Neurodegenerative diseases are characterized by selective neuronal vulnerability and subsequent degeneration in specific areas of the brain. Huntington’s Disease (HD) is inherited as an autosomal dominant mutation that primarily affects the cells of the striatum and the cerebral cortex, leading to a triad of symptoms that include the progressive loss of motor function, defects in cognitive ability and psychiatric manifestations. HD is caused by a CAG repeat expansion that exceeds 37 repeats in Exon1 of the ​HTT​ gene, manifesting as a pathogenic polyglutamine (polyQ) amino acid tract expansion in the huntingtin protein. HD is a late onset disorder, with disease onset around 40-50 years of age and symptoms that worsen over 10-20 years. Only a few symptomatic treatments are available and there is currently no cure for the disease. Therapeutics to target the huntingtin gene itself have only been in clinical trial in the past 2 years. The length of the expansion has an inverse relationship with the age of disease onset. Most patients that have repeats between 40-45 CAG, however, have varying age of disease onset. Recent genome-wide association studies (GWAS) have implicated DNA handling and repair pathways as modifiers of age of disease onset up to 6 years. Therapeutic approaches to modify and delay onset indefinitely through other genetic targets will require identification of pathological mechanisms that precede disease onset. Several hallmark phenotypes have been identified in cell and animal models, including pathogenic aggregate formation. These models are not reflective of human biology, using excessively large CAG repeats (>100) associated with the more aggressive, juvenile HD, overlooking the importance of GWAS results and the progression of disease with lower pathogenic CAG repeats (40-50 CAG). We have therefore generated novel, clinically-relevant human patient fibroblast cell lines and have characterized several disease phenotypes. My thesis presents a culmination of several projects that focus on disease modelling, primarily outlining phenotypic differences between wildtype and HD cells that will benefit our understanding of disease pathogenesis. / Thesis / Doctor of Philosophy (PhD)

The most thermodynamically stable biological structure is the cross-beta secondary structure of the "amyloid"or "prion". As a testament to its stability, the amyloid occurs naturally in 2 rare instances: as a mechanism to protect or destroy life. Pathogenic amyloids are the signature of neurological disorders such as Alzheimer's and Parkinson's disease and bovine spongiform encephalopathy (BSE), which have no effective treatments or known cures. Pathogenic amyloids appear as nanometer sized "plaques" that self-assemble over time. The plaques usually are well-organized crystalline/fibrous structures ~10-20 nm in diameter and >100 nm long. "Functional" amyloids are very rare in nature and serve the direct purpose to proliferate life. Stalks to protect eggs, fibers to coat spores, and adhesive proteins of bacteria, algae, fungi, and mollusks are examples. Functional amyloids can be larger than pathogenic amyloids by 1-2 orders of magnitude. There is a burgeoning research field based on emulating the amyloid. This is because it can be formed from a host of proteins or peptides simply by denaturing them enough to form a cross-beta secondary structure and has a modulus of >10 GPa. As a general reference, "protein" is usually a very high molecular weight, naturally occurring molecule and "peptide" is a much smaller portion of a natural protein or a non-natural molecule synthesized from a few amino acids. Researchers are increasingly attempting to take advantage of the functional amyloid. It is still not understood how the functional amyloid self-assembles or why it can be larger than the pathogenic amyloid. We have identified a potential pathway to large functional amyloids that involves a long alpha-helix containing protein (the "adder") undergoing an alpha to beta transition in the presence of a hydrophobic beta-sheet template. Testing our hypothesis against proteins found in natural large functional amyloids seems to suggest it is a ubiquitous process. The resulting material is a fiber composite similar to the native structures. / Master of Science

Healthy articular joints exhibit remarkable lubrication due in large part to the complex rheological and tribological behavior of the synovial fluid (SF) that lubricates the joints. Current approaches that seek to elucidate such remarkable lubrication usually focus on the roles of high molecular weight SF components such as lubricin and hyaluronic acid but frequently overlook the role of serum albumin (SA), although it represents 90% of the protein content of SF. In this thesis, we used the Surface Forces Apparatus to investigate in detail the structural and tribological response of SA thin films when sheared between model surfaces and subjected to a large range of shearing parameters. Our data indicate that, under shear, SA films reproduce closely the shear response previously reported for SF, i.e., film thickening and formation of numerous long-lived aggregates accompanied by low friction and efficient surface protection against damage. More specifically, our detailed investigation of shear parameters reveals that (i) strong anchoring of SA to surfaces promotes the formation of large rod-like shaped aggregates that enable rolling friction and keep surfaces far apart, preventing damage, (ii) aggregation mechanism is irreversible, which makes aggregates long-lived (though mobile) in the contact, and (iii) aggregate formation only occur when SA was sheared above a ‘critical’ amplitude Ac and a critical shear velocity Vc. Collectively, our results provide experimental evidence of the role of globular proteins, such as SA, in lubrication and establish a correlation between shearing parameters, formation and stability of aggregates, low friction and wear protection. Although our findings are based on experiments involving rigid, nonporous surfaces hence can hardly be generalized to compliant and porous cartilage surfaces, they are applicable to other rigid tribosystems such as artificial joints and will certainly advance our understanding of joint implants’ lubrication in SF mediated by protein aggregation, with implications for future design of artificial joints and therapeutic interventions.

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg·HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg·Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg·Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg·Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg·Glu on cell viability was compared against NaCl, Arg·HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg·Glu in the cell culture models studied, therefore Arg·Glu has the potential as an excipient as it reduces aggregation and is nontoxic. Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.

Protein aggregation is a frequently cited problem during the development of liquid protein formulations, which is especially problematic since each protein exhibits different aggregation behaviour. Aggregation can be controlled by judicious choice of solution conditions, such as salt and buffer type and concentration, pH, and small molecule additives. However, finding conditions is still a trial and error process. In order to improve formulation development, a fundamental understanding of how excipients impact upon protein aggregation would significantly contribute to the development of stable protein therapeutics. The underlying mechanisms that control effects of excipients on protein behaviour are poorly understood. This dissertation is directed at understanding how excipients alter the conformational and colloidal stability of proteins and the link to aggregation. This knowledge can be used for finding novel ways of either predicting or preventing/inhibiting protein aggregation. Experiments using static and dynamic light scattering, intrinsic fluorescence, turbidity and electrophoretic light scattering were conducted to study the effect of solution conditions such as pH, salt type and concentration on protein aggregation behaviour for three model systems: lysozyme, insulin and a monoclonal antibody. Emphasis is placed on understanding the effects of solution additives on protein-protein interactions and the link to aggregation. This understanding has allowed the rational development of stable formulations with novel additives, such as arginine containing dipeptides and polycations.

Going down the folding funnel, proteins may sample a wide variety of conformations, some being outright detrimental to the organism. Yet, the vast majority of polypeptide molecules avoid such pitfalls. Not only do they reach the native minimum of the energy landscape; they do so via blazingly fast, biased, routes. This specificity and speed is remarkable, as the surrounding solution is filled to the brim with other molecules that could potentially interact with the protein and in doing so stabilise non-native, potentially toxic, conformations. How such incidents are avoided while maintaining native structure and function is not understood.  This doctoral thesis argues that protein structure and function can be separated in the folding code of natural protein sequences by use of multiple partly uncoupled factors that act in a concerted fashion. More specifically, we demonstrate that: i) Evolutionarily conserved functional and regulatory elements can be excised from a present day protein, leaving behind an independently folded protein scaffold. This suggests that the dichotomy between functional and structural elements can be preserved during the course of protein evolution. ii) The ubiquitous charges on soluble protein surfaces are not required for protein folding in biologically relevant timescales, but are critical to intermolecular interaction. Monomer folding can be driven by hydrophobicity and hydrogen bonding alone, while functional and structural intermolecular interaction depends on the relative positions of charges that are not required for the native bias inherent to the folding mechanism. It is possible that such uncoupling reduces the probability of evolutionary clashes between fold and function. Without such a balancing mechanism, functional evolution might pull the carpet from under the feet of structural integrity, and vice versa. These findings have implications for both de novo protein design and the molecular mechanisms behind diseases caused by protein misfolding. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

In quiescent yeast, the widespread reorganization of cytosolic proteins into punctate has been observed (Narayanaswamy et al. 2009). We seek to better understand and describe this reorganization, which we hypothesize to be a protein aggregation phenomenon. To test this hypothesis, we examined mutant proteins (Ade4p protein variants) in yeast with predicted non-native aggregation propensities and measured their punctate formation kinetics. Monitoring punctate formation kinetics involved the validation of an automated quantification technique using an Amnis ImageStream imaging flow cytometer. The automated punctate counts were strongly correlated with the manual punctate counts, with usual R² values of 0.99 or better, but evaluated 50-fold more cells per run. Fitness evaluations of the mutant yeast in the form of growth curves and batch competition experiments revealed the slowed growth of the Ade4-1286 strain and the functional inequality to the wild type strain of the Ade4-mtoin2034, Ade4-mtoin2105, and Ade4-2800 strains in competition experiments, especially when the mutants were forced to generate their own adenine. Subsequent structural analysis of the mutant proteins revealed destabilizing mutations for 4 of the 6 mutant proteins with 2 of the mutations classified as significantly destabilizing ([delta][delta]G >2 kcal/mol). We concluded that the reduction in protein fitness was likely due to the destabilizing effects of the mutations. Evaluation of the punctate formation kinetics revealed little difference between strains in the rate of punctate formation. Further examination revealed the wild type Ade4p and all of the mutants (with the exception of the Ade4-1286 mutant) were predicted to have similar aggregation propensities according to a secondary aggregation predicting algorithm (Zyggregator, Pawar et al. 2005). Additionally, solvent accessibility calculations estimate ~3-19% of the side chain surface area to be solvent accessible, which indicates proximity of mutations to the protein surface. However, mutating buried amino acids likely would have generated a greater disturbance (Matthews 1993, Tokuriki et al. 2007). We concluded that the mutations, although destabilizing, altered the aggregation propensity very little. Deletion of chaperone proteins (Hsp82p, Hsc82p, and Ssa1p) revealed no difference in the Ade4-GFP punctate formation kinetics, although a slight kinetic difference was detected in the chaperone (Hsp82p) knockout, Gln1-GFP strain and the wild type strain. While further workup is necessary in the chaperone knockout, Gln1-GFP work, the initial results are promising and suggest the involvement of protein folding machinery in punctate formation. / text

The aggregation of alpha-synuclein (αS) protein from soluble monomer into solid amyloid fibrils in the brain is associated with a range of devastating neurodegenerative disorders such as Parkinson’s disease. Soluble oligomers formed during the aggregation process are highly neurotoxic and are thought to play a key role in the onset and spreading of disease. Despite their importance, these species are difficult to study by conventional experimental approaches owing to their transient nature, heterogeneity, low abundance and a remarkable sensitivity of the oligomerisation process to the chosen experimental conditions. In this thesis, well-established single-molecule techniques have been utilised to study the aggregation and oligomerisation of αS in solution.

The increasing demands for biopharmaceuticals to treat different diseases have raised concerns about controlling the quality and efficacy of such pharmaceuticals. The design and formulation of a stable protein or peptide based biopharmaceutical runs into the limitation that at high concentrations (> 100 mg/ml) or during long storage process the drug undergoes aggregation. During synthesis, purification, storage or packaging of these drugs different kinds of stresses like chemical, oxidative, thermal, shear, etc. are encountered. These stresses promote the non-native aggregation of protein and peptide based drugs. Injection or administration of such drugs if contaminated with aggregates causes patient discomfort or development of an antibody which can adversely affect patient’s conditions. This brings out the necessity of finding a way so that such aggregation is avoided. Nanoparticles have been used as vehicles for drug delivery and diagnostic agents in biology for a while. The surface of the nanoparticles is known to adsorb small as well as large molecules with different kinetics and energetics of interaction. I have used nanoparticles to adsorb proteins to protect them against aggregation when they are subjected to denaturing conditions. The effectiveness of the nanoparticles in stopping protein aggregation, recovery of the proteins and reversibility of the adsorption process, the catalytic activity of the proteins before and after adsorption on the surface have all been studied in details. The work described here has been divided in 8 chapters and the contents of each chapter are described below. In Chapter 1 I have provided a brief introduction to the protein aggregation problem. The motivation and scope of the current work has been presented in this chapter. Materials and methods have been described in Chapter 2. Synthesis of gold and silica nanoparticles, their characterization and stability under experimental conditions have been illustrated in this chapter. The spectroscopic assays and techniques which I have used to study the effect of gold and silica nanoparticles on protein aggregation have been discussed at lengths in this chapter. In Chapter 3 I have demonstrated the effect of gold nanoparticles on thermal aggregation of alcohol dehydrogenase (ADH). The size of the nanoparticle was varied in the range of 15-60 nm and the effect was measured by various spectroscopic assays and techniques. I have observed that gold nanoparticles prevent thermal aggregation of ADH and the efficiency is high. Gold nanoparticles in nanomolar or even picomolar concentrations are capable of preventing the aggregation of ADH at micromolar concentrations. In Chapter 4 the role of gold nanoparticles as suppressor of protein aggregation was extended to another protein, insulin. Chemically induced aggregation of insulin using dithiothreitol (DTT) in the presence of gold nanoparticles was studied in the same manner as was done for ADH. Similar prevention property of gold nanoparticles was established by making the observation independent of the method of denaturation or the type of protein used in the prevention experiments. In Chapter 5 huge second harmonic light scattering (SHS) signal from pure gold nanoparticles has been used to measure the free energy of interaction of ADH and insulin with nanoparticles in solution, for the first time. The change in the second harmonic scattered signal was monitored which decreased steadily as a function of added protein concentration to the aqueous solution of gold nanoparticles. The fitting of the second harmonic signal decay was done with a modified Langmuir adsorption isotherm to extract the free energy change in the interaction and the number of protein molecules adsorbed on the surface. In Chapter 6 I have demonstrated a way to recover the adsorbed ADH and insulin from the gold nanoparticle surface and tested the activity of ADH by an assay. The structure of the proteins in the adsorbed state has been probed by CD spectroscopy and described in this chapter. It is found that ADH retains its activity in the adsorbed state. Both the proteins retain the native secondary structures in their adsorbed state. However, the structures change drastically under denaturing conditions. In Chapter 7 the effect silica nanoparticles which are known to have hydrophilic surface has been examined on the aggregation of ADH and insulin in pretty much the same way as was done with gold nanoparticles. The efficiency of silica nanoparticle was found to be lower compared to gold nanoparticles. In addition, the size dependency of prevention efficiency of silica and gold nanoparticles was found to be completely opposite to each other. In Chapter 8 I have presented the overall summary and possible future directions of this work

In the past forty years advancements in fluorescence-based methods including imaging (e.g. confocal and multi-photon) and quantitative spectroscopies (e.g. Fluorescence Correlation Spectroscopy) have been applied to systems ranging from solutions to in vivo models: such methods possess the ability to monitor fluorescence intensity fluctuations and offer the potential to unravel biophysical and biochemical phenomena. A major disadvantage associated with these methods is their ever-increasing cost resulting in the development of image analysis tools that offer the potential to exploit hidden information contained in confocal images.The hypothesis pertaining to this thesis is that image analysis tools developed in recent years exemplified by Raster Image Correlation Spectroscopy (RICS), Spatial Intensity Distribution Analysis (SpIDA) and Fluorescence Intensity Gaussian Mixture Model Analysis (FIGMMA) will provide a new insight into current pharmaceutical problems. The application of these methods to the quantification of protein aggregation, monomer/dimer equilibria, p-glycoprotein efflux activity and transcytosis are presented in this thesis.Protein aggregation poses a major challenge to the biotechnology industry which currently lacks analytical capabilities to profile broad particle size ranges. An in-house RICS (ManICS) software was validated against Dynamic Light Scattering and Fluorescence Correlation Spectroscopy (FCS) to determine Bovine Serum Albumin (BSA) aggregate population distributions under accelerated stability conditions. Initial stages implicated in the growth of aggregates are vital to the mechanistic assessment of protein aggregation. Hence, real-time in situ examination of monomer loss and aggregation of BSA was performed at 50 °C to enable continuous assessment with imaging and subsequent SpIDA analysis. Results obtained from this study suggested reversible fluctuation between monomers and dimers for up to four hours.To correlate membrane receptor and transporter expression with activity and enable the comparison of expression in multiple cell lines, population densities of p-glycoprotein transporters and transferrin receptors were determined using SpIDA in samples subjected to immunofluorescence labelling.The Calcein retention assay is a routine approach to determining multidrug resistance associated with p-glcoprotein efflux and the traditional plate reader approach omits microscopic aspects of p-glycoprotein Calcein-AM uptake and efflux. Confocal microscopy and data obtained from image analyses supported the subcellular and intercellular assessment of Calcein accumulation in MDR1-transfected and control cell lines as a function of time and verapamil concentration. Finally, live cell imaging of transferrin vesicular transport and Cell TraceTM Calcein red-orange AM internalisation in combination with traditional Transwell® assays were assessed to compare their transcellular transport and intracellular concentrations in multiple cell lines. Images obtained enabled visualisation of internalisation and following analysis using SpIDA, RICS and FIGMMA the number of intracellular vesicles and dynamic parameters of Cell TraceTM Calcein red-orange diffusion and intracellular concentration were determined.In conclusion, image analysis tools were applied to providing new parametric insights into a number of pharmaceutically-relevant processes and in some instances this is the first example of such studies. Despite current phenomenal advances in image acquisition capabilities, there remains a broad scope for the validation of image analysis tools and their application to a multitude of areas of interest to pharmaceutical and biomolecular research.