No description available.

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<p>The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste.</p><p>In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster.</p><p>In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.</p>

The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste. In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster. In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.

No description available.

No / ß2-microglobulin (ß2m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How ß2m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on ß2m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (¿N6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that ¿N6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of ß2m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type ß2m with that of ¿N6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.

The self-assembly of proteins into filamentous structures underpins many aspects of biology, from dynamic cell scaffolding proteins such as actin, to the amyloid plaques responsible for a number of degenerative diseases. Typically, these self-assembly processes have been treated as nucleated, reversible polymerisation reactions, where dynamic fluctuations in a population of monomers eventually overcome an energy barrier, forming a stable aggregate that can then grow and shrink by the addition and loss of more protein from its ends. The nucleated, reversible polymerisation framework is very successful in describing a variety of protein systems such as the cell scaffolds actin and tubulin, and the aggregation of haemoglobin. Historically, amyloid fibrils were also thought to be described by this model, but measurements of their aggregation kinetics failed to match the model's predictions. Instead, recent work indicates that autocatalytic polymerisation - a process by which the number of growth competent species is increased through secondary nucleation, in proportion to the amount already present - is better at describing their formation. In this thesis, I will extend the predictions made in this mean-field, autocatalytic polymerisation model through use of kinetic Monte Carlo simulations. The ubiquitous sigmoid-like growth curve of amyloid fibril formation often possesses a notable quiescent lag phase which has been variously attributed to primary and secondary nucleation processes. Substantial variability in the length of this lag phase is often seen in replicate experimental growth curves, and naively may be attributed to fluctuations in one or both of these nucleation processes. By comparing analytic waiting-time distributions, to those produced by kinetic Monte Carlo simulation of the processes thought to be involved, I will demonstrate that this cannot be the case in sample volumes comparable with typical laboratory experiments. Experimentally, the length of the lag phase, or "lag time", is often found to scale with the total protein concentration, according to a power law with exponent γ. The models of nucleated polymerisation and autocatalytic polymerisation predict different values for this scaling exponent, and these are sometimes used to identify which of the models best describes a given protein system. I show that this approach is likely to result in a misidentification of the dominant mechanisms under conditions where the lag phase is dominated by a different process to the rest of the growth curve. Furthermore, I demonstrate that a change of the dominant mechanism associated with total protein concentration will produce "kinks" in the scaling of lag time with total protein concentration, and that these may be used to greater effect in identifying the dominant mechanisms from experimental kinetic data. Experimental data for bovine insulin aggregation, which is well described by the autocatalytic polymerisation model for low total protein concentrations, displays an intriguing departure from the predicted behaviour at higher protein concentrations. Additionally, the protein concentration at which the transition occurs, appears to be affected by the presence of salt. Coincident with this, an apparent change in the fibril structure indicates that different aggregation mechanisms may operate at different total protein concentrations. I demonstrate that a transition whereby the self-assembly mechanisms change once a critical concentration of fibrils or fibrillar protein is reached, can explain the observed behaviour and that this predicts a substantially higher abundance of shorter laments - which are thought to be pathogenic - at lower total protein concentrations than if self-assembly were consistently autocatalytic at all protein concentration. Amyloid-like loops have been observed in electron and atomic-force microscographs, together with non-looped fibrils, for a number of different proteins including ovalbumin. This implies that fibrils formed of these proteins are able to grow by fibrillar end-joining, and not only monomer addition as is more commonly assumed. I develop a simple analytic expression for polymerisation by monomer addition and fibrillar end-joining, (without autocatalysis) and show that this is not sufficient to explain the growth curves obtained experimentally for ovalbumin. I then demonstrate that the same data can be explained by combining fibrillar end-joining and fragmentation. Through the use of an analytic expression, I estimate the kinetic rates from the experimental growth curves and, via simulation, investigate the distribution of lament and loop lengths. Together, my findings demonstrate the relative importance of different molecular mechanisms in amyloid fibril formation, how these might be affected by various environmental parameters, and characteristic behaviour by which their involvement might be detected experimentally.

Thesis advisor: Daniel A. Kirschner / In the present study, the ability of small compounds to inhibit the fibrillogenesis of beta-amyloid 12-28 was explored. Beta-amyloid 12-28 is a synthetic fragment of Alzheimer's beta-amyloid, which contains the core hydrophobic residues thought to be significant for fiber formation. Using x-ray diffraction, preliminary screening of over sixteen compounds was performed. Cotinine, PTI-00703®, and tetracycline were chosen because of their ease of solubility, the effect on the coherent domain size of the beta-crystallite subunit in the presence of chosen small molecules as shown by x-ray diffraction, as well as their presence in previously published literature. This conformational-driven inhibition of fibrillogenesis was explored in the current research using circular dichroism spectroscopy and x-ray diffraction. Circular dichroism spectroscopy revealed the nascent beta-sheet structure of beta-amyloid12-28 when first dissolved and only cotinine, out of all three inhibitors, was able to shift the equilibrium away from the fibrillogenic beta-sheet structure toward a random coil secondary structure after 36 hours of incubation. X-ray diffraction in this study demonstrated no change in hydrogen bond spacing at ~4.7Å and intersheet spacing at ~10-12Å both alone and in the presence of all small molecules. With increasing concentration of inhibitor, however, the widths of these reflections increased, indicating a decrease in the coherent domain size. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Chemistry. / Includes bibliographical references (leaves 84-87).

The ultrastructural mechanism for strain rate sensitivity of collagenous tissue has not been well studied at the collagen fibril level. The objective is to reveal the mechanistic contribution of the collagen fibril to strain rate sensitivity. Collagen fibrils underwent significantly greater fibril strain relative to global tissue strain at higher strain rates. A better understanding of tendon mechanisms at lower hierarchical levels would help establish a basis for future development of constitutive models and assist in tissue replacement design. High rate mechanical property of tendon was also studied. Tendon was compressed under high strain rate (550 /s) using a polycarbonate split Hopkinson pressure bar (PSHPB). The objectives are to investigate the tissue behavior of porcine tendon at high rates. Tendon’s high rate behavior was compared with brain and liver at both hydrated and dehydrated states to investigate how water content and ultrastructural affect high rate responses of soft tissues.