Submitted by Nivaldo Melo (nivaldo.melo@ufvjm.edu.br) on 2015-11-30T17:13:26Z No. of bitstreams: 2 nathale_rodrigues_pinheiro.pdf: 3567634 bytes, checksum: 502f3816acde8f2f1b1ee7fe24e188f1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-12-03T16:14:53Z (GMT) No. of bitstreams: 2 nathale_rodrigues_pinheiro.pdf: 3567634 bytes, checksum: 502f3816acde8f2f1b1ee7fe24e188f1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2015-12-03T16:14:53Z (GMT). No. of bitstreams: 2 nathale_rodrigues_pinheiro.pdf: 3567634 bytes, checksum: 502f3816acde8f2f1b1ee7fe24e188f1 (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2014 / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / O sistema ADEPT (antibody-directed enzyme prodrug therapy) ? uma terapia antitumoral que envolve a ativa??o catal?tica de um pr?-f?rmaco, nas proximidades do s?tio tumoral, por uma enzima conjugada a um anticorpo monoclonal com afinidade para ant?genos espec?ficos das c?lulas tumorais. O sistema composto pela enzima Horseradish peroxidase (HRP) e ?cido indol-3-ac?tico (IAA) tem sido estudado para o emprego na terapia ADEPT, e associado ? indu??o de apoptose de c?lulas tumorais. A 2,4-pentanodiona (PD) tamb?m atua como substrato da HRP sendo oxidada por ela atrav?s de uma rea??o cuja cin?tica ? semelhante ? da cat?lise do IAA e, portanto, pode representar uma alternativa para essa terapia. Este trabalho teve como objetivo realizar uma avalia??o da citotoxicidade mediada pelos produtos provenientes da oxida??o da PD pela HRP frente a diferentes linhagens celulares, utilizando para isso diferentes metodologias que determinam a viabilidade celular como o azul de Trypan, MTT e vermelho neutro assim, como a an?lise microsc?pica das altera??es celulares induzidas por esses sistemas; estabelecer uma compara??o com a citotoxicidade mediada pela oxida??o do IAA catalisada pela mesma enzima; verificar a incid?ncia de morte celular por apoptose mediada pelos sistemas IAA/HRP/O2 e PD/HRP/O2; al?m de verificar a produ??o e os tipos de esp?cies reativas de oxig?nio (ERO) produzidas pelos dois sistemas. Os experimentos permitiram evidenciar que as combina??es PD/HRP/O2 e IAA/HRP/O2 levam a forma??o de ERO, sendo as esp?cies provavelmente formadas pela oxida??o da PD o radical ?nion super?xido e o per?xido de hidrog?nio (H2O2) e pela oxida??o do IAA o H2O2. Foi observado, somente para o IAA, um aumento na forma??o de ERO com o uso de uma maior concentra??o do substrato. Quanto ao estudo de viabilidade celular, esse permitiu evidenciar, atrav?s das tr?s metodologias, o efeito citot?xico dos sistemas PD/HRP/O2 e IAA/HRP/O2, no entanto, o ensaio do MTT mostrou-se mais sens?vel para esse estudo. A oxida??o do IAA pela HRP induziu apoptose, contudo n?o foi poss?vel identificar o tipo de morte celular mediada pelo sistema PD/HRP/O2, provavelmente devido a um problema t?cnico durante algumas an?lises em citometria de fluxo, o Quenhcing. Apesar de o sistema IAA/HRP/O2 ter apresentado uma destrui??o celular mais expressiva, o substrato IAA quando testado na aus?ncia da enzima mostrou-se t?xico, o que n?o foi visto para a PD quando testada nas concentra??es de 1; 1,5 e 2 mM, o que a torna um bom substrato para o emprego na terapia ADEPT. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT The system ADEPT (antibody-directed enzyme prodrug therapy) is an antitumor therapy that involves catalytic activation of a prodrug near the tumor site by an enzyme conjugated to a monoclonal antibody with affinity for specific antigens of tumor cells. The system composed by horseradish peroxidase (HRP) enzyme and indole-3-acetic acid (IAA) has been studied for the use in ADEPT therapy, and associated with apoptosis induction on tumor cells. The 2,4-pentanedione (PD) also acts as a substrate for HRP and being oxidized through a reaction whose kinetics is similar to the catalysis of IAA and, therefore, might represent an alternative to this therapy. This study aimed to conduct a evaluation of the cytotoxicity mediated by products from the oxidation of PD by HRP against different cell lines, using different methodologies that determine cell viability as Trypan blue, MTT and Neutral Red, as well as morphologic changes of the cell induced by these systems; establish a comparison with the cytotoxicity mediated by the oxidation of IAA catalyzed by the same enzyme; verify the incidence of apoptosis mediated by IAA/HRP/O2 and PD/HRP/O2 systems; besides verifying the production and types of reactive species oxygen (ROS) produced by the two systems. The experiments allowed to show that PD/HRP/O2 and IAA/HRP/O2 combinations lead to the formation of ROS, being the species probably formed by oxidation of PD the radical superoxide anion and hydrogen peroxide (H2O2) and by the oxidation of IAA the H2O2. It was observed only for the IAA, an increase in ROS production using a higher concentration of the substrate. Regarding the study of cell viability, this allowed to evidence, through the three methodologies, the cytotoxic effect of PD/HRP/O2 and IAA/HRP/O2 systems, however, the MTT assay proved more sensitive for this study. The oxidation of the IAA by HRP induced apoptosis, but could not identify the type of cell death mediated by PD/HRP/O2 system, The oxidation of by HRP the IAA induced apoptosis, but could not identify the type of cell death mediated by PD/HRP/O2 system, probably due to a technical problem for a few flow cytometric analyzes, the Quenching. Although IAA/HRP/O2 system have presented a more significant cell destruction, the IAA substrate when tested in the absence of enzyme was toxic, what has not seen for PD when tested in the concentrations of 1, 1.5, and 2 mM, making it a good substrate for employment in ADEPT therapy.

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2018-10-19T11:51:51Z No. of bitstreams: 1 Rafael Emídio Murata Rodrigues.pdf: 1071545 bytes, checksum: 468e0f7e27e278e12eed0dd52f4198cc (MD5) / Made available in DSpace on 2018-10-19T11:51:51Z (GMT). No. of bitstreams: 1 Rafael Emídio Murata Rodrigues.pdf: 1071545 bytes, checksum: 468e0f7e27e278e12eed0dd52f4198cc (MD5) Previous issue date: 2018-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In our lives, we constantly move in streets and neighborhoods. In general, we consider time and (or distance) when planning the route. However, their solutions may face complex problems arising from the different possibilities of solutions. Similar to route planning, the Vehicle Routing Problem was introduced by George B. Dantzig and John H. Ramser in 1959 and consists of delivering gasoline to several fuel stations; at first a mathematical proposal, later became an algorithmic approach, for planning of routes of delivery of products in an optimized way (searching the "shortest path"). Although, during the search of shortest path, they are limited to the use of the streets. In this context emerges the Human Routing Problem, such an approach is not limited to streets, but makes use of all possible paths, by vehicles and humans. Such a problem can be observed in route planning at airports, museums and a supply chain company that wants to optimize the route of delivery of its products and increase customer satisfaction. Based on the Vehicle Routing Problem, the Human Routing Problem will be proposed. Its problematic will be demonstrated in a prototype, capable of assisting in the planning of human routes and three use cases. Ideas of Human Routing Problem had inspiration in the collective foraging insects / Na nossa vida, nos locomovemos constantemente em ruas e bairros. Em geral, consideramos o tempo e (ou a distância), ao planejar a rota. Contudo, suas soluções podem enfrentar problemas complexos, decorrentes das diversas possibilidades de soluções. Semelhante a planejamento de rotas, o Problema de Roteamento de Veículos foi introduzido por George B. Dantzig, e John H. Ramser em 1959 e consiste em entregar gasolina diversos postos de combustível; a princípio uma proposta matemática, mais tarde tornou-se uma abordagem algorítmica, para planejamento de rotas de entrega de produtos de forma otimizada (buscando o “menor caminho”). Embora, durante a busca de menor caminho, limitam-se ao uso de ruas. Neste contexto emerge o Problema de Roteamento Humano, tal abordagem não se limita a ruas, mas faz uso de todos os caminhos possíveis, por veículos e humanos. Tal problemática, pode ser observada nos planejamentos de rotas em aeroportos, museus e em uma empresa de supply chain que deseja otimizar a rota de entrega dos seus produtos, e aumentar a satisfação dos seus clientes. Tomando como base central, o Problema de Roteamento de Veículos será proposto o Problema de Roteamento Humano. Sua problemática, será demonstrado em um protótipo, capaz de auxiliar no planejamento de rotas humanas e três casos de uso. As ideias do Problema de Roteamento Humano tiveram inspiração no forrageamento dos insetos coletivos

Depuis de nombreuses années, l’utilisation d’enzyme pour la synthèse de polymères naturels ou synthétiques a largement été développée en tant que procédé alternatif plus vert et plus respectueux de l’environnement. En effet, comparée aux méthodes conventionnelles de synthèse, les enzymes offrent une sélectivité élevée, une capacité à réagir dans des conditions de réaction douces, ainsi que la possibilité de recyclage du biocatalyseur. D’autre part, les polymères à empreintes moléculaires (MIPs) sont des matériaux synthétiques avec des propriétés de reconnaissance moléculaire spécifique envers une molécule cible. Récemment, les MIPs ont été utilisés dans les applications environnementales et biomédicales de part leur propriétés de reconnaissance moléculaire, leur spécificité et sélectivité. Cependant, leur application reste limitée en raison de leur faible biocompatibilité et de la présence de résidu de polymérisation potentiellement nocif. Ce travail de thèse a pour objectif de proposer une méthode alternative pour la synthèse de MIPs basée sur le concept de chimie verte. La peroxydase de raifort (HRP) est utilisée pour initier la co-polymérisation en milieux aqueux de monomères fonctionnels méthacrylates et d’agents réticulants en catalysant la génération des radicaux libres. Différents hydrogels ont été synthétisés et caractérisés, en particulier une cytotoxicité plus faible a été obtenue comparée à celle des polymères synthétisés traditionnellement. La synthèse a été optimisée afin de pouvoir contrôler la taille des particules et le rendement de polymérisation. Des MIPs sous forme de nanoparticules ont été préparés en milieu aqueux pour plusieurs molécules de faible poids moléculaire ainsi que pour des protéines par polymérisation radicalaire libre initiée par HRP. L’effet de la méthode d’initiation a été évalué en comparant les propriétés de ces MIPs à ceux préparées par les méthodes traditionnellement. L’immobilisation de l’HRP a été aussi effectuée pour synthétiser des hydrogels et des MIPs. L’enzyme immobilisée a pu être réutilisée pour synthétiser des MIPs avec les mêmes performances en termes de morphologie, rendement, spécificité et sélectivité. Ces nouveaux matériaux offrent de nombreuses perspectives pour des applications environnementales et biomédicales. / Enzyme-catalyzed synthesis of natural and synthetic polymers has been developed since several decades, as an eco-friendly process. Compared to the conventional methods, enzymes offer high selectivity, ability to operate under mild conditions and to recycle the catalyst. On the other hand, molecularly imprinted polymers (MIPs) are synthetic materials with specific recognition properties for target molecules. They have recently attracted increasing attention in environmental and newly in biomedical applications for their specificity and selectivity. However, concerns about MIP toxicity for human and environment safety are of great importance. Herein, carrying forward the concept of green chemistry, an enzyme-mediated synthesis approach is described to prepare molecularly imprinted nanoparticles (MIP-NPs) in aqueous media. Horseradish peroxidase (HRP) is used to initiate the polymerization of methacrylate-based monomers and cross-linkers by catalyzing the generation of free radicals. Different hydrogels are synthesized and characterized. “Greener” hydrogels are obtained with lower cytotoxicity than that of polymers synthesized by traditional way. The hydrogels synthesis is optimized in order to control the particles sizes and polymerization yields. Moreover, water-compatible MIP nanoparticles for the recognition of different small molecules and proteins are prepared in aqueous media by HRP-initiated free radical polymerization and compared to MIPs prepared by the thermal or photopolymerization methods. HRP immobilization is also performed for hydrogels synthesis as well as MIP preparation. The reusability of immobilized enzyme is investigated for the preparation of several MIP batches with the same morphology, yield as well as good specificity and selectivity. We believe that this new synthesis method for MIPs will provide new opportunities to enlarge the use of molecular imprinting technology in biomedical and environmental applications.

Le travail de recherche dans ce mémoire aborde les problèmes de l'identification des systèmes non linéaires et également de l'application de la théorie d'optimisation. Dans une première partie, nous proposons des méthodes efficaces et nouvelles afin d'identifier les systèmes linéaires dans le cas d'expérimentations multiples, les séries de Volterra à horizon infini et les systèmes quadratiques en l'état. Dans une seconde partie, nous appliquons la théorie d'identification à la modélisation de la locomotion humaine. Nous abordons ensuite l'optimisation des mouvements des robots humanoïdes, l'imitation des mouvements humains par un robot humanoïde et enfin le paramétrage temporel des chemins dans l'espace des configurations pour un robot humanoïde. Les résultats expérimentaux de nos méthodes sur la plate-forme HRP-2 ont révélé non seulement leur efficacité, mais aussi leurs bonnes performances qui dépassent largement celles des méthodes conventionnelles.

<p>The main hypothesis in this study is the development of a nanocomposite mediated amperometric biosensor for detection of hydrogen peroxide. The aim is to combine the electrochemical properties of both polyaniline and ferrocenium hexafluorophosphate into highly conductive nano composites capable of exhibiting electrochemistry in non acidic media / shuttling electrons between HRP and GCE for biosensor applications.</p>

<p>The main hypothesis in this study is the development of a nanocomposite mediated amperometric biosensor for detection of hydrogen peroxide. The aim is to combine the electrochemical properties of both polyaniline and ferrocenium hexafluorophosphate into highly conductive nano composites capable of exhibiting electrochemistry in non acidic media / shuttling electrons between HRP and GCE for biosensor applications.</p>

In biosensors, printing involves the transfer of materials, proteins or cells to a substrate. It offers many capabilities thatcan be utilized in many applications, including rapid deposition and patterning of proteins or other biomolecules.However, issues such as stability when using biomaterials are very common. Using proteins, enzymes, as biomaterialink require immobilizations and modifications due to changing in the structural conformation of the enzymes, whichleads to changes in the properties of the enzyme such as enzymatic activity, during the printing procedures andrequirements such as solvent solutions. In this project, an innovative approach for the fabrication of proteinmicroparticles based on cross-linking interchange reaction is presented to increase the stability in different solvents.The idea is to decrease the contact area between the enzymes and the surrounding environment and also preventconformation changes by using protein microparticles as an immobilization technique for the enzymes. The theory isbased on using a cross-linking reagent trigging the formation of intermolecular bonds between adjacent proteinmolecules leading to assembly of protein molecules within a CaCO3 template into a microparticle structure. TheCaCO3 template is removed by changing the solution pH to 5.0, leaving behind pure highly homogenous proteinmicroparticles with a size of 2.4 ± 0.2 μm, according to SEM images, regardless of the incubation solvents. Theenzyme model used is Horse Radish Peroxidase (HRP) with Bovine Serum Albumin (BSA) and Glutaraldehyde (GL)as a cross-linking reagent. Furthermore, a comparison between the enzymatic activity of the free HRP and the BSAHRPprotein microparticles in buffer and different solvents are obtained using Michaelis-Menten Kinetics bymeasuring the absorption of the blue product produced by the enzyme-substrate interaction using a multichannelspectrophotometer with a wavelength of 355 nm. 3,3’,5,5’-tetramethylbenzidine (TMB) was used as substrate. As aresult, the free HRP show an enzymatic activity variation up to ± 50 % after the incubation in the different solventswhile the protein microparticles show much less variation which indicate a stability improvement. Moreover, printingthe microparticles require high microparticle concentration due to contact area decreasing. However, usingmicroparticles as a bioink material prevent leakage/diffusion problem that occurs when using free protein instead.

策略性人力資源規劃(Strategic Human Rresource Planning, SHRP)是研究如何將組織策略與人力策略結合的一套整合性人力規劃理論，不僅重人力數量的探討，更重人力素質之研究。筆者以我國環保機關為研究對象，首先探討當代之SHRP理論，並依執行規制性政策之行政機關之特色，建構我國環保機關進行SHRP研究之理論架構，再經環境、工作、工作力調查分析找出目前我國環保機關所具有之優劣(SWOT)特性，最後根據SHRP方案之特點，以及各方案之可行性評估，選擇最能運用組織優點，匡正組織矛盾之人力資源策略。 　　全文約十七萬餘言，分七章二十一節： 　　第一章緒論，分三節。說明本文之研究動機與目的，研究方法範圍及限制，並針對人力資源規劃(Human Resource Planning, HRP)概念之發展及各時期其代表性學者之定義，與相關之名詞加以辯正釐清，說明當代SHRP之操作性定義與特性。 　　第二章策略性人力資源規劃理論，分三節。首先探討HRP理論之演進，並依理論之發展特性加以分期，說明自西元一九八○年代以來HRP理論發展之趨勢正走向如何使組織策略與人力策略相互聯結之SHRP理論。其次探討近十年間研究SHRP諸位具代表性學者之理論並評估其理論之特性，最後根據各學者之理論模式，經過對我國環保機關之組織特性分析建構本論文研究架構。 　　第三章我國環保機關人力資源環境分析，分三節。首先探討我國環保機關人力資源次級系統外之組織內環境，透過訪談找出當前組織內部人力資源相關之問題、未來發展趨勢，以及其對未來組織之工作、工作力之影響；其次探討我國環保機關之外環境，經實證調查分析，發現影響各環保機關人力資源運作之特定環境系統因素，並說明其對未來組織之工作、工作力之影響；最後對環境趨勢的發展加以評估。 　　第四章我國環保機關工作分析，分三節。經過工作分析，之實證調查分析，說明目前工作現況及及未來工作需求預測，最後對我國環保機關的組織的策略加以評估。 　　第五章我國環保機關工作力分析，分三節，經過實證調查分析，說明目前組織工作力現況及未來工作力需求預測，最後對我國環保機關當前之人力策略加以評估。 　　第六章我國環保機關未來人力方案之規劃與評估，分三節。首先整合工作、工作力與環境之優劣分析(Strengths, Weeknesse, Opportunities, Threats)，最後依此分析結果提出我國環保機關人力資源規劃方案之評估與建議。 　　第七章結論與建議。首先提出本文之研究發現與檢討，其次對實務之運作及未來研究提出建議。

<p>The main thesis hub was on development of two electrochemical biosensors for the determination of 17&beta / -estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17&beta / -estradiol endocrine disrupting compound / consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide.</p>

<p>The main thesis hub was on development of two electrochemical biosensors for the determination of 17&beta / -estradiol: an estrogenic endocrine disrupting compound. Endocronology have significantly shown that the endocrine disruptors contribute tremendously to health problems encountered by living species today, problems such as breast cancer, reproductive abnormalities, a decline in male population most significant to aquatic vertebrates, reduced fertility and other infinite abnormalities recurring in the reproductive system of mostly male species. The first biosensor developed for the detection of 17&beta / -estradiol endocrine disrupting compound / consisted of an electro-active polymeric 3-mercaptoprorionic acid capped zinc selenide quantum dots cross linked to horseradish peroxidase (HRP) enzyme as a bio-recognition element. The second biosensor developed was comprised of cysteamine self assembled to gold electrode, with 3-mercaptopropionic acid capped zinc selenide quantum dots cross linked to cytochrome P450-3A4 (CYP3A4) enzyme in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and succinimide.</p>