Le repliement des protéines est principalement gouverné par les interactions spécifiques des structures secondaires. 1, 2 Toutefois, il existe expérimentalement peu d’informations sur les propriétés mécaniques fondamentales des hélices α et des feuillets β isolées. Les recherches antérieures sur l'étude du déploiement des hélices sont peu concluantes 3-5 et à notre connaissance l'étude des propriétés mécaniques d'un feuillet β isolé, intramoléculaire est sans précédent. Les copolymères PEG114-b-poly(L-lysine)134-(2-pyridyl disulfure),PEG114-b-poly(L-lysine)-b-PEG114 et poly(L-acide glutamique)85-b-(2-pyridyldisulfure) été synthétisés et utilisés comme systèmes modèles pour tester les propriétés mécaniques des motifs secondaires de type hélice α et feuillet β. Les résultats obtenus se sont révélés être en bon accord avec les résultats théoriques obtenus en utilisant un modèle statistique basé sur AGAGIR 6. La différence de force de déroulement comparant les hélices de poly(L-Lysine) ≈ 30 pN et de poly(L-acide glutamique) ≈ 20 pN des copolymères diblocs a été attribuée à l'hydrophobicité différente des chaînes latérales. La plus grande hydrophobie dumotif lysine conduit à de plus grandes interactions entre les chaînes latérales qui empêchent les fluctuations aléatoires au sein de l’hélice, et conduisent à une stabilité supérieure de l'hélice α. Lorsque les expériences ont été conduites dans des conditions favorisant la solubilité des chaînes latérales de lysine, les interactions ont diminué à une force de ≈ 20 pN, similaire à la force des interactions observées pour le poly(L-acide glutamique). Nous supposons qu'un minimum de ≈ 20 pN est nécessaire pour rompre la liaison hydrogène en maintenant l'hélice α, car cette force a été obtenue dans des conditions où les interactions de la chaîne latérale étaient minimisées. La présence de plateaux de force constants et d'inflexions correspondantes démontre une force de dépliement indépendante de la longueur, qui supporte un mécanisme de déroulement tour-par-tour pour l'hélice. De plus, la plus grande hydrophobie des chaînes latérales a été suggérée non seulement pour stabiliser la structure en hélice, mais également pour inhiber la formation d'une structure de type β-turn métastable intermédiaire lorsque les forces entropiques dominent. Des études préliminaires ont été effectuées sur le système de PEG114-bpoly(L-Lysine)134-(2-pyridyl disulfure) après induction d’une transition - β par un traitement thermique dans des conditions basiques. Une inflexion à une force≈ 70 pN a été obtenue, ce qui suggère la formation d'une interaction de type feuillet β. Une stratégie bottom-up a ainsi été proposée avec succès, démontrant le potentiel d'utilisation de tels systèmes artificiels pour simplifier et modéliser des systèmes biologiques réels. La compréhension de ces modèles isolés plus simples aidera sans doute la compréhension de systèmes plus complexes. / Proteins fold by the initial, preferential folding of secondarystructures 1, 2, however surprisingly little is known about the basic mechanicalproperties of isolated α-helices and β-sheets from an experimental standpoint.Previous investigations into studying the generic unfolding behaviour of α-heliceshave proved inconclusive 3-5, and to our knowledge the study of an isolated,intramolecular β-sheet is unprecedented.Bioinspired PEG114-b-poly(L-glutamic acid)85-(2-pyridyl disulphide),PEG114-b-poly(L-lysine)134-(2-pyridyl disulphide) and PEG114-b-poly(Llysine)134–b-PEG114 were designed, synthesized and utilized as model systems toprobe the mechanical properties of α-helix and β-sheet secondary motifs. Theobtained results were shown to be in good agreement with theoretical resultsobtained by utilizing a AGAGIR-based statistical mechanical model 6. Thedifference in unravelling force comparing the helices of poly(L-Lysine) ≈30 pNand poly(L-glutamic acid) ≈20 pN diblock copolymers was attributed to thediffering hydrophobicity of the side chains. The greater hydrophobicity of thelysine allowed greater interactions between the side chains and sterically hinderedrandom helix-coil fluctuations, which lead to a superior α-helix stability. Whenexperiments were conducted in conditions promoting the solubility of the lysineside chains, the interactions decreased to a force of ≈20 pN, similar to the force ofinteractions observed for the poly(L-glutamic acid). We infer that a minimum of≈20 pN is needed to rupture the hydrogen bonding maintaining the α-helix as thisforce was obtained in conditions where the side chain interactions wereminimized.The presence of constant force plateaus and corresponding inflectionsdemonstrates a length independent unfolding force, which supports a turn-by-turnunfolding mechanism for the α-helix.In addition, the greater hydrophobicity of the side chains was suggestedto not only stabilize the α-helix structure, but also to inhibit the formation of anintermediate metastable β-hairpin-like structure when entropic forces dominate.Preliminary studies were also conducted on the PEG114-b-poly(LLysine)134-(2-pyridyl disulphide) system after a α-β transition had been inducedby heat in basic conditions, where an inflection at a much higher force of ≈ 70 pNwas obtained suggesting the formation of a β-sheet interaction.A bottom-up, investigative strategy has thus been successfully proposeddemonstrating the potential of utilizing such artificial systems to simplify andexemplify real biological systems. The comprehension of these simpler isolatedmodels will no doubt aid the understanding of more complex systems.

Orientadores: Cecília Amélia de Carvalho Zavaglia, Marcos Akira d'Ávila / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-19T00:23:40Z (GMT). No. of bitstreams: 1 RodriguezPerea_GeraldineNancy_M.pdf: 1196408 bytes, checksum: 1a7f7c5e1320ddfd713867dbc7a1d5b6 (MD5) Previous issue date: 2011 / Resumo: Este trabalho consiste na obtenção pelo método de eletrofiação, de microfibras poliméricas e microfibras reforçadas com nanopartículas de hidroxiapatita. Este método foi utilizado, pois propicia a produção de membranas microporosas que possuem um grande potencial de aplicação na área de engenharia tecidual, especificamente em aplicações para regeneração óssea. Este trabalho teve como objetivo principal produzir fibras poliméricas com a intenção de comparar suas características com o nanocompósito de fibras poliméricas e nanopartículas de hidroxiapatita como reforço. Trabalhou-se com o poli(L-ácido lático) (PLLA) e nanopartículas de hidroxiapatita (HA) produzidas pelo processo sol-gel. As fibras e os nanocompósitos foram caracterizados pelos seguintes métodos: microscopia eletrônica de varredura (MEV), análise termogravimétrica (TGA), calorimetria exploratória diferencial (DSC) e espectroscopia na região do infravermelho por transformada de Fourier (FTIR). As fibras obtidas apresentaram diâmetros na faixa de 1 a 10 micrômetros. O objetivo de produzir membranas a partir de soluções de PLLA e nanocompósito PLLA/HA por eletrofiação foi atingido / Abstract: This work consists in obtaining polymeric microfibers and microfibers reinforced with nanoparticles of hydroxyapatite by the method of electrospinning. This method was used because it allows the production of microporous membranes that have great potential like application in tissue engineering, specifically in applications for bone regeneration. This work aimed to produce polymer fibers with the intention to compare their characteristics with the nanocomposite fibers with hydroxyapatite nanoparticles as reinforcement. The polymer used was poly (L-lactic acid) (PLLA) and nanoparticles of hydroxyapatite (HA) produced by the sol-gel process. The fibers and nanocomposites were characterized by the following methods: scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), spectroscopy in the region of Fourier transform infrared (FTIR). The fibers obtained presented diameters in the range 1 to 10 micrometers. The goal of producing membranes from solutions of PLLA and nanocomposite PLLA / HA by electrospinning was reached / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

Hollow fiber membranes (HFMs) formed through phase inversion methods exhibit specific physicochemical characteristics and generally favorable surface and mechanical properties, supporting their use in diverse applications including ultrafiltration, dialysis, cell culture, bioreactors, and tissue engineering. Characterization of, and modifications to, such membranes are important steps in achieving desired characteristics for specific applications. HFMs subject to gas, irradiation, and chemical sterilization techniques were characterized based on several analytical techniques. It was revealed that these common sterilization techniques can cause inadvertent changes to HFM properties. While these changes may cause detrimental effects to HFMs used in filtration, the methods of sterilization are also presented as a facile means of tuning properties toward specific applications. Modifications to HFM surface chemistries were also sought as a method of adsorbing bacterial lipopolysaccharide (LPS) from solutions used in hemodialysis treatments and bioprocessing applications. It was found that additives such as polyvinylpyrrolidone (PVP), polyethyleneglycol (PEG), and poly-L-lysine (PLL) can facilitate adsorption capacities of HFMs toward LPS. Additionally, chemical changes are presented as a means of preferentially adsorbing LPS to specific locations on the HFM surface.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21682号 / 医博第4488号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 大森 孝一, 教授 別所 和久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Controlling the surface morphology and degree of crystallinity of poly(L-lactic acid) (PLLA) substrates have recently attracted considerable attention because of their applications in cell adhesion, tissue engineering, and drug delivery. Several techniques have been used to fabricate PLLA substrates, some of which may be invalid because PLLA can degrade during fabrication processes. This dissertation provides the Langmuir-Blodgett (LB) technique as a mechanism for fabricating PLLA substrates at temperatures where PLLA degradation is uncommon. In order to fully understand surface morphologies of PLLA LB-films, studies of Langmuir monolayers at the air/water (A/W) interface using surface pressure-area (Pi-A) isotherm and Brewster angle microscopy (BAM) are vital. PLLA exhibits a first-order liquid expanded to condensed (LE/LC) phase transition with molar mass dependent critical phenomena, the first such observation for a homopolymer Langmuir monolayer. Atomic force microscopy (AFM) images of PLLA LB-films prepared in the LC phase exhibit well-ordered lamellar structures. Molar mass scaling of lamellar dimensions, x-ray reflectivity, and reflection absorption infrared spectroscopy (RAIRS) measurements on PLLA LB-films are consistent with PLLA existing as single molecule 10/3-helices at the A/W interface. Morphologies observed after collapse of the LC monolayer are dependent upon the collapse mechanism and subsequent thermal treatment. For temperatures below the LE/LC critical temperature (Tc), two mechanisms are identified for the formation of three dimensional structures: a buckling and stacking of lamellar monolayers on top of existing lamellae during constant compression rate experiments, and a modified nucleation and growth mechanism during isobaric area relaxation experiments. PLLA LB-films prepared in different Langmuir film phases at temperatures below Tc all contain lamellae with different surface roughnesses and similar helical content. Conventional thermal annealing studies on PLLA LB-films reveal that well-ordered lamellar features are destroyed after annealing the LB-films at bulk crystallization temperature through a melting-recrystallization process, which is confirmed by RAIRS and AFM. Our results may prove useful for studying critical behavior and experimentally testing scaling predictions for two dimensions, the development and testing of theories for crystallization in confined geometries, and separating the roles that roughness and crystallinity play in cell adhesion and spreading on biocompatible polymer surfaces. / Ph. D.

The ultimate design of a microencapsulated AI dose is to continuously release sperm over a period of time in the female reproductive tract, thus alleviating the need for estrus detection. The objective of Trial 1 was to determine in vitro sperm release times for three microcapsule membranes. Semen was collected from four bulls, pooled, extended in 20% egg yolk TEST to a concentration of 80 = 10⁶ cells/ml, and encapsulated. Microcapsule membranes were constructed from isomers of polylysine: .1% poly-L-lysine (PLL), .1% poly-D-lysine (PDL), and a 50:50 mixture of the isomers (PLPD). Microcapsules were incubated at 37°C in a buffer containing .5% heparin or .5% trypsin and evaluated at 0.5, 1, 2, 4, 8, and 16 h post-encapsulation. For sperm encapsulated there were no significant differences in sperm motility. However, peak time of maximum sperm release differed between PLL and PDL membranes at 2 and 4 h of incubation. In Trial 2, sperm viability and microcapsule membrane stability were assessed post-thaw using PLL or PDL, two encapsulating temperatures (5°C or 23°C) and two times of glycerol addition (prior or post encapsulation at 5°C). Semen was extended to 80 = 10⁶ cells/ml and encapsulated. Capsules from all treatment combinations were incubated in .5% trypsin and evaluated as in Trial 1. In addition, motility was estimated at 1, 3, 6, and 9 h post-thaw. Motility from the unencapsulated control and capsules with glycerol addition prior to encapsulation, was superior (P < .05). Additionally, sperm release from capsules prepared at 5°C with glycerol addition post encapsulation was greater than all other treatments (P < .05). Time of peak sperm release for capsules was similar to the previous trial. There was a positive correlation between average capsule diameter and sperm release for both trials (P < .05). These data suggest that a combination of PLL and PDL capsules may complement each other in timing of sperm release and may be utilized in an inseminate mixture for extending the effective release in the female / Master of Science

Metal contamination is a considerable environmental problem because metals are persistent contaminants. Ion exchange is one of the most commonly used treatment options for trace metal removal. This research develops and evaluates a redox active modified ion exchange system that has the potential to reduce the ionic strength of ion exchange regeneration streams. Poly-L-cysteine (PLC) was selected as the redox active, adsorbing functional group on the surface of a reticulated vitreous carbon (RVC) electrode. PLC is an excellent soft acid metal chelator and is unique in that its thiol groups can form disulfide bonds with each other. The reduction of available thiols changes the metal binding capacity of the peptide since the thiol is the primary binding group. RVC provides a macroporous conductive monolithic resin to support the peptide. An experimental apparatus was designed to study the properties of this system and estimate performance. Distinct oxidized and reduced states of PLC on the surface of the RVC were confirmed by changes in metal binding characteristics. Adsorption edges showed a sharper pH dependence for the reduced electrode compared to the oxidized electrode from pH 3-7. Adsorption isotherms performed at pH 7 showed increased capacity for the reduced electrode. The change was reversible by chemical and electrical reduction. This difference was confirmed at the molecular level with Cd- EXAFS of oxidized and reduced electrodes. A greater degree of cadmium-sulfur coordination was observed on the reduced electrode and a greater cadmium-oxygen coordination was apparant on an oxidized electrode. A multidentate adsorption model was developed to model the pH dependent behavior of cadmium adsorption on the PLC-RVC surface. Nickel adsorption showed increased adsorption in the oxidized state. The most likely explanation is increased carboxylate complexation. The electronically switchable ion exchange system (ESIE) provides a framework for modifying traditional ion exchange processes. The system has 5 to 10 times less specifc capacity than current ion exchange systems, but uses solutions 10-100 times lower in ionic strength for regeneration. Further studies on the effect of ionic strength on adsorption and current usage are necessary to compare the cost of the ESIE process to traditional ion exchange. / text

This research is the first systematic investigation of solid-state orientation as a novel method for controlling drug release from biodegradable polymers. The effect of various degrees of polymer orientation was studied in oriented Poly (L-lactic acid) (PLA) films containing curcumin and theophylline as model drugs. Additionally, direction specific drug release was studied from oriented PLA rods containing paracetamol. The films oriented to 2X uniaxial constant width (UCW) or 2X2Y biaxial draw ratio showed retardation of drug release, when their nematic structure was stabilised by the presence of crystalline theophylline. Contrarily, the same films when contained solid solution of curcumin, shrunk in the release medium and exhibited a release profile similar to the un-oriented films. All films oriented to the UCW draw ratio ≥ 3X contained α crystalline form of PLA and showed acceleration of drug release proportionate to the draw ratio. According to the proposed mechanism augmented formation of water filled channels in these films was responsible for faster drug release. Similarly, the paracetamol loaded PLA rods die-drawn to uniaxial draw ratios ≥ 3X exhibited enhancement of drug release. Importantly, the amount of drug released along the oriented chain axis was significantly larger than that in the perpendicular direction. Drug release from the die-drawn rods was accelerated by a greater degree than that observed from the oriented films. This can be correlated to the differences in their size, geometry and the crystalline form of PLA. In conclusion, the current study provided substantial evidence that solid-state orientation can offer a control over drug release from PLA.

L'immobilisation de biomacromolécules à la surface de polymères peu réactifs est une voie de synthèse de nanomatériaux qui fait actuellement l'objet de nombreuses recherches pour le développement d'applications biologiques et médicales. Nous avons synthétisé de nouveaux nanomatériaux à base de polypropylène (PP) greffé par des dendrimères de lysine (DGL). Les DGL sont parfaitement solubles dans l'eau, biocompatibles, polycationiques à pH neutre et leur structure dendritique particulière font d'eux des macromolécules de plus en plus étudiées en interactions avec les milieux biologiques. Différents traitements par plasma ont permis de fonctionnaliser la surface du PP et plusieurs stratégies ont été adoptées pour greffer les DGL sous forme de monocouche, multicouche ou à partir de brosses de polymères : le greffage direct, les polymérisations non contrôlée et contrôlée de type RAFT associées au plasma d'iode et à la chimie click de surface. L'aptitude des matériaux PP fonctionnalisés par le DGL à interagir avec les milieux biologiques a été étudiée, en particulier l'immobilisation de l'ATP et le comportement vis-à-vis des bactéries et des virus. Les propriétés de ces nanomatériaux sont liées à la réactivité des groupements amine des DGL ainsi qu'à la structure régulière et sphérique des dendrimères. Les résultats obtenus ouvrent de nombreuses applications potentielles pour le traitement des eaux, le diagnostic et la prévention du développement des micro-organismes. / Great attention has been focused these last years on tailoring polymer surfaces by immobilizationof suitable molecules for biological and medical applications such as tissue engineering, drug delivery systems, antibacterial supports, and biosensors. In that context, we report the preparation of an original hybrid material based on polypropylene and poly-L-Lysine dendrigrafts (DGL) which are perfectly water soluble, and biocompatible. First, activation of the polypropylene surface (PP) was achieved using plasma treatment. Then, several strategies have been developed to graft DGL onto the PP surface such as (i) direct grafting of DGL after surface activation, (ii) the use of conventional radical polymerization or (iii) RAFT polymerization of monomers from the PP surface. The last methodology favored the increase of the DGL grafts density onto the surface. The ability of PP surface functionalized with DGL to interact with biological media was studied and the modified surfaces open the way to many potential applications in water treatment, diagnosis and prevention of the development of microorganisms.

The sensitivity of biological studies performed between 190 and 650 nm is greatly reduced due to the autofluorescence of biomolecules and impurities in this region. Therefore, the enhanced signal-to-noise ratios encountered at longer wavelengths makes biological analysis within the near infrared (NIR) region from 650 nm to 1100 nm far more advantageous. This dissertation describes the noncovalent binding interactions of near-infrared (NIR) carbocyanine dyes with human serum albumin (HSA) and poly-L-lysine (PLL) using UV-Vis/NIR absorption spectroscopy, emission spectroscopy, circular dichroism (CD), and fluorescence detected circular dichroism (FDCD). The optical spectroscopy methods used in this work are described in detail in Chapter 1. The various applications of NIR dyes in protein analysis are introduced in Chapter 2. In general, the sensitivity of cyanines to the polarity of their local environment makes them quite suitable for protein labeling schemes. In aqueous media, cyanines have a high propensity for self-association. Yet in the hydrophobic binding sites of globular proteins, these aggregates often dissipate. Absorption and emission spectroscopy can be utilized to observe the differential spectral properties of monomer, intra-molecular and intermolecular aggregates. In Chapter 3, the photophysical properties of bis(cyanine) NIR dyes containing di-, tri-, and tetraethylene glycol linkers were each examined in the presence of HSA are discussed. Variations in chain length as well as probe flexibility were demonstrated through distinct differences in absorption and emission spectra. The observed changes in the spectral properties of the NIR dyes in the presence and absence of HSA were correlated to the physical parameters of the probes' local environment (i.e., protein binding sites and self-association). All three bis-cyanines examined exhibited enhanced fluorescence in the presence of HSA. The bis-cyanine dye containing the tri(ethylene glycol) spacer allowed for a complete overlap of the benzene rings, to form π-π interactions which were observed as intra-molecular H-aggregate bands. The dye exhibited no fluorescence in buffer, owing to the H-aggregation observed in the absorption data. In the presence of HSA, the intra-molecular dimers were disrupted and fluorescence was then detected. The "cut-on" fluorescence displayed by the dye in the presence of HSA made it ideal for noncovalent labeling applications. The utility of several NIR dyes for use as secondary structural probes was investigated in Chapter 4. NIR dyes were screened thoroughly using UV-Vis/NIR absorption spectroscopy dyes with spectral properties which were sensitive to protein secondary structure models of such as PLL in basic solution. Two NIR dyes were found to be quite sensitive to the structural features of uncharged α- and β-PLL. The chiral discrimination of these probes for basic protein secondary structures was also evaluated through CD measurements within the NIR probes' absorption bands.