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.

While the catalytically powerful [Cu,Zn] superoxide dismutase (SOD1) possesses great potential as a therapeutic, unfavorable properties in circulation limit its use in clinical medicine. The small, water soluble dimer is rapidly excreted by the kidney. Previous initiatives have been used to increase the mass of the enzyme (PEGylation, liposome encapsulation). This has resulted in highly heterogeneous mixtures of modified SOD1, which are difficult to characterize. Furthermore, these modified proteins have utilized foreign material that has shown to elicit an inflammatory response. We developed an improved strategy that creates a homogenous high molecular weight SOD1 based on combinations of the protein itself. This was accomplished through the addition of a site-specific, azide functionalized cross-linker to unmodified SOD1, followed by the conjugation of SOD dimers using CuAAC and a bis-alkyne linker to form a 64 kDa SOD tetramer. The final product, bis-SOD, presents the fully catalytic activity of the combined proteins.

While the catalytically powerful [Cu,Zn] superoxide dismutase (SOD1) possesses great potential as a therapeutic, unfavorable properties in circulation limit its use in clinical medicine. The small, water soluble dimer is rapidly excreted by the kidney. Previous initiatives have been used to increase the mass of the enzyme (PEGylation, liposome encapsulation). This has resulted in highly heterogeneous mixtures of modified SOD1, which are difficult to characterize. Furthermore, these modified proteins have utilized foreign material that has shown to elicit an inflammatory response. We developed an improved strategy that creates a homogenous high molecular weight SOD1 based on combinations of the protein itself. This was accomplished through the addition of a site-specific, azide functionalized cross-linker to unmodified SOD1, followed by the conjugation of SOD dimers using CuAAC and a bis-alkyne linker to form a 64 kDa SOD tetramer. The final product, bis-SOD, presents the fully catalytic activity of the combined proteins.

Superoxide, a reduced form of dioxygen is produced in natural waters from abiotic thermal processes such as iron(II) oxygenation photochemically, and biologically in natural waters. Superoxide is highly redox-active due to the presence of an unpaired electron and plays an important role in the geochemical cycling of trace metals and degradation of organic pollutants in natural waters. It also has a significant effect on the physiology of aquatic microorganisms and has been implicated in the growth of toxic microalga in coastal areas of Japan, Australia and Canada. The generation of superoxide by both biotic and photochemical pathways is described in this thesis and attention given to selected reactions of superoxide in simulated natural waters. Particular attention in this work has been given to the interaction of superoxide and iron and the impact of this interaction on iron availability to Chattonella marina, a red-tide phytoplankton frequently associated with fish-mortalities in Australia and Japan. Superoxide production from both phototchemical and biological sources is measured using a highly sensitive chemiluminescence technique and the effect of superoxide production on iron transformation under various environmental conditions is investigated by employing spectrophotometric techniques with high sensitivity and temporal resolution. The intermediacy of superoxide in iron acquisition mechanism by C. marina is also investigated using a radioisotope labeling technique. Our experimental results show that superoxide in natural waters is produced via reduction of dioxygen. The reduction of dioxygen takes place by transfer of electrons by the photo-excited quinone moieties present in natural organic matter or by reductases located on the outer cell membrane of organisms' surface. The experimental results suggest that the fate of iron in marine waters is closely related to the superoxidedioxygen redox couple. Superoxide can reduce a wide range of organically complexed iron(III) species to the more soluble iron(II) redox state, thus affecting its bioavailability. A simple kinetic model for redox-cycling of iron in the presence of superoxide is developed. In addition, by coupling the model for redox-cycling of iron with the forms of iron acquired by C. marina, a generalized mathematical model for iron acquisition is presented which satisfactorily describes all results obtained.

Iron is an essential micronutrient that is required by some microorganisms in relatively large quantities. This is problematic for those inhabiting marine environments, where iron is highly insoluble and the dissolved fraction is predominantly strongly bound to organic compounds. Due to low supply and high demand, iron limits primary productivity in many oceanic waters, and may also limit growth of organisms in coastal waters under some circumstances. Recent incidents of explosive growth (???blooms???) of the noxious filamentous cyanobacterium Lyngbya majuscula in the coastal marine waters of Moreton Bay, Queensland, have prompted speculation that terrestrial human activities have increased iron availability to the organism, thus overcoming previous limitations on growth imposed by scarcity of the nutrient. This thesis describes work investigating the chemical form of iron in coastal waters under various environmental conditions and the way in which this influences its availability to L. majuscula. Chemical speciation of iron was investigated as a function of terrestrial-derived inputs of natural organic matter (NOM) of variable origin and sunlight in coastal marine waters, employing chemiluminescence-based and spectrophotometric techniques with high sensitivity and temporal resolution. These techniques allowed determination of iron and other chemical parameters at naturally occurring (typically nanomolar) concentrations. The mechanism of iron acquisition by L. majuscula was also investigated using a radioisotope-labelling labelling technique in addition to the other techniques described. Results indicated that iron speciation can be described by five classes: inorganic dissolved and organically complexed dissolved iron in both ferrous (reduced) and ferric (oxidised) forms, and precipitated inorganic iron. Simulation of laboratory results by numerical kinetic modelling of the processes investigated indicated that while the thermodynamic impetus is strongly towards precipitated iron, iron complexation by NOM and its reduction by sunlight-mediated processes and/or L. majuscula results in meta-stable dissolved species that are more readily available to L. majuscula. Superoxide is a critical intermediate in iron reduction by both sunlight and L. majuscula. Thus L. majuscula is capable of altering iron speciation to increase its availability, however uptake is also strongly dependent on environmental conditions and may be enhanced by increased inputs of iron, NOM and sunlight into coastal waters.

Thesis (Ph. D.)--University of New South Wales, 2005. / Also available online.

Thesis (Ph. D.)--Washington State University, December 2008. / Title from PDF title page (viewed on Apr. 15, 2009). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaf 193). Also available via the Internet.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 86-97). Also available via the World Wide Web.

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.