Tesi sul tema "PEG"

To take advantages of single-walled carbon nanotubes (SWNTs) for cellular delivery of chemotherapeutic agents (e.g. doxorubicin) in order to decrease doxorubicin toxicity and increase its efficacy, we aimed to develop a novel approach to aqueous disperse and stabilize SWNTs through consequent steps of oxidation (oxSWNT) and PEG-PEI complexation (PEG-PEI-SWNT). Doxorubicin was loaded onto the modified SWNTs in alkalione pH with more considerable capacity ( 900 %) than those previously reported, due to complex formation with PEI proved by UV-visible spectroscopy. The loaded carrier was stable in physiologic simulated medium. Drug release was prolonged and dilution independent, but exhibited pH-dependent burst release that makes SWNTs as suitable in vivo drug carriers in acidic tumor milieu. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/34928

The effectiveness of the PEG ratio as a valuation tool has been a topical debate between market commentators ever since being popularised by Lynch (1989). This study examines the appropriateness of the fair value criteria of 1.0 (PEGL) in comparison with a time-series based share specific benchmarking model (PEGT). Furthermore, influencing factors of analyst forecasting accuracy, namely: the number of analyst contributions, forecast dispersion and forecast horizon, were tested and compared using sub-set portfolios for each category with the objective of identifying a possible optimal PEG trading rule strategy. The outcome showed a consistent outperformance of PEGT portfolios compared to PEGL portfolios and the market benchmark. Unexpected results were obtained for the impact of analyst forecasts on the performance of the PEG ratio with additional literature review providing possible reasons that analyst optimism may have a more influencing impact on the PEG ratio than forecasting accuracy. Finally, an optimised PEG trading rule strategy delivered annual abnormal returns of 5.4% (CAGR: 19.7%) for a PEGL portfolio, versus that of 13.7% (CAGR: 28.5%) for a PEGT portfolio. The ensuing methodology appeared to single out small cap firms with above market growth prospects. Copyright
Dissertation (MBA)--University of Pretoria, 2010.
Gordon Institute of Business Science (GIBS)
unrestricted

Throughout the study of polymeric Langmuir monolayers at the air/water (A/W) interface, the Wilhelmy plate and Langmuir-Blodgett (LB) techniques along with Brewster angle microscopy (BAM) have been identified as key methods for acquiring structural, thermodynamic, rheological and morphological information. These techniques along with surface light scattering (SLS), a method for probing a monolayerâ s dynamic dilational rheological properties, will be used to characterize homopolymers, poly(ethylene oxide) (PEO) and poly(ethylene glycol) (PEG), and a new class of novel polymeric surfactants, telechelic (POSS-PEG-POSS) and hemi-telechelic (POSS-PEG) polyhedral oligomeric silsesquioxane (POSS) derivatives of PEG. PEO with number average molar mass, Mn > ~ 18 kgâ ¢mol-1 form stable spread Langmuir films at the A/W interface, while oligomeric PEG have ï -A isotherms that deviate from high molar mass PEO. Nonetheless, SLS reveals that the dynamic dilational viscoelastic properties of any Mn PEG(PEO) only depend on ï and not Mn. Likewise, POSS-PEG-POSS telechelics exhibit molar mass dependent ï -A isotherms, where low ï regimes (ï < 1 mNâ ¢m-1) have PEG-like behavior, but high ï regimes were dominated by POSS-POSS interactions. SLS studies reveal that the dynamic dilational moduli of POSS-PEG-POSS are greater than either PEO or an analogous POSS compound, trisilanolcyclohexyl-POSS. The ability to control rheological properties and the hydrophilic-lipophilic balance even allows one POSS-PEG-POSS (PEG Mn = 1 kgâ ¢mol-1) to form Y-type LB-multilayer films. For POSS-PEG systems, comparisons at comparable POSS:PEG ratios reveal short PEG chains (PEG Mn ~ 0.5 kgâ ¢mol-1) yield similar viscoelastic properties as POSS-PEG-POSS (PEG Mn ~ 1 kgâ ¢mol-1), while longer PEG chains (PEG Mn ~ 2 kgâ ¢mol-1) yield lower modulus films than comparable POSS-PEG-POSS. These differences are attributed to brush-like PEG conformations in short POSS-PEG versus mushroom-like PEG conformations in long POSS-PEG at the A/W interface. These results provide insight for designing PEG-based amphiphilic nanoparticles with controlled interfacial rheology.
Ph. D.

Warren Buffet started an investment partnership of $100 in 1956 and has gone on to accumulate a personal net worth of over $60 billion. He started primarily as a value investor, and gradually changed over time to a strategy which uses the PEG ratio as its main tool. Peter Lynch, one of the most successful fund managers in history and had a compound annual growth rate of 29% for 13 years, was the man to first introduce the world to the PEG ratio. With such prominence, however, widespread use of previously successful strategies tend to render them ineffective due to everyone using them, and today the PEG ratio’s effectiveness as a valuation tool remains a topical debate between market commentators.This study sets out to determine if the PEG ratio adds value using JSE Main Board data from 2002 to 2012. Returns from five portfolios constructed directly from share quintiles based on PEG ratio magnitude are compared to returns of a portfolio constructed from the optimum quintile of value shares. The PEG ratio portfolio returns are examined based on 3 rebalancing period strategies, and on relative performance between the quintiles within each strategy.It is found that a 24 monthly rebalancing strategy provides superior returns to that of 3 or 12 monthly rebalancing for PEG quintiles of selected stocks. Furthermore, the lowest PEG ratio quintile in this strategy outperforms the value portfolio by a compound annual growth rate of 4.3%. The second lowest PEG ratio quintile portfolio performs slightly better to ensure that 40% of stocks selected based on the PEG ratio produced sustained superior returns to the optimum quintile value portfolio.
Dissertation (MBA)--University of Pretoria, 2012.
Gordon Institute of Business Science (GIBS)
unrestricted

Les objectifs étaient d’étudier les effets de la pégylation sur les relations doses-concentrations-réponse à travers deux exemples qui sont le PEG-IFN beta 1a et le PEG-GRF, par la modélisation pharmacocinétique. La 1ère molécule étudiée était une glycoprotéine le PEG-IFN beta-1a. Une réduction de la clairance a été observée pour le PEG-IFN beta-1a par rapport à l’IFN beta-1a. L’effet du PEG-IFN beta-1a a été évalué à l’aide de 3 marqueurs pharmacodynamiques : beta2-microglobuline, néoptérine et 2’,5’-oligoadénylate synthétase. Aucune amélioration sur ces 3 marqueurs n’a été observée après l’injection du PEG-IFN beta-1a, comparé à l’IFN beta-1a, sur l’intensité et sur le délai de réponse. La 2ème molécule était un peptide le PEG-GRF. L’effet du PEG-GRF a été estimé par 2 marqueurs : IGF-1 et GH. Les constantes de vitesse d’apparition et de disparition de l’IGF-1 ne sont pas modifiées après injection du PEG-GRF, comparé au GRF. Le PEG-GRF présente une biodisponibilité supérieure à celle du GRF, vis-à-vis de l’IGF-1. Le PEG-GRF induit une réponse GH, représentée par des paramètres intégrés AUE sur différents intervalles de temps de 0 à 24h, plus importante par rapport au GRF
The objectives were to assess the effect of pegylation on the dose-concentration-response relationships throughout two examples PEG-IFN-beta-1a and PEG-GRF, using PK/PD modelling. The 1st molecule was a glycoprotein the PEG-IFN-beta-1a. A reduced clearance has been observed for PEG-IFN-beta-1a. The effect of PEG-IFN-beta-1a was assessed with 3 pharmacodynamic (PD) markers: beta2-microglobulin, neopterin and 2’,5’-oligo adenylate synthetase. By comparison with IFN-beta-1a, no improvement was observed in terms of magnitude and time course in the response of the PD markers following PEG-IFN-beta-1a injection. The 2nd molecule was a peptide the PEG-GRF. The effect of PEG-GRF-1a was assessed with 2 PD markers: GH and IGF-1. The absorption rate constant and the elimination rate constants remained unchanged following PEG-GRF administration, compared to GRF. Regarding the IGF-1 marker, PEG-GRF has a higher bioavailability compared to those of the GRF. The GH response, represented by AUE on different time intervals from 0 to 24 h, was higher following PEG-GRF injection, compared to GRF. Those 2 examples are representative of the variety in the properties of pegylation reported in literature

Peg solitaire is a game in which pegs are placed in every hole but one and the player jumps over pegs along rows or columns to remove them. Usually, the goal of the player is to leave only one peg. In a 2011 paper, this game is generalized to graphs. In this thesis, we consider a variation of peg solitaire on graphs in which pegs can be removed either by jumping them or merging them together. To motivate this, we survey some of the previous papers in the literature. We then determine the solvability of several classes of graphs including stars and double stars, caterpillars, trees of small diameter, particularly four and five, and articulated caterpillars. We conclude this thesis with several open problems related to this study.

In a 2011 paper by Beeler and Hoilman, the game of peg solitaire is generalized to arbitrary boards. These boards are treated as graphs in the combinatorial sense. An open problem from that paper is to determine the minimum number of edges necessary for a graph with a fixed number of vertices to be solvable. This thesis provides new bounds on this number. It also provides necessary and sufficient conditions for two families of graphs to be solvable, along with criticality results, and the maximum number of pegs that can be left in each of the two graph families.

In a paper by Beeler and Hoilman, the traditional game of peg solitaire is generalized to graphs in the combinatorial sense. One of the important open problems in this paper was to classify solvable trees. In this thesis, we will give necessary and sufficient conditions for the solvability for all trees with diameter four. We also give the maximum number of pegs that can be left on such a graph under the restriction that we jump whenever possible.

Denna rapport täcker arbetet och resultatet av en högnivåkontroller för en robotarmsombesitterintentionenattmonteraettcylinderformatverktygiettcirkulärt hål, så kallad peg-in-hole. Detta åstadkoms genom att implementera en kontroller som ett ROS paket. Styrning av roboten gjordes med hjälp av en impedans kontroller och kraftavläsning användes som externa sensorer för guidning av roboten. Utvecklingen gjordes både i simulering och på en riktig robot. Implemtationen visade sig vara lyckad att utföra uppgiften med hänsyn till de satta kraven. Förbättringspotentialen är bättre kraftavläsning samt ett tillägg av externa sensorer för en förbättrad avläsning av robotens omgivningen.
This thesis covers the work and result of a high level controller for a robot arm with the intention of inserting a cylinder shaped tool into a circular hole, a so called peg-in-hole insertion. This was done by implementing a controller as a ROS package. An impedance controller was used for controlling the robot and force feedback readings as external senses for guiding the robot tool. Development was done in simulation and on a real robot. The implementation proved to be successful at performing an insertion in regards to the set requirements, with the potential improvements being a better force reading as well as added external senses for improved environmental perception

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
? medida que demandas energ?ticas foram sendo apresentadas e requeridas pelo avan?o tecnol?gico, a s?ntese de novos materiais, capazes de suprir estas necessidades, foi e tem sido exaustivamente executada e estudada; dentre estes materiais, est?o filmes polim?ricos comp?sitos que t?m se destacado como uma alternativa qu?mica, f?sica, el?trica e econ?mica vi?vel. Neste trabalho filmes finos autossustent?veis de poli(metactrilato de metila) (PMMA) sob a forma de blendas, associado ao poli(etileno glicol) (PEG), e comp?sitos, dopados com tungstato de s?dio diidratado, foram obtidos pela associa??o dos m?todos sol-gel e ?casting?. A caracteriza??o el?trica, estrutural, qu?mica e morfol?gica dos filmes obtidos foi realizada usando-se espectroscopia de imped?ncia eletroqu?mica (EIS) para obten??o dos par?metros el?tricos, reflex?o total atenuada na regi?o do infravermelho (IV-ATR) e difra??o de raios X (DRX) para estudo da composi??o estrutural e qu?mica, enquanto que a estabilidade t?rmica foi analisada usando termogravimetria (TG/DTG), an?lise t?rmica diferencial (DTA) e calorimetria diferencial de varredura (DSC); as caracter?sticas morfol?gicas e topogr?ficas dos filmes foram analisadas por microscopia ?tica (MO) e eletr?nica de varredura (MEV). A adi??o do sal promoveu modifica??es do padr?o de disposi??o das cadeias polim?ricas, observado por difra??o de raios X e ratificado por altera??es das bandas relativas ? deforma??o angular ?CH2? em cadeia e estiramento assim?trico ?C?C? em cadeia, sugerindo mudan?as estruturais na cadeia polim?rica. A miscibilidade entre os pol?meros poli(metacrilato de metila) e poli(etileno glicol) foi resultado da presen?a de intera??es intermoleculares do tipo liga??o de hidrog?nio entre o ?CO? do PMMA e os grupos OH do PEG e do sal tungstato de s?dio, evidenciadas nas an?lises por IV-RTA, assim como, intera??es puramente eletrost?ticas. Os sistemas apresentaram melhores caracter?sticas capacitivas e maiores tempos de relaxa??o, favorecidos pelo aumento da concentra??o do tungstato de s?dio, resultado da melhora da sensibilidade do ac?mulo de cargas no eletrodo.
As energy demands were being presented and required by technological advances, the synthesis of new materials capable of meeting these needs, was and has been thoroughly studied and performed; among these materials are composite polymeric films that have stood out as an alternative chemistry, physics, electrical and economically viable. In this work self-supporting thin films of poly(methyl methacrylate) (PMMA) in the form of blends, linked to poly (ethylene glycol) (PEG), and composite doped with sodium tungstate dihydrate were obtained by combining the sol-gel methods and casting. The electrical, structural, chemical and morphological characterization of the films obtained was performed using electrochemical impedance spectroscopy (EIS) to obtain the electrical parameters, attenuated total reflection in the infrared (IR-ATR) and X-ray diffraction (XRD) to study the structural and chemical composition, while the thermal stability was evaluated using thermogravimetric analysis (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC); morphological and topographical characteristics of the films were analyzed by optical microscopy (OM) and scanning electron (SEM). The addition of the salt promoted standard modifications of the arrangement of the polymer chains, observed by X-ray diffraction and confirmed by the changes of the bands relative to the angular deformation -CH2- chain and asymmetric stretching -C-C-chain, suggesting structural changes in polymer chain. The miscibility between the polymers poly (methyl methacrylate) and poly (ethylene glycol) was a result of the presence of intermolecular interactions type hydrogen bonding between the PMMA and -CO- OH groups of the PEG and sodium tungstate salt, highlighted in analyzes by IR-ATR as well as purely electrostatic interactions. The systems showed better capacitive characteristics and larger relaxation times, favored by increasing the concentration of sodium tungstate, a result of improved sensitivity of the charges accumulated in the electrode.

Les composés polymacrocycliques présentent un grand intérêt grâce à leurs caractéristiques remarquables de coordination. Pour la première fois, l’approche synthétique fonctionnelle aux diverses composés bi- et polycycliques contenant plusieurs fragments d’éthers aza-couronnes, aux cryptands et aux supercryptands à base d’éthers aza-couronnes a été élaborée par Krakowiak et ses collaborateurs au début des années 1990, par l’usage de simples réactions de substitution nucléophile. Actuellement, les composés hétéropolymorphes polymacrocycliques, capables de former des complexes polynucléaires avec différents métaux présentent le plus grand intérêt. Dans la grande majorité des travaux la synthèse de presque toutes les composés polymacrocycliques a été réalisée à l’aide des méthodes non orthodoxes, à l’exception des molécules, composées de plusieurs macrocycles porphyriques (de la diade et de la triade), pour l’obtention desquelles ont été appliquées les réaction de Suzuki, le couplage de Sonogashira et la réaction de Heck. Le laboratoire de synthèse organominérale de la Faculté de chimie de l’Université d’Etat de Moscou a accumulé une riche expérience dans l’utilisation de l’amination pallado-catalysée pour la synthèse de diverses composés macrocycliques et polymacrocycliques, et, actuellement, on étudie activement l’arylation cupro-catalysée des di- et polyamines. À cet égard, l’étude de l’amination cupro-catalysée pour la synthèse de composés polymacrocycliques qui contiennent comme connecteurs des diamines et des polyamines a permis développer des méthodes de catalyse métallique et d’obtenir de nouveaux types de conjugués polytopiques et polymacrocycliques, qui comprennent dans leur composition structurelle des fragments éthers aza-couronnes, porphyrines et calixarènes, étudiés pour leur capacité à détecter des cations des métalliques.Le but de ces recherches est de développer des méthodes catalytiques de synthèse de conjugués polymacrocycliques qui contiennent dans leur composition structurale des fragments éthers aza-couronnes, des porphyrines et des calix[4]arènes et d’étudier leurs capacités à détecter des cations métalliques. Pour atteindre cet objectif, il est nécessaire de résoudre les problèmes suivants: 1) établir la généralité de l’amination catalysée par le Cu(I) des dérivés éthers aza-couronnes et des porphyrines contenant des halogènes et de synthétiser les dérivés aminés; 2) développer des méthodes de la macrocyclisation catalytique pour obtenir des composés macrobicycliques et macrotricycliques qui contiennent dans leur structure des fragments éthers diaza-couronnes, des tétraazamacrocycles ( cyclènes et cyclames) et des calix[4]arènes; 3) modifier les composés macrocycliques et macrobicycliques par des substituants fluorophores, y compris des porphyrines; 4) à l’aide de la spectroscopie UV et de la fluorescence, étudier la liaison cations métalliques-polymacrocycles et identifier les détecteurs potentiels moléculaires fluorescents et colorimétriques
Polymacrocyclic compounds are of great interest due to their unique coordination properties. The first convenient synthetic approach to various polycyclic compounds containing several azacrown-ether moieties, to cryptands and supercryptands, based on azacrown-ethers, has been developed by Krakowiak and coworkers in the beginning of 1990s using simple nucleophilic substitution reactions. At present time heteropolytopic polymacrocyclic compounds, capable of forming polynuclear complexes with various metals, attract the utmost interest. In the majority of publications dealing with the synthesis of polymacrocyclic compounds non-catalytic approaches were applied, except for several porphyrin dyads and triads, which were obtained using Suzuki, Sonogashira and Heck reactions. The laboratory of organoelement compounds of Chemistry Department of Lomonosov Moscow State University has a great experience of the application of Pd-catalyzed amination reactions for the synthesis of polymacrocyclic compounds, nowadays Cu-catalyzed arylation of di- and polyamines is under investigation. Bearing it in mind we have found the research for Cu-catalyzed amination to be important in synthesis of polymacrocyclic compounds containing di- and polyamine linkers; as well as the synthesis of new types of polytopic polymacrocyclic conjugates, bearing azacrown-ether, porphyrin and calixarene moieties, by means of Pd- and Cu-catalyzed reactions; and studying their properties as metal cations detectors.The aim of the research is to develop catalytic synthetic approaches to polymacrocyclic conjugates, bearing azacrown-ether, porphyrin and calix[4]arene moieties, and to study their abilities as detectors for metal cations. For this purpose it is necessary to carry out the following investigations: 1) to study the regularities of Cu(I)-catalyzed amination of halogen derivatives of azacrown-ethers and porphyrins and to synthesize corresponding amino derivatives; 2) to develop the methods for the catalytic macrocyclization aimed at the synthesis of macrobicyclic and macrotricyclic compounds, containing diazacrown-ether, cyclen, cyclam and calix[4]arene moieties; 3) to introduce fluorophoric fragments (including porphyrins) into macrocyclic and macrobicyclic compounds; 4) to investigate metal cations binding by thus synthesized polymacrocycles using UV and fluorescent spectroscopy, and to find possible fluorescent and colorimetric detectors among them

Os polietilenoglicóis (PEGs) são polímeros sintéticos não iônicos, solúveis em água, que possuem diferentes propriedades físico-químicas devido ao efeito de sua cadeia longa, conferindo assim uma vasta disposição na aplicação industrial do PEG. Como exemplos de aplicação destacam-se o uso como lubrificante solúvel em água para moldes de borracha, fibras têxteis e operações de formação de metais, sendo também usado em tintas à base de água, revestimento de papéis, polimento, na indústria cerâmica, na confecção de fases estacionárias para cromatografia, entre inúmeras outras. O PEG também pode ser adicionado às dietas de ruminantes baseadas em foragem com alto teor de taninos, onde ele se insere como um agente complexante do tanino. Contudo, devido à ampla aplicação do PEG, mostra-se uma maior preocupação com seu uso e disposição no meio ambiente, uma vez que sua concentração, seja no solo ou água, possa ser grande. No entanto, pouco se sabe acerca do seu destino no ambiente, principalmente em condições tropicais, sendo que não foram encontrados na literatura estudos de monitoramento de águas subterrâneas e superficiais, e nem mesmo estudos de impacto ambiental. Neste contexto, este projeto se insere na busca sobre o destino do PEG-4000 no ambiente, buscando informações sobre sua mobilidade, degradação e sorção em solos, utilizando técnicas radiométricas (14C-PEG). Complementando o estudo do PEG, foi avaliado o transporte de pesticidas na presença do PEG, uma vez que ele possui uma alta solubilidade, podendo atuar como co-solvente, avaliando assim o papel do PEG na presença de contaminantes
Polyethylene glycols (PEGs) are non-ionic synthetic polymer, soluble in water, which have different physical and chemical properties because of its long chain, thereby providing a broad industrial application of PEG. Examples of application are the use as water-soluble lubricant for rubber molds, textile fibers and metallurgy, also used in water-based paints, paper coating, polishing, ceramic industry, in construction of stationary phases for chromatography, and numerous others. PEG can also be added to the diets of ruminants based on forage with a high content of tannins, where it comes as a complexing agent of tannin. However, due to the wide application of PEG, it is a greater concern about its use and disposal in the environment, since their concentration, either in soil or water, can be large. However, little is known about their fate in the environment, especially in tropical conditions, which were not found in the literature studies for monitoring underground and surface water, and even environmental impact studies. In this context, this project falls in the search on the fate of PEG-4000 in the environment, seeking information on their mobility, degradation and sorption in soils, using radiometric techniques (14C-PEG). Complementing the study of PEG, was evaluated the pesticides transport in the presence of PEG, since it has a high solubility and can act as co-solvent, thereby assessing the role of PEG in the presence of contaminants

En 1998, le traitement de la co-infection VHC rarement discuté avant l’ère des HAART, compte tenu d’une réponse médiocre à la monothérapie par IFN et d’un pronostic de vie lié au VIH estimé en moyenne à 10 ans, fût reconsidéré. C’est ainsi que débuta en 2000, l’essai RIBAVIC HC02, essai randomisé et multicentrique comparant l’association de la ribavirine 800 mg/j à l’Interféron 3 MUI x3/semaine ou au PEG--2b Interféron 1,5 µg/kg/semaine pendant 48 semaines. Une cohorte des patients inclus dans l’essai RIBAVIC (cohorte RIBAVIC EP10) débuta en 2001 pour évaluer le devenir à long terme de ces patients. L’essai RIBAVIC et la cohorte RIBAVIC ont apporté les enseignements suivants : - la cinétique de la charge virale VHC peut différer selon la nature du traitement antirétroviral. - la prévalence et les facteurs de risque de la stéatose sont similaires à ceux observés dans la population mono-infectée VHC - le taux de réponse virologique soutenue est inférieur chez les patients co-infectés (27%) comparé aux patients mono-infectés VHC (50%) - le taux de non réponse virologique (diminution de la charge virale VHC inférieure à 2 log à S12) sous traitement par pegIFN plus ribavirine est plus élevé (33%) comparé aux patients monoinfectés VHC (14%). L’interaction entre la ribavirine et l’abacavir pourrait être un facteur de risque. - l’indétectabilité de l’ARN VHC dès S4 est prédictive de la réponse à long terme (valeur prédictive positive 97%) et la décroissance de la charge virale VHC est significativement plus lente chez les patients rechuteurs comparée aux patients répondeurs long terme à S2 et à S4 - Au cours du traitement anti-VHC :1- le risque d’anémie est élevé et majoré par la co-prescription de zidovudine et de ribavirine ; 2- l’amaigrissement est fréquent et sévère et peut être révélateur d’une toxicité mitochondriale ; 3- le risque bactérien n’est pas lié au taux des polynucléaires neutrophiles mais à la fibrose hépatique ; 4- le risque de toxicité mitochondriale, d’aggravation de la fibrose et de décompensation hépatique est majoré par l’interaction entre la didanosine et la ribavirine - une réponse virologique soutenue est associée à un bénéfice histologique et clinique.

Poly ethylene glycol (PEG) based microgels were synthesized and investigated. The PEG microgel has the same phase transition as the traditional poly N-isopropylacrylamide (PNIPAM). As a good substitute of PNIPAM, PEG microgel exhibits many advantages: it is easier to control the lower critical solution temperature (LCST) of the microgel by changing the component of copolymers; it has a more solid spherical core-shell structure to have a double thermo sensitivity; it is straightforward to add other sensitivities such as pH, magnetic field or organic functional groups; it readily forms a photonic crystal structure exhibiting Bragg diffraction; and, most importantly, the PEG microgel is biocompatible with human body and has been approved by FDA while PNIPAM has not. PEG microgels with core-shell structure are synthesized with a two-step free radical polymerization and characterized with DLS, SLS and UV–Vis. The dynamic mechanics of melting and recrystallizing of the PEG core-shell microgel are presented and discussed. Photonic crystals of PEG microgels were synthesized and characterized. The crystal can be isolated in a thin film or a bulk column. The phase transition of PEG microgel was simulated with the mean field theory. The enthalpy and entropy of phase transition can be estimated from the best fit to theoretical calculation with experimental data.

Le infezioni post-operatorie in campo ortopedico sono un fenomeno che si verifica frequentemente e che causa dolore nel paziente che è costretto alla sostituzione della protesi con una seconda operazione. Diventa fondamentale riuscire a prevenire tali infezioni e ciò può essere fatto migliorando le proprietà antibatteriche delle protesi grazie all’applicazione di rivestimenti superficiali. In questo lavoro sono stati sviluppati dei rivestimenti nanocompositi con attività antibatterica a base di glicole polietilenico (PEG) silanizzato e nanoparticelle di ossido di zinco (ZnO) da applicare su protesi di titanio così da diminuire la possibilità di infezioni post-operatorie nei pazienti. I coating sono stati prodotti tramite la tecnica di spray-coating su superfici di titanio. Diversi step sono serviti per la preparazione delle sospensioni usate per creare il rivestimento. Nel primo è stata utilizzata una reazione di silanizzazione per ottenere un PEG termoindurente, poi un pre-curing con una soluzione acida e, infine, aggiunta di nanoparticelle di ZnO per addizionare la proprietà antibatterica. Dopo l’applicazione sulle superfici di titanio tramite uno spray-coater, i campioni sono posti in forno per attivare il processo di reticolazione termicamente. Durante la procedura sono state effettuate alcune caratterizzazioni: lo studio della cinetica della reazione di silanizzazione tramite la spettroscopia infrarossa (FT-IR) e la valutazione del grado di reticolazione raggiunto dopo il curing termico grazie a prove gravimetriche di frazione estraibile. I coating realizzati differiscono tra loro per il diverso contenuto percentuale di nanoparticelle presenti (0%, 1%, 5%, 10%, 20%) e per ognuno di questi sono state determinate le proprietà di resistenza, tramite Scratch Test, e le proprietà antibatteriche, grazie ai test di cinetica di Time-killing. Tutte le prove sono state effettuate nelle così dette “dark conditions”, ovvero senza l’attivazione dello ZnO tramite luce UV.

The aim of this study was the development and the validation of two pharmacogenetic tests for determination of the personalized dosage of drugs used in clinical settings (Warfarin and PEG-Interferone/Ribavirin) in which genetic testing is necessary and recommended. Warfarin (Coumadin®) is a widely prescribed anticoagulant used for treatment and prevention of thrombotic disorders. Although highly efficacious, Warfarin’s narrow therapeutic index and wide inter-individual variability in dose response make its dosing difficult. The standard procedure to define the personalized dose is an iterative process that can take weeks or months and exposes the patients to an increased risk of over- or underanticoagulation and thus to serious side effects like thromboembolism or bleeding. The anticoagulation effect of the Coumadin® drug therapy is influenced by combination of genetic and non-genetic factors , which account for more than half of the inter-individual variation in Warfarin doses required to achieve a therapeutic level of anticoagulation or therapeutic INR (Prothrombin International Normalized Ratio). Since 2011, the FDA requires the product label to include a recommended daily Warfarin dose (mg/day) for therapeutic INR, which is to be based on the genotypes of CYP2C9 and VKORC1. In addition, Genome-Wide Association Studies (GWAS) have confirmed that CYP2C9, VKORC1 and CYP4F2 are the main genetic factors determining the effective Warfarin dose in Caucasians. Therefore, knowledge of the genetic profile provides a means to assess the differential risk of adverse reactions and allows the personalization of the therapy, e.g. lowering the dosage for patients with the alleles CYP2C9 *2 and *3 or that are homozygous for the A-allele (A/A) of the rs9923231 polymorphism in the VKORC1 gene. The goal of this study was to develop an innovative genotyping system assessing the gene polymorphisms that mainly determine the clinical response to Warfarin. Four polymorphisms were investigated: rs9923231 in the VKORC1 gene, rs1799853 and rs1057910 in the CYP2C9 gene and rs2108622 in the CYP4F2 gene. To evaluate already available systems and decide on the method to be used for the test we performed a benchmarking of the tests present in the market. The genotyping is performed using a multiplex PCR in combination with a Reverse Line Blot hybridization assay (mPCR/RLB). To ensure specific amplification the test uses the SSP-PCR (Sequence Specific Primer PCR) approach. The high specificity of this PCR method was confirmed by direct sequencing. The test identifies and differentiates SNPs using ASO probes (Allele Specific Oligonucleotide) that recognize the different alleles. The hybrid formed between the amplified target DNA sequence and the complementary probes is detected by a colorimetric reaction using biotin-streptavidin and alkaline phosphatase. In the first part of the study several parameters like assay temperature, typology of hybridization and washing solutions, incubation time and probe design were analyzed in order to assess their influence on the assay’s performance. Next, the amplification protocol was defined and the conditions of the RLB assay were set up. The kit prototype was then validated on 125 samples, which had been previously genotyped with a standard method (TaqMan® Drug Metabolism Genotyping Assay, Life Technologies), and the analytical sensitivity of the assay was determined. All tested samples were accurately genotyped (100% diagnostic specificity and sensitivity) and the method efficiently worked in a broad range of DNA concentrations. Sensitivity tests demonstrated that the method developed in this study, provided reliable results within a DNA concentration range of 1 ng/reaction to 500 ng/reaction. The shelf life of the reagents was determined to be at least twelve months. In addition, the assay was shown to work on/with the following combinations of probe support materials (a nylon membrane and a nitrocellulose blotting membrane) and PCR master mixes (three commercial available master mixes). A software was developed that automates and facilitates interpretation of the RLB (strip) patterns. After completion, the system, GENEQUALITY AB WARFARIN TYPE cod. 04-74A-20 (AB ANALITICA), was notified to the Ministero della Salute and was CE IVD marked for commercialization in the European Community. As this system is the only assay on the market that analyzes the CYP4F2 gene in combination with the CYP2C9 and the VKORC1 gene, it represents the most complete approach for determining the genetic factors influencing Warfarin dosage in Caucasians. The Hepatitis C virus (HCV) infection is a major health problem with more than 170 million infected individuals worldwide (2-2.5% of the world’s population). Approximately 30% of the infections are cleared spontaneously, whereas 40% of the patients develop a chronic infection that can lead to cirrhosis, hepatocellular carcinoma and end-stage liver disease. The current standard of care is a combination treatment with pegylated interferon (PEG-INF) and Ribavirin (RBV), but its efficacy and tolerability are limited. Many efforts have been made to identify the factors that influence host resistance to HCV infection and treatment outcome. Several Genome-Wide Association Studies (GWAS) have demonstrated independently that variations in the IL28B (Interleukin 28B) gene are strongly associated with spontaneous clearance of the virus, early viral kinetics and with the varying PEG-INF/RBV therapy response in different populations. Particularly, polymorphism IL28B rs12979860 influences the prospect of recovery from an HCV infection. Patients homozygous for allele rs12979860-C show a two- to threefold higher SVR (Sustained Virological Response) rate compared to persons with a non-CC genotype when undergoing Peg-INF/RBV treatment, have a higher probability of spontaneous clearance of the virus and, during therapy, display a stronger reduction of virus RNA levels, which is reflected in higher RVR (Rapid Virological Response) and EVR (Early Virological Response) rates. Other studies reported an association of certain functional variants of the ITPA (Inosine Triphosphatase) protein with the reduction of hemoglobin during Peg-INF/RBV therapy. These allelic variants of the ITPA gene decrease the enzymatic activity of ITPA, thus protecting from RBV-induced anemia. The aim was to develop an innovative system to genotype the most significant genetic polymorphisms important for the prediction of treatment response and risk of adverse reactions in patients infected with HCV. Four polymorphisms were investigated in this study: rs12979860 and rs8099917 in the IL28B gene and rs7270101 and rs1127354 in the ITPA gene. Human genomic DNA was extracted from peripheral blood using an automated system (EZ1, Qiagen). Genotyping of the genetic variants was performed combining multiplex PCR with a Reverse Line Blot hybridization assay (mPCR/RLB). For amplification (PCR) of the target sequences, biotinylated primers were used. The primers were designed and analyzed in silico using bioinformatics alignment tools like “blat” (UCSC) and ClustalW, excluding genomic sequences that contain SNPs, SINEs/LINEs or other repetitive elements and all primer binding sites that show a sequence similarity of more than 95% with other genomic sequences. Due to the fact that a sequence that is highly homologous to the target region in IL28B gene is located on the same chromosome 15.000 bp upstream, the SSP-PCR approach was adopted in order to ensure a high specificity of the amplification. To evaluate the efficacy of the SPP-PCR, the amplification products were analyzed by direct sequencing. Subsequently, the protocol for the multiplex amplification was set up. Probes recognizing the different alleles of each investigated SNP (two ASO, Allele Specific Oligonucleotide, per SNP) were designed and their specificity analyzed under varying experimental conditions. This allowed estimation of the effect of parameters like temperature and time of incubation on the assay performance. The analytical sensitivity of the assay was determined using a range of DNA concentrations between 0.5 ng/µl and 200 ng/µl. The diagnostic specificity of the test was assessed analyzing 160 samples of a known genotype. The test provided a result within a DNA concentration of 1 ng/reaction to 400 ng/reaction and all tested samples were accurately genotyped. This underlines the reliability and specificity of the assay. The shelf life of the reagents was determined to be at least eight months and the compatibility of the assay with other amplification master mixes or probe supports (membranes) was tested. The system, GENEQUALITY IL28B-ITPA TYPE cod. 04-47A-20 (AB ANALITICA), was notified to the Ministero della Salute and CE IVD marked for commercialization in the European Community. RLB is a convenient way to identify up to 8 targets in 20 individual specimens simultaneously. It is more flexible and less costly than DNA microarrays while providing the same specificity and sensitivity. The system developed in this study, GENEQUALITY IL28B-ITPA TYPE, code 04-47A-20 (AB ANALITICA), allows analysis of the most important host genetic factors influencing the efficacy of the Peg-INF/RBV therapy and determining the risk of RBV-induced anemia. It is the only assay on the market that analyzes ITPA and IL28B polymorphisms together and therefore represents the most complete approach for determining the genetic constitution in regard to the IL28B and the ITPA gene for personalized management of patients with Hepatitis C. A genotype-phenotype correlation analysis was performed to evaluate the association between IL28B and ITPA polymorphisms and the therapy outcome in a sub-group of patients (N=52). Genetic analysis of the IL28B polymorphism rs12979860 revealed that 27% of the patients carried the C/C genotype, whereas 56% and 17% carried the C/T and the T/T genotype, respectively. The T/T genotype of the IL28B polymorphism rs8099917 was in 40% of patients, whereas 48% carried T/G and 12% the G/G. No association was found between variants of the IL28B polymorphisms and SVR rates: 57% of patients with IL28B rs12979860-CC and 52% with IL28B rs8099917-TT achieved SVR and 43% and 48%, respectively, failed to clear the HCV virus. For analysis of the influence of ITPA, the patients were subdivided into the following three groups: normal ITPase activity (100% ITPA), mild ITPase deficiency (60% ITPA- 60% ITPase activity) and moderate to severe ITPase deficiency (≤30% ITPA, ≤30% ITPase activity). These groups reflected the individual Composite ITPase Deficiency Variable based on the genotypes of the ITPA polymorphisms rs7270101 and rs1127354. Patients with normal ITPase activity were compared to the groups of patients with mild ITPase deficiency and moderate to severe ITPase deficiency by statistical analysis. The results showed that the predicted ITPase deficiency correlated to a reduction of median Hb levels at 4, 12 and 24 weeks of treatment. A statistically significant difference was observed between the groups with normal ITPase activity and moderate to severe ITPase deficiency (≤30% ITPA) at 4 and 24 weeks (Dunnett’s Multiple Comparison Test: T4, p < 0.0001; T24, p < 0.0451). This clearly shows that patients with an at least by 70% decreased ITPase activity are protected from anemia throughout the treatment.
L’attività di ricerca ha avuto come obiettivo quello di sviluppare due test diagnostici di interesse commerciale, utili a predire il dosaggio di farmaci utilizzati in ambito clinico (Warfarin e PEG-IFN/Ribavirina), per i quali si rende necessaria la personalizzazione della terapia ed è raccomandato eseguire il test genetico. Il Warfarin (Coumadin®) è uno degli anticoagulanti più largamente utilizzati in clinica per la prevenzione e il trattamento di eventi trombotici, il cui utilizzo si associa però a dei rischi molto gravi per la salute del paziente a causa sia dell’indice terapeutico molto ristretto sia dell’ampia variabilità individuale nella risposta al farmaco. Questi aspetti rendono necessaria la personalizzazione della terapia, attualmente ottenuta a posteriori, con aggiustamenti posologici in base a misurazioni seriali dell’INR. Durante le fasi iniziali della terapia, tuttavia, è particolarmente elevato il rischio di eventi trombotici o emorragici dovuti rispettivamente a sotto- e sovra-dosaggio farmacologico. Recentemente numerosi studi di associazione genome-wide hanno messo in luce il ruolo chiave di alcuni polimorfismi nei geni CYP2C9 (rs1799853 e rs1057910), CYP4F2 (rs2108622) e VKORC1 (rs9923231) nell’influenzare l’efficacia anticoagulante del farmaco modificandone sia la farmacocinetica che la farmacodinamica. La conoscenza del profilo genetico permette di prevedere un rischio differenziale di manifestare reazioni avverse e quindi di personalizzare il dosaggio del farmaco, orientandosi verso dosaggi minori nel caso di pazienti che presentino le varianti alleliche *2 e *3 del gene CYP2C9 o siano omozigoti A/A per il polimorfismo rs9923231 nel gene VKORC1. L’utilità di associare alla prescrizione del farmaco un test genetico è stata sottolineata anche dall’FDA che nel 2010 ha revisionato il foglietto illustrativo del farmaco inserendo una tabella con i dosaggi (mg/die) raccomandati di farmaco per raggiungere un INR terapeutico sulla base del dato genetico. L’obiettivo di questo studio è stato quindi quello di sviluppare un test per l’identificazione dei più significativi polimorfismi associati alla risposta clinica al Warfarin e di preparare la documentazione necessaria per poter marcare il kit come dispositivo diagnostico in vitro commercializzabile nella Comunità Europea. A tal scopo si è proceduto effettuando un’attività di benchmarking per valutare i sistemi già presenti nel mercato e successivamente definendo la metodica del test. Considerando l’utilità che deriverebbe dall’analisi simultanea di tutte le varianti alleliche di interesse, si è deciso di sviluppare un test farmacogenetico basato sulla metodica Reverse Line Blot che combina una multiplex PCR ad un saggio di ibridazione su strip mediante oligonucleotidi sequenza-specifici adesi ad un supporto di membrana. Per garantire l’amplificazione univoca delle sequenze target, è stato utilizzato un approccio SSP-PCR (Sequence Specific Primer – PCR), la cui efficienza e specificità sono state valutate mediante sequenziamento diretto. Per la detection su strip di ciascun polimorfismo sono state poi progettate due sonde ASO (Allele-Specific Oligonucleotide), la cui funzionalità è stata valutata su campioni informativi per i polimorfismi indagati e precedentemente genotipizzati con un metodo di riferimento. Questa prima fase di definizione del prototipo si è conclusa con la messa a punto delle condizioni sperimentali del saggio, relative ai protocolli di amplificazione in multiplex e di rivelazione su strip. Tale fase ha richiesto un notevole impegno tecnico per superare alcuni dei limiti tecnici sulla costruzione degli oligonucleotidi, imposti dalle sequenze altamente omologhe dei citocromi e per la difficoltà di riuscire a individuare le giuste condizioni sperimentali che consentono di discriminare in modo univoco, specifico e sensibile, sequenze che differiscono tra loro di una singola base (sequenza wild-type Vs. sequenza polimorfica). La fase successiva è stata quella di definire la sensibilità analitica del metodo e le performance del test, analizzando 125 campioni precedentemente genotipizzati da un laboratorio di riferimento (Laboratorio di Biochimica Clinica e Biologia Molecolare Clinica dell’Azienda Ospedaliera di Padova), mediante sistema in Real-Time (TaqMan® Drug Metabolism Genotyping Assay, Life Technologies). Le prove effettuate con concentrazioni scalari di DNA di partenza hanno permesso di definire una sensibilità analitica di 0,5 ng di DNA/µl e un range di quantità ottimale di DNA di partenza che va da 10 ng/µl a 100 ng/µl, sebbene il test garantisca un risultato corretto anche per quantità di DNA pari a 500 ng/reazione. Inoltre, tutti i 125 campioni analizzati in fase di validazione sono stati correttamente genotipizzati; per ciascuno dei polimorfismi indagati: rs1799853 (c.430 C>T, Cys144Arg, CYP2C9*2), rs1057910 (c.1075 A>C, Ile359Leu, CYP2C9*3), rs2108622 (c.1297 C>T, Val433Met, CYP4F2*3) e rs9923231 (c.-1639 G>A, VKORC1) il dispositivo ha fornito un risultato concorde al 100% al dato ottenuto con il metodo di riferimento ed è stato possibile calcolare un valore di sensibilità e specificità diagnostica del 100%, esprimibile anche come accuratezza del 100%, ossia numero di genotipizzazioni corrette sul totale delle genotipizzazioni effettuate (osservato/atteso). Ai fini della marcatura CE IVD, è stata valutata la stabilità dei reagenti a intervalli di tempo regolari di 1 mese per 14 mesi e le prove condotte hanno permesso di definire una shelf life di 12 mesi; da studi di compatibilità effettuati variando le mix di amplificazione e i supporti su cui vengono fatte aderire le sonde è emerso, inoltre, che il dispositivo risulta compatibile con tre tipi di master mix di amplificazione disponibili in commercio e due tipologie di materiali usati per il blotting delle probes (una membrana in nylon e membrana in nitrocellulosa). Per facilitare l’interpretazione dei risultati, il kit è stato implementato con un software interpretativo conforme ai requisiti della norma IEC 62304 – “Medical Device Software – Software Life Cycle Processes” e rispondente alle esigenze di rendere automatico e ripetibile il processo di lettura e interpretazione del risultato della strip e di archiviare i risultati in un format stampabile. Il test sviluppato in questo lavoro di dottorato, GENEQUALITY AB WARFARIN TYPE cod. 04-74A-20 (AB ANALITICA), è stato notificato al Ministero della Salute, è stato marcato come dispositivo diagnostico in vitro CE IVD e può pertanto essere commercializzato. Il dispositivo rappresenta uno dei sistemi più completi per la personalizzazione della terapia in pazienti con disordini tromboembolici e in cura con Warfarin. Nessuno dei sistemi presenti nel mercato, infatti, analizza il polimorfismo rs2108622 C>T nel gene CYP4F2 che, però, soprattutto per la popolazione caucasica è associato in modo statisticamente significativo ad un incremento del dosaggio di anticoagulante, necessario per raggiungere un INR terapeutico; lo SNP rs2108622 spiega in media circa il 10% delle differenze di dosaggio osservate tra i portatori della variante CYP4F2 c.1297 T e i soggetti CYP4F2 c.1297 C/C e si conferma il terzo locus genico in ordine di importanza come predittore di risposta terapeutica al Warfarin e ai suoi derivati. L’epatite C cronica (CHC) è una delle maggiori cause di cirrosi epatica ed epatocarcinoma. Nel corso degli anni la terapia dell’epatite C ha subito una notevole evoluzione che ha portato a definire come Standard of Care (SOC) la combinazione di Interferone Peghilato e Ribavirina (PEG-IFN/RBV, duplice terapia). L’efficacia di tale associazione, tuttavia, è elevata (80%) nei casi di infezione da HCV di genotipo 2-3, ma non supera il 50% nei casi di infezione da HCV di genotipo 1. Grazie anche alla migliore comprensione dei meccanismi molecolari di replicazione virale e dei meccanismi patogenetici dell’infezione, negli ultimi decenni sono stati sviluppati agenti antivirali di nuova generazione da utilizzarsi in pazienti intolleranti alla terapia standard oppure da usarsi in combinazione con PEG-IFN e Ribavirina (triplice terapia) per aumentare l’efficacia della SOC, ridurne la durata e la tossicità e facilitare la compliance del paziente. Sia nel caso della duplice che della triplice terapia, però, il numero e la gravità delle reazioni avverse non sono trascurabili (anemia e neutropenia gravi, disturbi a carico del sistema nervoso centrale, reazioni di ipersensibilità) e hanno determinato un notevole interesse nell’identificazione di fattori prognostici in grado di predire l’efficacia della terapia. La probabilità di risposta alla terapia antivirale dipende in primis dal genotipo virale, ma sono emersi significativi anche fattori dell’ospite, sia clinici che genetici. In particolare, numerosi studi hanno evidenziato un’associazione statisticamente significativa tra i polimorfismi rs12979860 e rs8099917 nel gene IL28B e il tasso di SVR (Sustained Virological Response) identificando nel gene IL28B uno dei più importanti predittori pretrattamento di risposta alla terapia. E’ stato riportato che pazienti con genotipo rs12979860-CC e rs8099917-TT rispondono più efficacemente alla terapia antivirale rispetto agli eterozigoti o agli omozigoti rs12979860-TT e rs8099917-GG (tassi di SVR da due a tre volte maggiori). Il polimorfismo rs12979860 è correlato anche alla probabilità di clearance spontanea del virus, che è tre volte maggiore nei portatori della variante protettiva rs12979860-C e alla cinetica di declino virale durante la terapia; indipendentemente dall’etnia, i pazienti CC mostrano un’elevata riduzione dei livelli di RNA virale nel siero che si riflette in più alti tassi di RVR (Rapid Virological Response) ed EVR (Early Virological Response) rispetto a quelli osservabili in pazienti con genotipo rs12979860-CT o –TT. Da diverse evidenze scientifiche, inoltre, è emersa una correlazione tra il rischio di manifestare anemia emolitica cronica indotta da Ribavirina e due polimorfismi (rs1127354 e rs7270101) nel gene ITPA che codifica per l’inosina trifosfatasi, un enzima coinvolto nell’idrolisi di nucleotidi tri- e difosfato. Sia nel caso del gene IL28B che del gene ITPA, la conoscenza del dato genotipico può indirizzare quindi il clinico nella scelta del regime più adatto e fornisce un’ informazione aggiuntiva per valutare meglio il rapporto rischio/beneficio del trattamento rispetto al non trattamento. Con questo studio ci si è quindi proposti di sviluppare un test farmacogenetico per la discriminazione allelica delle varianti genetiche associate alla resistenza al trattamento antivirale (duplice o triplice terapia) e al rischio di manifestare anemia emolitica indotta da Ribavirina. Scopo di questo lavoro è stato anche quello di confermare tali correlazioni nel campione di pazienti analizzati in questo studio. Il test si basa sulla metodica del Reverse Line Blot e prevede una prima fase di amplificazione in multiplex delle regioni a cavallo dei polimorfismi di interesse, una seconda fase di rivelazione colorimetrica su strip e infine una fase di interpretazione dei dati. Per assicurare un appaiamento univoco dei primers al genoma, ciascun oligo è stato progettato ed analizzato in silico utilizzando tools bioinformatici di allineamento con il genoma, come “blat” (UCSC) e ClustalW, escludendo così dalle regioni candidate quelle contenenti SNPs, SINE, LINE o altri elementi ripetuti e tutte le regioni per le quali esistano sequenze con percentuali di identità superiori al 95%. Per quanto riguarda il gene IL28B, in particolare, la presenza di un blocco ripetuto, localizzato sullo stesso cromosoma circa 15000 nucleotidi più a monte, con omologia di sequenza estremamente elevata alla regione di interesse, ha portato a scegliere un approccio SSP-PCR (Sequence-Specific Primer PCR) come metodo di elezione per aumentare la specificità della reazione di amplificazione. Una volta messo a punto il protocollo di amplificazione, si sono settate le condizioni sperimentali del saggio RLB andando a valutare l’influenza di diversi parametri sperimentali (quantità e tipologia delle probes, temperatura, tipologia delle soluzioni di ibridazione e di lavaggio, tempi di incubazione) sulle performance del saggio, in termini di sensibilità analitica e specificità diagnostica. Il prototipo è stato quindi validato e a tal scopo sono stati analizzati 160 campioni precedentemente genotipizzati con sistemi di riferimento in Real Time (TaqMan Allelic Discrimination Assay, Life Technologies, LightMix in vitro diagnostics kit IL28B, LigthMix rs7270101 ITPA e LigthMix rs1127354 ITPA, Roche). Dai dati raccolti è stato possibile definire una sensibilità analitica di 0,5 ng/µl e un’accuratezza del test del 100%; per ciascuno dei campioni analizzati, infatti, i polimorfismi rs12979860 e rs8099917 nel gene IL28B e rs7270101 e rs1127354 nel gene ITPA sono stati correttamente identificati e genotipizzati dal dispositivo in esame, in accordo al dato ottenuto con il metodo di riferimento. In prospettiva di marcare il kit CE IVD, è stata poi valutata la stabilità dei reagenti e dalle prove effettuate finora è stato possibile definire una shelf life di 8 mesi, da implementare con i dati derivanti dal completamento delle prove di stabilità a 14 mesi, come indicato nel relativo piano di stabilità. Le prestazioni del dispositivo sono state analizzate anche variando i reagenti di amplificazione oppure il supporto su cui sono adese le sonde sequenza-specifica, mantenendo inalterati tutti gli altri parametri sperimentali; anche in tal caso, i risultati ottenuti in ciascuna prova sono stati del tutto equiparabili a quelli ottenuti con i reagenti usati in fase di validazione. Visti e considerati gli ottimi risultati ottenuti, il test sviluppato in questo lavoro di dottorato, GENEQUALITY IL28B-ITPA TYPE cod. 04-47A-20 (AB ANALITICA), è stato notificato al Ministero della Salute ricevendo la marcatura come dispositivo diagnostico in vitro CE IVD e può pertanto essere commercializzato. L’utilizzo di un saggio mPCR/RLB per l’identificazione dei polimorfismi nei geni IL28B e ITPA risulta essere un metodo rapido e altamente specifico che trova spazio in ambito diagnostico per la genotipizzazione dei pazienti in corso di terapia con agenti antivirali standard e di nuova generazione (PEG-IFN/RBV, Boceprevir, Telaprevir). Attualmente, inoltre, il dispositivo GENEQUALITY IL28B-ITPA TYPE cod. 04-47A-20 è l’unico kit sul mercato ad analizzare insieme tutti i polimorfismi più significativi per predire la risposta alla terapia antivirale dell’epatite C cronica e il rischio di reazioni avverse, pertanto rappresenta l’approccio più completo per la personalizzazione della terapia in pazienti con epatite C cronica. Per 52 dei campioni analizzati è stato possibile ottenere i dati clinici relativi ai parametri biochimici ed ematologici e all’outcome terapeutico. Il 63% dei soggetti presentava un’infezione a carico dei genotipi virali 1-4 più difficili da trattare e la terapia si è dimostrata efficace (raggiungimento della SVR, negativizzazione dell’RNA virale nel siero a 24 settimana dalla fine del trattamento) nel 27% dei pazienti con infezione da HCV GT 1-4 e nel 74% dei soggetti con infezione da HCV GT 2-3, riflettendo quanto riportato in letteratura. Nel campione analizzato la maggior parte dei pazienti, indipendentemente dal genotipo virale, era portatore in eterozigosi di almeno uno dei due polimorfismi del gene IL28B, rs12979860 e rs8099917 (27% dei pazienti era rs12979860-CC, mentre il 56% e il 17% era C/T o T/T; il 40% dei pazienti era rs8099917-TT, il 48% T/G e il 12% G/G). La valutazione dei polimorfismi in relazione alla risposta alla terapia non ha mostrato alcuna correlazione significativa. Su 14 soggetti rs12979860-CC, il 57 % ha raggiunto l’SVR, mentre il 43% non è riuscito a eradicare il virus e tale situazione di sostanziale equilibrio si è riscontrata anche nel gruppo di soggetti rs8099917-TT; nel 52% dei casi, infatti, la terapia ha determinato il raggiungimento della risposta virologica sostenuta, mentre nel 48% dei casi si è registrato un fallimento terapeutico. L’analisi di correlazione genotipo-fenotipo nel sottogruppo di pazienti indagati (N=52) ha messo in luce, invece, un’associazione tra la riduzione media dei livelli di emoglobina e l’aplotipo definito dalle varianti polimorfiche rs7270101-C e rs1127354-A del gene ITPA. Nei pazienti che presentavano un’attività ITPasica inferiore al 30% rispetto al basale, predetta su base genetica, la riduzione media di Hb è stata inferiore al 5% nelle prime 4 settimane di trattamento e non ha superato il 15% nell’arco delle 24 settimane, mentre nei soggetti con il 100% di attività enzimatica (rs7270101-AA e rs1127354-CC), il declino di Hb ha raggiunto quasi il 20% fin dal primo mese di trattamento. Il calo di emoglobina nei pazienti con attività enzimatica ridotta al 60% rispetto al wild-type (gruppo 60% ITPA) ha assunto nel tempo un andamento intermedio. I pazienti con deficit enzimatico severo (gruppo ≤30% ITPA) risultano quindi protetti dalla riduzione di Hb indotta da Ribavirina e tale associazione è stata confermata anche dall’analisi statistica, condotta effettuando un confronto multiplo a gruppi tra le tre classi di rischio (100% ITPA, 60% ITPA e ≤30% ITPA), in relazione ai livelli di emoglobina dopo 4, 12 e 24 settimane di trattamento (Dunnett’s Multiple Comparison Test, 100% ITPA Vs. 30% ITPA a 4 settimane: p < 0.001 con α= 0.05, a 24 settimane: p < 0.05 con α = 0.05).

Le mélanome métastatique reste à l’heure actuelle une pathologie dramatique avec une survie moyenne de 13 mois après diagnostic, démontrant l’échec des chimiothérapies. Ces travaux de thèse ont pour but de développer une stratégie alternative utilisant les siRNA (petits ARN interférents) encapsulés au sein de nanocapsules lipidiques (LNC) pour une injection intraveineuse. Les premiers travaux ont porté sur le développement et l’amélioration du procédé de formulation des LNC chargées en siRNA. Les résultats ont permis une encapsulation à hauteur de 35%, une stabilité prolongée supérieure à 3 mois et une efficacité d’extinction du gène cible dans les cellules de mélanome. La deuxième partie de la thèse s’est orientée sur les stratégies de ciblage du mélanome après administration systémique. Différentes modifications de surface des LNC à l’aide de polyéthylène glycol (PEG) et d’Affitins (peptide d’affinité) ont été mises au point. La biodistribution sur des animaux « sains » ou des animaux greffés en sous-cutanés (mélanome humain) a révélé des comportements distincts en fonction des recouvrements utilisés. Les formes pegylées ont montré une accumulation préférentielle au site tumoral en comparaison avec les formes non modifiées ou modifiées avec les Affitins. Dans une troisième partie, des études d’efficacité anti-tumorale ont été réalisées avec un siRNA ciblant la protéine Bcl-2 ou la sous unité alpha 1 de la pompe Na/K ATPase. Une réduction du volume tumoral de 25% est observée avec les LNC siRNA Bcl-2. Par ailleurs, l’association de nouvelles chimiothérapies, les ferrocifènes, et des siRNA Bcl-2, montrent grâce à leur co-encapsulation au sein des LNC, des effets prometteurs. Ces travaux démontrent ainsi la capacité des LNC à délivrer des siRNA par voie intraveineuse dans de nouvelles stratégies de ciblage du mélanome
Metastatic melanoma represents the most aggressive form of skin cancer with a median survival around 13 months. The low efficacy of actual chemotherapy is explained by important resistance phenomenon. The objective of this work consists in developing a new alternative strategy based on siRNA (small interfering RNA) encapsulated into lipid nanocapsules (LNCs) for intravenous injection. Firstly, the experiments were focused on development and optimization of formulation process for the encapsulation of siRNA into LNCs. The result demonstrated an encapsulation efficiency evaluated at 35% by spectrophotometer analysis, an important stability at 4°C (for at less 3 month) and an efficient gene inhibition in melanoma cells. The second part of this work studied the melanoma targeting potential of surface modified LNCs after systemic injection. In this way, pegylation with different polymers and Affitin grafting (affinity peptide) was performed on siRNA LNCs. The biodistribution on healthy animals and subcutaneous melanoma tumor bearing mice revealed the distinct behavior of various modified LNCs. All pegylated LNCs showed a preferential accumulation in tumor site in comparison with non-modified or Affitins LNCs. In a third part, the anti-cancer efficacy was tested with siRNA targeting Bcl-2, an anti-apoptotic member, or Alpha 1 subunit of Na/K ATPase. A reduction of tumoral volume evaluated at 25% was observed for Bcl-2 siRNA LNC. Moreover, the association with new promising anticancerous drug, ferrocifens, and Bcl-2 siRNA co-encapsulated into LNCs evidenced promising effects. This work demonstrated the capacity of LNC to deliver siRNA into melanoma cells and tumor after systemic administration thank to new targeting strategies in melanoma

Die Funktionalisierung von tetra-PEG Makromolekülen mit fotoreaktiven Gruppen und die anschließende Umsetzung zu Hydrogelen durch fotochemische Vernetzung werden beschrieben. Die Funktionalisierung der Makromoleküle wird mittels UV-Vis- und NMR-Spektroskopie nachgewiesen, während der Verlauf der Vernetzung über die dynamische Lichtstreuung und IR-Spektroskopie betrachtet wird. Die hergestellten Hydrogele werden hinsichtlich des Sol-Anteils und der Quelleigenschaften untersucht. Über den Umsatz wird die Konzentration der Netzketten theoretisch berechnet. Einen weiteren Schwerpunkt bildet die Charakterisierung der Hydrogele hinsichtlich der mechanischen Eigenschaften. Über den Speichermodul wird die Konzentration der Netzketten experimentell bestimmt. Mittels dynamischer Lichtstreuung werden die kooperativen Diffusionskoeffizienten und Maschenweiten der Hydrogele bestimmt.

The growing incidences of coronary artery bypass graft surgeries have triggered a need to engineer a viable small diameter blood vessel substitute. An ideal tissue engineered vascular graft should mimic the microenvironment of a native blood vessel, while providing the adequate compliance post-implantation. Current vascular graft technologies lack the ability to promote vascular ECM deposition, leading to a compliance mismatch and ultimately, graft failure. Hence, in order to engineer suitable vascular grafts, this thesis describes the synthesis and characterization of novel elastin mimetic peptides, EM-19 and EM-23, capable of promoting vascular ECM deposition within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel. By combining the material properties of a synthetic and bio-inspired polymer, a suitable microenvironment for cell growth and ECM deposition can be engineered, leading to improved compliance. As such, characterization of EM-19 and EM-23 was conducted in human vascular smooth muscle cell (SMC) cultures, and the peptides self-assembled with a growing elastic matrix. After grafting the peptides onto the surface of PEG-DA hydrogels, EM-23 increased SMC adhesion by 6000% over PEG-RGDS hydrogels, which have been the gold standard of cell adhesive PEG scaffolds. Moreover, EM-23 grafted surfaces were able to promote elastin deposition that was comparable to tissue cultured polystyrene (TCPS) surface even though TCPS had roughly 4.5 times more SMCs adhered. Once translated to a 3D model, EM-23 also stimulated increased elastin deposition and improved the mechanical strength of the scaffold over time. Moreover, degradation studies suggested that EM-23 may serve as a template that not only promotes ECM deposition, but also allows ECM remodeling over time. The characterization studies in this thesis suggest that this peptide is an extremely promising candidate for improving vascular ECM deposition within a synthetic substrate, and that it may be beneficial to incorporate EM-23 within polymeric scaffolds to engineer compliant vascular grafts.

The focus of this study is the change in accretion disk size in Dwarf Novae (DN), IP Peg. DN systems are a type of cataclysmic variable that experience periodic outbursts. These outbursts are caused by the release of gravitational potential energy from an increased rate of matter flow through the accretion disk. Throughout outburst, the radius of the accretion disk of the DN changes. Recent research done at Ball State University has suggested that the disk radius may not change as the disk instability model predicts. According to the disk instability model, the accretion disk should be at its largest radial size when the DN is at the peak of outburst. IP Peg in September and October of 2006 underwent outburst. It was found that during that particular outburst that the accretion disk was at its largest radial size on the decline from outburst and not peak. Further research into how the accretion disk changes with time is needed.
Department of Physics and Astronomy

ITC/USA 2009 Conference Proceedings / The Forty-Fifth Annual International Telemetering Conference and Technical Exhibition / October 26-29, 2009 / Riviera Hotel & Convention Center, Las Vegas, Nevada
This paper describes a psychophysical experiment to measure visibility thresholds (VT) for quantization distortion in JPEG2000 and an associated quantization algorithm for visually lossless coding of color aerial images. The visibility thresholds are obtained from a quantization distortion model based on the statistical characteristics of wavelet coefficients and the deadzone quantizer of JPEG2000, and the resulting visibility thresholds are presented for the luminance component (Y) and two chrominance components (Cb and Cr). Using the thresholds, we have achieved visually lossless coding for 24-bit color aerial images at an average bitrate of 4.17 bits/pixels, which is approximately 30% of the bitrate required for numerically lossless coding.

In this project, the interaction between polyethylene glycol modified (PEG-ylated) lipid nanoparticles and silica substrates was studied to find out how this interaction was affected by bulk concentration, temperature and the composition of particles. One kind of lipodisk and four kinds of PEG-ylated liposome were prepared from lipid films and characterized by quartz crystal microbalance with dissipation monitoring (QCM-D) instrument mounted with silica sensor. The detailed information of particle-silica interaction could be obtained from the raw data, frequency and dissipation values, and the adsorbed mass surface density calculated from the raw data. Lipodisks could be immobilized on the silica surface. Whether they would be rinsed away by PBS buffer was influenced by both the bulk concentration and temperature. The way of their binding could change and the changing process was affected by temperature. PEG-ylated liposomes could also be immobilized on the silica surface, and they could break and spread to form supported lipid bilayer in certain conditions, for example, the changing of temperature or the using of certain lipids. Supported lipid bilayers were created with high reproducibility in this project, which could be very useful to the future study of transmembrane proteins functions and lipodisk properties.

Les hydrogels, qui sont des réseaux de chaînes de polymères dans l’eau, sont caractérisés par des structures poreuses et hydrophiles qui en font en principe des matériaux avantageux utilisés dans le domaine de la filtration. Le contrôle du transport des solutés et des particules dans ces réseaux de polymères peut être réalisé en contrôlant leur morphologie microscopique et leur porosité. Dans cette thèse, nous concevons des membranes d’hydrogel composites autoportantes basées sur la polymérisation du poly(éthylène glycol) diacrylate PEGDA en présence de chaînes de poly(éthylène glycol) PEG. Nous étudions l'effet de la concentration et de la masse molaire du PEG sur les propriétés de perméabilité à l'eau ainsi que sur la sélectivité des membranes composites PEGDA/PEG et leur lien avec leurs propriétés structurelles. Nous montrons que les chaînes de PEG restent irréversiblement piégées dans la matrice du PEGDA, même après plusieurs cycles de filtration, ce qui contredit la littérature existante rapportant l'utilisation de chaînes de PEG comme agents de porogènes pour induire la porosité dans les matrices réticulées. Nous observons que l'ajout de chaînes de PEG, avec des concentrations et des masses molaires différentes, permet de régler la perméabilité à l’eau des systèmes d'hydrogel PEGDA/PEG sur plusieurs ordres de grandeur. Nous montrons que la perméabilité présente un maximum avec la concentration de recouvrement des chaînes de PEG, ce qui est un phénomène robuste observé pour les différentes masses molaires de PEG étudiées. En plus, nous étudions la sélectivité de membranes de PEGDA/PEG en filtrant des particules de polystyrène de différentes tailles. Nos résultats suggèrent que la présence de chaînes de PEG dans la matrice du PEGDA crée des défauts locaux à l'échelle nanométrique dans la densité de réticulation qui peuvent contrôler la perméation des particules et de l'eau à travers les échantillons
Hydrogels, which are networks of polymer chains in water, are characterized by porous and hydrophilic structures that make them in principle advantageous materials used in the field of filtration. Controlling the transport of solute and particle in such polymer networks can be achieved by controlling their microscopic morphology and porosity. In this thesis, we design free-standing composite hydrogel membranes based on the polymerization of poly(ethylene glycol) diacrylate PEGDA in the presence of poly(ethylene glycol) PEG chains. We investigate the effect of PEG concentration and molecular weight on the water permeability properties as well as the selectivity of PEGDA/PEG composite membranes and their link with their structural properties. We show that the PEG chains remain irreversibly trapped in the PEGDA matrix even after several filtration cycles which contradicts existing literature reporting the use of PEG chains as templating agents to induce porosity in cross-linked matrices. We observe that the addition of PEG chains, with different concentrations and molecular weights, allows to tune the water permeability of the PEGDA/PEG hydrogel systems over several orders of magnitude. We show that the permeability presents a maximum with the overlap concentration of PEG chains, which is a robust phenomenon observed for several molecular weights. In addition, we investigate the selectivity of the PEGDA/PEG membranes by filtering polystyrene particles of different sizes. Our results suggest that the presence of PEG chains in the PEGDA matrix provide some local nanoscale defects in the cross-linking density that may control the permeation of particles and water across the samples

O Monometoxi-polietilenoglicol succinimidil ácido propiônico (mPEG-SPA 5 e 20 kDa) foi analisado como adjuvante e inibidor da atividade neurotóxica da toxina tetânica (TxT) adsorvida ou não em Al(OH)3, à qual o polímero foi conjugado. Avaliou-se a toxicidade das amostras por DL50, demonstrando que a atividade neurotóxica da TxT foi inibida. A via subcutânea foi mais efetiva na indução de resposta à TxT tratada pelo mPEG-SPA e o efeito adjuvante do Al(OH)3 se deu pela intramuscular. Trinta cavalos foram submetidos a esquema de imunização seletiva, dividindo-se os dezoito escolhidos em grupos para imunização com TxT conjugada ao mPEG-SPA 5.000 e 5.000(2X) e TxT adsorvida ou não. Os soros dos cavalos foram analisados por ToBI Teste, que avaliou a evolução da resposta imune. Os soros também foram analisados por imunodifusão, eletroforese e immunoblotting, tendo este indicado uma provável superioridade antigênica da TxT Fluida relativamente aos adjuvantes. A conjugação mPEG-SPA provou ser efetiva na produção do soro antitetânico terapêutico para uso humano.
Monometoxypolyethylene glycol succinimidyl propionic acid (SPA-mPEG 5 and 20 kDa) was analyzed as adjuvant and inhibitor of tetanus toxin neurotoxic activity (TxT) adsorbed or not by Al(OH)3, to which the polymer was conjugated. The samples toxicity was evaluated by DL50, disclosing that TxT neurotoxic activity was inhibited. The subcutaneous inoculation was more effective in induction of response to TxT treated with SPA-mPEG and the adjuvant effect of Al(OH)3 was evidenced by the intramuscular. Thirty horses were submitted to a selective scheme of immunization and eighteen were divided in groups to be immunized with TxT conjugated to SPA-mPEG 5,000 and 5,000(2X) and TxT adsorbed or not. The horses sera were analysed by ToBI Test, which evaluated the immune response development. The sera were also analysed through immunodifusion, electrophoresis and immunoblotting and the last one indicates a probable antigenic superiority of TxT fluid relatively to the adjuvants. The SPA-mPEG conjugation proved to be effective for anti-tetanus human therapeutic serum production.

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Mimosa caesalpiniifolia Benth., known by sabiá in the brazilian Northeast, is a forest species native to the Caatinga and belonging to the Fabaceae family. It has recently been included in the list of vulnerable species due to their use as wood, firewood and charcoal. Seeds of species that develop in soils of arid and semi-arid regions, such as sabiá, commonly find unsuitable conditions for germination, such as soils affected by water deficiency and abundance of saline soils. The objective of this study was to evaluate the effect of water and saline stress on the germination and seed vigor of three lots of M. caesalpiniifolia from matrices located in the municipalities of Luziania - GO (Lot 1), Vazante - MG (Lot 2) and Mountains - RN (Lot 3). In the simulation of the water restriction, two osmotic agents, mannitol and polyethylene glycol 6000 (PEG 6000), adjusted for the osmotic potentials of 0; -0.2; -0.4; -0.6 and -0.8 MPa. To simulate salt stress, NaCl solutions were used in the electrical conductivities of 0,0; 5.0; 10.0; 15.0; 20.0; 25.0; 30.0 dS m-1. Were analyzed: germination, germination velocity index, shoot and root length and dry mass of shoot and root of seedlings. The experimental design was completely randomized, with four replicates of 25 seeds for each treatment. The results demonstrated that mannitol-induced water stress did not influence seed germination and root length of sage seedlings, reduced IVG and seedlings dry length and dry mass, and increased dry mass of seedlings. The water restriction simulated with PEG, in turn, was more critical, reducing percentage and speed of germination, aerial and root length, as well as dry mass of shoot and root of seedlings. The three batches responded similarly to water stress, but Lot 3, with seeds from Montanhas – RN, was less vigorous than the others. The salinity reduced the germination and vigor of M. caesalpiniifolia seeds, reducing the parameters evaluated in the higher electrical conductivities (25.0 and 30.0 dS m-1), and affecting in a more expressive way, IVG and length of seedlings. Lot 2 was more tolerant of salt stress
Mimosa caesalpiniifolia Benth., conhecida por sabiá no Nordeste brasileiro, é uma espécie florestal nativa da Caatinga e pertencente à família Fabaceae. Recentemente foi incluída na lista de espécies vulneráveis em função de sua utilização como madeira, lenha e carvão vegetal. Sementes de espécies que se desenvolvem em solos de regiões áridas e semiáridas, a exemplo de M. caesalpiniifolia, comumente encontram condições inadequadas para a germinação, como solos afetados pela deficiência hídrica e abundância de solos salinos. Assim, objetivou-se avaliar o efeito dos estresses hídrico e salino na germinação e vigor de sementes de três lotes de M. caesalpiniifolia provenientes de matrizes localizadas nos municípios de Luziania – GO (Lote 1), Vazante – MG (Lote 2) e Montanhas – RN (Lote 3). Na simulação da restrição hídrica foram utilizados dois agentes osmóticos, manitol e polietilenoglicol 6000 (PEG 6000), ajustados para os potenciais osmóticos de 0; -0,2; -0,4; -0,6 e -0,8 MPa. Para simular o estresse salino, foram utilizadas soluções de NaCl, nas condutividades elétricas de 0,0; 5,0; 10,0; 15,0; 20,0; 25,0; 30,0 dS m-1. Foram avaliados: germinação, índice de velocidade de germinação, comprimento da parte aérea e da raiz e massa seca da parte aérea e da raiz das plântulas. O delineamento experimental foi inteiramente casualizado, com quatro repetições de 25 sementes para cada tratamento. Os resultados demonstraram que o estresse hídrico induzido por manitol não influenciou a germinação e o comprimento da raiz das plântulas de sabiá, reduziu o IVG e o comprimento e massa seca da parte aérea das plântulas, e aumentou a massa seca da raiz das plântulas. A restrição hídrica simulada com PEG, por sua vez, se mostrou mais crítica, reduzindo a porcentagem e a velocidade de germinação, o comprimento da parte aérea e da raiz, além da massa seca da parte aérea e da raiz das plântulas. Os três lotes responderam de maneira semelhante ao estresse hídrico, porém o Lote 3, com sementes provenientes de Montanhas – RN, foi menos vigoroso que os demais. A salinidade reduziu a germinação e o vigor das sementes de M. caesalpiniifolia, diminuindo os parâmetros avaliados nas condutividades elétricas mais elevadas (25,0 e 30,0 dS m-1), e afetando de maneira mais expressiva, o IVG e o comprimento da parte aérea das plântulas. O Lote 2 se mostrou mais tolerante ao estresse salino
2017-05-22

Moderne Methoden für die Einzelzellanalyse werden dank der fortschreitenden Weiterentwicklung immer sensitiver. Dabei steigen jedoch auch die Anforderungen an das Probenmaterial. Viele Aufbereitungsprotokolle adhärenter Zellen beinhalten eine enzymatische Spaltung der Oberflächenproteine, um die Ablösung vom Zellkultursubstrat zu ermöglichen. Verschiedene Methoden, wie die Patch-Clamp-Technik oder eine auf der Markierung extrazellulärer Domänen von Membranproteinen basierende Durchflusszytometrie können dann nur noch eingeschränkt eingesetzt werden. Daher ist die Etablierung neuer Zellablösemethoden dringend notwendig. In der vorliegenden Arbeit werden erstmals PEG-basierte thermo-responsive Oberflächen erfolgreich für die Zellkultur eingesetzt. Dabei wird das zerstörungsfreie Ablösen verschiedener Zelllinien von den Oberflächen durch Temperatursenkung realisiert. Die Funktionalität der Oberflächen wird durch Variation der Polymerstruktur, sowie der Konzentration der Beschichtungslösung, durch Beschichtung der Oberflächen mit einem zelladhäsionsfördernden Protein (Fibronektin) und durch Adsorption zelladhäsionsvermittelnder Peptide (RGD) optimiert. Um den Zellablösungsprozess detaillierter zu untersuchen, wird hier zum ersten Mal der direkte Zellkontakt mit thermo-responsiven Oberflächen mittels oberflächensensitiver Mikroskopie (TIRAF) sichtbar gemacht. Mit dieser Technik sind die exakte Quantifizierung und die Analyse der Reduktion der Zelladhäsionsfläche während des Abkühlens möglich. Hierbei werden in Abhängigkeit von der Zelllinie Unterschiede im Zellverhalten während des Ablösens festgestellt: Zellen, wie eine Brustkrebszelllinie und eine Ovarzelllinie, die bekanntermaßen stärker mit ihrer Umgebung in Kontakt treten, vergrößern im Verlauf des Beobachtungszeitraumes den Abstand zwischen Zellmembran und Oberfläche, reduzieren jedoch ihre Zell-Substratkontaktfläche kaum. Mausfibroblasten hingegen verkleinern drastisch die Zelladhäsionsfläche. Der Ablösungsprozess wird vermutlich aktiv von den Zellen gesteuert. Diese Annahme wird durch zwei Beobachtungen gestützt: Erstens verläuft die Reduktion der Zelladhäsionsfläche bei Einschränkung des Zellmetabolismus durch eine Temperatursenkung auf 4 °C verzögert. Zweitens hinterlassen die Zellen Spuren, die nach dem Ablösen der Zellen auf den Oberflächen zurückbleiben. Mittels Kombination von TIRAF- und TIRF-Mikroskopie werden die Zelladhäsionsfläche und die Aktinstruktur gleichzeitig beobachtet. Die Verknüpfung beider Methoden stellt eine neue Möglichkeit dar, intrazelluläre Prozesse mit der Zellablösung von thermo-responsiven Oberflächen zu korrelieren.
Modern methods for single-cell analysis are becoming increasingly sensitive. At the same time, requirements for the sample material are on the rise. Today, sample preparation of adherent cells usually includes steps of enzymatic treatment to digest surface proteins thus, inducing cell detachment from culture substrates. This strongly limits the application of different techniques like patch clamp or labelling of extracellular domains of membrane proteins for flow cytometry. Therefore, a new cell detachment method is urgently required. In the present work, new PEG-based thermo-responsive polymers are used for cell culture for the first time. Here, non-destructive detachment of different cell lines from polymer-coated surfaces is realised by controlled temperature reduction. The surface functionality is systematically optimised by varying the concentration of the coating solutions, by artificial surface coating of a cell adhesion-mediating protein (fibronectin) and by co-adsorption of a cell adhesion-mediating peptide (RGD). For detailed analysis of the cell detachment process, TIRF microscopy is used to directly visualise the cell contacts on the thermo-responsive surfaces. Using this technique allows both the quantification and analysis of the reduction of the cell adhesion area during sample cooling. Furthermore, for several cell lines, different behaviours in cell detachment are observed. Cells that have close contact to their substrate like MCF-7 breast cancer cell line and CHO-K1 ovary cells increase the distance between cell membrane and surface, but there is only little decrease of cell-substrate adhesion area. In contrast, L929 fibroblasts reduce the cell adhesion area drastically. Furthermore, the hypothesis that the cell detachment is an active process is shown by lowering the cell metabolism by temperature reduction to 4 °C and by the cell traces that are left behind after rinsing the surfaces. A combination of TIRAF and TIRF enables visualising the cell adhesion area and actin structures. Measuring both parameters simultaneously opens up new possibilities to correlate intracellular and cell detachment processes on thermo-responsive surfaces.

碩士
國立臺灣大學
醫學工程學研究所
92
Abstract Hydrogels are capable of containing large amounts of water and resemble nature living tissue so it can be used in drug delivery for low molecular hydrophobic drugs, hydrophilic drugs, peptides, proteins and genes, and can be used as scaffold in tissue engineering. Due to its biocompatibility and biodegradablility, hydrogels could be easily applied in in vivo experiments. This research is to synthesize PEG-PLGA-PEG triblock copolymer with the polyethylene glycol and poly(lactide-co-glycolide). This polymers is gel in body temperature and solution in room temperature. It can be used as an implantable drug delivery system which no any surgical operation is needed after drug depleted. The polymers can also be completely metabolized. These polymers are called temperature-sensitive hydrogels. They are formed by the phase transformation of micelles, the same mechanism as that of pluronics and tetronics. Different temperature makes the solubility change due to the methyl and ethyl functional organic group. PEG-PLGA-PEG triblock copolymer were synthesized by bulk reaction. The characteristics of polymers were validated by H-NMR. Sol-gel phase diagrams were established by the tube reverse method. Vancomycin in vitro release was conducted in 37℃.Comparing the drug delivery curve verse with the different temperature at 32℃ and 40℃, we proposed a gelation mechanism of these copolymer gels.

碩士
國立清華大學
生醫工程與環境科學系
105
Gene therapy is a process of introducing foreign genomic materials into target cells to elicit a therapeutic benefit. A diverse array of inherited and acquired diseases are targets of gene therapy. To overcome the multidrug resistance (MDR), one of the major impediment against curative cancer chemotherapy, the application of small interfering RNA (siRNA) provide a new opportunity for specific gene-silencing of MDR-associated proteins. In this study, a simple and novel approach is presented for constructing a dual delivery vector through a one-pot hydrothermal reaction, using graphene oxide (GO), polyethylenimine (PEI) and polyethylene glycol (PEG) as starting materials. The resulting nanoplatform, rGO-PEI/PEG exhibited minimal toxicity and could effectively complex siRNA at a W/W ratio above 3.4. Additionally, rGO-PEI/PEG was capable of high drug loading (doxorubicin, ~0.49 mg/mg) and photothermally triggered cytosolic drug delivery. With optimal near-infrared laser irradiation, the drug-loaded rGO-PEI/PEG demonstrated an enhanced antitumor efficacy in cancer cells through combined photothermal effect and chemotherapy. The synergistic potential of dual drugs (doxorubicin and siRNA)-loaded rGO-PEI/PEG in combination with laser irradiation will next be explored to augment the therapeutic effect in MDR cancer cells. The advances described above will complement our knowledge of graphene functionality and serve to guide its application in gene/drug delivery.

碩士
國立中山大學
機械與機電工程學系研究所
92
Both position and angular errors during the insertion process, which cannot be easily predicted because of indeterminate collision situations, may cause failure of the assembly. One of the frequently applied strategies is to use a passive remote center compliance. We break the insertion problem down in to two phases: chamfer-crossing, and inserting (after chamfer-crossing)phase. In this article, the relationship between the position and angular errors during chamfer-crossing with different chamfer size and locality are thoroughly analysis. We also try to design a technological processes of minimizing the angular errors during chamfer-crossing. Besides single round peg insertion, two dimensional dual peg-in-hole insertion problems are briefly analysis.

博士
中原大學
化學研究所
97
The present study is aimed to observe osteogenesis in-vivo when a composite hydrogel of biodegradable thermosensitive poly(ethylene glycol) - block poly(lactide-co-glycolide) - block poly(ethylene glycol) triblock copolymer (PEG-PLGA-PEG) and hydroxyapatite (HA) is injected subcutaneously. The composite hydrogel was also mixed with recombinant bone morphologenesis protein 2 (BMP-2) for the osteoinduction. PEG-PLGA-PEG was synthesized using ring opening polymerization technique whereas HA was synthesized in a chemical co-precipitation method. The hydrogel was formed by vortexing predetermined amount of PEG-PLGA-PEG and HA and injected into skin throught a syringe in rat. Tissue sections at various time point post injection were prepared and In-vivo biocompatibility of injected materials was evaluated accordingly. In the part of chemical synthesis of PEG-PLGA-PEG, the structure was determined by 1H nuclear magnetic resonance spectrophotometer and Fourier-transform infrared spectrum. The thermosensitivity and in-vitro degradation of PEG-PLGA-PEG and its composites with HA were studied by following the viscometer of hydrogels and in-vitro degradation test, respectively. On the other hand, the crystal structure of HA was measured by powder X-ray diffraction, scanning electron microscope - energy dispersive spectrometer and inductively coupled plasma atomic emission spectrometry, respectively. Results show that 2-theta degree appeared a peak at 25.8° which is the obviously characteristic peak of HA and the Ca/P ratio is close to composition of nature bone. Secondly, the quantitative assessment of the injectability of composite hydrogel was conducted using in syringe. Finally, In-vivo injection of the composite hydrogels (PEG-PLGA-PEG hydrogel, HA disc, composite hydrogel of PEG-PLGA-PEG and HA, PEG-PLGA-PEG, HA and BMP-2 composite hydrogel) under skin of the rat was conducted for a research period of 8 weeks. Results demonstrated that osteoinductive BMP-2 can be injected through biocompatible hydrogel of PEG-PLGA-PEG and HA. The biocompatibility of materials can be observed by the optical microscope. After eight weeks, fibroblasts not only grow but also osteocytes. The adding bone morphogenetic proteins-2 can be clearly observed more of osteocytes. Then conclusion is the as-prepared composite hydrogel of PEG-PLGA-PEG and HA, with small additive amount (e.g. 0.01 wt%) of BMP-2 as bone promoter, is a suitable material for osteoinduction.

碩士
國立交通大學
生物科技學系
103
CR1 is a derivative of the plant lignan nordihydroguaiaretic acid (NDGA). It has been proposed that the parent compound and its derivatives inhibit the interaction between the transcription factor Sp1 and its DNA-binding site via DNA groove-binding. Furthermore, Sp1 has highly related to colorectal cancer development and progression. Therefore, CR1 is a potential anticancer agent in colorectal cancer therapy, but CR1 also has side effect to normal tissues. Because of its high cytotoxicity, liposome encapsulation may reduce the cytotoxicity of CR1 to normal tissues, but keep the cytotoxicity for tumor cells. Colorectal carcinoma, cancer of the colon and rectum, is the second leading cause of cancer death worldwide in women and the third leading cause of cancer death worldwide in men. Nowadays, therapy of colorectal cancer is only moderately successful for late-stage and is often limited by severe side effects and dose limiting toxicity. Hence, it is needed a new chemotherapy agent in colorectal cancer. My purpose is to use Lipo-PEG-PEI complex (LPPC) encapsulated CR1 to reduce cytotoxicity in normal tissues, and improve therapeutic efficacy for colorectal cancer. In my results, the protocol for the formation of LPPC/CR1 was optimal. LPPC/CR1 had an average size less than 200 nm and a zeta potential of approximately 35 mV. In addition, this study also showed the antitumor effects of LPPC/CR1 for colorectal cancer in vitro and in vivo. These results indicate that the LPPC/CR1 complex may supply a feasible strategy for the advanced colorectal cancer therapy in the future.

碩士
中原大學
數學研究所
100
Existing palm-recognition machines require using pegs for image acquisition. The main purpose of this study is to acquire high-quality and high-accuracy palm features in a peg-free environment in order to reduce environmental constraints during image acquisition. The experiments use three peg-free pictures acquired from the same individual, and use computer programs to automatically extract features on palm. Extracted features may be different due to muscle scaling and the direction fingers pointing to. The final features are derived by averaging features from those three samples. The final features are compared with the original features and the error ranges are (1) length feature error: 1.52%, (2) area feature error: 1.60%, and (3) perimeter feature error: 1.15%.

碩士
國立成功大學
化學系碩博士班
91
Molecular dynamics simulation have been used to study the temperature effects on the ionic diffusion, conductivity, coordination, and aggregation in polyelectrolytes Li(CF3SO3)/poly(ethylene glycol) and LiN(CF3SO2)2/poly(ethylene glycol). The simulations are examined against the experimental data from the literature. The polymer under study is hydroxyl-terminated poly(ethylene glycol)(PEG), with a MW of 400 g/mol. Simulations were conducted with O:Li (the ratio for the number of the ether oxygen to that of lithium ion) = 10:1 at 323, 333 and 353 K to mimic the experiment of Johansson et al. 　　The simulated diffusion coefficients of Li+, anion, and polymer increase with temperature. Since the motions of the lithium ion and polymer are, owing to coordination, strongly coupled, their diffusion coefficients are quite similar. The simulated diffusion coefficients agree well with experimental values, especially for LiN(CF3SO2)2PEG system. It is known that the number of oxygen atoms in the first coordination sphere of the lithium ion is about 5.4, which is in accord with experiment. The higher temperature facilitates the coordination of lithium ion with the oxygen atom of PEG, but Li+ coordination with anion is favored at low temperature. For the Li(CF3SO3)PEG, most of the oxygen atoms that coordinate with Li+ come from PEG. But the result is reversed when the salt is changed to LiN(CF3SO2)2. The number of anions around the Li+ ion in about 2 for the former, but 3 for the latter, which indicates that the probability to form ion pair or ion aggregate is higher for the latter.

碩士
國立臺灣大學
材料科學與工程學研究所
93
The study focuses on the effect of PEG with different end groups blending PLLA to prepare membranes by wet casting method. In this work, owning to the miscibility of two polymers, PLLA and PEG were thought as one phase, 1,4-dioxane was the solvent and n-Heptane was the non-solvent. Via the phase diagram, it was realized how liquid-liquid de-mixing and solid-liquid de-mixing varied with the end groups of PEG. The structure of membranes prepared with different end groups of PEG and the concentration of casting solutions can be observed clearly by SEM. The structures of PLLA/PEG(2CH3), (1OH-1CH3), and(2OH) were observed varying with the concentration of casting solutions; nevertheless, the structures of PLLA/PEG(2NH2) were found dissimilar with others. Besides, the viscosity of PLLA/PEG(2NH2) casting solution measured by the Ubbelohde viscosmeter was extreme lower than others. Through the above work, it was found how PEG end groups affected the structures of PLLA/PEG membranes.