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Amyotrophic lateral sclerosis (ALS) is a devastating, progressive, and fatal neurodegenerative disease. Despite the fact that ALS is the most common motor neuron disease in adulthood, there is no effective treatment for the disease. Although most ALS cases (90-95%) are sporadic (sALS), the remaining cases (5-10%) are dominantly inherited and referred to as familial ALS (fALS). Because sALS and fALS show indistinguishable disease symptoms, a common disease mechanism has been proposed. After the discovery of the link between fALS and mutants of cytosolic Cu/Zn superoxide dismutase (SOD1), over 140 mutations in SOD1 have been identified to account for ~20% of fALS. The location of these mutants are scattered throughout the primary and tertiary structures of the protein. It is widely accepted that fALS-linked mutations in SOD1 result in a gain of toxic function to cause the disease, rather than a loss of physiological function, although the nature of the toxic mechanism remains unclear. SOD1 is a -rich, homodimeric metalloenzyme that catalyzes the dismutation of superoxide radicals to O2 and H2O2. The protein is ubiquitously expressed and the mature form of SOD1 (holo SOD1) contains one catalytic Cu ion, one structural Zn ion, one intra-molecular disulfide bond (between C57 and C146) and two free Cys residues (C6 and C111) per 153 residue subunit. Analogous to many different human diseases in which protein aggregation is a hallmark, aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in the pathogenesis of ALS. This thesis reports the first observation of aggregation of the most abundant form of SOD1 in vivo, the native, metallated (holo) dimer, under physiologically relevant conditions (37 °C and pH 7.8). The medical relevance of aggregates is demonstrated by structural and tinctorial analyses as well as the novel observation of binding of an anti-SOD1 antibody that specifically recognizes pathological aggregates in ALS. Additionally, ALS-associated SOD1 mutations promote aggregation but are not required, supporting a common mechanism in familial and sporadic ALS. The aggregation is characterized by a lag phase, which is diminished by self- and cross-seeding where heterogeneous nucleation is the underlying mechanism. Moreover, multiple pathways of aggregation are elucidated including dimer dissociation and metal loss. It is shown that if holo SOD1 loses more Zn ions than Cu ions, the aggregation profiles have shorter duration and lower final intensity, whereas when holo SOD1 loses more Cu ions than Zn ions, the aggregation profiles have longer duration and higher intensity. Taken together, the data in this thesis establish a valuable system for understanding SOD1 aggregation and toxicity mechanisms which can be used for developing therapeutic strategies targeting protein aggregation.

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An increasing number of diseases are associated with protein misfolding, one type of which results in the formation of amyloid fibrils. This research has addressed the hypothesis that all proteins can form amyloid fibrils and investigates what factors protect proteins from forming these macromolecular assemblies. Most analyses of the aggregation propensity of proteins have been limited to the properties of the amino acid sequence, thus fail to consider the roles that higher levels of organisation play in protecting polypeptides from misfolding. The (α/β)8 barrels are a common class of proteins and have never been shown to form amyloid fibrils. This thesis aims to elucidate the characteristics that prevent (α/β)8 barrels from misfolding using Escherichia coli dihydrodipicolinate synthase (DHDPS), a homotetrameric (α/β)8 barrel protein, as a model. It is widely accepted that the precursor of amyloid fibrils is a partially folded species. It is hypothesised in this thesis that the (α/β)8 barrel fold protects the protein against this partial unfolding. This was tested by generating a catalogue of site-directed mutants of DHDPS and screening each of these in a range of pHs and ionic strengths. Amorphous aggregation propensity was assessed by monitoring light scattering at 340 nm and β-sheet specific aggregation was assessed using ThT. Thermal stability was monitored using DSF and CD spectroscopy. Crystallography was used to assess tertiary and quaternary structures and in the cases where crystal structures were not obtained, kinetics was used as a proxy indicator of correct folding and monomeric association. CD spectroscopy was also used to investigate the secondary structure of the DHDPS variants and analytical gel permeation liquid chromatography and AUC were used to confirm quaternary structure. The stability and aggregation propensity of DHDPS and its variants were assessed under a range of pH and salt conditions. It was established through the characterisation of the wild-type protein that the predominant determinant of stability was, unsurprisingly, pH. This was a trend observed for all the variants described. Affinity tags were used during the course of this research to facilitate and expedite the production of the protein variants. The introduction of tags containing a polyhistidine motif to DHDPS significantly altered some biophysical properties. Whilst the secondary and quaternary structures were found to be similar to the wild-type enzyme, the catalytic properties were changed. In addition to this, the propensity to aggregate was altered. The full-length polyhistidine tags increased the propensity of DHDPS to form β-sheet-specific aggregate, although this did not result in the formation of amyloid fibrils for most of the variants. The Zyggregator algorithm was used to predict amino acid substitutions that would increase the aggregation propensity of DHDPS. It identified several amino acids, three of which were chosen for mutation and two of which were expressed in sufficient quantity for further study. DHDPS Q90L and A207V were characterised and the amino acid substitutions did not significantly alter the kinetic parameters of the enzyme. The crystal structure of A207V was solved and confirmed the results of the kinetic analysis demonstrating unchanged tertiary and quaternary structures. Both variants exhibited tertiary and quaternary structures similar to the wild-type enzyme, although Q90L contained more disorder than the wild-type enzyme. The thermal denaturation temperatures and aggregation propensities were also similar to wild-type, although the propensity for both variants to form β-sheet-specific aggregates was reduced. The combinatorial effects of Q90L, A207V and the polyhistidine tags were assessed. This revealed that whilst most biophysical properties were unaffected, the β-sheet-specific aggregation propensity for pET M11 and pET 151/D-TOPO DHDPS Q90L and pET M11 DHDPS A207V, were significantly increased compared to the wild-type enzyme. The evolutionary forces driving the association of the monomeric and dimeric subunits of DHDPS are undetermined. Investigation of two quaternary structure mutants (DHDPS Y107W and L197Y) revealed that the tetrameric nature of E. coli DHDPS is important for protein activity, stability and the prevention of aggregation. The combinatorial affects of the disrupted quaternary structure and the polyhistidine tags further increased the predisposition of DHDPS to form β-sheet-specific aggregates, resulting in the formation of linear aggregates with some characteristics of amyloid fibrils. The additive affect of Q90L, Y107W and a polyhistidine tag was assessed and revealed that the major determinant in protein stability and prevention of amorphous and β-sheet specific aggregation is the quaternary structure. This study demonstrates that the destabilisation of the quaternary structure of DHDPS can result in the formation of amyloid-like aggregates by an (α/β)8 barrel, the first example of an (α/β)8 barrel misfolding in such a way. This finding supports the assertion that all proteins can form amyloid fibrils.

Although numerous hazard models exist to assess possible ash fallout from explosive volcanic eruptions around the world, these models frequently neglect to consider ash aggregation or use a simple percent proxy to represent aggregation, without considering the varying processes at work throughout the volcanic flow. Eruption dynamics are sensitive to ash aggregation, and ash aggregates are commonly found in eruptive deposits, yet few experiments have been conducted on aggregation phenomena using natural materials. In this work, experiments were developed to produce both probabilistic and process-based relationships for the efficiency of ash aggregation with respect particle size, collision kinetic energy, atmospheric water vapor and residence time. A synthetic ash proxy, ballotini, and ash from the 2006 eruption of Tungurahua, Ecuador, and the 1980 eruption of Mount St. Helens, WA, were examined for their aggregation potential. Two aggregation regimes, wet and dry, were identified based on their potential for aggregation. The wet flow regime occurs when particles are circulated in high relative humidity environments long enough to develop a water layer with a thickness that exceeds the particle roughness scale. Hydrodynamic forces control aggregation in the wet flow regime. The dry flow regime includes particles in low relative humidity environments as well as those that circulate too briefly in high humidity environments to fully develop a water layer. Electrostatic forces control aggregation in the dry flow regime. Aggregation efficiency in both regimes was dominantly controlled by collision kinetic energy; however, this effect is significantly dampened in the wet flow regime. Equations governing the relationships between aggregation efficiency, collision kinetic energy and the related forcings in the wet or dry flow regimes have been developed for implementation into large-scale numerical volcanic models. The results of this experimental work have been developed into a probability distribution that has been integrated and incorporated into a multifluid numerical model. The numerical simulation was tested on a range of explosive depths and overpressure estimates from the 1790 eruption of Kilauea volcano, HI. The model output was compared to field data collected on the deposit thickness moving away from the source and the distribution, including both size and density, of aggregates. The mass fraction of ash removed from the eruption column in the form of aggregates was also calculated to examine how efficiently aggregation processes remove ash throughout the eruption. Cumulatively, the work presented here furthers our understanding of aggregation processes and the role they play in volcanic eruptions.

Data exploration is considered one of the major processes that enables the user to analyze massive amount of data in order to find the most important and relevant informa- tion needed. Aggregation and Ranking are two of the most frequently used tools in data exploration. The interaction between ranking and aggregation has been studied widely from different perspectives. In this thesis, a comprehensive survey about this interaction is studied. Holistic Ranking Aggregation which is a new interaction is introduced. Finally, various algorithms are proposed to efficiently process ad-hoc holistic ranking aggregation for both monotone and generic scoring functions.

Zugl.: Wuppertal, Univ., Diss., 2005

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Christopher J. Roberts, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.