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<p> The formation of soluble amyloid oligomers by polypeptide chains is the main pathogenic mechanism underlying several neurodegenerative disorders including some of the most common debilitating and aging-related illnesses such as Alzheimer's and Parkinson's diseases. However, the molecular basis of polypeptide oligomerization and amyloid formation is currently not fully understood. In this thesis the focus will be on the early steps of oligomer formation that precede the nucleation of amyloid fibrils, that are still reversible. The reversibility of these initial self-association equilibria makes them an attractive target for therapeutic intervention in the treatment of amyloid diseases. Specifically three general questions will be addressed: (a) What are the residues within a given polypeptide chain that mediate self-recognition? (b) What are the driving forces for self-association? (c) Is self-recognition coupled with conformation changes? </p> <p> The objective of this thesis is to provide initial responses to these key questions using as prototypical system the Ap (12-28) peptide, which has been previously proposed as a model for the initial self-association events that are linked to Alzheimer's disease. Given the flexibility of this peptide the main tool for its investigation will be Nuclear Magnetic Resonance (NMR) spectroscopy. Specifically, both classical (i.e., TOCSY and NOESY) and more novel (i.e. saturation transfer difference and off-resonance relaxation) NMR experiments were used to probe the soluble oligomers through the comparative analysis of samples with different monomer/oligomer distributions. The combined analysis of this integrated set of experiments reveals that while the residues in the central hydrophobic core (CHC) drive self-recognition, stable oligomers require a conformational change towards more folded structures that affects residues well outside the CHC. The conformational change occurring upon self-association thus effectively couples CHC and non-CHC residues. This model may also explain why mutations outside the CHC (i.e. E22, D23) can affect significantly the kinetics of self-association. </p> <p> / Thesis / Master of Science (MSc)

Current dogma suggests modulating or preventing amyloid assembly will prove critical to the armamentarium of therapeutic interventions that will likely be required to overcome the multifaceted pathology associated with amyloid diseases. The work described in this dissertation reveals substantial gains in understanding key aspects relating to the anti-amylin amyloid activities associated with both individual and broad groups of small molecule amyloid inhibitors. A main observation was the important role that the catechol functional group plays in modulating and preventing amyloid formation. In this context, each chapter provides unique yet complementary mechanistic insight that delineates a wide range of anti-amyloid activities associated with preventing amylin amyloid formation by mainly catechol-containing structural scaffolds. Structure activity studies show that the catechol moiety present within baicalein, oleuropein and rosmarinic acid are critical for their anti-amyloid functions, including exerting cell rescue effects against amylin induced cytotoxicity. We also demonstrate that in general, autoxidation enhances the anti-amyloid potency associated with many catechol containing amyloid inhibitors that may be mechanistically linked to a covalent mode of action. For example, we demonstrate that the O-quinone form of baicalein conjugates with amylin via a Schiff base mechanism. In contrast, we also show that catechol mediated formation of protein denaturant resistant aggregates, which requires autoxidation and that also stems from a predicted covalent mode of action, does not necessarily correlate with the enhanced anti-amyloid activities that occur upon catechol autoxidation. Regardless of the chemical mechanism(s) that drive catechol mediated anti-amyloid activity in vitro, the observed cell rescue effects exhibited by catechol containing molecules against amylin amyloid induced cytotoxicity is congruent with several recent in vivo studies that indicate polyphenols prevent toxic amyloid deposition as well as decades of population based studies that show regular consumption of diets rich in polyphenols are linked to a reduce incidence of age-related neurodegenerative amyloid disease. Indeed, advances in structure based drug discovery against amyloid formation may provide new avenues to optimize various catechol containing scaffolds that could be readily leveraged into improving diagnostic tools or perhaps accelerate the effort of discovering anti-amyloid therapeutics. / Ph. D. / From causing dementia in diseases like Alzheimer’s disease (AD) to potentiating type 2 diabetes (T2D), amyloid diseases represent some of the most devastating and increasingly more common human diseases. Amyloids themselves mainly consist of an aggregated mass of a specific type of protein that is believed to be either directly or indirectly toxic. Currently, there are no known cures for preventing amyloid diseases, and so far, efforts to discover anti-amyloid therapeutics have been largely unsuccessful. Many studies indicate regular consumption of plant-based diets, like the Mediterranean diet, that includes foods such as olives, vegetables and red wine leads to reduced incidence of age related amyloid diseases. Guided by these data, scientists have begun to uncover specific molecules within these diets that are able to prevent amyloid formation. A main emphasis in this dissertation was to understand the details of how these molecules prevent toxic amyloid formation. The insights gained from these studies have elucidated key chemical structural features present within these molecules that convey unique effects on perturbing amyloid formation. Excitingly, we also found that the presence of oxygen within the air we breathe, interacts with and enhances the ability of these compounds to exhibit stronger anti-amyloid functions! These data can be used to engineer better amyloid inhibitors that could lead to drugs.

The proteins wheat gluten and gelatin were tested for use in biocomposites and soft actuating materials, respectively. In Chapter II, the self-assembly mechanism of trypsin hydrolyzed wheat gluten (THWG) into rigid β-sheets was applied to an aqueous polyvinyl alcohol (PVA) environment. Aqueous PVA was used in order to determine the effects of an aqueous environment other than pure water on THWG self-assembly kinetics and to realize the potential use of THWG as a nanofiller in polymer matrices. THWG was able to self-assemble into anisotropic spikes and agglomerates of spikes called "pompons" through hydrophobic interactions. THWG self-assembly kinetics were retarded in aqueous PVA solutions compared to water, with the highest molecular weight PVA solution showing the slowest self-assembly kinetics. Chapters III and IV explore the potential of gelatin hydrogels for use in soft actuators. A gelatin bilayer system was designed where an active layer swelled more than a passive layer to cause the system to bend/actuate in response to an environmental stimulus. In Chapter III, gelatin layers were chemically crosslinked to different degrees with glutaraldehyde to achieve bilayer bending when placed in water. Curvature of the bilayer system was found to be dependent on the difference in volume swell ratio between the two layers. It was determined that maximum bending occurred when the passive layer swelled to 60% of the swelling of the active layer. Addition of pre-gelatinized starch to the active layer increased layer swelling and bilayer curvature. Treating the starch containing bilayer with -amylase returned the bilayer to its original shape. In Chapter IV, a pH responsive gelatin bilayer was constructed using Type A and Type B gelatin. Type A and Type B gelatin gels had different chemical properties and swelled to different volumes based on the gel solution pH. Bilayers constructed from Type A and Type B gelatin exhibited different degrees of bending when placed in various pH solutions with maximum curvature occuring at pH 10. A cyclic actuator could be formed when the bent bilayers were placed in a minimum of 0.01M NaCl solution. Placement in salt solution resulted in the unbending of the bilayer. Overall, this work demonstrated the various applications of proteins as functional and green materials. / Doctor of Philosophy / The majority of plastics consist of synthetic polymers derived from oil that cannot be broken down by the environment (i.e., not biodegradable). Research is underway to develop sustainable, biodegradable materials. Proteins are a biological polymer that have a wide range of chemical, structural, and functional properties; for this reason they are an excellent source material for use in the design of environmental friendly materials. In Chapter II, the ability of wheat gluten protein to self-assemble into rigid, nanosized structures is used to explore the potential of the protein to be used as a biodegradable nanofiller. A nanofiller is added to various materials in order to improve the overall mechanical properties of the material. Wheat gluten is self-assembled in an aqueous polymer environment. The results show that the polymer environment stunts or slows down the self-assembly rate of the protein compared to a pure water environment. Nanometer sized spikes form in the polymer solutions, indicating wheat gluten could be used as a nanofiller in certain materials. Chapters III and IV explore the use of gelatin proteins for applications in soft robotics. Soft robots and their moveable parts, called soft actuators, are deformable and respond to changes in the environment such as pH, light, temperature, etc. For this reason, soft robots are considerable adaptable compared to traditional rigid robots. Designing a soft actuator from gelatin gels would result in a “smart” material that is biocompatible and biodegradable. A gelatin soft actuator is created using a bilayer design in which one layer of the bilayer swells more than the other layer causing the entire system to bend/actuate. Depending on how the bilayer system was fabricated, bending could be achieved based on stimuli such as the presence of water, the presence of a substrate and enzyme, and changes in pH. Overall, this dissertation demonstrates the extraordinary potential for the use of proteins in designing sustainable materials.

Sporadic Inclusion Body Myositis (sIBM) is an inflammatory muscle disease that strikes individuals at random and accounts for approximately 1/3 of all idiopathic inflammatory myopathies. It is characterized by progressive weakness of distal and proximal muscles and is the most common muscle disorder in individuals over 50 years of age. Currently, there is no known cause, cure, or enduring treatment for sIBM, although a number of theories as to its cause have been proposed. One theory proposes that activation of the inflammatory/ immune response is the primary trigger resulting in muscle degeneration and protein abnormalities, while an alternative theory suggests that sIBM is a degenerative muscle disease with abnormal pathogenic protein accumulation, in particular Abeta, being a primary cause that triggers an inflammatory/ immune response. Mitochondrial abnormalities have been observed in skeletal muscle from patients diagnosed with the disease, however the role of the mitochondria in disease pathology is still unclear. The aim of this dissertation was to evaluate: 1) the role of the mitochondria in the development of sIBM and 2) the role of amyloid beta on mitochondrial function in skeletal muscle. A better understanding of the role of the mitochondria in the development of sIBM may help to identify novel prevention and/ or treatment strategies. / Ph. D.

Yes / Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer’s disease. These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease modifying therapies for Alzheimer’s disease, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of Alzheimer’s disease. A small number of the four hundred transporter superfamilies’ are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in Alzheimer’s disease. Here we review the current evidence for six astrocytic transporter subfamilies involved in Alzheimer’s disease, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling Alzheimer’s disease. / BBSRC, Alzheimer's Society, Motor Neuron Disease Association