Chemical and Biomolecular Engineering - Theses
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ItemInhibitors of amyloid fibril formation( 2013)Despite significant recent advances in medical technology, there is still no commercially available treatment for the amyloid diseases that were first observed by Nicolaus Fontanus in 1639. These amyloids are fibrillar aggregates of misfolded proteins that form plaques in various organs and are the hallmarks of a number of incurable diseases such as Alzheimer’s disease and Parkinson’s disease among others. Alzheimer’s disease is the most well-known amyloid disease and was first described by Alois Alzheimer in 1906. It is widely believed that the amyloids or their precursors are responsible for the tissue damage that eventually leads to these diseases, though there is debate in the literature regarding the poor relationship between amount of fibrils and disease progression. Nonetheless, there is a general consensus that the fibrils are related to the progression of these amyloid diseases. Several strategies to prevent or cure amyloid diseases include reducing the amount of amyloid beta (Aβ) produced and the removal of amyloid plaques. Inhibitors that prevent the formation of amyloid fibrils can prevent amyloid diseases from occurring. Hence, this thesis is concerned with the design and testing of amyloid inhibitors as possible therapeutics for these diseases. In order to achieve this objective, the key universal physical properties of amyloid fibrils involved in their self-assembly have been used to design a generic class of fibril inhibitors. A design of an amphiphilic polymer with a hydrophobic backbone and hydrophilic side chains was proposed as a generic amyloid inhibitor and several compounds with this design were obtained. 3 model amyloid forming proteins: bovine insulin (BI), hen egg white lysozyme (HEWL) and Aβ were used to study the effect of these compounds on amyloid fibril formation. Suitable amyloid-forming conditions for these proteins were identified and fibril formation was monitored using Thioflavin T (ThT) fluorescence and other techniques. A naturally occurring compound that fits the proposed inhibitor design was identified and found to be effective at inhibiting amyloid fibril formation in all 3 protein systems. Unusually large fibrils were formed when incubating BI and HEWL with this natural compound and this has potential nanotechnological applications as nanowire templates. To further test the proposed structure, synthetic polymers based on the proposed structure with different chemical groups were produced and one of them, FA-diacid (“FA” was a designation used by the polymer science group of the University of Melbourne for divinylcyclopentane polymers), showed promising inhibitory capabilities. The FA-diacid was improved with the addition of larger hydrophilic side chains to produce the more effective inhibitors named PNGA and PNGE. Finally, glycoproteins based on the structure of AGP were produced and tested using the 3 proteins and were found to have some inhibitory ability. However, they were not as effective as PNGA or PNGE. The results show that compounds with the proposed inhibitor structure can be effective as a generic amyloid inhibitor, but further modification of the current compounds is needed to improve the effectiveness of these compounds as drugs. Further development on this class of chemicals can lead to the production of a new class of generic amyloid inhibitor that can be used to prevent and halt the progression of presently incurable amyloid diseases such as Alzheimer’s disease.
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ItemProtein stability in shear flow( 2010)Given that globular proteins show a strong conformation-function relationship, the stability of a native protein structure is essential for its function. Aberrant proteins, resulting from structural instabilities in native protein conformations, and consequent aggregation, evolve a gain-of-function pathogenesis which has serious implications in industry and medicine. Therefore, it is important to appreciate the key factors that perturb the solution conformation of protein systems leading to aggregation. Considering the fact that proteins generally function in solution form, and those solutions have an inherent tendency to flow, knowledge of the stability of protein solutions in shear flow is essential. This thesis employs a combination of spectroscopic and microscopic techniques to study the conformational dynamics and morphological transformations of bulk peptide/polypeptide solutions in both uniform and heterogeneous velocity gradients. Preliminary studies in this thesis demonstrate that protein denaturation and subsequent aggregation can be probed using intrinsic protein fluorescence. The induction of protein aggregation was found to be greatly enhanced in heterogeneous flow regimes. Studies in a well defined flow field, Couette flow, revealed that the hydrodynamic stress generated in such flow regimes induce the unfolding of the helical segments of natively folded insulin; a prerequisite for aggregation and amyloid fibril formation. Further analysis of the shear-effect on α-helical conformations was performed using the homopolypeptide poly-L-lysine as a model protein system. The results reveal that the shear-induced unfolding of α-helical segments depends on both the shear rate and the duration of its application. An assessment of the chain-length-dependence of this phenomenon revealed that, contrary to classical theory, the strain in a given flow field varies inversely with the chain-length of α-helical poly-L-lysine. Collectively, the results provide new insight into existing theories in polymer physics. More importantly, it provides quantitative information on the conformational dynamics of peptide/polypeptide solutions in shear flow. This report is relevant to quality control measures during the commercial isolation and purification of protein products, and might help explain the role of shear stress, originating from pulsatile blood flow, in protein misfolding diseases and vascular disorders.