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.

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

Apertureless near-field scanning infrared microscopy is a technique in which an impinging infrared beam is scattered by a sharp atomic force microscopy (AFM) tip oscillating at the resonant frequency of the cantilever in close proximity to a sample. Several advantages offered by near-field imaging include nanoscale imaging with high spatial resolution (near-field imaging is not restricted by the diffraction limit of light) and the ability to differentiate between chemical properties of distinct compounds present in the sample under study due to differences in the scattered field. An overview of the assembly, tuning, and implementation of the near-field instrumentation is provided, as well as detailed descriptions about the samples probed and other instrumentation used. A description of the near-field phenomena, a comparison between aperture and apertureless-type near-field microscopy, and the coupled dipoles model explaining the origin of the chemical contrast present in near-field infrared imaging was discussed. Simultaneous topographic and chemical contrast images were collected at different wavelengths for the block copolymer thin film, polystyrene-b-poly(methyl ethacrylate) (PS-b-PMMA) and for amyloid fibrils synthesized from the #21-31 peptide of β2-microglobulin. In both cases it was observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum, and using near-field microscopy, nanoscale structural and/or compositional variations were observed, which would not have been possible using ensemble FTIR measurements. Lastly, tip-enhanced Raman spectra of the #21-31 and #16-22 peptide fragments from the β2-microglobulin and Aβ(1-40) peptide were collected, examined, and an outline of the optimization conditions described.

<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.

Apertureless near-field scanning infrared microscopy is a technique in which an impinging infrared beam is scattered by a sharp atomic force microscopy (AFM) tip oscillating at the resonant frequency of the cantilever in close proximity to a sample. Several advantages offered by near-field imaging include nanoscale imaging with high spatial resolution (near-field imaging is not restricted by the diffraction limit of light) and the ability to differentiate between chemical properties of distinct compounds present in the sample under study due to differences in the scattered field. An overview of the assembly, tuning, and implementation of the near-field instrumentation is provided, as well as detailed descriptions about the samples probed and other instrumentation used. A description of the near-field phenomena, a comparison between aperture and apertureless-type near-field microscopy, and the coupled dipoles model explaining the origin of the chemical contrast present in near-field infrared imaging was discussed. Simultaneous topographic and chemical contrast images were collected at different wavelengths for the block copolymer thin film, polystyrene-b-poly(methyl ethacrylate) (PS-b-PMMA) and for amyloid fibrils synthesized from the #21-31 peptide of β2-microglobulin. In both cases it was observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum, and using near-field microscopy, nanoscale structural and/or compositional variations were observed, which would not have been possible using ensemble FTIR measurements. Lastly, tip-enhanced Raman spectra of the #21-31 and #16-22 peptide fragments from the β2-microglobulin and Aβ(1-40) peptide were collected, examined, and an outline of the optimization conditions described.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23218号 / 工博第4862号 / 新制||工||1759(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 田中 庸裕, 教授 跡見 晴幸, 准教授 菅瀬 謙治, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No / Beta 2-microglobulin (ß2m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues; by the addition of pre-assembled seeds; or in the presence of Cu2+. Based on these observations, and other structural data, models for fibril formation of ß2m have been proposed that involve the fraying of the N and C-terminal ß-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of ß2m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven ß-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of ß2m.

Some diagnostic methods have been used for a very long time. Congo red stain saw the light of day in 1883, and quickly became important in many fields of use. Nowadays we recognize the importance of Congo red in diagnose of amyloid diseases. However, the technique and experience needed throughout the process from a suspected case to the diagnose is of greate importance. When diagnostic difficulties appeared in a few patient cases at the local hospital in Gävle, Sweden, a solution was needed. A delayed diagnose could have a potential devastating outcome seen in the perspective of the patient. Therefore it is crucial to have both sensitive and specific diagnostic methods that are optimized against the sought pathogenesis. This study aspired to find the solution to the difficulties in diagnostic work, brought to light by a pathology doctor at the hospital. Several different methodical procedures are used throughout the process, and were evaluated with focus lying on the thickness of the tissue, the staining method and the microscopes used in diagnostics. Different thickness of the tissue was cut and stained. The results demonstrated the importance of proper techniques and methods in preparing the tissue, and the tools to analyse it with. The thickness of tissue and the lightsource in the microscope played a cruicial role in diagnostics. Additionally it showed the importance to continue to raise the quality of work and make progress in the diagnostic and scientific field, possibly by finding new applications for old methods.

No description available.