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New nanomaterials: amyloid fibrils from waste proteinsDomigan, Laura Joy January 2012 (has links)
The current landscape of nanotechnology has focussed attention on materials that self-assemble. The search for such materials has unsurprisingly led to the biological world, where functional nanoscale biomolecular assemblies are in abundance. Amyloid fibrils are one such self-assembling biological structure, formed when native proteins misfold into insoluble fibrous quaternary structures. This research has explored the use of amyloid fibrils formed from waste proteins, namely crude crystallin proteins from fish eye lenses, as biological nanowires.
The use of amyloid fibrils as nanowires was investigated by examining the ability to control their dimensions and arrangement, along with analysis of their properties, such as stability and conductivity. TEM and AFM studies on the model amyloid forming protein, bovine insulin, showed that a number of fibril length distributions can be achieved, by systematically altering fibril growth and storage conditions. Although the same set of conditions cannot be directly applied to crystallin fibrils, these fibrils can also be produced on a range of length scales. Amyloid fibrils can be manipulated and aligned in a controlled manner by dielectrophoresis; this tool could later be used to incorporate amyloid fibrils into a biosensing or bioelectronics device.
Dielectrophoresis was also used to immobilise crystallin fibrils between electrode pairs, in order to investigate the conductivity of small numbers of fibrils. These experiments complemented work carried out on the conductivity of amyloid fibril networks, using fabricated interdigitated electrodes. In the unmodified state, amyloid fibrils formed from bovine insulin, fungal hydrophobins, and crude crystallins were all shown to have low conductivity, with current values in the range of 10⁻⁸–10⁻¹⁰ A recorded at bias voltages of 0–2 V. Amyloid fibrils were used as a template for the synthesis of conductive nanowires, by modification with the conducting polymers polyaniline and polypyrrole, increasing conductivity by one and four orders of magnitude respectively.
The functionalisation of fibrils with glucose oxidase enabled the creation of a very simple glucose sensing device. This device, consisting of a gold electrode modified with the glucose oxidase functionalised fibrils, showed an electrochemical response in the presence of glucose and the mediator FcOH. Future work is necessary to optimise the use of amyloid fibrils in this way; however, this study confirms a role for amyloid fibrils from a low cost source in bionanotechnology.
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STRUCTURE OF PRION PROTEIN AMYLOID FIBRILS AS DETERMINED BY HYDROGEN/DEUTERIUM EXCHANGELu, Xiaojun 25 March 2008 (has links)
No description available.
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Incorporating glucose oxidase activity into amyloid fibrilsPilkington, Sarah January 2009 (has links)
Amyloid fibrils are a misfolded state formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them an excellent target for enzyme immobilisation and their strength, stability and nanometre size are attractive features for exploitation in the creation of new bionanomaterials. The aim of this thesis was to functionalise amyloid fibrils by conjugation to glucose oxidase (GOD). GOD is a relatively stable glycoprotein that catalyses the oxidation of glucose and the release of hydrogen peroxide. The consumption of glucose can be measured to assess glucose levels, and the release of hydrogen peroxide is cytotoxic to cells and is thus an effective antibacterial agent. Three methods of attachment were used: cross-linking using glutaraldehyde, periodate oxidation of the glycoprotein shell, and cross-linking using glutaraldehyde following deglycosylation. GOD retained activity upon attachment by all three methods. These attachment methods were assessed using electrophoresis, centrifugation, sucrose gradient centrifugation and TEM. Gel electrophoresis indicated a high degree of cross-linking and TEM showed no significant change of fibril morphology upon cross-linking. Centrifugation experiments suggested a non-covalent interaction was occurring between amyloid fibrils and GOD, and a covalent attachment was occurring upon addition of glutaraldehyde. Sucrose gradient centrifugation provided increased separation of cross-linked material compared to other separation methods, and showed greater cross-linking to crystallin amyloid fibrils than insulin fibrils. Cross-linking native GOD using glutaraldehyde was chosen for further experiments, as it was found to be most effective for GOD attachment to amyloid fibrils. The resulting functionalised enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The distribution of the functionalised fibrils through the film was characterised using SEM and confocal microscopy, where film components were found to be unevenly dispersed. The antibacterial effect of the functionalised film was then tested on E. coli and the antifungal effect of the film was tested on Fusarium, Rhizopus and Penicillium. Growth of E. coli was inhibited around functionalised film circles, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. However, no growth inhibition of fungal species was observed. This work is of significance as it demonstrates the ability to convert a waste material, bovine lens crystallin, to high value protein nanofibres and incorporate functionality via GOD attachment. The incorporation of the GOD-functionalised amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalised amyloid fibril scaffold. Future development of this model system has the potential to lead to the production of a novel biomaterial for use in food packaging due to the antimicrobial properties of GOD.
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Strain rate effects on structure-property relationship in the rabbit patellar tendonDavis, Deborah D 13 December 2008 (has links)
This study quantified mechanical and structural responses to loading conditions at subtendon hierarchical levels. Tensile tests were performed at three strain rates on three groups of rabbit patellar tendon specimens. For each rate, tangent modulus (E) was computed from the stress-strain curves and the following structural responses were evaluated: (i) Area percent of collagen fibrils (FAR) and (ii) Skewness angle formed between proteoglycans and collagen fibrils. For 0.1%/s, 10%/s, and 70%/s, E was 48.8±20.3MPa, 64.7±29.3MPa, and 78.6±31.7MPa, respectively. For control, 0.1%/s, 10%/s, and 70%/s, the mean FAR was 0.7552±0.1476, 0.6628±0.1190, 0.6335±0.1013, and 0.6047±0.0384, respectively; and proteoglycan skewness angles were 14.70º±11.01º, 12.76º±10.13º, 15.08.0º±11.66º, and 16.68º±12.07º, respectively. For increased E, interfibrillar components had less time for effective fluid flow, energy dissipation, and structural rearrangement. The inverse relationship of FAR to strain rate may be due to broken fibrils and the Poisson effect. Proteoglycan skewness angle increase is likely due to stretched fibrils.
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Exploring the mechanisms of fibrillar protein aggregationRyan, Morris January 2013 (has links)
The aim of this thesis is to investigate and better understand the mechanisms of protein self-assembly. Specifically, I study three protein systems which form morphologically and structurally distinct brillar protein aggregates. The first of these studies is concerned with the self-assembly of amyloid brils formed from bovine insulin. Amyloid brils are associated with human diseases such as Alzheimers Disease and type-2 diabetes, and are also garnering interest in biomaterial applications. Fragmentation-dominated models for the self-assembly of amyloid brils have had important successes in explaining the kinetics of amyloid bril formation but predict bril length distributions that do not match experimental observations. Here I resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. I provide evidence for a structural transition demarcated by a critical bril mass concentration, or CFC, above which fragmentation of the brils is suppressed. Our simulations predict the formation of distinct bril length distributions above and below the CFC, which I confirm by electron microscopy. These results point to a new picture of amyloid bril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC. I further show that the CFC can be modulated by environmental conditions, pointing to possible in vivo strategies for controlling cytotoxicity. I probe the structural nature of the transition by performing small angle neutron scattering. Secondly, I study the formation of amyloid-like brils from the protein ovalbumin. I undertake kinetic experiments of self-assembly and find two key features emerge: the lack of a lag time and the existence of a slow growth regime in the long-time limit. I observe, using TEM, that these brils are worm-like in nature and form closed-loops. I find the growth kinetics are intimately connected to this particular morphology. I present a simple kinetic model which captures the features of the kinetics found in experiments by incorporating end-to-end association of brils. I comment on the ramifications this type of amyloid bril assembly may have on oligomeric toxicity. Thirdly, the DNA-mimic protein ocr is highly charged (-56e at pH 8) and forms non-amyloid brillar assemblies at very high ammonium sulphate concentrations (3.2M). The fact that ocr forms translucent brillar gels at such high salt concentrations is extremely unique. Typically under such high salt conditions, non-specific amorphous aggregates are formed. In order to better understand the mechanism of why ocr forms specific bril aggregates, I used variants of the wile-type protein in which extensive regions of surface have been removed or modified. The structural characteristics of gels formed from the variants were probed using microrheological techniques. I find that non-specific electrostatic charge screening plays an important role in ocr aggregation. However, I also locate a potentially important α-helical region which may play a part in establishing specific interactions so that ocr may form ordered brillar assemblies.
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Intercellular adhesion in resin canal tissue isolated from slash pine chlorite holocelluloseKibblewhite, R. Paul 01 January 1969 (has links)
No description available.
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Scaling up the production of protein nanofibresWong, Kang Yuon January 2011 (has links)
Protein nanofibres, commonly known as amyloid fibrils, are emerging as potential biological nanomaterials in a number of applications. Protein nanofibres are a highly ordered insoluble form of protein, which results when a normally soluble protein aggregates via a self-association process. However, researchers are currently faced with several challenges such as finding a cheap source of proteins that can be obtained without expensive purification and optimizing a scalable method of the manufacturing of protein nanofibres. This thesis has identified crude mixtures of fish lens crystallins as a cheap protein source and has optimized methods for large scale production of protein nanofibres of varying morphologies. Results show that by varying the conditions of fibre formation, individual protein fibres can be used as building blocks to form higher order structures. This ability to control the morphology and form higher ordered structures is a crucial step in bottom up assembly of bionanomaterials and opens possibilities for applications of protein nanofibres.
The method of formation of protein nanofibres was optimized on a bench scale (1.5 mL Eppendorf tubes) and successfully scaled-up to 1 L volume. For larger scale-up volume (i.e. greater than 10 ml), internal surface area was important for the formation of protein nanofibres. The crude crystallin mixture prepared at 10 mg/mL was heated at 80oC in the presence of 10% v/v TFE at pH 3.8 for 24 hours and stored for an additional of 24 hours at room temperature for storage process. Aggregation and precipitation of proteins were observed as the protein solution was added to the pre-heated TFE. The resulting protein nanofibres were characterised using ThT dye binding, TEM and SEM. The TEM images show a network of long and criss-crossing protein nanofibres with individual fibres of approximately 10 to 20 nm in diameter and 0.5 to 1 μm long. These protein nanofibres were prepared in 1 mL centrifuge tubes and were left on the laboratory bench at room temperature. After 5 months, fresh TEM grids of the sample were prepared and visualized using TEM. Interestingly, TEM images show that a number of individual fibres had self-assembled in an intertwining fashion to form large bundles and higher order structures containing bundles of nanofibres up to 200 nm thick.
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Scanning Probe Microscopy Methods to Study Electrostatic Properties within BiosystemsMoores, Bradley Adam James January 2010 (has links)
Many proteins are known to actively interact with biological, as well as inorganic and synthetic surfaces that are widely used in nano- and bio-technology as biosensing platforms and in tissue engineering. Amyloid fibrils are insoluble protein aggregates in beta-sheet conformation that are implicated in at least 20 diseases for which no cure is currently available. The molecular mechanism of fibril formation, as well as the mechanism of fibril clusters interacting with lipid membrane surfaces is currently unknown. The lipid membrane surface has a complex biochemical composition and is also electrostatically non-homogeneous. Currently, the experimental data available for amyloid fibril formation both on lipid and artificial surfaces is limited. The goal of our study is to investigate how the physical properties of the surfaces affect binding of amyloid peptides and affect the fibril formation. We seek to elucidate the effect of electrostatic interactions of amyloid peptides with surfaces using Atomic Force Microscopy (AFM) and Kelvin probe force microscopy (KPFM). We show using KPFM that electrostatic domains readily form within biological systems such as lung surfactant and lipid monolayers. We compared three different implementations of KPFM to demonstrate that frequency modulated (FM-) KPFM provides significant advantages over other modes. We also present a study of Amyloid beta (1-42) fibril formation on model surfaces, which are uniformly charged or possess periodicity of charges and hydrophobic functionality based on thiol self-assembly. Effect of membrane composition, surface charge, and presence of steroids will be discussed.
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Disentangling structural complexity in proteins by decomposing SAXS data with chemometric approaches / Détermination de la complexité structurale des protéines en décomposant les données SAXS avec des approches chimiométriquesHerranz-Trillo, Fatima 29 September 2017 (has links)
De nombreux systèmes biologiques sont intrinsèquement polydispersés, présentant de multiples espèces coexistantes, de taille, de forme ou de conformation différentes (c'est-à-dire, mélanges oligomèriques, des complexes faiblement liés se dissociant en composantes individuelles ou des espèces apparaissant lors de processus amyloïdogéniques). L'étude de tels systèmes complexes est une tâche difficile en raison de l'instabilité des espèces concernées, de leurs concentrations relatives faibles et interdépendantes et des difficultés rencontrées pour l'isolation des composantes pures. Dans cette thèse, j'ai développé des approches méthodologiques pour appliquer la diffusion des rayons X aux petits angles (SAXS), une technique de biologie structurale, à l'étude de systèmes polydispersés. SAXS est une technique additive et par conséquent, le diagramme de diffusion mesuré pour un échantillon polydispersé correspond à la somme pondérée en concentration des contributions de chacune des composantes individuelles du mélange. Cependant, la décomposition des données de SAXS en des spectres spécifiques des espèces et de leurs concentrations relatives est extrêmement laborieuse et ambigue. Dans cette thèse, je présente d'abord une approche objective pour solidement décomposer les jeux de données de SAXS en composantes individuelles. Cette approche adapte la méthode chimiométrique « Multivariable Curve Resolution Alternate Least Squares » (MCR-ALS) aux spécificités des données de SAXS. Notre méthode permet une décomposition rigoureuse et robuste des données de SAXS en introduisant simultanément différentes représentations de ces données et par conséquent, en mettant l'accent sur des changements moléculaires à différentes plages de temps et de résolution structurale. Nous avons appliqué cette approche, que nous appelons COSMiCS (Analyse structurelle objective complexe des systèmes multi-composants) pour étudier deux systèmes polydispersés: la fibrillation des protéines, et les fluctuations conformationnelles de protéines grâce à l'analyse de données obtenues à l'aide d’une technique de couplage de chromatographie d'exclusion de taille (SEC) avec le ligne de SAXS (SEC-SAXS). L'importance d'étudier les processus de fibrillation réside dans leur implication dans des pathologies amyloïdogéniques telles que les maladies de Parkinson ou d'Alzheimer. Il existe de fortes indications que les espèces oligomériques solubles, et non les fibrilles matures, sont la cause principale de la cytotoxicité et des dommages neuronaux. Cette observation souligne l'importance de caractériser les premiers stades des processus de fibrillation. Notre approche COSMiCS a permis d'étudier les processus amyloïdogéniques de l'insuline et du mutant familial E46K de l'α-synucléine, une protéine associée à la maladie de Parkinson. Cette analyse permet la caractérisation structurale des espèces présentes (y compris les espèces oligomériques) et la caractérisation cinétique de leurs transformations.La deuxième partie de la thèse est consacrée à l'utilisation de COSMiCS pour analyser des données de SEC-SAXS. Le SEC-SAXS est extrêmement populaire et a été implémenté sur plusieurs lignes de SAXS à travers le monde. En utilisant des données synthétiques, je démontre la capacité des approches chimiométriques à décomposer des profils chromatographiques complexes. À l'aide de cette approche, j'ai décomposé l’ensemble des données SEC-SAXS mesurés pour la Prolyl OligoPeptidase (POP).En résumé, cette thèse présente une nouvelle approche chimiométrique qui peut être généralement appliquée à tout mélange macromoléculaire pouvant subir une modifacation de son équilibre et pouvant être abordé par SAXS. Les complexes biomoleculaires transitoires, les processus de repliement, les réarrangements structuraux dépendants d’un ligand ou la formation de grands ensembles supramoleculaires peuvent être sondés de façon structurale en utilisant l'approche COSMiCS. / Many biological systems are inherently polydisperse, presenting multiple coexisting species differing in size, shape or conformation (i.e. oligomeric mixtures, weakly bound complexes, and species appearing along amyloidogenic processes). The study of such complex systems is challenging due to the instability of the species involved, their low and interdependent relative concentrations, and the difficulties to isolate the pure components. In this thesis, I have developed methodological approaches to apply Small-Angle X-ray Scattering (SAXS), a low-resolution structural biology technique, to the study of polydisperse systems. As an additive technique, the SAXS pattern measured for a polydisperse sample corresponds to the concentration-weighted sum of the contributions from each of the individual components. However, decomposition of SAXS data into species-specific spectra and relative concentrations is laborious and burdened by ambiguity. In this thesis, I present an approach to decompose SAXS datasets into the individual components. This approach adapts the chemometrics Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method to the specificities of SAXS data. Our method enables the rigorous and robust decomposition of SAXS data by simultaneously introducing different representations of these data and, consequently, emphasizing molecular changes at different time and structural resolution ranges. We have applied this approach, which we name COSMiCS (Complex Objective Structural analysis of Multi-Component Systems), to study two polydisperse systems: amyloid fibrillation by analysing time-dependent SAXSdata, and conformational fluctuations through the analysis of data obtained using on-line size-exclusion chromatography coupled to SAXS (SEC-SAXS). The importance of studying fibrillation processes lies in their implication in amyloidogenic pathologies such as Parkinson’s or Alzheimer’s diseases. There exist strong indications that soluble oligomeric species, and not mature fibrils, are the main cause of cytotoxicity and neuronal damage emphasizing the importance of characterizing early stages of fibrillation. The first application of our COSMiCS approach has allowed the study of the amyloidogenic mechanisms of insulin and the familial mutant E46K of ↵-synuclein, a Parkinson’s disease related protein. The analysis enables the structural characterization of all the species present as well as their kinetic transformations. The second part of the thesis is dedicated to the use of COSMiCS to analyze on-line SEC-SAXS experiments. Using synthetic data, I demonstrate the capacity of chemometric approaches to decompose complex chromatographic profiles. Using this approach, I have studied the conformational fluctuations in prolyl oligopeptidase (POP), a protein related to synaptic functions and neuronal development. In summary, this thesis presents a novel chemometrics approach that can be generally applied to any macromolecular mixture with a tuneable equilibrium that is amenableto SAXS. Transient biomolecular complexes, folding processes, or ligand-dependent structural rearrangements can be probed structurally using COSMiCS.
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Nanoparticle-induced Changes in Insulin Fibrillation BehaviorKhosravi, Zahra January 2020 (has links)
No description available.
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