This dissertation describes the work done on two distinct projects. In the first part I sought to unravel the mechanisms that underlie the activity-dependent modulation of response in the excitation-secretion coupling of the neurohypophysis. In the second part, I optically monitored and analyzed the secondary structure changes accompanying amyloid fibril formation along multiple pathways, under both denaturing and near-physiological conditions. Neuronal plasticity plays an important role in regulating various biological systems by modulating release of hormones or neurotransmitters. The changing response to the same stimulus, depending on the context and previous stimulation events, is also the basis of learning and all higher order brain functions. The mechanisms behind this modulation are widely varied, and are often poorly understood in specific tissues. In this work, we examined excitation-secretion coupling in the neurohypophysis, a tissue composed of densely packed axons that secretes the hormones arginine vasopressin and oxytocin. The release of hormones depends not only on the overall level of activity in the gland, but also upon the specifics of the temporal pattern of stimulation. By optically monitoring the electrical activity using voltage sensitive dyes, we were able to investigate this plasticity in the intact gland. Varying extracellular potassium concentration in the bath, increasing interstitial space via hypertonic saline, and retarding potassium reuptake with ouabain all showed that extracellular potassium accumulation drives the depression of excitability. This effect is hidden from glass micro-electrode recordings because of the inevitable damage sustained by the surrounding tissue. Furthermore, no calcium mediated release mechanism played any significant role in the depression. Numerical simulations confirmed the findings and give more insight to the details of the mechanism. Deposits of amyloid fibrils, long, unbranched polymeric protein aggregates, are the molecular hallmark for a variety of human diseases, including Alzheimer's disease, Parkinson's disease, and type II diabetes. While the amyloid fibrils all share a characteristic cross-beta sheet structure, the proteins that make up the aggregates have no unifying theme in either native structure or function. In this research, I characterized the structural reordering that accompanies this aggregation using Fourier transform infrared spectroscopy (FTIR). Hen egg white lysozyme forms fibrillar aggregates with two distinct morphologies, depending on the growth conditions. At acidic pH with low ionic concentrations, lysozyme forms the fibrils with standard amyloid morphology. These aggregates are long and stiff but with the cross sectional area of a single monomer. At higher salt concentrations, the aggregation follows another pathway, under which oligomers initially form and later assemble into protofibrils. The oligomeric protofibrils are thicker than the monomeric filaments, but are much more curvilinear. These fibrils are not universally recognized as amyloidogenic aggregates. Using FTIR, I showed that both this aggregates are indeed amyloid structures, but that they are structurally distinct. While it is generally accepted that partial unfolding of the protein is a prerequisite for amyloid fibril formation, we found that native protein can be the substrate for amyloid growth when seeded with preformed oligomeric or protofibrillar aggregates. These seeded fibrils grown under near-physiological conditions are structurally indistinguishable from those grown from partially unfolded protein under denaturing conditions. This incorporation and restructuring of native monomers is characteristic of prion-like assembly.

The broad objective of my research is to investigate the physical characteristics and interactions of macromolecules and nanoparticles, and the corresponding effects on their phase separation behavior using static and dynamic light scattering (SLS & DLS). Light scattering provides a non-invasive technique for monitoring the in-situ behavior of solutes in solution, including solute interactions, sizes, shapes, aggregation kinetics and even rheological properties of condensed phases. Initially, we investigated lysozyme solutions for the presence of preformed aggregates and clusters that can distort the kinetics of protein crystal nucleation studies in this important model system for protein crystallization. We found that both undersaturated and supersaturated lysozyme solutions contained population of large, pre-existing protein aggregate. Separating these clusters and analyzing their composition with gel chromatography indicated that these clusters represented pre-formed lysozyme aggregates, and not extrinsic protein contamination. We investigated the effect of chaotropic versus kosmotropic ions (water structure breakers vs. structure makers) on the hydration layer and hydrodynamic interactions of hen egg white lysozyme. Surprisingly, neither chaotropic nor kosmotropic ions affected the protein hydration layer. Salt-effects on direct and hydrodynamic protein interactions were determined as function of the solutions ionic strength and temperature. Using both static and dynamic light scattering, we investigated the nucleation of gold nanoparticles forming from supersaturated gold sols. We observed that two well separated populations of nuclei formed essentially simultaneously, with sizes of 3nm vs. several tens of nanometer, respectively. We explore the use of lysozyme as tracer particle for diffusion-base measurements of electrolyte solutions. We showed that the unusual stability of lysozyme and its enhanced colloidal stability enable viscosity measurement of salts solutions at high salt concentration, over a wide range of pH values and temperatures for the common tracer particle polystyrene flocculates. We applied dynamic light scattering to measure the viscoelastic responses of polystyrene probe particles embedded in solutions and gels of two different polymers: polyacrylamide (PAAm) and poly-N-isopropylacrylamide (poly-NiPAAm).

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Glycation is a series of chemical interactions occurring in food and biological systems between reducing sugars and proteins leading to the formation of Advanced Glycation End products (AGEs). Ingestion of dietary AGEs and/or their formation in-vivo are mainly associated with cardiovascular and other age-related diseases and complications of long term diabetes. Potential strategies to prevent AGE formation can help to reduce risk factors associated with thermal processing of many foods. The overall objective of this research was focused on the identification of potential AGE inhibitors and investigation of their activity in glucose and ribose-based model systems containing lysozyme. The carboxylic acid functional group was chosen as a potential candidate based on their ability to interact with Schiff bases in addition to their ability to form amide bonds and carboxylate salts with the lysine side chains of proteins. The model systems were incubated with and without selected carboxylic acids (maleic, acetic, oxalic and citraconic) at 50°C for 12, 24 and 48h at pH 6.5. The effect of carboxylic acids on the glycation of lysozyme was studied by electrospray ionization mass spectrometry (ESI-MS). The experimental results showed that none of the carboxylic acids were able to form amide linkages with lysozyme under the experimental conditions and only maleic acid was able to form carboxylate salts, however, oxalic acid was the only acid able to interact with the Schiff base and form 1,3-oxazolidine-4,5-dione intermediate and thus hinder its rearrangement into Amadori product and consequently inhibit glycation. As a result the percentage of free or unreacted lysozyme was the highest in oxalic acid model systems (9.4% in the case of glucose, 7.1% in the case of ribose system) and was even higher than the control systems (6.0% in the case of glucose, 1.2% in the case of ribose system) of both glucose and ribose. In addition, all carboxylic acids were able to modify the relative distribution of different glycoforms generated during the incubation period however oxalic acid was the most efficient in shifting the distribution of glycoforms to lower molecular weight clusters which can additionally contribute to its anti-glycation activity.

In vitro assays were developed using small intestinal contents from broilers fed maltodextrin in preparation for shipping. This was done to establish an effective bactericidal dose of allicin or lysozyme as ingredients in maltodextrin-based feed. The antimicrobials were added to overnight cultures of gut material bacteria from maltodextrin fed broilers and a pure culture of Salmonella. Following this, lysozyme was incorporated into maltodextrin feed at 0, 10 and 20 g.kg-1 of feed and offered for 9 h to 4 pens of 20 birds per treatment. Bacterial numbers were analyzed using Proc Mixed of SAS. Allicin and lysozyme inhibited Enterobacteriaceae and Clostridium perfringens, respectively, in vitro. Lysozyme showed the most promise; it reduced bacterial numbers in nutrient broth. Feeding lysozyme-enriched maltodextrin for 9 h inhibited bacilli growth (P<0.05) when evaluated using next generation sequencing. Lysozyme was effective in reducing specific bacterial numbers in the gut of market-aged broilers / The project focused on ways to ensure poultry meat safety by controlling bacteria population in the gastrointestinal tract of market-aged broilers prior to shipping.

Dietary supplements were provided during 24 hour transport from hatchery and growth of turkeys in two trials. Female poults (768 and 825 respectively) were used in two 3 x 4 factorial analyses (transportation supplement x post placement supplement) with treatment provided during transport (no supplement, Oasis® and Oasis® + lysozyme (0.01%)) and as dietary supplements post-placement (no supplement, Bacitracin Methylene Disalicylate (BMD)(ANTI), lysozyme (LYS), BMD + lysozyme (AL)) as the main effects. Growth, incidence of mortality, gastrointestinal size, strength and histology and behavioural data was collected. Transport supplementation of poults did not improve growth or reduce mortality, but influenced early feeding and drinking behaviour at placement. Body weight and feed consumption increased and percent mortality decreased for birds fed AL. Gizzard and proventriculus weight increased when birds consumed ANTI and jejunal breaking strength was highest for birds consuming LYS. Villi morphology and bird behaviour were not affected by dietary supplementation.

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The reactions of the early and intermediate stages of protein glycation were studied using bovine serum albumin and hen egg white lysozyme as model proteins, and D-glucose and D-fructose as model reducing sugars. The reactions were studied under low moisture and limiting sugar conditions in the presence and absence of oxygen in a closed system. Several analytical methods, including the fluorescamine assay, total Maillard fluorescence and solubility measurement, size exclusion chromatography, and electrospray ionization mass spectrometry were used to follow the glycation reaction. The effect of glycation on the structure of proteins was also studied by electrospray ionization mass spectrometric peptide mapping. The results showed that under low moisture conditions, the initial rate of glucation was 5 to 6 times higher than the initial rate of fructation, both in the presence and in the absence of oxygen. The presence of oxygen in the reaction system induced selectivity of the glycation of the protein amino groups during the initial stages of the reaction, due to competition between glycation and glycoxidation. The reaction, however, occurred at a higher rate in the presence of oxygen than in its absence, especially during the latter stages of the reaction. The more reactive glycoxidation products, formed during the initial stages of the reaction also participated in the glycation reaction and increased the involvement of arginine residues in the glycation reaction. The glycation reaction also resulted in a heterogeneous mixture of protein glycoforms with varying degrees and extent of glycation. The heterogeneity of the glycated species was further complicated by the involvement of glycoxidation products in the glycation reaction, resulting in a distribution of different classes of glycoconjugates. Limited glycation improved protein solubility and increased solvent accessibility into the interior of the protein, thereby, increasing protein digestibility. Electrospr