Degradation and stabilisation of diisocyanate cured polybutadiene

Hydroxyl terminated polybutadiene (HTPB) has been used as a rocket propellant binder which is required to be stored for at least twenty years. It is found that the excellent stress-strain characteristics of this propellant can be totally lost, during this long storage, due to the deterioration of the polybutadiene chains. As a result, the propellant can not stand the service loads, which may lead to a catastrophe. The study of the HTPB binder degradation, below 80°C, has been carried out by investigating the environmental factors and the changes which occur along the macromolecular chains. Results have shown that oxygen is the main factor which causes the crosslinking and chain scission reactions. The former is the predominant reaction and proceeds rapidly under oxygen sufficient environment. The unsaturation of polymer chain, which provides the desired physical properties to the binder, was lost with the increase in crosslink density. At the same time hydroperoxides were found to form and decompose along the polymer chains. Therefore, the deterioration of the binder results from the oxidation of polymer chains. Since the oxidation reaction occurred at higher rate than oxygen diffusion rate and oxygen diffusion rate is inversely proportional to the crosslink density, the binder, below the surface layer in a thick section container, could be naturally protected under an oxygen deficient condition for a long time. Investigation of the effectiveness of antioxidants in HTPB binder has shown that the efficiency of an antioxidant depends on its ability to scavenge radicals. Generally, aromatic amines are the most effective binder antioxidants. But when a peroxide decomposer is combined with an aromatic amine at the appropriate ratio, a synergistic effect is obtained, which gives the lowest binder gel increase rate.

Fatigue in rubber vulcanisates

A study has been made of the effect of single extensions and continuous fatigue on the structures of various natural rubber networks. The change in network structure of a conventional vulcanisate on a single extension manifests itself as permanent set. The change in network structure has been assessed by the use of the chemical probes, propan-2-thiol/piperidine, hexane-thiol/piperidine and triphenyl phosphine, which determine the polysulphide and disulphide crosslink densities and main chain modification respectively. The permanent set induced on a single extension of a conventional sulphur vulcanisate has been shown to result from the destruction and reformation of polysulphide crosslinks. The magnitude of the effect was dependent upon the degree of extension and showed a maximum at extensions corresponding to the onset of stress-induced crystallisation. The incorporation of a reinforcing filler, HAF-carbon black, magnified the effect. Vulcanisates that possessed only mono and disulphide crosslinks did not show any significant permanent set. The continuous changes in network structure during fatigue have also been determined, and the effects of carbon black and antioxidants on these changes and the fatigue life have been assessed. During fatigue the overall crosslink density increased slightly, which resulted from the destruction of polysulphide crosslinks. and their replacement by principally disulphide crosslinks. Antioxidants reduced the rate of destruction of polysulphide crosslinks and increased the fatigue life of the rubber network. The fatigue life of the network also depended upon the concentration of free chain ends. These chain ends were incorporated into the network by masticating rubber under nitrogen in the presence of bis (diisopropyl)thiophosphoryl disulphide, which improved the fatigue resistance by up to 9%.

A sulphonate based hydrogel for nucleus pulposus repair

Introduction: Lower back pain treatment and compensation costs >$80 billion overall in the US. 75% of back pain is due to disc degeneration in the lumbar region of the spine. Current treatment comprises of painkillers and bed rest or as a more radical solution – interbody cage fusion. In the early stages of disc degeneration the patient would benefit from addition of an injectable gel which polymerises in situ to support the degenerated nucleus pulposus. This involves a material which is an analogue of the natural tissue capable of restoring the biomechanical properties of the natural disc. The nucleus pulposus of the intervertebral disc is an example of a natural proteoglycan consisting of a protein core with negatively charged keratin and chondroitin sulphate attached. As a result of the high fixed charge density of the proteoglycan, the matrix exerts an osmotic swelling pressure drawing sufficient water into support the spinal system. Materials and Methods: NaAMPs (sodium 2- acrylamido 2-methyl propane sulphonic acid) and KSPA (potassium 3- sulphopropyl acrylate) were selected as monomers, the sulphonate group being used to mimic the natural sulphate group. These are used in dermal applications involving chronic wounds and have acceptably low cytotoxicity. Other hydrophilic carboxyl, amide and hydroxyl monomers such as 2-hydroxyethyl acrylamide, ß-carboxyethyl acrylate, acryloyl morpholine, and polyethylene glycol (meth)acrylate were used as diluents together with polyethyleneglycol di(meth)acrylate and hydrophilic multifunctional macromers as cross-linker. Redox was the chosen method of polymerisation and a range of initiators were investigated. Components were packaged in two solutions each containing a redox pair. A dual syringe method of injection into the cavity was used, the required time for polymerisation is circa 3-7 minutes. The final materials were tested using a Bohlin CVO Rheometer cycling from 0.5-25Hz at 37oC to measure the modulus. An in-house compression testing method was developed, using dialysis tubing to mimic the cavity, the gels were swelled in solutions of various osmolarity and compressed to ~ 20%. The pre-gel has also been injected into sheep spinal segments for mechanical compression testing to demonstrate the restoration of properties upon use of the gel. Results and Discussion: Two systems resulted using similar monomer compositions but different initiation and crosslinking agents. NaAMPs and KSPA were used together at a ratio of ~1:1 in both systems with 0.25-2% crosslinking agent, diacrylate or methacrylate. The two initiation systems were ascorbic acid/oxone, and N,N,N,N - tetramethylethylenediamine (TEMED)/ potassium persulphate. These systems produced gelation within 3-7 and 3-5 minutes respectively. Storage of the two component systems was shown to be stable for approximately one month after mixing, in the dark, refrigerated at 1-4oC. The gelation was carried out at 37oC. Literature values for the natural disc give elastic constants ranging from 3-8kPa. The properties of the polymer can be tailored by altering crosslink density and monomer composition and are able to match those of the natural disc. It is possible to incorporate a radio-opaque (histodenz) to enable x-ray luminescence during and after injection. At an inclusion level of 5% the gel is clearly visible and polymerisation and mechanical properties are not altered. Conclusion: A two-pac injection system which will polymerise in situ, that can incorporate a radio-opaque, has been developed. This will reinforce the damaged nucleus pulposus in degenerative disc disease restoring adequate hydration and thus biomechanical properties. Tests on sheep spine segments are currently being carried out to demonstrate that a disc containing the gel has similar properties to an intact disc in comparison to one with a damaged nucleus.

Structural effects in photopolymerized sodium AMPS hydrogels crosslinked with poly(ethylene glycol) diacrylate for use as burn dressings

Synthetic hydrogel polymers were prepared by free radical photopolymerization in aqueous solution of the sodium salt of 2-acrylamido-2-methylpropane sulfonic acid (Na-AMPS). Poly(ethylene glycol) diacrylate (PEGDA) and 4,4'-azo-bis(4-cyanopentanoic acid) were used as the crosslinker and UV-photoinitiator, respectively. The effects of varying the Na-AMPS monomer concentration within the range of 30-50% w/v and the crosslinker concentration within the range of 0.1-1.0% mol (relative to monomer) were studied in terms of their influence on water absorption properties. The hydrogel sheets exhibited extremely high swelling capacities in aqueous media which were dependent on monomer concentration, crosslink density, and the ionic strength and composition of the immersion medium. The effects of varying the number-average molecular weight of the PEGDA crosslinker from = 250 to 700 were also investigated. Interestingly, it was found that increasing the molecular weight and therefore the crosslink length at constant crosslink density decreased both the rate of water absorption and the equilibrium water content. Cytotoxicity testing by the direct contact method with mouse fibroblast L929 cells indicated that the synthesized hydrogels were nontoxic. On the basis of these results, it is considered that photopolymerized Na-AMPS hydrogels crosslinked with PEGDA show considerable potential for biomedical use as dressings for partial thickness burns. This paper describes some structural effects which are relevant to their design as biomaterials for this particular application. © 2013 Copyright Taylor and Francis Group, LLC.

Elevated ε-(γ-glutamyl)lysine in human diabetic nephropathy results from increased expression and cellular release of tissue transglutaminase

Introduction: Diabetic nephropathy (DN) is the leading cause of chronic kidney failure, however the mechanisms underlying the characteristic expansion of the extracellular matrix (ECM) in diabetic kidneys remain controversial and unclear. In non-diabetic kidney scarring the protein crosslinking enzyme tissue transglutaminase (tTg) has been implicated in this process by the formation of increased ε-(γ-glutamyl)lysine bonds between ECM components in both experimental and human disease. Studies in db/db diabetic mice and in streptozotocin-treated rats have suggested a similar mechanism, although the relevance of this to human disease has not been addressed. Methods: We have undertaken a retrospective analysis of renal biopsies from 16 DN patients with type 2 diabetes mellitus using an immunohistochemical and immunofl uorescence approach, with tTg and ε-(γ-glutamyl)lysine crosslink quantified by confocal microscopy. Results: Immunofl uorescent analysis of human biopsies (confocal microscopy) showed increases in levels of tTg (+1,266%, p <0.001) and ε-(γ-glutamyl)lysine (+486%, p <0.001) in kidneys with DN compared to normal. Changes were predominantly in the extracellular periglomerular and peritubular areas. tTg staining correlated with e-(?-glutamyl)lysine (r = 0.615, p <0.01) and renal scarring (Masson's trichrome, r = 0.728, p <0.001). Significant changes in e-(?-glutamyl)lysine were also noted intracellularly in some (=5%) tubular epithelial cells. This is consistent with cells undergoing a novel transglutaminase-mediated cell death process in response to Ca influx and subsequent activation of intracellular tTg. Conclusion: Changes in tTg and ε-(γ- glutamyl)lysine occur in human DN. Cellular export of tTg may therefore be a factor in the perpetuation of DN by crosslinking and stabilisation of the ECM, while intracellular activation may lead to cell death contributing towards tubular atrophy. Copyright © 2004 S. Karger AG, Basel.

Tissue transglutaminase:a mediator and predictor of chronic allograft nephropathy?

Background. The precise mechanisms underlying the development of chronic allograft nephropathy (CAN) and the associated renal fibrosis remain uncertain. The protein-crosslinking enzyme, tissue transglutaminase (tTg), has recently been implicated in renal fibrosis. Methods. We investigated the involvement of tTg and its crosslink product, [epsilon]-([gamma]-glutamyl) lysine, in 23 human kidney allografts during the early posttransplantation period and related these to changes of CAN that developed in 8 of them. Sequential biopsies were investigated using immunohistochemical, immunofluorescence, and in situ enzyme activity techniques. Results. From implantation, tTg (+266%) and [epsilon]-([gamma]-glutamyl) lysine crosslink (+256.3%) staining increased significantly (P <0.001) in a first renal biopsy performed within 3 months from transplantation. This was paralleled by elevated tTg in situ activity. The eight patients who developed CAN had further increases in immunostainable tTg (+197.2%, P <0.001) and [epsilon]-([gamma]-glutamyl) lysine bonds (+465%, P <0.01) that correlated with interstitial fibrosis (r=0.843, P =0.009 and r=0.622, P =0.05, respectively). The staining for both was predominantly located within the mesangium and the renal interstitium. Both implantation and first biopsies showed tTg and [epsilon]-([gamma]-glutamyl) lysine crosslinking levels in patients who developed CAN to be twice the levels of those with stable renal function. Cox regression analysis suggested the intensity of the early tTg staining was a better predictor of inferior allograft survival that other histologic markers (hazard ratio=4.48, P =0.04). Conclusions. tTg and [epsilon]-([gamma]-glutamyl) lysine crosslink correlated with the initiation and progression of scarring on sequential biopsies from renal-allograft recipients who experienced CAN. Elevated tTg may offer an early predictor of the development of CAN, whereas tTg manipulation may be an attractive therapeutic target

Tissue transglutaminase and the progression of human renal scarring

ABSTRACT. Experimental renal scarring indicates that tissue transglutaminase (tTg) may be associated with the accumulation of extracellular matrix (ECM), both indirectly via TGF-β1 activation and directly by the formation of ε(γ-glutamyl) lysine dipeptide bonds within the ECM. The latter potentially accelerates deposition and confers the ECM with resistance to proteolytic digestion. Studied were 136 human renal biopsy samples from a range of chronic renal diseases (CRD) to determine changes in tTg and ε(γ-glutamyl) lysine crosslinking. Immunofluorescence for insoluble tTg showed a 14-fold increase in the kidneys of CRD patients (5.3 ± 0.5 versus 76 ± 54 mV/cm2), which was shown to be active by a similar 11-fold increase in the ε(γ-glutamyl) lysine crosslink (1.8 ± 0.2 versus 19.3 ± 14.2 mV/cm2). Correlations were obtained with renal function for tTg and crosslink. In situ hybridization for tTg mRNA showed that tubular epithelial cells were the major source of tTg; however, both mesangial and interstitial cells also contributed to elevated levels in CRD. This mRNA pattern was consistent with immunohistochemistry for soluble tTg. Changes in renal tTg and its product, the ε(γ-glutamyl) lysine crosslink, occur in progressive renal scarring in humans independently of the original etiology and in a similar manner to experimental models. tTg may therefore play a role in the pathogenesis of renal scarring and fibrosis in patients with CRD and can therefore be considered a potential therapeutic target. E-mail: T.Johnson@sheffield.ac.uk

Increases in renal ε-(γ-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy:Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, e-(γ-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney e-(γ-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p <0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p <0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular e-(γ-glutamyl) lysine (+ 361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular e-(γ-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Importance of tissue transglutaminasenitrosylation in fibroblasts migration and MMPregulation

As an extracellular second messenger, nitric oxide (NO) mediates the modification of proteins through nitrosylation of cysteine andtyrosine residues. Tissue Transglutaminase (TG2) is a Ca2+ activated, sulfhydryl rich protein with 18 free cysteine residues, which catalyzes ε-(γ glutamyl)lysine crosslink between extracellular and intracellular proteins. NO can nitrosylate up to 15 of the cysteine residues in TG2, leading to the irreversible inactivation of the enzyme activity. The interplay between these two agents was revealed for the first time by our study showing that NO inhibited the TG2-induced transcriptional activation of TGFb1and extracellular matrix (ECM) protein synthesis by nitrosylating TG2 in an inactive confirmation with inert catalytic activity. However, nitrosylated TG2 was still able to serve as a novel cell adhesion protein. In the light of our previous findings, in this study we aim to elucidate the NO modified function of TG2 in cell migration using an in vitro model mimicking the tissue matrix remodeling phases of wound healing. Using transfected fibroblasts expressing TG2 under the control of the tetracycline-off promoter, we demonstrate that upregulation of TG2 expression and activity inhibited the cell migration through the activation of TGFβ1. Increased TG2 activity led to arise in the biosynthesis and activity of the gelatinases, MMP-2 andMMP-9, while decreasing the biosynthesis and activity of the col-lagenases MMP-1a and MMP-13. NO donor S-Nitroso-N-acetyl-penicillamine (SNAP) treatment relieved the TG2 obstructed-cellmigration by blocking the TG2 enzyme activity. In addition,decrease in TG2 activity due to nitrosylation led to an inhibition of TGFβ1, which in turn affected the pattern of MMP activation. Recent evidence suggests that, once in complex with fibronectin in the ECM, TG2 can interact with syndecan-4 or integrinβ-1and regulate the cell adhesion. In the other part of this study, the possible role of nitrosylated TG2 on the regulation of cell migration during wound healing was investigated with respect to its interactions with integrin β1 (ITGβ1) and syndecan-4 (SDC4). Treatment with TG2 inhibitor Z-DON resulted in a 50% decrease in the TG2 interaction with ITGB1 and SDC4, while increasing concentrations of SNAP firstly led to a substantial decrease and then completely abolished the TG2/ITGβ1 and TG2/SDC4 complex formation on the cell surface. Taken together, data obtained from this study suggests that nitrosylation of TG2 leads to a change not only in the binding partners of TG2 on cell surface but also in TGFβ1-dependent MMP activation, which give rise to an increase in the migration potential of fibroblasts.

Mechanismus der CR-Vernetzung mit Ethylenthio-harnstoff und Zinkoxid

Es wurde eine Untersuchung zum Mechanismus der Vernetzung von Polychloropren durch Ethylenthioharnstoff in Kombination mit Zinkoxid durchgeführt. Dies wurde mit einer Überprüfung der Vernetzungsmechanismen von Polychloroprenkautschuk mit Ethylenthioharnstoff und Zinkoxid getrennt bzw. gemeinsam erreicht. Dabei kamen spektroskopische und physikalische Charakterisierungsverfahren zum Einsatz, um die Vernetzungsmechanismen von  CR mit anderen Standardvulkanisationsbeschleunigern und Modellverbindungen – mit ETU-analogen Strukturen und Funktionalitäten – zu erforschen. Aus den Untersuchungen resultierte der Vorschlag zu einem neuen Mechanismus, nach dem ETU und ZnO Polychloropren synergistisch vernetzen. Zusätzlich wurden neue Hinweise gewonnen, die gleichzeitig bestehende Mechanismen, die schon zur Vernetzung von Polychloropren veröffentlicht wurden, untermauern. An investigation into the mechanism by which ethylene thiourea crosslinks polychloroprene in combination with zinc oxide was undertaken. This was achieved through an examination of the mechanisms of crosslinking polychloroprene rubber with ETU and ZnO separately and in unison. Spectroscopic and physical characterisation techniques were employed to probe the crosslinking mechanisms of CR using other standard rubber accelerators and model compounds with analogous structures and functionalities to ETU. These investigations have resulted in the proposal of a new mechanism by which ETU and ZnO can synergistically crosslink polychloroprene, in addition to providing new evidence to support concomitant mechanisms already published for crosslinking polychloroprene.

Mechanismen kleiner regulatorischer RNAs in Dictyostelium discoideum: Charakterisierung und Anwendung

Die RNA-Interferenz (RNAi) ist ein in Eukaryoten weit verbreiteter Mechanismus, der die transkriptionelle oder posttranskriptionelle Stilllegung von Genen beschreibt. Die Spezifität wird dabei durch die Sequenz einer kleinen, nicht-kodierenden RNA gewährleistet. Diese RNA leitet einen Effektorkomplex, dessen Zentrum immer von einem Argonautenprotein gebildet wird, üblicherweise zu einer komplementären mRNA. In der Folge kommt es zum Abbau des Transkripts oder zur Inhibierung der Translation. Aktuelle Veröffentlichungen konnten zudem das Aktivitätsprofil der Argonautenproteine beträchtlich erweitern: Im Zellkern vorkommende Argonautenproteine wurden beispielsweise mit Spleißvorgängen, der Promotorkontrolle von Genen und der DNA-Reparatur in Verbindung gebracht. In den letzten Jahren konnten weitreichende Kenntnisse bezüglich der Kontrolle einiger transposabler Elemente durch RNAi sowie der Biogenese kleiner regulatorischer RNAs in Dictyostelium discoideum und anderen Organismen gewonnen werden. Ein Fokus dieser Arbeit lag zunächst auf der Charakterisierung des Argonautenproteins AgnB und der Identifikation von Interaktionspartnern. Es konnte gezeigt werden, dass AgnB zumindest partiell im Zellkern der Amöbe lokalisiert und dort vermutlich regulatorische Aufgaben wahrnimmt. Gestützt wurde diese Annahme durch die massenspektrometrische und Western Blot basierte Detektion nukleärer Bindungspartner. Weiterführende Analysen konnten AgnB zudem als positiven Regulator für drei entwicklungsregulierte Gene beschreiben und so die Verbindung zum Prozess der RNA activation in der Amöbe herstellen. Identifizierte posttranslationale Modifikationen könnten diesbezüglich die Aktivität des Argonauten steuern. Mit Hilfe von Crosslink-RNA-Immunopräzipitation und anschließender Hochdurchsatz-sequenzierung oder der Northern Blot basierten Auswertung konnte eine Assoziation von AgnB und der Class I RNAs gezeigt werden. Diese Klasse umfasst nicht-kodierende RNAs mit einer Länge von etwa 42 bis 65 Nukleotiden und wurde bisher nicht als Substrat für die RNAi-Maschinerie in D. discoideum angesehen. Ein weiterer Teil dieser Arbeit beschäftigte sich mit dem Einfluss von AgnA und AgnB auf die Promotorbereiche des D. discoideum Retrotransposons DIRS-1. Im Verlauf der Untersuchungen konnte beobachtet werden, dass die Anordnung entgegengesetzt operierender DIRS-1 Promotor-sequenzen für die Stilllegung eines Reportergens ausreichend war. Darauf aufbauend konnte ein DIRS-1 basiertes knockdown System etabliert werden. Mit Hilfe dieses Systems konnten die Proteinmengen ausgewählter Zielgene so effektiv reduziert werden, dass die entsprechenden Stämme den Phänotyp des korrespondierenden knockout Stammes zeigten. Darüber hinaus war es möglich die Proteinreduktion zu modulieren, um so beispielsweise dosisabhängige Effekte zu registrieren. Vorangegangene Arbeiten zur Biogenese von micro (mi)RNAs konnten das RNA-bindende Protein RbdB als eine Hauptkomponente für die Prozessierung maturer miRNAs identifizieren. Der miRNA defiziente RbdB- Stamm wurde in dieser Arbeit zur Identifikation putativer miRNA Ziele genutzt. Dazu wurde sowohl das Transkriptom des D. discoideum Wildtyps als auch des rbdB knockout Stammes in hohem Durchsatz sequenziert, um so differentiell exprimierte Gene zu detektieren. Vielversprechende Kandidaten wurden mittels qRT-PCR verifiziert. Dabei wurde unter anderem ein putativer Transkriptionsfaktor als miRNA target identifiziert, der eine Vielzahl weiterer Gene regulieren könnte. Abschließend konnte in dieser Arbeit gezeigt werden, dass RbdB ebenfalls für die Generierung von small interfering (si)RNAs aus strukturierten Loci von Bedeutung ist. Dies weist auf mindestens zwei unterschiedliche Mechanismen zur siRNA Prozessierung in D. discoideum hin.

Développement de matériaux à base de protéines extraites du byssus de la moule bleue Mytilus edulis

Le byssus est un amas de fibres que les moules produisent afin de s’ancrer aux surfaces immergées sous l’eau. Ces fibres sont pourvues de propriétés mécaniques impressionnantes combinant rigidité, élasticité et ténacité élevées. De plus, elles possèdent un comportement d’auto-guérison de leurs propriétés mécaniques en fonction du temps lorsque la contrainte initialement appliquée est retirée. Les propriétés mécaniques de ces fibres sont le résultat de l’agencement hiérarchique de protéines de type copolymère blocs riches en collagène et de la présence de métaux formant des liens sacrificiels réversibles avec certains acides aminés comme les DOPA et les histidines. Bien que cette fibre soit très intéressante pour la production de matériaux grâce à son contenu élevé en collagène potentiellement biocompatible, cette ressource naturelle est traitée comme un déchet par les mytiliculteurs. L’objectif de cette thèse était de valoriser cette fibre en extrayant les protéines pour générer une nouvelle classe de matériaux biomimétiques. Un hydrolysat de protéines de byssus (BPH) riche en acides aminés chargés, i.e. ~30 % mol, et permettant de former des films a pu être généré. Lorsque solubilisé à pH 10.5, le BPH forme un hydrogel contenant des structures en triple hélice de collagène et des feuillets β anti-parallèles intra- et inter-moléculaires. Suite à l’évaporation de l’eau, le film de BPH résultant est insoluble en milieu aqueux à cause des structures secondaires très stables agissant comme points de réticulation effectifs. Les propriétés mécaniques des films de BPH sont modulables en fonction du pH. Au point isoélectrique (pI = 4.5), les interactions électrostatiques entre les charges opposées agissent comme points de réticulation et augmentent la rigidité des films et leur contrainte à la rupture sans affecter la déformation à la rupture. À pH plus élevé ou plus bas que le pI, les performances mécaniques des films sont plus faibles à cause de la répulsion entre les groupements fonctionnels de même charge qui interagissent plutôt avec les molécules d’eau et causent le gonflement de la matrice protéique des films. Le BPH contenant un nombre élevé d’acides aminés chargés et réactifs, nous avons pu réticuler les films de manière covalente à l’aide d’EDC ou de glutaraldéhyde. Les propriétés mécaniques des films sont modulables en fonction de la concentration d’EDC utilisée lors de la réticulation ou en employant du glutaraldéhyde comme agent réticulant. Les films sont à la fois plus rigides et plus forts avec un degré de réticulation élevé, mais perdent leur extensibilité à mesure que les segments libres de s’étirer lors d’une traction deviennent entravés par les points de réticulation. La réticulation augmente également la résistance à la dégradation enzymatique par la collagénase, les films les plus fortement réticulés lui étant pratiquement insensibles. La spectroscopie infrarouge montre enfin que la réticulation entraîne une transition de feuillets β anti-parallèles inter-moléculaires vers des structures de type hélices de collagène/PPII hydratées. Des liens sacrificiels ont été formés dans les films de BPH par traitement au pI et/ou avec différents métaux, i.e. Na+, Ca2+, Fe3+, afin de moduler les propriétés mécaniques statiques et d’évaluer le rôle de ces traitements sur le comportement d’auto-guérison lors de tests mécaniques cycliques avec différents temps de repos. Plus la valence des ions métalliques ajoutés augmente, plus les propriétés mécaniques statiques affichent un module, une contrainte à la rupture et une ténacité élevés sans toutefois affecter la déformation à la rupture, confirmant la formation de liens sacrificiels. Les tests mécaniques cycliques montrent que les traitements au pI ou avec Ca2+ créent des liens sacrificiels ioniques réversibles qui mènent à un processus d’auto-guérison des performances mécaniques dépendant du pH. L’ajout de Fe3+ à différentes concentrations module les performances mécaniques sur un plus large intervalle et la nature plus covalente de son interaction avec les acides aminés permet d’atteindre des valeurs nettement plus élevées que les autres traitements étudiés. Le Fe3+ permet aussi la formation de liens sacrificiels réversibles menant à l’auto-guérison des propriétés mécaniques. Les spectroscopies Raman et infrarouge confirment que le fer crée des liaisons avec plusieurs acides aminés, dont les histidines et les DOPA. Les résultats dans leur ensemble démontrent que les films de BPH sont des hydrogels biomimétiques du byssus qui peuvent être traités ou réticulés de différentes façons afin de moduler leurs performances mécaniques. Ils pourraient ainsi servir de matrices pour des applications potentielles dans le domaine pharmaceutique ou en ingénierie tissulaire.

Obtenção de um hidrogel proveniente de proteínas da corvina (micropogonias furnieri) e solubilização das proteínas fibrosas residuais

Com o aumento na captura de pescado e da poluição do meio ambiente, esta-se à margem de exceder a estimativa do limite da sustentabilidade, e obviamente isto faz com se utilize os recursos marítimos com mais inteligência e precaução. Aplicando tecnologia enzimática ou química é possível recuperar as proteínas do processamento do pescado, produzindo hidrolisados e isolados protéicos. Uma grande quantidade de proteínas insolúveis está disponível em escamas, peles e ossos, subprodutos do processamento do pescado, que podem ser solubilizadas através de fungos e bactérias. Utilizando isolados protéicos é possível obter biopolímeros, estes têm chamado a atenção nos últimos anos, pois são biodegradáveis, não-tóxicos e geralmente biocompatíveis. Os hidrogéis protéicos são polímeros que podem absorver uma quantidade de água a partir de 10 até centenas de vezes o seu peso seco. O objetivo deste trabalho foi desenvolver um hidrogel protéico, com propriedades superabsorventes, a partir das proteínas solúveis e insolúveis da corvina (Micropogonias furnieri). Para a produção dos hidrolisados a partir das proteínas solúveis foi utilizado processo enzimático (Alcalase e Flavourzyme) e químico (solubilização ácida e alcalina). Nos processos de solubilização das proteínas insolúveis foram utilizados microrganismos (bactérias e fungos). Tanto as bactérias como os fungos avaliados apresentaram capacidade de solubilizar as proteínas insolúveis presentes nos resíduos (escamas, ossos, cartilagens e outros). A bactéria que atingiu a maior atividade proteolítica foi a Bacillus velesensis (47,56 U mL-1) e o fungo foi o Penicillium sp. (E20) (31,20 U mL-1). Para a produção dos hidrogéis, foram utilizados isolados protéicos provenientes de solubilização ácida ou alcalina, produzidos a partir de resíduos da industrialização de pescado, modificados quimicamente com dianidrido etilenodiamino tetraacético (EDTAD) e adicionados de agente de ligação cruzada (glutaraldeído). Algumas proteínas modificadas ainda foram submetidas a tratamento com etanol. Foram realizadas análise estrutural das proteínas modificadas e estudo da capacidade de retenção de água dos hidrogéis assim obtidos. Os hidrogéis produzidos apresentaram alta capacidade de retenção de água. A máxima absorção de água foi alcançada pelo hidrogel ácido sem o tratamento com etanol foi de 103,25 gágua/ggel seco, enquanto que a mesma amostra tratada com etanol alcançou 216,05 gágua/ggel seco. Os hidrogéis produzidos podem ser utilizados em diversas indústrias, tais como, farmacêutica, alimentícia, médica, agroindústria, entre outras, que necessitem de hidrogéis com alta capacidade de retenção de água.

Dielectric properties of thermosetting material nanocomposites

The dieletric relaxation properties of thermosetting material nanocomposites based on spherosilicate nanoplatforms were studied from room temperature to 170 degrees C, varying the frequency from 10 to 1000 KHz. Permittivity (epsilon'), dielectric loss (epsilon ''), and activation energy (E-a) were calculated. The results of dielectric relaxation were confirmed by those of the final properties. The dielectric loss amplitude decreases with increasing ODPG content until about 70-73 wt % and slightly increases at higher ODPG content. This means that the increasing of the ODPG content in the composite samples decreases the number of pendants groups and/or increases crosslink densitv, causing decreased motion of organic tethers, and subsequently decreasing of the dipolar mobility. The results of apparent activation energy, fracture toughness and tensile modulus mechanical properties show the same profile with respect to ODPG content, in the sense that they exhibit maxima around 70 wt % ODPG. For the ODPG/MDA composites, this formulation of 70 wt % ODPG containing excess of amine is not composition where the highest crosslinked density is reached. This implies that the best mechanical properties and E-a are provided by some degree of chain flexibility. (c) 2007 Wiley Periodicals, Inc.

Développement de matériaux à base de protéines extraites du byssus de la moule bleue Mytilus edulis

Le byssus est un amas de fibres que les moules produisent afin de s’ancrer aux surfaces immergées sous l’eau. Ces fibres sont pourvues de propriétés mécaniques impressionnantes combinant rigidité, élasticité et ténacité élevées. De plus, elles possèdent un comportement d’auto-guérison de leurs propriétés mécaniques en fonction du temps lorsque la contrainte initialement appliquée est retirée. Les propriétés mécaniques de ces fibres sont le résultat de l’agencement hiérarchique de protéines de type copolymère blocs riches en collagène et de la présence de métaux formant des liens sacrificiels réversibles avec certains acides aminés comme les DOPA et les histidines. Bien que cette fibre soit très intéressante pour la production de matériaux grâce à son contenu élevé en collagène potentiellement biocompatible, cette ressource naturelle est traitée comme un déchet par les mytiliculteurs. L’objectif de cette thèse était de valoriser cette fibre en extrayant les protéines pour générer une nouvelle classe de matériaux biomimétiques. Un hydrolysat de protéines de byssus (BPH) riche en acides aminés chargés, i.e. ~30 % mol, et permettant de former des films a pu être généré. Lorsque solubilisé à pH 10.5, le BPH forme un hydrogel contenant des structures en triple hélice de collagène et des feuillets β anti-parallèles intra- et inter-moléculaires. Suite à l’évaporation de l’eau, le film de BPH résultant est insoluble en milieu aqueux à cause des structures secondaires très stables agissant comme points de réticulation effectifs. Les propriétés mécaniques des films de BPH sont modulables en fonction du pH. Au point isoélectrique (pI = 4.5), les interactions électrostatiques entre les charges opposées agissent comme points de réticulation et augmentent la rigidité des films et leur contrainte à la rupture sans affecter la déformation à la rupture. À pH plus élevé ou plus bas que le pI, les performances mécaniques des films sont plus faibles à cause de la répulsion entre les groupements fonctionnels de même charge qui interagissent plutôt avec les molécules d’eau et causent le gonflement de la matrice protéique des films. Le BPH contenant un nombre élevé d’acides aminés chargés et réactifs, nous avons pu réticuler les films de manière covalente à l’aide d’EDC ou de glutaraldéhyde. Les propriétés mécaniques des films sont modulables en fonction de la concentration d’EDC utilisée lors de la réticulation ou en employant du glutaraldéhyde comme agent réticulant. Les films sont à la fois plus rigides et plus forts avec un degré de réticulation élevé, mais perdent leur extensibilité à mesure que les segments libres de s’étirer lors d’une traction deviennent entravés par les points de réticulation. La réticulation augmente également la résistance à la dégradation enzymatique par la collagénase, les films les plus fortement réticulés lui étant pratiquement insensibles. La spectroscopie infrarouge montre enfin que la réticulation entraîne une transition de feuillets β anti-parallèles inter-moléculaires vers des structures de type hélices de collagène/PPII hydratées. Des liens sacrificiels ont été formés dans les films de BPH par traitement au pI et/ou avec différents métaux, i.e. Na+, Ca2+, Fe3+, afin de moduler les propriétés mécaniques statiques et d’évaluer le rôle de ces traitements sur le comportement d’auto-guérison lors de tests mécaniques cycliques avec différents temps de repos. Plus la valence des ions métalliques ajoutés augmente, plus les propriétés mécaniques statiques affichent un module, une contrainte à la rupture et une ténacité élevés sans toutefois affecter la déformation à la rupture, confirmant la formation de liens sacrificiels. Les tests mécaniques cycliques montrent que les traitements au pI ou avec Ca2+ créent des liens sacrificiels ioniques réversibles qui mènent à un processus d’auto-guérison des performances mécaniques dépendant du pH. L’ajout de Fe3+ à différentes concentrations module les performances mécaniques sur un plus large intervalle et la nature plus covalente de son interaction avec les acides aminés permet d’atteindre des valeurs nettement plus élevées que les autres traitements étudiés. Le Fe3+ permet aussi la formation de liens sacrificiels réversibles menant à l’auto-guérison des propriétés mécaniques. Les spectroscopies Raman et infrarouge confirment que le fer crée des liaisons avec plusieurs acides aminés, dont les histidines et les DOPA. Les résultats dans leur ensemble démontrent que les films de BPH sont des hydrogels biomimétiques du byssus qui peuvent être traités ou réticulés de différentes façons afin de moduler leurs performances mécaniques. Ils pourraient ainsi servir de matrices pour des applications potentielles dans le domaine pharmaceutique ou en ingénierie tissulaire.