Ο όρος ‘Δίκτυα Επόμενης Γενιάς’ αναφέρεται σε μελλοντικά δίκτυα πρωτοποριακής λογικής και δομής, προσανατολισμένα στην υποστήριξη σύγχρονων απαιτητικών εφαρμογών και στη βελτίωση της λειτουργικότητας της τερματικής συσκευής, όπως την αντιλαμβάνεται ο χρήστης. Πολύ σημαντικά στοιχεία της νέας τηλεπικοινωνιακής πραγματικότητας που επιφέρουν τα δίκτυα αυτά, και με τα οποία σχετίζεται άμεσα η παρούσα εργασία, είναι η διάθεση προώθησης της γενικευμένης κινητικότητας των ασύρματων χρηστών και η ενθάρρυνση της σύγκλισης επιμέρους τεχνολογιών διαφορετικών δικτύων και της δημιουργίας υβριδικών ετερογενών δικτύων, με στόχο την επίτευξη καλύτερης αξιοποίησης του φάσματος και βελτίωσης των ρυθμών μετάδοσης δεδομένων. Σκοπός της παρούσας διπλωματικής εργασίας είναι η παρουσίαση της δομής και των λειτουργιών των Δικτύων Επόμενης Γενιάς, καθώς και ορισμένων υπαρχόντων τύπων ασύρματων δικτύων, η συνεργασία των οποίων θα μπορούσε να προσφέρει τα επιθυμητά πλεονεκτήματα. Δύο τέτοια είδη δικτύων είναι το δίκτυο κινητής τηλεφωνίας UMTS και τα τοπικά δίκτυα τεχνολογίας WLAN, τα οποία εξετάζονται ενδελεχώς ως προς τα χαρακτηριστικά τους και, κυρίως, ως προς τους μηχανισμούς διευθέτησης της περιαγωγής των χρηστών. Περιλαμβάνεται επίσης μία περιγραφή της λειτουργίας του Mobile IP, πρωτοκόλλου που θεωρείται ιδιαίτερα χρήσιμο για τη διαχείριση της κινητικότητας χρηστών ανάμεσα σε περιοχές εξυπηρέτησης διαφορετικών δικτύων. Η εργασία καταλήγει στη μοντελοποίηση των παραπάνω συστημάτων σε περιβάλλον εξομοίωσης, επιδιώκοντας την αξιολόγηση της αποτελεσματικότητας του ενδεχόμενου συνδυασμού των συστημάτων UMTS και WLAN και της χρησιμότητας του Mobile IP. Το λογισμικό που χρησιμοποιείται γι’ αυτόν το σκοπό είναι το OPNET Modeler ®, ένα πρόγραμμα που αναδεικνύεται τα τελευταία χρόνια σε εργαλείο όλο και περισσότερο πολύτιμο, τόσο σε ερευνητικό όσο και σε επιχειρησιακό επίπεδο. / The term ‘Next Generation Networks’ refers to future networks of revolutionary concept and structure, oriented to the support of demanding applications and the upgrade of the terminal device’s functionality, as perceived by the user. Some very important aspects of the new telecommunications reality that is brought on by these networks, and to which this project is directly related, is the intention of promoting generalized mobility for the wireless users and the encouragement of the convergence of distinct network technologies and of the foundation of new hybrid heterogeneous networks, in order to achieve better spectrum utilization and improvement of data transmission rates. The purpose of the current diploma thesis project is to present the structure and the functions of the Next Generation Networks, as well as of some existing types of wireless networks, the cooperation of which could provide the desirable advantages. Two such network types are the UMTS mobile telephony network and the local networks of WLAN technology, that are examined thoroughly towards their characteristics and, foremost, towards their roaming arrangement mechanisms. Also included is a description of the operation of Mobile IP, a protocol that is considered particularly convenient for the management of users’ mobility between service areas of different networks. The project concludes to the modeling of the above mentioned systems in a simulation environment, aiming to evaluate the efficiency of the prospective combination of the UMTS and WLAN systems and the utility of Mobile IP. The software used for this purpose is OPNET Modeler ®, a program that has emerged during the last years as an increasingly valuable research and business tool.

Ce rapport de thèse présente l’étude de la technologie des empreintes moléculaires pour le développement de polymères spécifiques et sélectifs envers des biomarqueurs urinaires nucléosidiques du cancer colorectal chez l’Homme. L’objectif principal était de développer des polymères à empreintes moléculaires compatibles aux milieux aqueux en utilisant la technique du « dummy template », l’approche non-covalente and la polymérisation radicalaire en masse. Nous nous sommes concentrés principalement sur la qualité des polymères à partir de leur formulation, c’est-à-dire la spécificité et la sélectivité. Cela a été mené de façon empirique d’abord par la production de poudres issues de polymères monolithiques. Ainsi, pour atteindre les objectifs fixés, nous avons exploré le choix de la molécule « template ». Une étude de modèle est présentée au chapitre 3, en utilisant trois nucléosides 2’,3’,5’-peracétylés comme molécule empreinte dans une approche « dummy template ». Ensuite, en s’appuyant sur la connaissance apportée par le chapitre 3, nous avons développé des polymères à empreintes moléculaires (MIPs) sélectifs de la pseudouridine et de la N7-méthylguanosine dans les chapitres 4 et 5, respectivement, en utilisant la 2’,3’,5’-tri-O-acétylpseudouridine et la 2’,3’,5’-tri-O-acétylguanosine comme templates. L’étude de la rétention des nucléosides recherchés et de leurs analogues structuraux menée par chromatographie en phase liquide et par analyse frontale a permis de déterminer la capacité des différents polymères et de connaître leur comportement dans de l’urine synthétique. Finalement, pour évaluer la possible application de ces polymères dans un échantillon réel, l’urine humaine, la technique de l’extraction sur phase solide à empreintes moléculaires ou MISPE a été développée. Ainsi, une purification sélective des biomarqueurs cibles, tels que la pseudouridine et la N7-méthylguanosine, dans des échantillons d’urines a pu être démontrée. / This thesis report presents the exploration of molecularly imprinted polymer (MIP) technology for developing of a sensitive and selective polymers used in urinary nucleoside biomarker recognition. The main goal was to develop water compatible MIPs prepared by a “dummy template” imprinting technology, using a non-covalent approach and radical-polymerization in bulk. We were focusing mostly on the polymer quality in the formulation (rigidity, stability and repeatability). This was chosen empirically first by production of powders from monolithic MIP. Thus, to accomplish the stated goals, we have explored the choice of the template molecule. A model study presented by Chapter 3, using three 2’3’5’-tri-Operacylateduridine nucleosides as templates in a “dummy” template approach was first developed. Then, applying the knowledge of the type of template choice, we developed a selective MIP for recognition of pseudouridine and N7-methylguanosine in the studies presented in Chapter 4 and Chapter 5 respectively. By using 2’3’5’-tri-O-acetylpseudouridine and 2’3’5’-tri-O-acetylguanosine as templates. Chromatographic methods like HPLC retention and frontal analysis were used in the interest of determining the binding capacity of synthesized polymers, and the behavior in synthetic urine. Finally, to evaluate the possible application of these polymers in urine, molecularly imprinted solid phase extraction (MISPE) was developed. Selective purification of urine samples containing pseudouridine and N7-methylguanosine obtained in the end.

The fall armyworm, Spodoptera frugiperda (J. E. Smith 1797) (Lepidoptera: Noctuidae), is considered the main insect-pest of corn in Brazil. Its control, in recent years, is mainly accomplished through the use of Bt plants and spray insecticides. Aiming to evaluate the efficiency of Bt proteins expressed by maize commercially available, as well as their interaction with the use of insecticides for the control of S. frugiperda to subsidize IPM and IRM programs in Bt maize, experiments were carried under field condition during two crop seasons in the 2015-16. Eight technologies of Bt maize and one non-Bt hybrid were evaluated, as well as seven insecticide application programs. Damage assessments were performed every five days, counting the number of damaged plants and assigning a score according to the Davis Scale (1992). For the application of the insecticides, a control level was established in which the spraying was carried out whenever 10% of the plants showed a damage score > 3. In the 1st season 2015/16, Agrisure TL (Cry1Ab), Herculex (Cry1F) and Optimum Intrasect (Cry1Ab/ Cry1F) and non-Bt maize required 1 to 3 sprays of insecticides to complement the control of S. frugiperda. In this season, all tested insecticides were efficient in control. In the case of YieldGard VT PRO (Cry1A.105 / Cry2Ab2), YieldGard VT PRO 3 (Cry1A.105 / Cry2Ab2 / Cry3Bb1), PowerCore (Cry1A.105 / Cry2Ab2 / Cry1F), Agrisure Viptera (Vip3Aa20) and Agrisure Viptera 3 (Vip3Aa20 / Cry1Ab) the use of insecticides was not necessary. In the second crop year 2015/16, with the exception of Agrisure Viptera and Agrisure Viptera 3, all other hybrids of Bt and non-Bt maize required 1 to 4 sprays of insecticides. The results obtained in the present work demonstrate that some Bt maize technologies (expressing genes that encode Cry1 proteins) present low control efficiency, thus making insecticides indispensable to complement the control of S. frugiperda. In contrast, the Vip3Aa20 protein shows high toxicity to S. frugiperda, showing insignificant damage to the culture. Among the insecticides tested in the work, spinetoram (12 g of a.i./ha), in general presented the highest control efficiency for fall armyworm. / A lagarta-do-cartucho, Spodoptera frugiperda (J. E. Smith 1797) (Lepidoptera: Noctuidae), é considerada o principal inseto-praga da cultura do milho no Brasil. Seu controle, nos últimos anos, é realizado principalmente por meio do uso de plantas Bt e pulverização de inseticidas. Visando avaliar a eficiência das proteínas Bt expressas pelos milhos disponíveis no mercado, bem como sua interação com o uso de inseticidas para o controle de S. frugiperda para subsidiar programas de MIP e MRI em milho Bt, foram realizados experimentos, em condição de campo, durante duas épocas de cultivo na safra 2015-16. Foram avaliadas oito tecnologias de milho Bt e um híbrido não-Bt, bem com sete programas de aplicação de inseticidas. A cada cinco dias, foram realizadas avaliações de dano, contabilizando o número de plantas danificadas e atribuindo uma nota de acordo com a Escala de Davis (1992). Para a aplicação dos inseticidas, foi estabelecido um nível de controle em que era realizada a pulverização sempre que 10% das plantas apresentassem nota de dano >3. Na 1a safra 2015/16, Agrisure TL (Cry1Ab), Herculex (Cry1F) e Optimum Intrasect (Cry1Ab/Cry1F) e milho não-Bt necessitaram de 1 a 3 pulverizações de inseticidas para complementar o controle de S. frugiperda. Nesta safra, todos os inseticidas testados foram eficientes no controle. Já em milho YieldGard VT PRO (Cry1A.105/Cry2Ab2), YieldGard VT PRO 3 (Cry1A.105/Cry2Ab2/Cry3Bb1), PowerCore (Cry1A.105/Cry2Ab2/ Cry1F), Agrisure Viptera (Vip3Aa20) e Agrisure Viptera 3 (Vip3Aa20/Cry1Ab) não foi necessário o uso de inseticidas. Na 2a safra 2015/16, com exceção de Agrisure Viptera e Agrisure Viptera 3, todos as demais híbridos de milho Bt e não-Bt necessitaram de 1 a 4 pulverizações de inseticidas. Os resultados obtidos no presente trabalho demonstram que algumas tecnologias de milho Bt (expressando genes que codificam proteínas Cry1) apresentam baixa eficiência de controle, tornando-se assim indispensável o uso de inseticidas para complementar o controle de S. frugiperda. Em contraste, a proteína Vip3Aa20 apresenta alta toxicidade para S. frugiperda, apresentando danos insignificantes à cultura. Dentre os inseticidas testados no trabalho, spinetoram (12 g de i.a./ha), de modo geral apresentou a maior eficiência de controle para lagarta-do-cartucho.

Made available in DSpace on 2016-06-02T19:52:05Z (GMT). No. of bitstreams: 1 6045.pdf: 14667118 bytes, checksum: f13a2c0983ea271f2e60ed298b158806 (MD5) Previous issue date: 2014-05-26 / Agência Nacional de Petróleo / This work studies the ship routing and scheduling problem in oil transportation from offshore platforms to inland terminals. It is motivated by a real situation in a Brazilian oil company. Brazil is one of the world's greatest oil producers and has around 80% of its oil explored in offshore mode. Thus, transportation costs play an important role in achieving operational excellence, and the recent growth trends for oil exploration in Brazil has transformed its operations and demanded agile and effective decision support systems for addressing the oil sector dynamism. This work's goal consists in developing and applying an optimization-based approach using a mixed integer linear programming model in real decision-making situations, along with a solution method based on mathematical programming (MIP-heuristics) in order to solve the model, such as relax-and-fix. The proposed model is inspired in a problem formulation for pickup and delivery with time windows (PDPTW) and heterogeneous fleet, where costs incurred for fuel consumption and fleet contracts is the objective function to be minimized. The pickup and delivery pairs are predetermined and the model's main decision refers to ship allocation to these pairs compounding a route. Furthermore, some additional constraints are modeled and proposed, such as terminal access and platform mooring limitation according to ship types, as well as product blend incompatibility. The model was implemented in a modeling language along with an optimizarion software. Computational experiments with the model and the heuristics are presented for different data sets supplied by the case study company. These experiments show the potential benefits of this approach for finding good solutions for the problem as well as the dificulty in finding solutions for realistic instances due to its NP-hard characteristics. / Este trabalho estuda o problema de roteirização e programação de navios que realizam o escoamento de petróleo das plataformas marítimas para terminais terrestres, motivado por uma situação real de uma empresa brasileira da indústria petrolífera. O Brasil é um dos maiores produtores mundiais de petróleo, e cerca de 80% de seu petróleo é explorado no mar. Dentro deste contexto, os custos de transporte desempenham um papel importante na busca pela excelência operacional e as tendências de crescimento da exploração de petróleo no Brasil têm tornado as operações mais complexas e demandantes de sistemas de apoio à decisão ágeis e eficazes que contemplem o dinamismo do setor petrolífero. O objetivo deste trabalho consiste em desenvolver e aplicar uma abordagem de otimização baseada em um modelo de programação linear inteira mista em situações reais de tomada de decisão, em conjunto com métodos de solução baseados em programação matemática (MIP-Heuristics) para resolver o modelo, como relax-and-fix. O modelo proposto é inspirado em uma formulação de problemas de coleta e entrega com janelas de tempo (pickup and delivery with time windows PDPTW) e frota heterogênea, no qual busca-se minimizar os custos decorrentes do consumo de combustível dos navios e contratos de afretamento. O modelo é do tipo origem-destino, no qual os pares coleta/entrega são pré-determinados e a decisão do modelo refere-se à alocação de navios para os diferentes pares, compondo uma rota. Além disso, são propostas restrições adicionais que contemplam limitações de acesso a terminais e de atracação em plataformas de acordo com os tipos de navio, além da incompatibilidade de mistura de produtos, entre outros. O modelo foi implementado utilizando uma linguagem de modelagem em conjunto com um software de otimização. Experimentos computacionais com o modelo e as heurísticas são apresentados para diferentes conjuntos de dados fornecidos pela empresa e comprovam o potencial das abordagens para encontrar boas soluções para o problema, mas também suas dificuldades para encontrar soluções para exemplares de tamanho realista, por tratar-se de um problema NP-difícil do ponto de vista de teoria de complexidade.

Made available in DSpace on 2016-12-23T14:37:30Z (GMT). No. of bitstreams: 1 Leandro Pin Dalvi.pdf: 1271308 bytes, checksum: 029f08fb27575fe178e1d7fbfa5e6720 (MD5) Previous issue date: 2008-02-23 / A cafeicultura é a maior geradora de empregos e renda para as famílias do Espírito Santo. Dentre os problemas que esta atividade enfrenta, destaca-se a broca-do-café Hypothenemus hampei, uma praga que causa prejuízos em todos os estágios de maturação dos frutos, assim como no pós-colheita. O objetivo do presente trabalho foi coletar, caracterizar molecularmente e selecionar isolados do fungo entomopatogênico Beauveria bassiana com potencial para utilização em programas de manejo fitossanitário da broca-do-café. O trabalho foi realizado no Setor de Entomologia e Fitopatologia NUDEMAFI, localizado no Centro de Ciências Agrárias da Universidade Federal do Espírito Santo (CCA-UFES), e nos laboratórios de Entomologia e Biotecnologia da Embrapa-Soja localizados em Londrina, Paraná. As coletas resultaram em 28 isolados classificados como B. bassiana, tendo sido um dos isolados descartado dos testes por apresentar baixo crescimento. As amplificações utilizando 12 primers (iniciadores) deram origem a 82 bandas. O coeficiente de similaridade Dice variou de 93 a 51%. Foi evidente, salvo algumas exceções, que o local de coleta foi peça chave na formação de grupos muito relacionados. Ocorreu grande amplitude na virulência. Foram pré-selecionados os isolados CCA-UFES/Bb4, CCA-UFES/Bb11, CCA-UFES/Bb15, CCA-UFES/Bb18 que produziram mortalidade confirmada acima de 60%. Posteriormente, o isolado CCA-UFES/Bb15 apresentou melhor resposta, diferenciando-se do isolado utilizados como padrão - ESALQ-447 - cujos CL50 foram de 4,0 x 104 e 11,85 x 104 conídios/mL, respectivamente. Para todos os isolados selecionados, assim como para o padrão, o aumento na concentração representou aumento na mortalidade. A conidiogênese sobre os cadáveres da broca-do-café foi inversamente proporcional à virulência dos isolados, sendo ambas as características importantes para o sucesso de um programa de Manejo Integrado de Pragas (MIP). Os resultados obtidos são promissores, dando subsídio a novas pesquisas preferencialmente a campo / The coffee plantation is the largest source of jobs and familiar income of the State of Espírito Santo (Brazil). The coffee berry borer, Hypothenemus hampei, is widely considered one of the most important coffee pests because the losses in all stages of coffee grain as well as in post-harvest in this activity. The objective of this study was the collect, molecular characterization and the select of entomopathogenic fungus Beauveria bassiana strains, with potential use in integrated pest management in coffe plantations. The work was carried out at NUDEMAFI, located in the Centro de Ciências Agrárias da Universidade Federal do Espírito Santo, in municipality of Alegre, Brazil and the Biotechnology and Biological laboratories in the Embrapa Soja, in municipality of Londrina, State of Paraná, Brazil. A collection of 28 B. bassiana strains,isolated from different geographic regions were established. Only one Bassiana bassiana strain was discarded for low growth in laboratory. The amplifications using 12 "primers" (initiators) led to 82 bands. Dices coefficient ranged from 51 to. 93% with few exceptions Groups of B. bassiana strains related were forming due to the place colleted and high levels of virulence was observed. The Beauveria bassiana strains CCA-UFES/Bb4, CCA-UFES/Bb11, CCA-UFES/Bb15, CCA-UFES/Bb18 were pre-selected which showed confirmed mortality higher than 60Only The LC50, of the CCA-UFES/Bb15 strains was better response with significant difference between the standard, with 4.0 x 104 in 11.85 x 104 conidia / ml, respectively,. All isolates selected, as well as for the standard, the increase in the concentration resulted an increase of mortality of H. hampei. The conidiogenesis on coffee berry borers cadavers was inversely proportional to the virulence of the B. bassiana strains, and both this characteristics are important to the success of a program of Integrated Pest Management (IPM)

O objetivo principal deste trabalho foi criar um modelo matemático para determinar, em nível estratégico, os locais no Brasil mais apropriados à instalação de tanques de álcool combustível (anidro e hidratado) e seus respectivos volumes. O modelo de programação inteira-mista desenvolvido baseou-se na organização do sistema de distribuição de álcool, enfocando sua logística, e considerando questões de oferta, demanda, infra-estrutura de transporte e armazenagem, além de custos de transporte, armazenagem e investimentos em tanques. O modelo foi formulado considerando o horizonte temporal dos meses do ano-safra canavieiro de 2006/2007. Essa formulação reflete as sazonalidades de produção, demanda e estoques do álcool. O modelo de transporte foi enfatizado na minimização dos custos logísticos da cadeia distribuição de álcool combustível dos produtores aos consumidores. Dois cenários e a análise de sensibilidade de suas respostas abordaram a questão estocástica do problema. O primeiro analisou o panorama atual do mercado de álcool, logo não considerou a possibilidade de criação de novos tanques. A idéia desse cenário foi apresentar a consistência da modelagem e ressaltar as condições de infra-estrutura existente de transporte e armazenagem para álcool combustível. Foi feita uma análise de sensibilidade em relação a custos de transporte e restrições de armazenagem para checagem das respostas e para a comparação das práticas atuais de mercado. No segundo cenário, considerou-se a possibilidade de criação de novos tanques procurando identificar os locais mais apropriados para construção dessas estruturas e seu dimensionamento. A análise de sensibilidade em relação a custos de transporte e restrições de armazenagem foi feita para confirmar o potencial de cada localização. Os resultados indicaram a localização inapropriada das bases de distribuição de álcool no país. Destacaram-se também os baixos níveis de fretes de transferência em função das limitações de infraestrutura do sistema de distribuição de álcool. Tanto que as principais localizações de novos tanques disseram respeito a bases no interior da região Centro-Sul, destinos cujos custos de transporte de coleta e entrega são mais competitivos. Em relação aos novos tanques de álcool hidratado houve a indicação das cidades de: Cascavel - PR, Umuarama - PR, Maringá - PR, Lages - SC, Sinop - MT, Limeira - SP e Sorocaba - SP. Para o caso do álcool anidro os novos investimentos sugeridos foram nas cidades de: Londrina - PR, Cascavel - PR, Guarapuava - PR, Lajes - SC, Santa Maria - RS, Araçatuba - SP, Sinop - MT, Vilhena - RO, Montes Claros - MT, Dourados - MS, Gurupi - TO e Teresina - PI. Somado a isso houve a alocação de praticamente todo o custo de armazenagem às usinas. Finalmente, as soluções para a localização de novos investimentos dos tanques de álcool foram todas em regiões de bases de distribuição, já que as usinas estão bem servidas em relação à capacidade de armazenagem. / The main purpose of this research is to develop a mathematical model intended for strategic analysis of the optimal location and considering suitable volumes for storage ethanol (anhydrous and hydrous) tanks. The Mixed Integer Program - MIP model was based on Brazilian ethanol distribution system. The model considered market parameters as supply, demand, and infrastructure parameters on transportation, storage values as well as their expenses. New construction ethanol tanks expenses also were considered. The months along the sugarcane crop year period of 2006/2007 were referred into the modeling formulation. This formulation allows a seasonal storage, production and demand patterns analysis. Transportation model is the main concern in the total logistics cost minimization from producers to consumers. The model stochastic formulation was elaborated by creating two simulated scenarios and developing a sensitivity analysis. The purpose of the first scenario was to check the model consistency and explore the current ethanol transport and storage infrastructure without considering the possibility of new tank installation. Based on these results, a sensitivity analysis regarding transportation expenses and storage restrictions was elaborated in order to make a comparison with current market practices. In the second scenario, it was considered the construction of new ethanol tanks and the identification of the most suitable places bearing in mind volume capacities. Based on these results, a sensitivity analysis regarding transportation expenses and storage restrictions was elaborated in order to check each location consistency. Results indicated that mills are mostly responsible for ethanol (anhydrous and hydrous) storages maintenance types and that the existing geographic organization of terminals and fuel distributors is inappropriate for ethanol distribution in Brazil. Transportation low flows among terminals and fuel distributors also indicated lack of a better infrastructure for ethanol distribution. The model indicated that main location results for installation of new tanks would be located especially in the countryside of the centersouth states, where allocation and distribution of ethanol from mills to the consumer market would be more competitive. In relation to the new hydrous ethanol tanks, the model indicated appropriated locations for the cities of: Cascavel - PR, Umuarama - PR, Maringá - PR, Lages - SC, Sinop - MT, Limeira - SP e Sorocaba - SP. In the other hand, for anhydrous ethanol, new investments suggested in: Londrina - PR, Cascavel - PR, Guarapuava - PR, Lajes - SC, Santa Maria - RS, Araçatuba - SP, Sinop - MT, Vilhena - RO, Montes Claros - MT, Dourados - MS, Gurupi - TO e Teresina - PI. Finally, the model indicated that the best locations for the establishment of new ethanol tanks would be located in fuel distributors\' bases, once results confirmed that mills have enough storage capacity.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-23T19:00:13Z No. of bitstreams: 1 Peçanha, Bruna Rachel de Britto [Dissertação, 2012].pdf: 3907379 bytes, checksum: f2acabc3c1c39363b86f0d651d6b5936 (MD5) / Made available in DSpace on 2017-03-23T19:00:13Z (GMT). No. of bitstreams: 1 Peçanha, Bruna Rachel de Britto [Dissertação, 2012].pdf: 3907379 bytes, checksum: f2acabc3c1c39363b86f0d651d6b5936 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polímeros de impressão molecular (MIPs) foram sintetizados e aplicados como adsorventes na técnica de extração em fase sólida (EFS). O método de polimerização por precipitação foi utilizado para a síntese dos polímeros, devido à simplicidade de preparo, altos rendimentos e obtenção de partículas mais uniformes, devido a não trituração do polímero. O MIP foi sintetizado com ácido metacrílico (MAA) como monômero funcional, trimetacrilato de trimetilolpropano (TRIM) e dimetacrilato de etilenoglicol (EDMA) como agentes de reticulação e o cloridrato de amilorida (AMI) foi escolhido como molécula-molde. Diferentes proporções de MAA, TRIM, EDMA, volume e tipo de solvente foram utilizadas para ajuste das condições ideais de síntese. Os MIP foram avaliados quanto à capacidade de adsorção comparando-se a polímeros sintetizados na ausência da molécula-molde (NIP, polímeros não impressos). O solvente de elevada polaridade empregado na síntese (THF:MeOH:H2O) permitiu o emprego da técnica para moléculas polares como AMI. O controle no volume de solvente permitiu a obtenção de partículas maiores, de modo que a EFS foi realizada em condições usuais, o que confere um potencial para aplicação dessa técnica de polimerização na preparação de adsorventes para EFS. O polímero que apresentou maior capacidade adsortiva no ensaio realizado em tampão citrato-acetato pH 6,5 foi o MIP/NIP 12 (AMI:MAA:TRIM 1:8:10), com uma taxa média de adsorção de 83 e 88% para NIP e MIP, respectivamente. A adsorção foi elevada devido a interação iônica entre MAA e AMI promovida pelo controle de pH, porém foi não específica. O polímero MIP/NIP 12 foi aplicado como adsorvente na EFS, onde a recuperação de AMI foi avaliada nos resíduos de carregamento e eluição com solventes. O carregamento com tampão citrato-acetato pH 6,5 foi o ideal, favorecendo a interação iônica do polímero com o analito. A eluição total de AMI do cartucho somente ocorre após lavagem com o solvente na presença de ácido, que protona os grupos carboxila do polímero, rompendo assim a interação iônica com o analito / Molecularly imprinted polymers (MIPs) were synthesized and applied as adsorbents in solid-phase extraction technique (SPE). The polymers have been synthesized by precipitation polymerization method because of its simplicity, high yields and good control of final size and shape of particles. MIP was synthesized using methacrylic acid (MAA) as functional monomer, trimethylolpropane trimethacrylate (TRIM) and ethyleneglycol dimethacrylate (EDMA) as cross-linker and amiloride hydrochloride (AMI) was chosen as template. Different ratios of MAA, TRIM and EDMA, volume and type of solvent were used to adjust the optimal synthesis conditions. The MIP were tested for adsorption capacity compared to the polymers synthesized in the absence of template molecule (NIP, non-imprinted polymers). The polar solvent mixture used (THF:MeOH:H2O) allowed the synthesis of MIP of polar molecules as AMI. The solvent volume control afforded the larger particles so the SPE was performed in the usual conditions, giving a potential application for this polymerization technique in the preparation of adsorbents for SPE. The polymers with higher adsorption capacity at the test performed in citrateacetate buffer pH 6,5 was MIP/NIP 12 (AMI:MAA:TRIM 1:8:10) with adsorption rate of 83 and 88% for NIP and MIP, respectively. The recognition of MIP was due to ionic interaction between MAA and AMI promoted by pH control, but was not specific. The polymer MIP/NIP 12 was used as a solid-phase extraction sorbent and the recoveries of AMI was evaluated using different loading and elution conditions. The loading with buffer citrate-acetate pH 6,5 was optimal, due to ionic interaction of the polymer with the analyte. Total elution of AMI bound to the polymers only occurs after washing with a acid-containing solvent, because of protonation of the carboxyl groups of the polymer and disrupting the ionic interaction with the analyte

Cette thèse décrit la synthèse de nanoparticules de polymères à empreintes moléculaires (MIP, de l’anglais molecularly imprinted polymer) pour la reconnaissance de protéines, par une approche de synthèse en phase solide. Les polymères à empreintes moléculaires sont des récepteurs biomimétiques synthétisés sur mesure par un processus de nanomoulage du polymère autour de la molécule unique. Ils possèdent ainsi des cavités de reconnaissance spécifiques pour leur molécule cible. La technique de l'impression moléculaire pour les petites molécules cibles est bien établie, alors que l'impression de protéines reste encore un défi en raison de la flexibilité et complexité de leur structure native et de leurs nombreux sites fonctionnels, mais aussi en raison de leur faible stabilité dans des conditions inhabituelles. Par conséquent, une approche de synthèse en phase solide a été développée ici où la protéine est immobilisée sur un support avant la synthèse de nanoparticules hydrosolubles de MIP par polymérisation radicalaire. Les MIPs obtenus ont des affinités comparables à celles des anticorps, et des réactivités croisées faibles. Ils possèdent des avantages tels qu'une stabilité meilleure, un coût plus faible et peuvent potentiellement être régénérés et réutilisés, devenant ainsi des alternatives prometteuses aux anticorps naturels. Nous avons fabriqué des MIPs contre des protéases à sérine, telles la trypsine et la kallikréine, mais aussi contre un épitope peptidique de la protéine gp41 du VIH. Des nanogels de MIP thermosensibles ont été synthétisés dans un réacteur sous la forme d’une colonne thermostatée ou une boîte de Pétri, par polymérisation radicalaire initiée par voie thermique ou photochimique. Un simple changement de la température permet de libérer les MIPs de la protéine immobilisée. Ces MIPs sont hydrosolubles en fonction de la température et ont un diamètre inférieur à 100 nm. Leur affinité pour leur cible est élevée, avec un Kd du nano ou picomolaire. Ces 'anticorps synthétiques' ont été appliqués dans des tests d'adsorption sur microbalance à cristal de quartz, mais également comme 'chaperons synthétiques'. Des études préliminaires de la protection des protéines d'une dénaturation thermique ou par un pH défavorable ont été effectuées. L'utilisation d'un iniferter pour initier la photopolymérisation vivante du MIP a permis de synthétiser des nanogels de type core-shell. En introduisant des marqueurs fluorescents dans les MIPs, les tests d’immunoessai dans des fluides biologiques ont été démontrés, ce qui indique le grand potentiel de ces MIPs dans le diagnostic clinique. En conclusion, nous avons développé une nouvelle approche de synthèse de nanoparticules de MIP hydrosoluble ayant une haute affinité pour une protéine, utilisables à la place des anticorps dans des applications dans le monde réel tel que la détection de protéines biomarqueurs dans des échantillons complexes, et potentiellement comme principe actif in vivo. / This thesis describes the synthesis, by a solid-phase synthesis approach, of nanoparticles of molecularly imprinted polymers (MIPs) for the recognition of proteins. Molecularly imprinted polymers are biomimetic receptors synthesized by a nanomolding process of the polymer around single molecules. They therefore possess specific recognition cavities for their target molecule. The technique of molecular imprinting for small target molecules is well established, while protein imprinting remains a challenge due to the flexibility and complexity of their native structure and functional sites, but also because of their low stability under unusual conditions. Therefore, a solid-phase synthesis approach has been developed where the protein is immobilized on a support before the synthesis of water-soluble MIP nanogel particles by radical polymerization. The MIPs obtained have affinities comparable to those of antibodies, and low cross-reactivities. They have advantages such as better stability, lower cost, and can potentially be regenerated and reused, thus becoming promising alternatives to real antibodies. We have synthesized MIPs against serine proteases such as trypsin, and kallikrein, but also against a peptide epitope of the HIV gp41 protein. Thermosensitive MIP nanogels were synthesized in a thermostated column-type reactor or a petri dish, by thermally or photo-initiated radical polymerization. Their thermosensitivity allows the MIPs to be released from the immobilized protein by a simple temperature change. They are water-soluble as a function of temperature and have a diameter of less than 100 nm. Their affinity for their target is strong, with a Kd in the nano or picomolar range. These 'synthetic antibodies' have been applied in binding assays with quartz crystal microbalance, but also as 'synthetic chaperones'. Preliminary studies of the protection of proteins from thermal denaturation or from denaturation by an unfavorable pH have been carried out. The use of an iniferter to initiate the living photopolymerization of MIP made it possible to synthesize nanogels of core-shell type. By introducing fluorescent markers into MIPs, immunoassay applications in biological fluids have been demonstrated, indicating the great potential of these MIPs in clinical diagnostics. In conclusion, we have developed a novel approach to the synthesis of soluble MIP nanoparticles having high affinity for a protein, usable in place of antibodies in real world applications such as the detection of biomarker proteins in complex samples, and potentially as an active principle in vivo.

L'isosorbide et ses dérivés sont des diols chiraux issus de l'amidon de maïs. L'utilisation de ce dernier en tant que monomère pour l'élaboration de polymères s'est révélée efficace, ces derniers égalant les propriétés des polymères conventionnels. Dans le cadre de cette thèse, ces diols sont employés pour synthétiser de nouvelles plateformes des monomères biosourcés AA et AB à partir des 1,4 :3,6- dianhydrohexitols. La synthèse d'intermédiaires à base de dinitriles ou de mononitriles et leurs dérivés à partir des trois isomères ainsi que les résultats d'essais de polymérisation de l'un de ces monomères prometteurs ont été décrits. Le polymère obtenu a révélé semi-cristallin grâce à l'agencement stéréorégulier des monomères AB. Ce travail est aussi le premier à décrire l'utilisation des réticulants chiraux à base de 1,4 :3,6-dianhydrohexitols participant à la formation du réseau tridimensionnel pour l'élaboration de polymères à empreintes (MIP) biosourcés pour la détéction de la Méthyltestostérone (MT). Les polymères synthétisés par polymérisation par précipitation ont été caractérisés. Les propriétés de rétention ont été évaluées en mode batch par HPLC-MS/MS. Ces MIPs présentent de bonnes propriétés d'adsorption vis-à-vis la MT avec des facteurs d'empreinte supérieurs à 1 montrant l'efficacité de l'impression. Ces matériaux présentent une bonne capacité d'adsorption comparée à la littérature. Les polymères non imprimés (NIP) ont même montré des capacités d'adsorption supérieure aux MIP classiques. La capacité d'adsorption la plus élevée a été observée pour cMIP-Is à base d'isosorbide, pour les concentrations importantes (500 mg L-1). Les données expérimentales ont été étudiées selon les modèles d'adsorption de Langmuir et Freundlich pour interpréter les phénomènes d'adsorption. Ces cMIP développés ont été adaptés pour l'extraction de la méthyltestotérone sur phase solide (SPE). Une procédure d'extraction a été développée en menant au travers d'une optimisation complète finalisée par une application aux eaux usées / The isosorbide and its derivatives are chiral diols obtained from cornstarch. The use of the latter as a monomer for the development of polymers has proved to be effective. The diols match the properties of conventional polymers. As part of this thesis, the diols are used to synthesize new platforms of bio based AA and AB from the 1,4: 3,6 - dianhydrohexitols monomers. The synthesis of intermediaries based on dinitriles or mononitrilies and their derivaties starting from the three isomers as well as the test results from one of these promising monomers polymerization which have been described. The resulting polymer revealed semi-cristallin through stereoregulier AB monomers layout. This work is also the first to describe the use of the reticulants chiral at base of 1.4: 3, 6-dianhydrohexitols participating in the formation of three-dimensional network for the development of polymers to footprints (MIP) Excelsior for detection of Methyltestosterone (MT). The polymers synthesized by polymerization have been characterized by precipitation. The properties of retention were evaluated in batch mode by HPLC-MS/MS. These MIPs present good properties of adsorption towards the MT with factors of footprint greater than 1 showing the effectiveness of printing. These materials have a good ability of adsorption compared to literature. Unprinted polymers (PIN) have shown even greater adsorption capacity than the conventional MIP. The high adsorption capacity was observed in cMIP-Is based on isosorbide for the concentrations (500 mg L-1). The experimental data have been studied according to Langmuir and Freundlich adsorption models to interpret the phenomena of adsorption these developed cMIP have been adapted for the methyltestoterone on the phase of extraction (SPE) solid. An extraction procedure has been developed leading through a full optimization finalized by an application in wastewater

Dans cette thèse, nous avons démontré la faisabilité de la préparation de polymères à empreinte moléculaires (MIP) hautement sélectifs pour la reconnaissance dans des matrices aqueuses complexes avec des applications dans les cosmétiques et en technologie alimentaire. Les MIP (de l'anglais molecularly imprinted polymers) sont des récepteurs synthétiques comparables aux anticorps, qui sont synthétisés par co-polymérisation de monomères fonctionnels et réticulants en présence d'un gabarit moléculaire. Leurs propriétés de reconnaissance moléculaire, associées à leur grande stabilité, robustesse mécanique, faible coût et leur synthèse facile les rendent extrêmement intéressants comme matériaux de capture sélective, avec des applications dans les séparations analytiques, la détection et la vectorisation des médicaments. Cependant, leur reconnaissance sélective dans des milieux aqueux reste toujours problématique et c'est l'une des raisons de leur expansion commerciale restreinte. Dans une première partie, nous avons développé un MIP fonctionnant en milieu aqueux pour son application comme ingrédient actif dans un déodorant. Les odeurs corporelles sont principalement dues à des acides gras volatils générés à partir de leurs précurseurs, des conjugués de glutamine par des enzymes hydrolytiques produites à partir de bactéries présentes sur la peau. La plupart des anti-transpirants et des déodorants actuellement commercialisés contiennent des sels d'aluminium et des agents antibactériens non spécifiques, respectivement. Cependant, l'utilisation extrêmement étendue de ces produits nécessite des solutions alternatives en ce qui concerne divers problèmes (environnement, respect de l'écosystème de la peau, toxicité, etc.). Pour cette raison, un MIP a été synthétisé pour capturer les précurseurs conjugués de glutamine afin qu'ils ne soient plus disponibles aux bactéries, empêchant ainsi leur transformation en composés malodorants. Afin de générer des liaisons sélectifs dans des environnements aqueux, un monomère à base d'amidinium qui peut former une interaction électrostatique stoechiométrique forte avec les groupes carboxyle sur le gabarit moléculaire a été synthétisé. Le MIP, mélangé dans une formulation dermo-cosmétique, pourrait capter sélectivement les précurseurs conjugués de glutamine, au milieu d'une multitude d'autres molécules présentes dans la sueur humaine. En outre, le MIP n’affecte pas les bactéries de la peau, ouvrant la voie à des déodorants innovateurs de nouvelle génération, moins problématiques pour la santé. Dans une deuxième partie, nous avons développé une procédure rapide et efficace basée sur l'extraction en phase solide à empreinte moléculaire (MISPE) pour la purification sélective de la bétanine et de son stéréoisomère l’isobétanine à partir d'extraits de betterave. La bétanine est un pigment naturel ayant un fort pouvoir antioxydant et dont les propriétés pharmacologiques sont de plus en plus étudiées. Ce pigment est actuellement utilisé comme simple colorant alimentaire. Dans notre étude, l'acide dipicolinique a été utilisé comme gabarit moléculaire pour la synthèse de MIP, en raison de sa similarité structurelle avec le groupe chromophore de la bétanine. Les procédures MISPE ont été optimisées permettant l'élimination presque complète des glucides issus de la matrice végétale ainsi que la majorité des protéines, ce qui permet d'obtenir un rendement élevé d'extraction de la bétanine / isobétanine en une seule étape. De plus, toute la procédure d'extraction a été réalisée dans des solvants respectueux de l'environnement, tels que l'éthanol ou l'eau. Pour conclure, nous sommes convaincus que ce travail pave le chemin au développement d'une nouvelle génération des MIP fonctionnant en milieu aqueux avec des propriétés de reconnaissance améliorées dans des environnements complexes, qui pourra s'appliquer également à d'autres domaines biotechnologiques et biomédicaux. / In this thesis, we have demonstrated the feasibility of preparing highly selective molecularly imprinted polymers (MIPs) for recognition in complex aqueous matrices with applications in cosmetics and food technology. MIPs are synthetic tailor-made receptors, with binding affinities and specificities comparable to those of natural antibodies. Their molecular recognition properties, combined with their high stability, mechanical robustness, low cost and easy synthesis make them extremely attractive as selective capture materials with applications in analytical and preparative separations, sensing and drug delivery, among others. However, their selective recognition in aqueous samples still remains problematic and is one of the reasons for their so far lilited commercial expansion. In the first part, we developed a water compatible MIP for its application as an active ingredient in a deodorant. Body odors are mainly due to volatile fatty acids generated from their glutamine conjugate precursors by hydrolytic enzymes from bacteria present on the skin. Most currently marketed anti-perspirants and deodorants contain, respectively aluminum salts and unspecific antibacterials. However, the extremely wide use of these products requires alternative solutions with regard to various problems (environmental, respect of skin ecosystem, toxicity, etc.). For this reason, a MIP was developed to capture the glutamine conjugate precursors so that they are no longer available to the bacteria, thus preventing their transformation to malodorous compounds. In order to generate binding selectivity in aqueous environments, an amidinium-based monomer which can form a strong stoichiometric electrostatic interaction with the carboxyl groups on the template, was synthesized. The MIP, blended in a dermo-cosmetic formulation, could capture selectively the glutamine precursors, amidst a multitude of other molecules present in human sweat. Furthermore, the MIP did not affect the skin bacteria, paving the way to an innovative and 'safer ' future-generation deodorant. In the second part, we developed a fast and efficient procedure based on molecularly imprinted solid­ phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from red beetroot extracts. Betanin is a natural pigment with significant antioxidant and biological activities currently used as food colorant. Dipicolinic acid was used as template for the MIP synthesis, because of its structural similarity to the chromophore group of betanin The MISPE procedures were optimized allowing the almost complete removal of carbohydrates and the majority of proteins, resulting in high extraction recovery of betanin / isobetanin in a single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with either ethanol or water. To conclude, we believe that this study paves the way towards the development of a new generation of water compatible MIPs with improved recognition properties in highly complex aqueous environments, and should be applicable to other biotechnological and biomedical areas as well.