelera il processo di riconoscimento dalla scala dei micro-millisecondi a quella dei nanosecondi. Nel presente lavoro è stata fatta un’implementazione di questa algoritmica per studiare il processo di riconoscimento peptide-proteina.

La conoscenza della struttura tridimensionale di un potenziale target farmacologico apre la via a nuove strategie terapeutiche (ad esempio tramite structure-based drug design (SBDD)) ed è requisito fondamentale per la bioinformatica strutturale. In questo contesto, durante la mia tesi di dottorato, sono state studiate due proteine di interesse biomedico. La prima è una proteina di membrana, l’isoforma 1 dell’Acid Sensing Ion Channel (ASIC), implicata in diverse malattie neurodegenerative. In studi precedenti il diminazene aceturato (DA) si era dimostrato un potente inibitore del canale. Diversi analoghi di DA sono stati progettati su base strutturale e la loro affinità per ASIC analizzata tramite docking molecolare. Le molecole migliori sono state testate in saggi cellulari per valutarne l’efficacia. Per caratterizzare la capacità inibitoria degli analoghi sintetizzati in vitro, è stato messo a punto un protocollo per la produzione della proteina ASIC1, utilizzando diversi sistemi di espressione eterologa. La purificazione della proteina è stata effettuata usando un approccio high-throughput per supportare successivamente la cristallizzazione della proteina, al fine di ottenere informazioni più dettagliate sul meccanismo d’azione degli analoghi del DA e, di conseguenza, disegnare nuovi farmaci, isoforma-selettivi e in grado di attraversare la barriera emato-encefalica. In secondo luogo, ho studiato la proteina Gelsolin (GSN), responsabile di una malattia familiare degenerativa (detta amiloidosi AGel). Lo scopo di questo progetto era quello di investigare l'effetto della mutazione D187N sulla struttura di GSN e la sua propensione ad aggregare e/o degradarsi in maniera anomala. Il D187N GSN è stato il primo mutante ad essere identificato, ma, ad oggi, non si avevano informazioni sulla sua struttura. In uno studio precedente, era stato identificato un nanobody (Nb11) in grado di proteggere la proteina dalla degradazione, ma il meccanismo di protezione non era stato chiarito. Nel mio lavoro ho risolto la struttura del complesso Nb11:D187N a 1.9 Å, permettendo la caratterizzazione molecolare del meccanismo di azione del Nb11. I dati strutturali ottenuti sono stati completati con una caratterizzazione biofisica e biochimica, estesa anche ad altre due varianti patologiche della GSN, recentemente identificate (G167R e N184K).
Knowledge of the three-dimensional structure of therapeutically relevant proteins paves the way for novel strategies in pharmacological research (such as the structure-based drug design (SBDD) method) and establishes the foundations for structural bioinformatics. In this context, during my PhD Thesis, two therapeutically relevant proteins have been studied. First, a membrane protein, Acid Sensing Ion Channel (ASIC) isoform 1, a validated target in neurodegenerative disorders, was selected. Previous studies showed that diminazene aceturate (DA) is a potent small-molecule inhibitor of ASIC channels. Here, several DA analogues were screened by molecular docking and the best binders were tested in cell-based assays to further assess their efficacy. In order to determine the inhibitory capability of the synthesized analogues in vitro on the purified protein, the expression of ASIC1 was undertaken, using different organisms of expression. The protein purification was performed in a high-throughput approach in order to recover enough protein for crystallization, with the final aim of studying the mechanism of action of DA analogs, and support the design of new, isoform-selective and brain-penetrant drugs. Secondly, the soluble protein Gelsolin (GSN), responsible for a familial degenerative disease (AGel amyloidosis) was studied. Aim of this project was to understand the impact of the D187N mutation on GSN structure and its propensity to aberrant aggregation and/or degradation. D187N GSN mutant was the first identified in man, but its crystal structure had until now eluded any characterization. Conversely, a nanobody (Nb11) was shown to protect GSN from aberrant proteolysis, but its mechanism of protection remained unclear. Here, the structure of the Nb11:D187N complex was solved at 1.9 Å resolution, enabling the characterization of the Nb11action mechanism. The structural data were complemented with biophysical and biochemical characterisations. These studies were then extended to two recently identified pathological variants of GSN (G167R and N184K).

97% des articles publiés sur les études climatiques racontent que le réchauffement climatique est entièrement causé par les activités humaines. Les gaz émis lors de la production d'énergie électrique ainsi que d'autres gaz rejetés par les voitures ont un réel impact sur l'atmosphère. Une solution consiste à mettre au point des composants présentant des pertes de conduction plus faibles et des caractéristiques de claquage plus élevées qui pourraient être utilisés dans des centrales nucléaires, des cellules de commutation à haute puissance, des voitures hybrides (électriques), etc.De nos jours, les composants à base de silicium contrôlent environ 95% des dispositifs électroniques. Le carbure de silicium SiC et le nitrure de gallium GaN sont actuellement à l’étape de R&D, et commencent à être intégrés dans certains circuits électroniques. D'autres matériaux tels que Ga2O3, AlN ou le diamant sont encore à l’étape de recherche. Les derniers sont connus sous le nom de matériaux à bande ultra large et semblent être la solution requise pour les faibles pertes de puissance. Le diamant est reconnu comme le matériau ultime pour la prochaine génération de composants de puissance en raison de ses propriétés physiques exceptionnelles telles qu'un champ de claquage élevé (>10 MV/cm) permettant d'utiliser le dispositif pour une commande de puissance élevée, une mobilité de porteurs élevée (2 000 cm^2/V.s pour les trous), une vitesse de saturation élevée, une conductivité thermique élevée (22 W/cm.K) pour une parfaite dissipation de chaleur et une faible constante diélectrique. Théoriquement, le diamant est le semi-conducteur offrant le meilleur compromis entre résistance à l'état passant et tension de claquage. En particulier, en raison de l'ionisation incomplète des dopants, il est encore plus efficace à haute température. Diverses diodes Schottky en diamant (SBD) avec de bonnes performances à l’état passant et bloqué (7,7 MV/cm) ont été rapportées. En plus des SBDs, des transistors à effet de champ (FET) ont également été étudiés à travers des oxyde-métal semi-conducteur FETs (MOSFETs) utilisant une surface hydrogénée avec des densités de courant élevées à l'état passant ou des surface oxygéné avec de bonnes caractéristiques de blocage. Pour les composants de haute-tension, il est nécessaire de changer l’architecture de l’électrode afin d’éviter un claquage prématuré due à l’encombrement du champ électrique aux bords. Dans ce but, les techniques de terminaison de bord sont utilisées pour atteindre les caractéristiques idéales. La tâche évidente avant toute fabrication de composant est la partie simulation qui prédit l’optimisation de l’architecture et les caractéristiques attendues. Une bonne prédiction nécessite la connaissance des paramètres du matériau. Les paramètres importants pour le claquage sont les coefficients d'ionisation par impact. Plusieurs coefficients ont été publiés pour le diamant. Toutefois, ils ont été extraits en « fittant » des structures non optimisées, d'où un manque de précision.Dans cette étude, deux structures de terminaisons de bord pour des diodes Schottky, appelées plaque de champ et anneaux à champ flottant, ont été étudiées. Leur efficacité de distribution du champ de surface par analyse de courant induit par faisceau d'électrons (EBIC) a été observée. De plus, des FETs ont été fabriqués et caractérisés, un MESFET et un RB-MESFET. Les FETs présentent un claquage élevé, jusqu’à 3 kV et une faible résistance. Le développement des transistors est indissociable de la diode Schottky, car ils sont tous deux nécessaires à la fabrication de cellules de commutation. Et enfin, les coefficients d'ionisation par impact pour les électrons ont été mesurés à l'aide d’EBIC pour un champ >0,5 MV/cm dans une région sans défaut. Les valeurs mesurées sont (sous l’equation de Chynoweth) an = 971 /cm et bn=2,39x10^6 V/cm. Ces valeurs sont proches des coefficients mesurés expérimentalement et rapportés dans la littérature
97% of the published climate studies articles agree with the fact that recent global warming is entirely caused by human activities. The gases emitted to produce electrical energy plus other gases rejected by cars impact considerably on the atmosphere by greenhouse effect (without referring other factors). A solution to this problem is the development of components with lower power conduction losses and higher breakdown characteristics that could be used in nuclear power plants, high power commutation cells, hybrid (electric) cars and so on.The choice of the material to reach low power conduction losses and higher breakdown is of great importance. Nowadays, silicon-based devices control about 95% of all electronic components. Silicon carbide SiC and gallium nitride GaN are at present under research and development and start to be integrated into some electronic circuits. Other materials like Ga2O3, AlN or diamond are under research for power electronic application. The last ones are known as ultra wide bandgap materials and they seem to be the required solution to low power losses. Diamond is recognized as the ultimate material for the next next-generation of power devices owing to its exceptional physical properties such as high breakdown field (>10 MV/cm) to use the device for high power control, high carrier mobility (2000 cm^2/V.s for holes) for fast switching and high frequency devices, high saturation velocity, high thermal conductivity (22 W/cm.K) for a perfect heat dissipation and low dielectric constant. Theoretically, diamond is the best semiconducting material showing the best trade-off between on-resistance and breakdown voltage. Especially, due to the incomplete ionization of the dopant, it is even more efficient at high temperature. Various diamond Schottky barrier diodes (SBDs) with good forward and reverse performances (7.7 MV/cm) were reported. In addition to SBDs, switches diamond field effect transistors (FETs) were also investigated through metal-oxide-semiconductor FETs (MOSFETs) using either an H-terminated diamond surface with high current densities in on-state or an O-terminated one with high blocking characteristics. For the high blocking voltage devices, one needs to properly terminate the edge of the electrode at the surface in order to avoid premature breakdown of the devices due to electric field crowding at the borders. In that aim, edge termination (ET) techniques are used to push the limit of the devices and reach ideal features. The obvious task before any device fabrication if the simulation part that predicts the device optimization and expected characteristics. A good device prediction requires knowledge of the material parameters. Important parameters for device breakdown in the off-state are the impact ionization coefficients. At present, several ionization coefficients were reported for diamond, however, they were extracted by fitting non-optimized structures and hence there is a lack of accuracy.In this study, two edge terminations structures for Schottky barrier diodes called field plate (FP) oxide and floating field rings were investigated. Their effectiveness in surface field distribution via electron beam induced current (EBIC) analysis was observed. In addition, normally-on FETs were fabricated and characterized, a MESFET and a reverse blocking (RB)-MESFET. The FETs exhibited a high BV, up to 3 kV and a low on-resistance. The development of transistors is inseparable from the Schottky diode since both are required to fabricate commutation cells. And finally, impact ionization coefficients for electrons were measured using EBIC for a field >0.5 MV/cm in a defect-free region. The measured values are (in a Chynoweth form) an = 971 /cm and bn = 2.39x10^6 V/cm. These values are close to the experimentally measured coefficients reported in the literature

La progressivité du claquage des oxydes de grille d'épaisseurs inférieures à 20 nm permet d'envisager une prolongation de la durée de vie des circuits. Cet enjeu majeur de la fiabilité contemporaine requiert des modèles adaptés afin de contrôler la variabilité des paramètres induites par le claquage. Après avoir étudié l'impact d'une fuite de courant sur une couche chargée, nous avons mis au point un modèle bas niveau de simulation par éléments finis, capable de reproduire la dérive des paramètres mesurée sur des dispositifs du nœud 45 nm. Des lois empiriques de ces dérives ont été injectées dans un modèle compact du transistor dégradé, simplifié par nos observations originales de la dépolarisation du canal et de la répartition des courants. Finalement nous avons simulé l'impact du claquage sur le fonctionnement de circuits simples et estimés la dérive de leurs paramètres tels que l'augmentation de la consommation due au claquage.

Human-in-the-loop simulators have long been used in the research community as well as in industry. The aviation field has been the pioneers in the use of simulators for design purposes. In contrast, corresponding activities in the automotive area have been less widespread. Published reports on experimental activities based on human-in-the-loop simulations have focused on methods used in the study, but nobody seems to have taken a step back and looked at the wider methodological picture of Simulator-Based Design. The purpose of this thesis is to fill this gap by drawing, in part, upon the author’s long experience in this field. In aircraft and lately also in ground vehicles there has been a technology shift from pure mechanics to computer-based systems. The physical interface has turned into screen-based solutions. This trend towards glass has just begun for ground vehicles. This development in vehicle technology has opened the door for new design approaches, not only for design itself, but also for the development process. Simulator-Based Design (SBD) is very compatible with this trend. The first part of this thesis proposes a structure for the process of SBD and links it to the corresponding methodology for software design. In the second part of the thesis the focus changes from methodology to application and specifically to the design of three-dimensional situation displays. Such displays are supposed to support the human operator with a view of a situation beyond the more or less limited visual range. In the aircraft application interest focuses on the surrounding air traffic in the light of the evolving free-flight concept, where responsibility for separation between aircraft will be (partly) transferred from ground-based flight controllers to air crews. This new responsibility must be supported by new technology and the situational view must be displayed from the perspective of the aircraft. Some basic design questions for such 3D displays were investigated resulting in an adaptive interface approach, where the current situation and task govern the details of information presentation. The thesis also discusses work on situation displays for ground vehicles. The most prominent example may be the Night Vision system, where the road situation ahead is depicted on a screen in the cab. The existing systems are based on continuous presentation, an approach that we have questioned, since there is strong evidence for negative behavioral adaptation. This means, for example, that the driver will drive faster, since vision has been enhanced, and thereby consume the safety margins that the system was supposed to deliver. Our investigation supports a situation-dependant approach and no continuous presentation. In conclusion, the results from our simulator-based studies showed advantages for adaptive interface solutions. Such design concepts are much more complicated than traditional static interfaces. This finding emphasizes the need for more dynamic design resources in order to have a complete understanding of the situation-related interface changes. The use of human-in-the-loop simulators and deployment of Simulator-Based Design will satisfy this need.

Le cancer représente l'un des problèmes majeurs en santé publique. Jusqu'à présent, en parallèle de l'intervention chirurgical, plusieurs traitements ont été mis au point et largement utilisés en thérapie clinique telles que les chimiothérapies. Cependant, leur efficacité est parfois limitée et couplée à des effets secondaires très néfastes, laissant les patients dans une impasse thérapeutique. Par conséquent, de nouvelles approches thérapeutiques doivent être développées sur de nouvelles cibles avérées en oncologie afin d'apporter des soins personnalisés aux patients. La première partie de mon travail de thèse a été dédiée à la compréhension des mécanismes moléculaires de la nucléotidase cytosolique de type II (cN-II), une enzyme du métabolisme des purines dont l'implication dans des phénomènes de résistance à des traitements anticancéreux a pu être démontrée. Aussi, une étude sur la cinétique enzymatique à l'état pré-stationnaire et stationnaire a été entreprise sur la forme sauvage et une forme mutée de l'enzyme lui conférant une activité accrue fortement impliquée dans les cas de résistance. Par cette approche, il a été possible de décortiquer le mécanisme cinétique, de définir l'étape cinétiquement limitant afin d'identifier les intermédiaires prépondérants de la réaction pouvant être ciblés pour le développement de nouveaux inhibiteurs. Cette étude cinétique est présentée dans ce premier volet de la thèse. En second lieu, mon travail s'est focalisée sur un second membre de cette famille d'enzyme qui est l'ecto-5'-nucléotidase (CD73). Cette enzyme exprimée sous forme dimérique à la surface extracellulaire régule la concentration en adénosine extracellulaire (par hydrolyse de l'adénosine monophosphate), ce dernier étant un puissant immunosuppresseur de la réponse immune anticancéreuse. L'objectif de mon travail de thèse a été de développer de nouveaux inhibiteurs de type allostérique en utilisant une approche basée sur la structure tridimensionnelle et la dynamique moléculaire. Une des étapes clés a été tout d'abord de mettre au point un système expression hétérologue afin d'obtenir l'enzyme recombinante en quantité suffisante pour les études enzymatiques ultérieures. Différents systèmes d'expression ont été testés et seul le système en cellules d'insecte infectées par le baculovirus a permis d'obtenir l'enzyme active en grande quantité. En parallèle, une étude in silico a permis de reproduire la dynamique fonctionnelle de l'enzyme requise pour sa fonction. A partir de ses données, un criblage virtuel d'une chimiothèque de 324 000 molécules a été réalisé sur le site de dimérisation et a permis d'identifier 33 composés chefs de files. Parmi, ces composés, dix molécules se sont avérés être de puissants inhibiteurs de CD73 (Ki < 1 µM) avec un mécanisme d'inhibition de type allostérique ou non-compétitif. La cytotoxicité des composés a été évaluée sur des lignées cellulaires transformées ou tumorales montrant un effet uniquement à des concentrations très élevées (supérieures à 100 µM). L'étude des relations structure-fonction devrait permettre à présent de proposer de voies d'optimisation afin d'améliorer l'efficacité des composés les plus actifs afin d'aboutir à de nouveaux candidats médicaments
Cancer burden still remains a major worldwide health problem. To date, several types of conventional anticancer treatments are widely used in clinical. However, the alternative effects of these treatments often leave patients impaired. Therefore, it is required to understand the unique medical needs of individual patients and to conduct effective, high–quality research focusing on the not yet identified oncotargets.The first part of my thesis is dedicated to decipher molecular basis of cN-II reaction. This study characterizes the steady state and transient state kinetics of cN-II wild type and hyperactive mutant which involved in cancer treatment resistance. Furthermore, the characterization of the rate-limiting step and reaction intermediates gave insights into the binding mechanisms and the development of small molecules inhibitors of cN-II.In the second part of this work, we aimed to investigate allosteric inhibitors of CD73 using structure-based drug design approach. In this study the suitable protein expression system was established for the production of sufficient quantities of fully active CD73. This work followed by in silico studies, including molecular dynamics, virtual screening, and hits identification and in vitro hits validations and kinetics characterizations. The cytotoxicity of the most powerful inhibitors exhibited on different cell types was determined. SAR studies gave insights into the binding mode of best compounds and function

This thesis deals with the topic of  model-based design and application of simulation in system design. In the introduction, the thesis discusses the history of software development process and outlines the current status. The aim is to demonstrate a model-driven design on a case study of conference system. There will be presented formalisms of DEVS and OOPN  together with experimental tools PNtalk and SmallDEVS that allow to work with these formalisms. The resulting model of conference system will be deployed as part of a web application using a framework Seaside in the Squeak environment.

碩士
國立成功大學
微電子工程研究所碩博士班
91
Schottky barrier diodes (SBDs) with low forward voltage drop (VF), low reverse leakage current (IR), low power loss and high breakdown voltage (VBD), etc., have been urgently required in electronic industry. Essentially, VF and IR of SBDs are key factors in determining the power loss of SBDs for power applications, which strongly depend on the Schottky barrier height (SBH). In general, a larger SBH would result in a lower IR but a larger VF, while a lower SBH shows an inverse situation. How to solve or release the trade-off problem between VF and IR, how to improve the breakdown voltage of SBDs to approach its theoretical value, and how to minimize the power loss of SBDs without sacrificing other device properties, are still open problems in the SBDs fabrication. In this thesis, a novel device design with a RESURF type lateral super-junction for edge termination, a novel polysilicon (poly-Si) guarding ring and related fabrication process including Boron ion implantation for IR reduction and VBD enhancement are presented to tackle the open problems mentioned above. Both theoretical and experimental studies including optimum device design and device fabrication process have been conducted in this study. Special emphasis for the theoretical study is focused on the design and simulation of edge termination with super-junction, poly-Si floating ring, guard ring, and field plate. Influence of device structural parameters used in the device geometry was investigated in detail. In this thesis, technology related to high breakdown voltage device design and fabrication process has been established. It is found that the device and fabrication technology developed in the present study could be successfully applicable to the realization of SBDs with VBD > 110V, IR < 10uA/cm2,VF < 0.5V @1A/cm2 and an adjustable SBH (0.778~0.796 V).

碩士
國立雲林科技大學
技術及職業教育研究所碩士班
91
The purpose of this study was to develop a vocational school’s new business curriculum by applying a school-based curriculum development (SBCD) model. An action research approach was employed to conduct the study. The uniqueness of this study is that it treats a business program, instead of the whole school’s curriculum, as the study unit for curriculum reform practice. Another feature of this study is that it is a bottom-up , spontaneous-oriented curriculum reform action. The literature review shows the successive changes of business department curriculum. It also shows the theory of technological and vocational education curriculum and the developing mode. The school-based curriculum theory practice, and related methods are presented as well develops the suitable theory basis and application modes to develop a new business curriculum. Then this study applies the develop of mode to the real situation. At the final stage, the researcher examines the feasibility of the curriculum develop of mode and the effectiveness of the finding in this study by helding a conference with experts. The study takes place in the school in which researcher teaches. The participants of the study include teachers, school staffs, students and community members. Data used in the study comprises students’ questionnaires, interview records, meeting records, observation records, etc. After a thorough research and cross analysis ,this study aims to obtain objective interpretations. The six conclusions of the study are as follows. I.The successive changes of vocational business curriculum is modified with the demand of economical composition. In the 89th academic year credit curriculum structure the "school-decided curriculum" appears for the first time. It’s a new age era for school-based curriculum of vocational senior high school II.The modes that are applicable to business curriculum development in senior high vocational school are those of Lo-dah harn’s , Li-long cherng’s (APIE), Lin-chun yen, Weng-shang chin, and Yang-min rong,Huang-meng liang’s (PPDEE), and Yang-min rong’s . III.There are five stages of “the mode of school-based curriculum developing for business departments”. The five stages and they are strategy planning, curriculum planning, curriculum designing, curriculum implementation, and curriculum evaluation. IV.The purpose of the strategy planning is to organize a work team for planning curriculum of business departments, analyzing the difference between the current and future curriculum, evaluating the demands, and deciding the future features of the business departments. As for the stage of curriculum planning, it aims to establish the committee for developing business department curriculum, to set up the curriculum framework of the school-decided curriculum, including the way of implementation, teachers human resources planning of business departments, and space and facility planning. V.The practical achievement of curriculum developing contain of the decision of curriculum name after transforming (Business Information Department), goals and features of the department, 3-year curriculum flowchart, teaching outlines of some subjects, weekly teaching hours sheet. Total 13 documents and forms of all the stages are presented in this study. VI.The problems the researcher encountered in the process of the curriculum developing can be categorized into three parts: educational policy, business department curriculum development, and the design of consistent curriculum. A. ational policy (1)It’s too hasty to give subordinates the authority of curriculum planning for technological and vocational education system. (2)There’s no network system of curriculum developing B.Business department curriculum development (1)Tachers don’t have the ability to develop the curriculum. (2)Teachers don’t have enough time. (3)Teachers don’t have enthuiasm about the curriculum development, and they think that it is an extra burden besides teaching. (4)It’s hard to evaluate the quality of curriculum developing. (5)The leader of curriculum developing should possess the professional ability and persistent personality. (6)Affected by the College Entrance Exam, the curriculum developing is exam-oriented. C.Design of continued curriculum, (1)The problems caused by school-decided curriculum, (2)The problems of the required curriculum by Education Ministry only for single semester, (3)Confusion about teaching new curriculum to business department of vocational high school, such as “Case Study”. According to the conclusions mentioned above, this study offer the following suggestions I.Suggestions for the administration authorized (1)The concept of school-based curriculum has to be broadcast widely to make the senior high vocational schools prepare the curriculum developing in advance. 2)Teaches rofessional ability for curriculum developing should be developed, including a work team, seed teachers and holding continuous speech or committee for curriculum developing. (3) Teacher education institutes should emphasize on developing pre-teachers' curriculum design competence. (4) Take consideration of reducing teaching hours of senior high vocational school curriculum planning team members, which can upgrade the quality of school-decided curriculum. (5) The curriculum developing centers should have their own websites, on which it provides the relevant documents or tools to solve the problems in the process of senior high curriculum developing. II. Suggestions for vocational senior high schools (1) If the principal or assistant principal can play an aggressive role in the process of curriculum developing, it will be helpful to decrease the related resistance. (2) Encourage teachers to participate in the committees for curriculum developing or refresher courses to equip some teachers with the curriculum develop of competence. III. Suggestions for further studies (1) The further studies of the process of action research can be extended to practical teaching and the evaluation. (2) The members of school-based curriculum developing can involve parents and community members. However, the plan should be formulated cautiously in order to make the members participate willingly.

碩士
國立中央大學
電機工程研究所
99
Lateral AlGaN/GaN Schottky Barrier Diodes (SBDs) on Si substrate have been fabricated and characterized. AlGaN/GaN hetero-junction layers were grown on 4-inch p-type Si (111) substrate with 2 ?m buffer layer. The measurement of etching pit density (EPD) of GaN films on Si substrate is about 1.92×109 cm-2 by atomic force microscopy (AFM). The full width at half maximum value (FWHM) of x-ray diffraction rocking curve for the GaN film on Si (111) substrate is 536 arc-sec (002 reflection), which is related to the screw type dislocation and resulted leakage current. The Hall measurement showed the mobility of 1430 cm2 /V-s with a sheet carrier density of 9.8?1012 cm-2 for the AlGaN/GaN structure across the wafer. The AlGaN/GaN SBDs were implemented by Ti/Al/Ni/Au Ohmic and Ni/Au Schottky contacts. The Ohmic contacts were deposited on both side of Schottky contact with equal distance. The Schottky-to-Ohmic contact distance (LGS) was varied from 10 to 30 ?m in this study. The specific on-state resistance (RON) was 1.3 m?-cm2, while the forward turn-on voltage was 1.4 V at the current density of 100 A/cm2 for device with LGS = 10 ?m. The measured reverse breakdown voltage (VB) at room temperature was up to 600 V without edge terminal scheme. The measured VB is not function of LGS, which mainly depends on the buffer layer structure. The figure-of-merit is defined (VB)2/RON, that was 277 MWcm-2. And reverse recovery time was < 10 ns for device (without package) switched from a forward current density of ~720 A/cm2 (1 A) to a reverse bias of 30 V with di/dt of 100 A/?s.

Yes
Although batch reactive distillation process outperforms traditional reactor-distillation processes due to simultaneous reaction and separation of products for many reaction systems, synthesis of Methyl lactate (ML) through esterification of lactic acid (LA) with methanol in such process is very challenging due to difficulty of keeping the reactants together when one of the reactants (in this case methanol) has the lowest boiling point than the reaction products. To overcome this challenge, two novel reactive distillation column configurations are proposed in this work and are investigated in detail. These are: (1) integrated conventional batch distillation column (i-CBD) with recycled methanol and (2) integrated semi-batch and conventional batch distillation columns (i-SBD) with methanol recovery and recycle. Performances of each of these configurations are evaluated in terms of profitability for a defined separation task. In i-SBD column, an additional constraint is included to avoid overflow of the reboiler due to continuous feeding of methanol into the reboiler as the reboiler is initially charged to its maximum capacity. This study clearly indicates that both integrated column configurations outperform the traditional column configurations (batch or semi-batch) in terms of batch time, energy consumption, conversion of LA to ML, and the achievable profit.

Yes
Methyl Decanoate (MeDC) is a Fatty Acid Methyl Ester (FAME) and is an important chemical compound with global production of 31 million tons per year. However, synthesis of methyl decanoate (MeDC) via esterification of Decanoic Acid (DeC) with methanol by reactive distillation is operationally challenging due to difficulty of keeping the reactants together in the reaction zone as methanol being the lightest component in the mixture can separate itself easily form the other reactant deteriorating significantly the conversion of DeC using either conventional batch or continuous distillation column. This is probably the main reason for not applying the conventional route for MeDC synthesis. Whether Semi-batch Distillation column (SBD) and the recently developed Integrated Conventional Batch Distillation column (i-CBD) offer the possibility of revisiting such chemical reactions for the synthesis of MeDC is the focus of this paper. The minimum energy consumption (Qtot) as the performance measure is used to evaluate the performances of each of these reactive column configurations for different range of methyl decanoate purity and the amount of product. It is observed that the use of i-CBD column provides much better performance than SBD column in terms of the production time and the maximum energy savings when excess methanol is used in the feed. However, the SBD column is found to perform better than the i-CBD column when both reactants in the feed are in equal amount. Also, the optimization results for a given separation task show that the performance of two-reflux intervals strategy is superior to the single-reflux interval in terms of operating batch time, and energy usage rate in the SBD process at equimolar ratio.

Silicon carbide (SiC) has higher breakdown field strength than silicon (Si), which enables thinner and more highly doped drift layers compared to Si. Consequently, the power losses can be reduced compared to Si-based power conversion systems. Moreover, SiC allows the power conversion systems to operate at high temperatures up to 250 oC. With such expectations, SiC is considered as the material of choice for modern power semiconductor devices for high efficiencies, high temperatures, and high power densities. Besides the material benefits, the typeof the power device also plays an important role in determining the system performance. Compared to the SiC metal-oxide semiconductor field-effect transistor (MOSFET) and bipolar junction transistor (BJT), the SiC junction field-effect transistor (JFET) is a very promising power switch, being a voltage-controlled device without oxide reliability issues. Its channel iscontrolled by a p-n junction. However, the present JFETs are not optimized yet with regard to on-state resistance, controllability of threshold voltage, and Miller capacitance. In this thesis, the state-of-the-art SiC JFETs are introduced with buried-grid (BG) technology.The buried grid is formed in the channel through epitaxial growth and etching processes. Through simulation studies, the new concepts of normally-on and -off BG JFETs with 1200 V blocking capability are investigated in terms of static and dynamic characteristics. Additionally, two case studies are performed in order to evaluate total losses on the system level. These investigations can be provided to a power circuit designer for fully exploiting the benefit of power devices. Additionally, they can serve as accurate device models and guidelines considering the switching performance. The BG concept utilized for JFETs has been also used for further development of SiC junctionbarrier Schottky (JBS) diodes. Especially, this design concept gives a great impact on high temperature operation due to efficient shielding of the Schottky interface from high electric fields. By means of simulations, the device structures with implanted and epitaxial p-grid formations, respectively, are compared regarding threshold voltage, blocking voltage, and maximum electric field at the Schottky interface. The results show that the device with an epitaxial grid can be more efficient at high temperatures than that with an implanted grid. To realize this concept, the device with implanted grid was optimized using simulations, fabricated and verified through experiments. The BG JBS diode clearly shows that the leakage current is four orders of magnitude lower than that of a pure Schottky diode at an operation temperature of 175 oC and 2 to 3 orders of magnitude lower than that of commercial JBS diodes. Finally, commercialized vertical trench JFETs are evaluated both in simulations andexperiments, while it is important to determine the limits of the existing JFETs and study their performance in parallel operation. Especially, the influence of uncertain parameters of the devices and the circuit configuration on the switching performance are determined through simulations and experiments.
Kiselkarbid (SiC) har en högre genombrottsfältstyrka än kisel, vilket möjliggör tunnare och mer högdopade driftområden jämfört med kisel. Följaktligen kan förlusterna reduceras jämfört med kiselbaserade omvandlarsystem. Dessutom tillåter SiC drift vid temperatures upp till 250 oC. Dessa utsikter gör att SiC anses vara halvledarmaterialet för moderna effekthalvledarkomponenter för hög verkningsgrad, hög temperature och hög kompakthet. Förutom materialegenskaperna är också komponenttypen avgörande för att bestämma systemets prestanda. Jämfört med SiC MOSFETen och bipolärtransistorn i SiC är SiC JFETen en mycket lovande component, eftersom den är spänningsstyrd och saknar tillförlitlighetsproblem med oxidskikt. Dess kanal styrs an en PNövergång. Emellertid är dagens JFETar inte optimerade med hänseende till on-state resistans, styrbarhet av tröskelspänning och Miller-kapacitans. I denna avhandling introduceras state-of-the-art SiC JFETar med buried-grid (BG) teknologi. Denna åstadkommes genom epitaxi och etsningsprocesser. Medelst simulering undersöks nya concept för normally-on och normally-off BG JFETar med blockspänningen 1200 V. Såvä statiska som dynamiska egenskper undersöks. Dessutom görs två fallstudier vad avser totalförluster på systemnivå. Dessa undersökningar kan vara värdefulla för en konstruktör för att till fullo utnyttja fördelarna av komponenterna. Dessutom kan resultaten från undersökningarna användas som komponentmodeller och anvisningar vad gäller switch-egenskaper. BG konceptet som använts för JFETar har också använts för vidareutveckling av så kallade JBS-dioder. Speciellt ger denna konstruktion stora fördelar vid höga temperature genom en effektiv skärmning av Schottkyövergången mot höga elektriska fält. Genom simuleringar har komponentstrukturer med implanterade och epitaxiella grids jämförst med hänseende till tröskelspänning, genombrottspänning och maximalt elektriskt fält vid Schottky-övergången. Resultaten visar att den epitaxiella varianten kan vara mer effektiv än den implanterade vid höga temperaturer. För att realisera detta concept optimerades en komponent med implanterat grid med hjälp av simuleringar. Denna component tillverkades sedan och verifierades genom experiment. BG JBS-dioden visar tydligt att läckströmmen är fyra storleksordningar lägre än för en ren Schottky-diod vid 175 oC, och två till tre storleksordningar lägre än för kommersiella JBS-dioder. Slutligen utvärderas kommersiella vertical trench-JFETar bade genom simuleringar och experiment, eftersom det är viktigt att bestämma gränserna för existerande JFETar och studera parallelkoppling. Speciellt studeras inverkan av obestämda parametrar och kretsens konfigurering på switchegenskaperna. Arbetet utförs bade genom simuleringar och experiment.

QC 20150915

In many scenarios, events such as singularities and transients that carry important information about a signal undergo spreading during acquisition or transmission and it is important to localize the events. For example, edges in an image, point sources in a microscopy or astronomical image are blurred by the point-spread function (PSF) of the acquisition system, while in a speech signal, the epochs corresponding to glottal closure instants are shaped by the vocal tract response. Such events can be extracted with the help of techniques that promote sparsity, which enables separation of the smooth components from the transient ones. In this thesis, we consider development of such sparsity promoting techniques. The contributions of the thesis are three-fold: (i) an auditory-motivated continuous wavelet design and representation, which helps identify singularities; (ii) a sparsity-driven deconvolution technique; and (iii) a sparsity-driven deconvolution technique for reconstruction of nite-rate-of-innovation (FRI) signals. We use the speech signal for illustrating the performance of the techniques in the first two parts and super-resolution microscopy (2-D) for the third part. In the rst part, we develop a continuous wavelet transform (CWT) starting from an auditory motivation. Wavelet analysis provides good time and frequency localization, which has made it a popular tool for time-frequency analysis of signals. The CWT is a multiresolution analysis tool that involves decomposition of a signal using a constant-Q wavelet filterbank, akin to the time-frequency analysis performed by basilar membrane in the peripheral human auditory system. This connection motivated us to develop wavelets that possess auditory localization capabilities. Gammatone functions are extensively used in the modeling of the basilar membrane, but the non-zero average of the functions poses a hurdle. We construct bona de wavelets from the Gammatone function called Gammatone wavelets and analyze their properties such as admissibility, time-bandwidth product, vanishing moments, etc.. Of particular interest is the vanishing moments property, which enables the wavelet to suppress smooth regions in a signal leading to sparsi cation. We show how this property of the Gammatone wavelets coupled with multiresolution analysis could be employed for singularity and transient detection. Using these wavelets, we also construct equivalent lterbank models and obtain cepstral feature vectors out of such a representation. We show that the Gammatone wavelet cepstral coefficients (GWCC) are effective for robust speech recognition compared with mel-frequency cepstral coefficients (MFCC). In the second part, we consider the problem of sparse blind deconvolution (SBD) starting from a signal obtained as the convolution of an unknown PSF and a sparse excitation. The BD problem is ill-posed and the goal is to employ sparsity to come up with an accurate solution. We formulate the SBD problem within a Bayesian framework. The estimation of lter and excitation involves optimization of a cost function that consists of an `2 data- fidelity term and an `p-norm (p 2 [0; 1]) regularizer, as the sparsity promoting prior. Since the `p-norm is not differentiable at the origin, we consider a smoothed version of the `p-norm as a proxy in the optimization. Apart from the regularizer being non-convex, the data term is also non-convex in the filter and excitation as they are both unknown. We optimize the non-convex cost using an alternating minimization strategy, and develop an alternating `p `2 projections algorithm (ALPA). We demonstrate convergence of the iterative algorithm and analyze in detail the role of the pseudo-inverse solution as an initialization for the ALPA and provide probabilistic bounds on its accuracy considering the presence of noise and the condition number of the linear system of equations. We also consider the case of bounded noise and derive tight tail bounds using the Hoe ding inequality. As an application, we consider the problem of blind deconvolution of speech signals. In the linear model for speech production, voiced speech is assumed to be the result of a quasi-periodic impulse train exciting a vocal-tract lter. The locations of the impulses or epochs indicate the glottal closure instants and the spacing between them the pitch. Hence, the excitation in the case of voiced speech is sparse and its deconvolution from the vocal-tract filter is posed as a SBD problem. We employ ALPA for SBD and show that excitation obtained is sparser than the excitations obtained using sparse linear prediction, smoothed `1=`2 sparse blind deconvolution algorithm, and majorization-minimization-based sparse deconvolution techniques. We also consider the problem of epoch estimation and show that epochs estimated by ALPA in both clean and noisy conditions are closer to the instants indicated by the electroglottograph when with to the estimates provided by the zero-frequency ltering technique, which is the state-of-the-art epoch estimation technique. In the third part, we consider the problem of deconvolution of a specific class of continuous-time signals called nite-rate-of-innovation (FRI) signals, which are not bandlimited, but specified by a nite number of parameters over an observation interval. The signal is assumed to be a linear combination of delayed versions of a prototypical pulse. The reconstruction problem is posed as a 2-D SBD problem. The kernel is assumed to have a known form but with unknown parameters. Given the sampled version of the FRI signal, the delays quantized to the nearest point on the sampling grid are rst estimated using proximal-operator-based alternating `p `2 algorithm (ALPAprox), and then super-resolved to obtain o -grid (O. G.) estimates using gradient-descent optimization. The overall technique is termed OG-ALPAprox. We show application of OG-ALPAprox to a particular modality of super-resolution microscopy (SRM), called stochastic optical reconstruction microscopy (STORM). The resolution of the traditional optical microscope is limited by di raction and is termed as Abbe's limit. The goal of SRM is to engineer the optical imaging system to resolve structures in specimens, such as proteins, whose dimensions are smaller than the di raction limit. The specimen to be imaged is tagged or labeled with light-emitting or uorescent chemical compounds called uorophores. These compounds speci cally bind to proteins and exhibit uorescence upon excitation. The uorophores are assumed to be point sources and the light emitted by them undergo spreading due to di raction. STORM employs a sequential approach, wherein each step only a few uorophores are randomly excited and the image is captured by a sensor array. The obtained image is di raction-limited, however, the separation between the uorophores allows for localizing the point sources with high precision. The localization is performed using Gaussian peak- tting. This process of random excitation coupled with localization is performed sequentially and subsequently consolidated to obtain a high-resolution image. We pose the localization as a SBD problem and employ OG-ALPAprox to estimate the locations. We also report comparisons with the de facto standard Gaussian peak- tting algorithm and show that the statistical performance is superior. Experimental results on real data show that the reconstruction quality is on par with the Gaussian peak- tting.

Trois protéines de la famille TRIM (Motif TRIpartite), TIF1α, β (Transcriptional Intermediary Factor 1) et PML (ProMyelocytic Leukaemia¬), font l’objet de cette étude. TIF1α est connu comme un coactivateur des récepteurs nucléaires et TIF1β comme le corépresseur universel des protéines KRAB-multidoigt de zinc dont le prototype étudié ici est ZNF74. PML possède divers rôles dont le plus caractérisé est celui d’être l’organisateur principal et essentiel des PML-NBs (PML-Nuclear Bodies), des macrostructures nucléaires très dynamiques regroupant et coordonnant plus de 40 protéines. Il est à noter que la fonction de TIF1α, β et PML est régulée par une modification post-traductionnelle, la sumoylation, qui implique le couplage covalent de la petite protéine SUMO (Small Ubiquitin like MOdifier) à des lysines de ces trois protéines cibles. Cette thèse propose de développer des méthodes utilisant le BRET (Bioluminescence Resonance Energy Transfert) afin de détecter dans des cellules vivantes et en temps réel des interactions non-covalentes de protéines nucléaires mais aussi leur couplage covalent à SUMO. En effet, le BRET n’a jamais été exploré jusqu’alors pour étudier les interactions non-covalentes et covalentes de protéines nucléaires. L’étude de l’interaction de protéines transcriptionnellement actives est parfois difficile par des méthodes classiques du fait de leur grande propension à agréger (famille TRIM) ou de leur association à la matrice nucléaire (ZNF74). L’homo et l’hétérodimérisation de TIF1α, β ainsi que leur interaction avec ZNF74 sont ici testées sur des protéines entières dans des cellules vivantes de mammifères répondant aux résultats conflictuels de la littérature et démontrant que le BRET peut être avantageusement utilisé comme alternative aux essais plus classiques basés sur la transcription. Du fait de l’hétérodimérisation confirmée de TIF1α et β, le premier article présenté ouvre la possibilité d’une relation étroite entre les récepteurs nucléaires et les protéines KRAB- multidoigt de zinc. Des études précédentes ont démontré que la sumoylation de PML est impliquée dans sa dégradation induite par l’As2O3 et dépendante de RNF4, une E3 ubiquitine ligase ayant pour substrat des chaînes de SUMO (polySUMO). Dans le second article, grâce au développement d’une nouvelle application du BRET pour la détection d’interactions covalentes et non-covalentes avec SUMO (BRETSUMO), nous établissons un nouveau lien entre la sumoylation de PML et sa dégradation. Nous confirmons que le recrutement de RNF4 dépend de SUMO mais démontrons également l’implication du SBD (Sumo Binding Domain) de PML dans sa dégradation induite par l’As2O3 et/ou RNF4. De plus, nous démontrons que des sérines, au sein du SBD de PML, qui sont connues comme des cibles de phosphorylation par la voie de la kinase CK2, régulent les interactions non-covalentes de ce SBD mettant en évidence, pour la première fois, que les interactions avec un SBD peuvent dépendre d’un évènement de phosphorylation (“SBD phospho-switch”). Nos résultats nous amènent à proposer l’hypothèse que le recrutement de PML sumoylé au niveau des PML-NBs via son SBD, favorise le recrutement d’une autre activité E3 ubiquitine ligase, outre celle de RNF4, PML étant lui-même un potentiel candidat. Ceci suggère l’existence d’une nouvelle relation dynamique entre phosphorylation, sumoylation et ubiquitination de PML. Finalement, il est suggéré que PML est dégradé par deux voies différentes dépendantes de l’ubiquitine et du protéasome; la voie de CK2 et la voie de RNF4. Enfin une étude sur la sumoylation de TIF1β est également présentée en annexe. Cette étude caractérise les 6 lysines cibles de SUMO sur TIF1β et démontre que la sumoylation est nécessaire à l’activité répressive de TIF1β mais n’est pas impliquée dans son homodimérisation ou son interaction avec la boîte KRAB. La sumoylation est cependant nécessaire au recrutement d’histones déacétylases, dépendante de son homodimérisation et de l’intégrité du domaine PHD. Alors que l’on ne connaît pas de régulateur physiologique de la sumoylation outre les enzymes directement impliquées dans la machinerie de sumoylation, nous mettons en évidence que la sumoylation de TIF1β est positivement régulée par son interaction avec le domaine KRAB et suggérons que ces facteurs transcriptionnels recrutent TIF1β à l’ADN au niveau de promoteur et augmentent son activité répressive en favorisant sa sumoylation.
Three TRIM proteins (TRIpartite Motif), TIF1α, β (Transcriptional Intermediary Factor 1) and PML (ProMyelocytic Leukaemia¬), were studied in this thesis. TIF1α is a nuclear receptor coactivator and TIF1β is the universal corepressor of the KRAB-zinc finger repressor family of which, ZNF74 is studied here as a prototypic member. PML functions as a tumor suppressor and is the essential organiser of PML-NBs (PML-Nuclear Bodies) which are very dynamic nuclear macrostructures containing more than 40 proteins. The function of these three TRIM proteins is regulated by sumoylation, a post-translational modification involving the covalent linkage of SUMO (Small Ubiquitin like MOdifier) to specific targets lysine. In this thesis, we propose to develop new methods based on BRET (Bioluminescence Resonance Energy Transfer) to detect non-covalent nuclear protein interactions but also covalent linkage to SUMO in real time in living cells. To date, BRET was never used to assess non-covalent or covalent nuclear protein interactions. Studying transcriptionally active protein interactions represents a challenge by classical methods in particular when proteins have a tendency to aggregate (TRIM family) or when characterizing nuclear matrix proteins (ZNF74). In the first article, homo- and heterodimerisation of TIF1 α and β as well as their interaction with ZNF74 was assessed by BRET using full length proteins in living mammalian cells. We ascertained the heterodimerisation of TIF1α and β. Whereas ZNF74 interacts strongly with TIF1β, no interaction was detected with TIF1α. However, we unravelled the existence of ternary complexes involving ZNF74, TIF1α and TIF1β. This suggested that a mechanisms for cross-talk between nuclear receptors and KRAB-zinc finger proteins. Thus, we showed that BRET can be advantageously used as a non-transcription-based interaction system for studying transcriptionally active proteins, including nuclear matrix proteins, in living cells. Previous studies have shown that the sumoylation of PML (a tumour suppressor) is involved in its proteasome degradation that is As2O3-inducible and dependent on the polySUMO E3 ubiquitin ligase, RNF4. In the second article, we describe the development of a new application of the BRET method for the detection of covalent and non-covalent interactions with SUMO. Owing to this SUMO BRET assay, we established that the As2O3 / RNF4-mediated degradation of PML, not only depends on PML sumoylation as previously demonstrated, but also on the integrity of its SUMO binding domain. We also demonstrated that As2O3 which increases PML sumoylation, also enhances PML / RNF4 interaction. Our study revealed that most PML SBD non covalent interactions with sumoylated proteins required the phosphorylation of serines within PML SBD that were previously described as target sites for CK2 kinase and involved in PML degradation. Despites the involvement of PML SBD in RNF4-mediated degradation, these serines which function as an SBD phospho-switch, were not required for RNF4-mediated degradation. This suggested that CK2- and RNF4-mediated PML degradation represents two distinct pathways triggering PML ubiquitin / proteasome-dependent degradation. At last, our study led to the hypothesis that the recruitment of sumoylated PML at PML-Nuclear Bodies subnuclear structures via the PML SBD and / or possibly an E3 ubiquitin ligase activity other than RNF4 (PML itself being candidate) may favour PML degradation. Our study also stresses the dynamic involvement of three PML post-translational modifications, phosphorylation, sumoylation and ubiquitination in its degradation. A third article addressing the role of TIF1β sumoylation is presented in the Appendix. We characterized the 6 SUMO targets lysine of TIF1β and demonstrated that sumoylation is required for TIF1β transcriptional repressive activity. This is in part explained by the fact that TIF1β sumoylation is a pre-requisite for histone deacetylases recruitment since TIF1β repressive activity is partly dependent on histone deacetylases. We found that TIF1β sumoylation does not influence its homodimerisation or interaction with the KRAB box of KRAB zinc finger proteins recruiting TIF1β to promoters. TIF1β sumoylation is however relying on the integrity of TIF1β PHD finger and on its self-oligomerisation. Interestingly, we demonstrated that TIF1β sumoylation is positively regulated by its interaction with KRAB domain. It is thus suggested that KRAB-zinc finger proteins recruit TIF1β at DNA promoters where they trigger increase of TIF1β sumoylation and thus enhance its repressive activity.