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Investigating the early events in proteasome assemblyRamamurthy, Aishwarya January 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to
characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.
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The Impact of a Digestive Inflammatory Environment and Genipin Crosslinking on the Immunomodulatory Capacity of an Injectable Musculoskeletal Tissue ScaffoldShortridge, Colin D. January 2019 (has links)
No description available.
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Burst Pressure Properties and Ex Vivo Analysis of Alginate-Based Hydrogels for Tissue Sealant ApplicationsCharron, Patrick Nelson 01 January 2015 (has links)
Lung diseases, cancers, and trauma can result in injury to the connective tissue lining the lung, i.e., the pleura. Pleural injuries lead to pneumothoraxes or pleural effusions, i.e., air or fluid leaking out of the lung respectively, and potential lung collapse - an immediately life threatening condition. While several bioengineered soft tissue sealants exist on the market, there is only one sealant FDA-approved for use in pulmonary surgery. In addition, very limited techniques are presented in the literature for characterizing the burst properties of hydrogel tissue sealants. For my thesis, I proposed to develop a protocol for characterizing the burst properties of hydrogel sealants using a novel burst pressure test chamber. I further proposed a novel combination of oxidation and methacrylation reactions of alginate for tissue sealant applications, with a particular focus on developing a pulmonary sealant. The proposed research objectives are: 1) To develop protocol for testing hydrogel sealants for soft tissue applications; 2) To verify alginate as a potential for tissue sealant applications; and 3) To optimize an alginate hydrogel sealant and perform ex vivo analysis for a pleural sealant application. Alginate materials with varying degrees of oxidation and methacrylation were synthesized and characterized. Oscillatory rheometry was used to characterize material properties such as viscosity, hydrogel gelation kinetics, and complex moduli. Burst pressure measurements properties and failure mechanisms, i.e. delamination or material failure, were collected for a liquid and dry-state application. Preliminary ex vivo mouse lung model testing demonstrated that methacrylated alginate hydrogels are able to withstand physiological pressures associated with breathing, and failure occurs within the hydrogel for adhesive alginate-based tissue sealants.
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Membranes PBI pour pile à combustible haute température / PBI membranes for high temperature fuel cellKreisz, Aurélien 11 April 2016 (has links)
Cette thèse débute par une courte introduction traitant des principes et de l'état de l'art des PEMFC dans le but de situer le contexte des travaux. Le but des travaux présentés dans ce manuscrit est de développer une nouvelle méthode de préparation de membrane pour les piles à combustible haute température (> 120 °C). Le polybenzimidazole dopé à l'acide phosphorique est devenu la référence des PEM haute température. Un degré de dopage élevé est essentiel pour minimiser les pertes ohmiques dans la cellule. Malheureusement un degré de dopage élevé entraine aussi une plastification de la membrane détériorant aussi sa résistance mécanique. Il est donc essentiel d'atteindre un compromis entre conductivité protonique élevée et résistance mécanique en contrôlant le degré de dopage. Dans ce travail, nous avons développé une nouvelle méthode de préparation de membrane, basée sur la gélation thermoréversible d'une solution de PBI dans l'acide phosphorique ou polyphosphorique, dans le but d'obtenir des degrés de dopage élevés. Une modification chimique a été réalisée dans l'état dopé afin d'induire une réticulation du polymère. De plus, les résistances mécaniques ont été améliorées en introduisant dans la membrane un mat de PBI réticulé obtenu par filage électrostatique. La faisabilité des approches suivies dans ces travaux a été démontrée par des tests en cellule de pile à combustible jusqu'à une température de 180 °C. Les AMEs élaborés à partir de ces membranes ont montré une stabilité satisfaisante durant 900 - 1000 heures de fonctionnement sous conditions statiques (opération continue à 0.2 A.cm-2) et sous conditions dynamiques (cyclage en tension et courant) avec une décroissance de la tension de la cellule au cours du temps de 0.7 - 0.8 µV.h-1 à 0.2 A.cm-2. Les caractéristiques I-V de ces AMEs ont été comparées à des assemblages de référence PBI/H3PO4 commerciaux et ont présenté des performances améliorées par rapport aux assemblages commerciaux. / The thesis begins with a short overview of the principles and the current state at the art of the PEMFC in order to give a background on the specific subject of high temperature PEM fuel cell. The aim of the work presented in this thesis is to develop a new method of preparation of membrane for high temperature fuel cell (T > 120 °C). Phosphoric acid doped PBI has become the reference for high temperature PEM. A high phosphoric acid content is essential to minimize the ohmic voltage loss in the fuel cell for high current density. Unfortunately high phosphoric acid content exerts a strong plasticizing effect resulting in poor mechanical properties of the membrane. Consequently the doping level of the membrane should be a compromise between the highest proton conductivity and mechanical strength. In this work we have presented a new method of preparation of membrane based on the thermoreversible gelation of a PBI solution in phosphoric or polyphosphoric acid in order to obtain high acid doping. The chemical modification of the membrane has been performed in the doped state in order to induce a chemical crosslinking. The mechanical strength of the membrane has been further improved by the introduction of PBI electrospun mat as reinforcement. The feasibility of the approaches followed in this work was demonstrated in fuel cell tests at temperature up to 180 °C. The MEA based on those membranes have shown a stability up to 900 - 1000 hours either under static (continuous operation at 0.2 A.cm-2) or dynamic (voltage and current cycling) operation with a small voltage decay of 0.7 - 0.8 µV.h-1 at 0.2 A.cm-2. The I-V characteristics of these MEA have been compared with reference commercial PBI/H3PO4 MEAs and shown improved performances.
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Development of in vitro iCLIP techniques to study spliceosome remodelling by RNA helicasesStrittmatter, Lisa Maria January 2019 (has links)
Pre-mRNA (precursor messenger RNA) splicing is a fundamental process in eukaryotic gene expression. In order to catalyse the excision of the intervening intronic sequence between two exons, the spliceosome is assembled stepwise on the pre-mRNA substrate. This ribonucleoprotein machine is extremely dynamic: both its activation and the progression through the catalytic stages require extensive compositional and structural remodelling. The first part of this thesis aims at understanding how the spliceosome is activated after assembly. When this work was started, the GTPase Snu114 was thought to activate the helicase Brr2 to unwind the U4/U6 snRNA duplex, which ultimately leads to the formation of the spliceosome active site. To explore the role of Snu114, a complex built from Snu114 and a part of Prp8 was expressed and analysed in its natural context, bound to U5 snRNA. However, before I was able to obtain highly diffracting crystals, the structure of Snu114 was determined in the context of a larger spliceosomal complex by electron cryo-microscopy by competitors. Regardless, the role of Snu114 in spliceosome activation remains elusive. In a short section of this thesis, genetic and biochemical analysis suggest Snu114 to be a pseudo-GTPase, precluding a role for Snu114-catalyzed GTP hydrolysis in activation. The second and larger part of the thesis describes the development of a novel, biochemical method to analyse spliceosome remodelling events that are caused by the eight spliceosomal helicases. Purified spliceosomes assembled on a defined RNA substrate are analysed by UV crosslinking and next-generation sequencing, which allows for the determination of the RNA helicase binding profile at nucleotide resolution. In vitro spliceosome iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) was initially developed targeting the helicase Prp16 bound to spliceosomal complex C. The obtained binding profile shows that Prp16 contacts the intron, about 15 nucleotides downstream of the branch in the intron-lariat intermediate. Our finding supports the model of Prp16 acting at a distance to remodel the RNA and protein interactions in the catalytic core and thereby it promotes the transition towards a conformation of the spliceosome competent for second step catalysis. Control experiments, which locate SmB protein binding to known Sm sites in the spliceosomal snRNAs, validated the method. Preliminary results show that in vitro spliceosome iCLIP can be adapted to analyse additional spliceosomal helicases such as Prp22. Finally, I performed initial experiments that give promising directions towards time-resolved translocation profiles of helicases Brr2 and Prp16.
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Biosuperabsorbent from proteinsBarghi, Hamidreza, Majdejabbari, Sara January 2009 (has links)
The present work is synthesizing novel protein-based superabsorbent hydrogels from albumin protein (AP) and isolated zygomycetes protein (IZP) via modification with an acylating reagent and a bifunctional crosslinker, also investigation on their swelling behaviors under some conditions. The hydrophilic acylating reagent was introduced into albumin and zygomycetes proteins and hydrophylicity of albumin and isolated zygomycetes protein were increased by ethylenediaminetetraacetic dianhydride (EDTAD). Provided protein hydrogels through this method include modification of lysyl residues in the unfolded proteins via adding of one or more hydrophilic carboxyl groups. The reaction was developed with a dialdehyde crosslinking reagent (glutaraldehyde) in order to stabilize the modified protein configuration. The maximum capacity of EDTAD-AP hydrogel swelling was observed 296 g water per g of dry gel, and for EDTAD-IZP hydrogel was 87 g water per g of dry gel.In this study, effect of selected physical and chemical parameters such as protein structure, extent of modification, protein concentration, various pH, ionic strength, gel particle size, temperature as important factors affecting the water uptake behavior of superabsorbent hydrogel were investigated. In addition, the effect of some organic solvents particularly absolute ethanol for increasing the swelling properties was studied. / Uppsatsnivå: D
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Desenvolvimento de filmes à base de proteínas extraídas da torta de mamona (Ricinus communis L.) reticuladas com glutaraldeído e glioxal / Development of films based on proteins extracted from the castor bean cake (Ricinus communis L.) crosslinked with glutaraldehyde and glyoxalMakishi, Gisele Lourenço da Aparecida 13 April 2012 (has links)
O processo de extração do óleo de mamona gera, como subproduto, uma torta rica em proteínas, que tem sido utilizada como adubo e contra fitonematóide, devido à sua toxicidade. Entretanto, as proteínas da torta de mamona são ricas em aminoácidos que permitem modificação química com aldeídos bifuncionais, como o glutaraldeído e o glioxal, o que pode ser interessante para a produção de filmes biodegradáveis com propriedades físicas adequadas ao emprego na agricultura. Assim, o objetivo geral deste trabalho foi a produção e caracterização de filmes à base de proteínas extraídas da torta de mamona e reticuladas com glioxal ou glutaraldeído e plastificados com glicerol. Especificamente, estudaram-se o efeito da concentração de proteína e do tipo de reticulante, e o efeito da concentração do reticulante sobre algumas propriedades dos filmes. Os filmes foram produzidos com uma técnica conhecida como tipo \"casting\", que consiste na desidratação de uma solução filmogênica (SF). Os filmes foram submetidos a testes para determinação da umidade, espessura, propriedades mecânicas (testes de tração e perfuração), permeabilidade ao vapor de água, solubilidade em água, cor, opacidade, brilho, além da microestrutura, por microscopia eletrônica de varredura. A concentração da proteína e o tipo de reticulante não influenciaram a umidade, espessura, microestrutura e propriedades óticas. Mas, de maneira geral, os filmes produzidos com o glioxal apresentaram melhores propriedades mecânicas e menor solubilidade em água que aqueles produzidos com glutaraldeído, sendo que a concentração de 6g de proteínas/100g de SF foi a formulação que apresentou os melhores resultados para os testes de tração e de solubilidade em água. Este resultado é muito importante para o emprego dos filmes na agricultura. Por outro lado, a concentração do glioxal não teve efeito sobre a umidade, espessura, propriedades óticas, microestrutura e nem sobre a permeabilidade ao vapor de água, influenciando apenas a solubilidade e as propriedades mecânicas. O teor de glioxal que proporcionou melhores propriedades mecânicas e baixa solubilidade em água foi de 5g/100g de proteínas. Filmes produzidos nessas melhores condições foram submetidos a análises complementares envolvendo testes de hidrofobicidade, propriedades térmicas, espectroscopia de infravermelho com transformada de Fourier, isoterma de sorção e biodegradabilidade. Finalmente, pode-se concluir que as proteínas da torta de mamona são capazes de formar uma matriz bem estruturada e que suas propriedades podem ser melhoradas com a adição de glioxal. / The extraction process of castor oil produces, as a co-product, a cake rich in proteins which has been used as a fertilizer and against plant parasitic nematode, because of its toxicity. However, the proteins of castor bean cake are rich in amino acids which allow chemical modification with bifunctional aldehydes such as glutaraldehyde and glioxal. This may be interesting for the production of biodegradable films with adequate physical properties to the use in agriculture. The objective of this work was the production and characterization of films based on proteins extracted from the castor bean cake and crosslinked with glyoxal or glutaraldehyde and plasticized with glycerol. Specifically, we studied the effect of protein concentration and type of crosslinker and the effect of the crosslinker concentration on some properties of the films. The films were produced with a technique known as \"casting\", which consists of drying a film solution (FS). The films were tested for determination of moisture content, thickness, mechanical properties (tensile and puncture tests), water vapor permeability, water solubility, color, opacity, gloss, and the microstructure by scanning electron microscopy. The protein concentration and type of crosslinking did not affect the moisture content, thickness, microstructure and optical properties. But, in general, films made with glyoxal showed improved mechanical properties and lower water solubility than those produced using glutaraldehyde. And, the concentration of 6g proteins/100 g of FS was the formulation that presented the best results for the tensile tests and water solubility. This result is very important for the use of films in agriculture. Moreover, the concentration of glyoxal had no effect on the moisture content, thickness, optical properties, or on the microstructure and permeability to water vapor, influencing only the solubility and mechanical properties. The concentration of glyoxal that provided the best mechanical properties and low water solubility of proteins was 5g/100g of gelatin. Films produced in the best conditions were subjected to further analyzes involving tests of hydrophobicity, thermal properties, spectroscopy, Fourier transform infrared spectroscopy, water vapor sorption isotherm and biodegradability. Finally, it can be concluded that the protein of castor bean cake are capable of forming a matrix structured and that their properties can be improved with the addition of glyoxal.
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Desenvolvimento de micropartículas de xilana utilizando reticulante não tóxico visando a liberação cólon-específicaCosta, Silvana Cartaxo da 23 May 2014 (has links)
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Previous issue date: 2014-05-23 / The development of a colon-specific delivery system using polymeric microparticles
has received great attention in the pharmaceutical field. An interesting group of
polymers with potential properties in this area are the hemicellulose. Xylan is a
hemicellulose that has the ability to pass through the digestive tract unchanged and its
complex structure requires enzymes that are produced specifically by the human colonic
microflora, which makes it an interesting raw material in the production of target drug
delivery systems. The microparticulate systems can be developed by various techniques.
The interfacial crosslinking polymerization is one of the major techniques to produce
polysaccharide based microparticles. However, the use of highly toxic crosslinkers often
makes the use of this technique limited. The sodium trimetaphosphate (TSTP), a low
toxic crosslinking agent, has no adverse effects reported on human beings. The aim of
this study was to develop xylan microparticles using sodium trimetaphosphate. The
microparticles were characterized by optical microscopy, SEM, XRD and FT -IR. The
influence of different parameters on the diameter of the microparticles was analyzed
during their development. Toxicity studies against Artemia saline Leach were made to
compare the microparticles produced with terephthaloyl chloride and sodium
trimetaphosphate. Xylan microparticles showed to be spherical shape, well
individualized and with a smooth surface. All different parameters influenced the in size
of the microparticles. The FT-IR spectrum of microparticles was similar to xylan, but
with the presence of the peak at 1258 cm
-1
, which is typical of phosphate ester bonds,
that can be attributed to the bond between TSTP and xylan during the crosslinking
process. The xylan microparticles produced in this work showed no toxicity at the
concentration studied. It be concluded that TSTP was able to produce xylan
microparticles with well defined physicochemical characteristics and low toxicity. / O desenvolvimento de um sistema de liberação cólon-específica utilizando
micropartículas poliméricas têm recebido grande atenção no campo farmacêutico. Um
grupo interessante de polímeros com potenciais propriedades nessa área são as
hemiceluloses. A xilana é uma hemicelulose que tem a capacidade de passar através do
trato digestivo inalterada e sua complexa estrutura requer enzimas que são produzidas
especificamente pela microflora colônica humana, o que a torna uma interessante
matéria-prima na área produção de sistemas de liberação de fármacos. Os sistemas
microparticulados podem ser desenvolvidos por várias técnicas. A reticulação
polimérica interfacial é uma das principais técnicas para produção de micropartículas à
base de polissacarídeos. Porém o uso de reticulantes de alta toxicidade muitas vezes
torna o uso desta técnica limitada. O trimetafosfato de sódio (TSTP) é um reticulante de
baixa toxicidade, sem efeitos adversos relatados em seres humanos. Esse trabalho teve
como objetivo desenvolver micropartículas de xilana utilizando TSTP. As
micropartículas foram caracterizadas por microscopia óptica, MEV, DRX e FT-IR.
Estudos de toxicidade frente à artemia salina Leach foram feitos para comparar as
micropartículas produzidas com cloridrato de tereftaloíla e trimetafosfato de sódio. As
micropartículas de xilana apresentaram forma esférica, bem individualizada e com
superfície bem definida. Todos os diferentes parâmetros influenciaram no tamanho das
micropartículas. O espectro de FT-IR das micropartículas foi semelhante ao da xilana,
porém com a presença de um pico em 1258 cm
-1
, que é típico de ligações éster-fosfato,
que pode ser atribuído a ligação entre TSTP e a xilana durante o processo de
reticulação. As micropartículas de xilana produzidas neste trabalho não apresentaram
toxicidade na concentração estudada. Podemos concluir que o TSTP foi capaz de
produzir micropartículas de xilana com cracterísticas fisico-químicas bem definidas e de
baixa toxicidade.
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Synthetic peptides derived from decorin as building blocks for biomaterials based on supramolecular interactionsFederico, Stefania January 2011 (has links)
In this work, the development of a new molecular building block, based on synthetic peptides derived from decorin, is presented. These peptides represent a promising basis for the design of polymer-based biomaterials that mimic the ECM on a molecular level and exploit specific biological recognition for technical applications.
Multiple sequence alignments of the internal repeats of decorin that formed the inner and outer surface of the arch-shaped protein were used to develop consensus sequences. These sequences contained conserved sequence motifs that are likely to be related to structural and functional features of the protein. Peptides representative for the consensus sequences were synthesized by microwave-assisted solid phase peptide synthesis and purified by RP-HPLC, with purities higher than 95 mol%. After confirming the desired masses by MALDI-TOF-MS, the primary structure of each peptide was investigated by 1H and 2D NMR, from which a full assignment of the chemical shifts was obtained. The characterization of the peptides conformation in solution was performed by CD spectroscopy, which demonstrated that using TFE, the peptides from the outer surface of decorin show a high propensity to fold into helical structures as observed in the original protein. To the contrary, the peptides from the inner surface did not show propensity to form stable secondary structure. The investigation of the binding capability of the peptides to Collagen I was performed by surface plasmon resonance analyses, from which all but one of the peptides representing the inner surface of decorin showed binding affinity to collagen with values of dissociation constant between 2•10-7 M and 2.3•10-4 M. On the other hand, the peptides representative for the outer surface of decorin did not show any significant interaction to collagen.
This information was then used to develop experimental demonstration for the binding capabilities of the peptides from the inner surface of decorin to collagen even when used in more complicated situations close to possible appications. With this purpose, the peptide (LRELHLNNN) which showed the highest binding affinity to collagen (2•10-7 M) was functionalized with an N-terminal triple bond in order to obtain a peptide dimer via copper(I)-catalyzed cycloaddition reaction with 4,4'-diazidostilbene-2,2'-disulfonic acid. Rheological measurements showed that the presence of the peptide dimer was able to enhance the elastic modulus (G') of a collagen gel from ~ 600 Pa (collagen alone) to ~ 2700 Pa (collagen and peptide dimer). Moreover, it was shown that the mechanical properties of a collagen gel can be tailored by using different molar ratios of peptide dimer respect to collagen.
The same peptide, functionalized with the triple bond, was used to obtain a peptide-dye conjugate by coupling it with N-(5'-azidopentanoyl)-5-aminofluorescein. An aqueous solution (5 vol% methanol) of the peptide dye conjugate was injected into a collagen and a hyaluronic acid (HA) gel and images of fluorescence detection showed that the diffusion of the peptide was slower in the collagen gel compared to the HA gel.
The third experimental demonstration was gained using the peptide (LSELRLHNN) which showed the lower binding affinity (2.3•10-4 M) to collagen. This peptide was grafted to hyaluronic acid via EDC-chemistry, with a degree of functionalization of 7 ± 2 mol% as calculated by 1H-NMR. The grafting was further confirmed by FTIR and TGA measurements, which showed that the onset of decomposition for the HA-g-peptide decreased by 10 °C compared to the native HA. Rheological measurements showed that the elastic modulus of a system based on collagen and HA-g-peptide increased by almost two order of magnitude (G' = 200 Pa) compared to a system based on collagen and HA (G' = 0.9 Pa).
Overall, this study showed that the synthetic peptides, which were identified from decorin, can be applied as potential building blocks for biomimetic materials that function via biological recognition. / In dieser Arbeit wird das Design, die Synthese und Analyse neuer molekularer Bausteine für Biomaterialien basierend auf synthetischen, von Decorin abgeleiteten Peptiden beschrieben. Diese Peptide sind deshalb als Baustein für polymer-basierte Biomaterialien von besonderem Interesse, da sie die extrazelluläre Matrix (ECM) auf molekularer Ebene nachempfinden und spezifische, biologische wichtige Interaktionen für technische Anwendungen nutzbar machen.
Das Alignment multipler Sequenzen der internen Repeats von Decorin, die jeweils die innere bzw. äußere Seite des sichelförmigen Decorins bilden, wurde genutzt, um Konsensus-Sequenzen zu definieren. Diese Sequenzen beinhalten stark konservierte Sequenzmotive, die wahrscheinlich wichtig für Struktur und Funktion des Proteins sind. Ausgewählte Peptide, die repräsentativ für die Konsensus-Sequenzen sind, wurden dann mittels Mikrowellen unterstützter Festphasensynthese synthetisiert und mit RP-HPLC aufgereinigt, so dass Peptide mit Reinheiten ≥ 95 mol% erhalten wurden. Die Peptide wurden per MALDI-TOF-MS sowie 1D und 2D NMR Spektroskopie charakterisiert, wobei die Zuordnung der chemischen Verschiebungen zu einzelnen Protonen und Kohlenstoffen aus den 2D NMR Experimenten erfolgte. In Lösung wurden die Peptide zudem mit CD Spektroskopie untersucht, wobei gezeigt werden konnte, dass nur Peptide, die von der äußeren Seite des Decorins abgeleitet wurden, sich durch Zugabe von 2,2,2-Trifluorethanol zu α-Helices falten. Diese Faltung ist auch in der Röntgenstruktur bei den korrespondierenden Abschnitten zu finden. Im Gegensatz dazu zeigten Peptide, die von der inneren Seite des Decorins abgeleitet wurden, keine stabilen Sekundärstrukturen in Lösung (β-Faltblattstruktur in der Röntgenstruktur). Bindungsstudien der Peptide zu Kollagen I wurden mit Oberflächenplasmonenresonanz durchgeführt, wobei gezeigt werden konnte, dass alle bis auf ein Peptid, die von der innneren Seite abgeleitet wurden, an Kollagen mit Dissoziationskonstanten von 2•10-7 M bis 2.3•10-4 M binden, während Peptide, die für die äußere Seite von Decorin repräsentativ sind, keine Bindung an Kollagen I zeigten.
Diese Information wurde genutzt, um experimentelle Demonstrationsobjekte dieser Interaktion in komplexeren, einer späteren Anwendung näheren Situation, zu entwickeln. Dazu wurde das Peptide LRELHLNNN, welches die stärkste Bindung zu Kollagen I zeigte (KD = 2•10-7 M), N-terminal mit einer Alkinbindung funktionalisiert, so dass durch Kupfer (I) katalysierte Reaktion mit 4,4'-Diazidostilben-2,2'-disulfonsäure ein Peptid-Dimer erhalten werden konnte. Rheologische Untersuchungen zeigten, dass durch Zugabe des Peptid-Dimers der Elastizitätsmodul G' von Kollagen-Gelen von ~ 600 Pa (nur Kollagen) auf ~ 2700 Pa (Kollagen und Peptide-Dimer) gesteigert werden konnte. Darüber hinaus konnte gezeigt werden, dass die Veränderung der mechanischen Eigenschaften der Gele durch Veränderung des Kollagen:Peptid-Dimer Verhältnisses angepasst werden konnten.
Das gleiche, mit einer Alkin-Bindung funktionaliserte Peptid wurde dann zur Darstellung eines Peptid-Fluorescein Konjugats genutzt, indem es mit N-(5'-azidopentanoyl)-5-aminofluorescein umgesetzt wurde. Eine wässrige Lösung des Peptid-Farbstoff-Konjugats wurde dann in Kollagen- bzw. Hyaluronsäuregele injiziert. Die Diffusion des Peptid-Farbstoff-Konjugats war in Kollagengelen im Vergleich zu Hyaluronsäuregelen deutlich verlangsamt.
Das dritte Demonstrationsobjekt wurde erhalten, indem das Peptid LSELRLHNN, welches die geringste Bindung an Kollagen zeigte (KD = 2.3•10-4 M), auf Hyaluronsäure (HA) gegrafted wurde. Die Reaktion wurde durch Carbodiimid-mediierte Kupplung erreicht, und ein Funktionalisierungsgrad von 7 ± 2 mol% wurde durch Integration der 1H-NMR Spektren bestimmt. Das erfolgreiche Grafting wurde durch FTIR- und TGA-Untersuchungen bestätigt. In letzteren wurde gezeigt, dass der thermische Abbau durch das Grafting bei etwas niedrigeren Temperaturen beginnt als der Abbau reiner Hyaluronsäure (ΔT = 10 °C). Rheologische Untersuchungen zeigten, dass ein System aus Kollagen und HA-g-Peptid ein um zwei Größenordnungen höheren Elastizitätsmodul G' hat (G' = 200 Pa) als Systeme, die aus einer physikalischen Mischung von Kollagen und HA bestehen (G' = 0.9 Pa).
Zusammenfassend konnte gezeigt werden, dass die Peptide, die von Decorin abgeleitet wurden, als Kollagen-bindende Bausteine für biomimetische Materialien genutzt werden können.
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Design and development of an elastin mimetic stent with therapeutic delivery potentialMartinez, Adam W. 11 November 2011 (has links)
Stenting remains a common treatment option for atherosclerotic arteries. The main drawback of early stent platforms was restenosis, which has been combated by drug eluting stents; however, these stents have suffered from a higher incidence of late stage thrombosis. To address current stenting limitations, the major research focuses have been the development of the next generation of drug eluting stents and first generation bioabsorbable stents. The main objective of this dissertation was the design and development of a new class of bioabsorbable stent composed of elastin mimetic protein polymers. The first phase explored different stent design schemes and fabrication strategies. Successfully fabricated stents were then mechanically tested to ensure they possessed sufficient mechanical strength. Additionally, described herein is the potential to modulate the properties of the elastin mimetics through different crosslinking strategies. We have demonstrated that chemical crosslinking allows for the tailoring of the physical, mechanical, drug delivery, and endothelialization properties of these materials. The potential for drug delivery from this elastin mimetic stent was benchmarked as was the potential to endothelialize these stents. Furthermore, we developed the necessary delivery systems to allow for deployment in the rat aorta model.
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