Parkinson disease (PD) is an incurable disease, second only to Alzheimer’s disease as the most common neurodegenerative disease in adults. Unfortunately, the course of disease is significantly longer for individuals diagnosed at an early age (20-40 years of age). Although early-onset, recessively inherited cases represent a small subset of individuals with PD (~5- 10%), their clinical presentation is unique, with symptoms being almost exclusively motor-related. The expressivity of early-onset PD is partially explained by post-mortem neuropathological findings, which demonstrate a specific loss of dopamine synthesizing cells in brainstem nuclei that also produce neuromelanin (i.e. Substantia nigra and Locus coeruleus). With the majority of early-onset PD cases being caused by homozygous and biallelic heterozygous mutations in the PRKN gene, its gene product, parkin, has been extensively studied. It is generally accepted that loss of its E3 ligase function leads to neurodegeneration by either one of the following two mechanisms: i) toxic substrate accumulation from the loss of target protein ubiquitination (and related degradation), or ii) accumulation of dysfunctional mitochondria due to impaired mitophagy initiation. However, whether these mechanisms lead to selective neuronal loss within the human brain remains unknown. This thesis represents a body of work that supports a novel role for parkin as a thiol-based anti-oxidant and redox homeostasis regulator, which helps explain the cell-specificity observed in recessive, PRKN-linked PD. These findings include: i) evidence that human brain parkin uniquely and natively undergoes age-associated aggregation beginning at 40 years of age (Chapter 2); ii) identification of multiple, reversible and irreversible oxidative modifications of parkin cysteines, both in cells and tissues, including dopamine-adduct conjugation on primate sequence-specific cysteine 95 (Chapter 2); iii) the demonstration that irreversible oxidation of parkin cysteines causes aggregate formation ii in cells and mice exposed to exogenous and/or endogenous sources of oxidative and dopamine stress (Chapter 2 and 3); iv) evidence that parkin functions as a thiol-dependent anti-oxidant similar to glutathione (Chapter 2), which lowers oxidation state in cells and tissues under native and stress conditions (Chapter 2 and 3); v) the demonstration that parkin cysteines, notably C95, directly bind glutathione and regulate glutathione redox homeostasis in cells and tissues in a dynamic fashion (Chapter 3); and vi) the development of novel, human-specific, anti-parkin monoclonal antibodies that preferentially detect oxidized and aggregated forms of parkin found associated with neuromelanin and lysosomal storage vesicles within neurons of human Substantia nigra (Chapter 2 and 4). Future studies focusing on further validation of in situ oxidative modifications of parkin cysteines and their effect on protein structure, notably the poorly studied linker region that contains C95, will provide insight into how these oxidative modifications affect the function of parkin in vivo, including in adult human brain. Also, identifying the bona fide intracellular redox partners of parkin will be crucial to understanding how this protein regulates cellular redox state. Of clinical importance, the findings presented here indicate a potential, human-specific link between parkin and neuromelanin formation, which deserves to be further explored, such as with parkin mouse models engineered to produce neuromelanin. Finally, designing clinical trials using anti-oxidants specifically in individuals affected by PRKN-associated PD represents a logical, translational treatment approach to explore.

Background: Although alcohol intake has been positively associated with breast cancer risk in epidemiologic studies, the mechanisms mediating this association are speculative. Objective: The Postmenopausal Women's Alcohol Study was designed to explore the effects of moderate alcohol consumption on potential risk factors for breast cancer. In the present analysis, we evaluated the relationship of alcohol consumption with antioxidant nutrients and a biomarker of oxidative stress. Design: Participants (n = 53) consumed a controlled diet plus each of three treatments (15 or 30 g alcohol/day or a no-alcohol placebo beverage), during three 8-week periods in random order. We measured the antioxidants, vitamin E (alpha (α)- and gamma (γ-tocopherols), selenium, and vitamin C in fasting blood samples which were collected at the end of diet periods, treated and frozen for assay at the end of the study. We also measured 15-F2t-IsoP isoprostane, produced by lipid peroxidation, which serves as an indicator of oxidative stress and may serve as a biomarker for conditions favorable to carcinogenesis. Results: After adjusting for BMI (all models) and total serum cholesterol (tocopherol and isoprostane models) we observed a significant 4.6% decrease (P=0.02) in α-tocopherol and a marginally significant 4.9% increase (P = 0.07) in isoprostane levels when women consumed 30 g alcohol/day (P = 0.06 and 0.05 for overall effect of alcohol on α-tocopherol and isoprostanes, respectively). The other antioxidants were not significantly modified by the alcohol treatment. Conclusions: These results suggest that moderate alcohol consumption increases some biomarkers of oxidative stress in postmenopausal women.

This thesis set out to investigate how heat stress may affect the oxidative balance and influence the activity patterns of animals. The use of wild animals has been scarce in testing the effects of heat stress on the oxidative balance of animals, especially under a simulated heat wave condition. Animals are expected to differ in their oxidative stress in response to heat stress, depending on their thermotolerance and the life history trait of activity is tested here as an indirect measure of thermotolerance. Therefore, two rodent species within the same family were used for experiments, one with a nocturnal activity rhythm and the other with a crepuscular/diurnal activity rhythm. Animals were tested firstly in a laboratory whole-body hyperthermia experiment, followed by a 3-day heat stress test with extreme temperatures. Following these experiments, animals were subjected to a simulated heat wave to determine how ecologically relevant temperatures may influence not just the oxidative balance of individuals, but also their activity patterns and water drinking in the absence of a microclimate. In order to determine the oxidative balance of individuals under control and heat treatment conditions, markers of oxidative damage and antioxidant defense were used. Markers for lipid and protein oxidation and a marker of enzymatic antioxidant activity and total non-enzymatic antioxidant activity were investigated in the liver, kidney, brain and heart. During the simulated heat wave, activity was measured with video recordings, with the videos analysed for water drinking behaviour as counts and time spent drinking water. During the whole-body hyperthermia experiment without the extreme temperatures, both species demonstrated no significant changes in liver oxidative markers except for high oxidative damage in the kidney for R. dilectus and high oxidative damage in the brain for M. namaquensis. During the 3-day acclimation experiment with extreme temperatures, both species were oxidatively stressed in the heart, with significant oxidative damage in the kidney for R. dilectus and significant oxidative damage in the liver for M. namaquensis. During the simulated heat wave, both species showed no significant changes in liver oxidative balance. The kidney was oxidatively stressed for M. namaquensis, with both species exhibiting signs of oxidative stress in the brain. Animals did not shift their activity during the heat wave, but rhythms demonstrated signs of disruption during the simulated heat wave. Activity increased during the day for both species during the heat wave. This was due to animals being uncomfortable and drinking more water. For the nocturnal species, the frequency of drinking water increased during the day and during the night during the simulated heat wave. The crepuscular species only increased in the amount of time spent drinking water during the day during the simulated heat wave. Overall, the nocturnal species was more susceptible to heat stress inducing either increased oxidative damage or reduced antioxidant defense compared to the crepuscular species, but this was tissue specific and also dependant on the experimental condition. It is recommended that future studies directly measure the reduced: oxidised state of individuals in addition to markers of defense and damage. / Thesis (PhD (Zoology))--University of Pretoria, 2019. / Zoology and Entomology / PhD (Zoology) / Unrestricted

Due to rapid urbanisation, people in metropolises spend the majority of their time indoors. Indoor mould contamination, as one of the most pungent biohazards in built environments, can ubiquitously present in humid areas and potentially compromise the health of occupants. Governmental institutions like the World Health Organisation and United States Environmental Protection Agency have put forward guidelines for indoor mould prevention. However, these guidelines normally require occupants to maintain a low indoor humidity (<75% or even 40%), and thus, in tropical and subtropical areas, one of the most widely used approaches to prevent indoor mould contamination is to continuously operate air-conditioners or dehumidifiers (AC/D). The 24 h operation of AC/D, however, conflicts with the requirement of energy sustainability, and hence posits a trade-off between sustainability and indoor mould hygiene. The aim of this study was to facilitate the development of sustainable and effective mould prevention strategies for indoor environments. The literature on currently adopted mould prevention strategies including that target moisture (24 h AC/D), temperature (air-conditioning system and cool wall paint) and nutrient (dust removal) elements as well as new nanoparticles technology (Ag, TiO 2 and MgO nanoparticles), was reviewed and the main limitations of these strategies were discussed. It was found that none of these current mould prevention measures has addressed both sustainability and mould hygiene on balance, urging further investigations. Therefore, the objective of the first phase investigation was to develop sustainable cause-specific mould control measures in built environments. A case study of a mould contaminated site was conducted to illustrate the micro-environments that contribute to mould contamination in buildings. The currently used 24-h AC/D approach was compared with and ranked against other sustainable alternatives. The results of this case study suggest that determining an effective non-24 h AC/D management regime tends to be a sustainable and user-friendly solution. To develop such a regime, understanding the critical mechanisms regulating indoor mould responses to water dynamics is essential. Thus, the objective of the second phase was to characterise the critical mechanism regulating the growth of common indoor moulds under water dynamics. It was hypothesised that oxidative stress is associated with the growth of indoor moulds under water dynamics. Using Cladosporium cladosporioides as a model, both its pre-germination and germinated spores were exposed to daily wet-dry cycles. Afterwards, the growth was assessed and cellular H 2 O 2 concentration and catalase activity were measured. It was found that under water dynamics, the longer growth delay in C. cladosporioides was associated with a higher encountered oxidative stress, with 12-12 wet-dry cycle (12 h wet, 12 h dry) showing the longest delay and highest oxidative stress. Pearson correlation and linear regression analysis suggest a positive correlation between growth delay and oxidative stress under water dynamics (R 2 =0.85, P<0.0001). Moreover, pre- germination spores generally exhibited shorter growth delay, lower cellular H 2 O 2 concentration and higher catalase activity. Collectively, these results suggest that the growth of C. cladosporioides is associated with oxidative stress under water dynamics. After revealing the association between the growth of C. cladosporioides and oxidative stress under water dynamics, at the third phase, this finding was extrapolated to different mould species (C. cladosporioides, Aspergillus niger and Aspergillus penicillioides), water activity (a w ) (0.4 a w , 0.6 a w and 0.8 a w ) and temperature levels (19 °C and 28 °C). In addition, the antioxidant responses of treated moulds, including antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase) were monitored. The results showed that lower water activity levels imposed higher oxidative stress to moulds, and A. penicillioides exhibited the highest tolerance which displayed the highest antioxidant activities and encountered lowest oxidative damage under water dynamics. Moreover, no significant difference was measured in terms of the survival, oxidative stress and antioxidant responses between these two temperature levels. The third phase of the study, for the first time discovered the reason contributing to the different resistance towards water dynamics among common indoor moulds, and further confirmed the important role of oxidative stress adaptation in withstanding transient water supply. In conclusion, this study reveals the critical role of oxidative stress adaptation in helping moulds to cope with changing water conditions, which may shed light on a new perspective for the future development of indoor mould prevention strategies. It also indicates that longer operation time of AC/D each day may not necessarily lead to better prevention of mould contamination, suggesting that in order to sustainably prevent mould contamination, one should operate reasonable non-24 h AC/D each day (12 h/day according to the examined species in this study) to yield a more stressful wet-dry cycle to moulds. The outcomes of this study foster the development of novel and sustainable indoor mould prevention strategies

Due to rapid urbanisation, people in metropolises spend the majority of their time indoors. Indoor mould contamination, as one of the most pungent biohazards in built environments, can ubiquitously present in humid areas and potentially compromise the health of occupants. Governmental institutions like the World Health Organisation and United States Environmental Protection Agency have put forward guidelines for indoor mould prevention. However, these guidelines normally require occupants to maintain a low indoor humidity (<75% or even 40%), and thus, in tropical and subtropical areas, one of the most widely used approaches to prevent indoor mould contamination is to continuously operate air-conditioners or dehumidifiers (AC/D). The 24 h operation of AC/D, however, conflicts with the requirement of energy sustainability, and hence posits a trade-off between sustainability and indoor mould hygiene. The aim of this study was to facilitate the development of sustainable and effective mould prevention strategies for indoor environments. The literature on currently adopted mould prevention strategies including that target moisture (24 h AC/D), temperature (air-conditioning system and cool wall paint) and nutrient (dust removal) elements as well as new nanoparticles technology (Ag, TiO 2 and MgO nanoparticles), was reviewed and the main limitations of these strategies were discussed. It was found that none of these current mould prevention measures has addressed both sustainability and mould hygiene on balance, urging further investigations. Therefore, the objective of the first phase investigation was to develop sustainable cause-specific mould control measures in built environments. A case study of a mould contaminated site was conducted to illustrate the micro-environments that contribute to mould contamination in buildings. The currently used 24-h AC/D approach was compared with and ranked against other sustainable alternatives. The results of this case study suggest that determining an effective non-24 h AC/D management regime tends to be a sustainable and user-friendly solution. To develop such a regime, understanding the critical mechanisms regulating indoor mould responses to water dynamics is essential. Thus, the objective of the second phase was to characterise the critical mechanism regulating the growth of common indoor moulds under water dynamics. It was hypothesised that oxidative stress is associated with the growth of indoor moulds under water dynamics. Using Cladosporium cladosporioides as a model, both its pre-germination and germinated spores were exposed to daily wet-dry cycles. Afterwards, the growth was assessed and cellular H 2 O 2 concentration and catalase activity were measured. It was found that under water dynamics, the longer growth delay in C. cladosporioides was associated with a higher encountered oxidative stress, with 12-12 wet-dry cycle (12 h wet, 12 h dry) showing the longest delay and highest oxidative stress. Pearson correlation and linear regression analysis suggest a positive correlation between growth delay and oxidative stress under water dynamics (R 2 =0.85, P<0.0001). Moreover, pre- germination spores generally exhibited shorter growth delay, lower cellular H 2 O 2 concentration and higher catalase activity. Collectively, these results suggest that the growth of C. cladosporioides is associated with oxidative stress under water dynamics. After revealing the association between the growth of C. cladosporioides and oxidative stress under water dynamics, at the third phase, this finding was extrapolated to different mould species (C. cladosporioides, Aspergillus niger and Aspergillus penicillioides), water activity (a w ) (0.4 a w , 0.6 a w and 0.8 a w ) and temperature levels (19 °C and 28 °C). In addition, the antioxidant responses of treated moulds, including antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase) were monitored. The results showed that lower water activity levels imposed higher oxidative stress to moulds, and A. penicillioides exhibited the highest tolerance which displayed the highest antioxidant activities and encountered lowest oxidative damage under water dynamics. Moreover, no significant difference was measured in terms of the survival, oxidative stress and antioxidant responses between these two temperature levels. The third phase of the study, for the first time discovered the reason contributing to the different resistance towards water dynamics among common indoor moulds, and further confirmed the important role of oxidative stress adaptation in withstanding transient water supply. In conclusion, this study reveals the critical role of oxidative stress adaptation in helping moulds to cope with changing water conditions, which may shed light on a new perspective for the future development of indoor mould prevention strategies. It also indicates that longer operation time of AC/D each day may not necessarily lead to better prevention of mould contamination, suggesting that in order to sustainably prevent mould contamination, one should operate reasonable non-24 h AC/D each day (12 h/day according to the examined species in this study) to yield a more stressful wet-dry cycle to moulds. The outcomes of this study foster the development of novel and sustainable indoor mould prevention strategies

The work presented in this dissertation has focused on synthesizing complexes of relevance to dioxygenase enzymes that oxidatively cleave aliphatic carbon-carbon bonds. The goal of this research was to elucidate mechanistic aspects of the activation of aliphatic carbon-carbon bonds towards cleavage by reaction with oxygen, and also investigate the regioselectivity of these reactions. The oxidative cleavage of a variety of enolizable substrates has been explored by utilizing several transition metal complexes supported by an aryl-appended tris(pyridylmethyl)amine ligand. In order to probe the widely-accepted “chelate hypothesis” for how changes in regiospecificity are achieved as a function of metal ion, we synthesized the compound [(6Ph2TPA)Fe(PhC(O)COHC(O)Ph)]OTf. Based on UV-vis and IR spectroscopy, the acireductone enolate was found to bind via a six-membered chelate ring. By comparison with the reactivity of [(6Ph2TPA)Ni(PhC(O)COHC(O)Ph)]ClO4, we determined that the chelate hypothesis was an insufficient explanation of the observed regioselectivity. Rather, ferrous ion-mediated hydration of a vicinal triketone intermediate was the key factor in determining the regioselectivity of the C-C cleavage reaction. We have developed a high-yielding synthetic route to protected precursors of C(1)H acireductones. Preparation of the complexes [(6Ph2TPA)M(PhC(O)COCHOC(O)CH3)]ClO4 (M = Fe, Ni) followed by judicious choice of deprotecting conditions allowed us to investigate the oxygen reactivity of a mono-nuclear complex with a dianionic acireductone substrate for the first time. This provides a promising strategy to continue investigations of complexes of relevance to the enzyme- substrate adduct of the acireductone dioxygenases. Divalent late first-row transition metal complexes have been used to investigate some new strategies for the activation of dioxygen and subsequent cleavage of C-C bonds. We have utilized photoreduction of a Ni(II) center to generate a highly O2-reactive Ni(I) fragment that leads to cleavage of a chloro-diketonate substrate. Additionally, we have found a Cu(II)-mediated thermal cleavage of chloro-diketonate substrates at room temperature. This reaction is interestingly accelerated by the addition of a catalytic amount of chloride ion.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 1 and Type 2 diabetes (T1D/T2D) share a common etiology that involves an increase in oxidative stress that leads to dysfunction and subsequent β cell death. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids to form lipid metabolites involved in a variety of biological functions including cellular oxidative stress response. On the other hand, Interleukin 6 (IL-6) signaling has been demonstrated to be protective in islets. In this study, we explored the effect of lipoxygenase enzymes 12-Lipoxygenase, 12/15 Lipoxygenase and IL-6 on β cell function and survival in mice using both STZ and high-fat diet (HFD) models of diabetes. Alox12-/- mice showed greater impairment in glucose tolerance following STZ and HFD compared to wild-type mice (WT), whereas Alox15-/- were protected against dysglycemia. These findings were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice showed a compensatory increase in Alox15 gene expression and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. IL-6 was able to significantly attenuate the generation of reactive oxygen species by proinflammatory cytokines in human pancreatic islets. Furthermore, we find that IL-6 regulates the master antioxidant response protein NRF2. Collectively these results show that loss of Alox12 activates a compensatory increase in Alox15 that sensitizes β cells to oxidative stress and signaling by IL-6 is required for maximal antioxidant response under conditions of increased ROS formation, such as obesity.

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The impact of N-heterocyclic carbenes (NHC) as ligands for transition metal catalysis has been rigorously investigated since their isolation by Arduengo in 1991. NHCs have become abundant in late-transition metal chemistry. This is attributed to NHCs being stronger sigma-donors than even the strongest phosphine analogues, thus constructing a transition metal-NHC complex with improved stability, catalytic reactivity, and selectivity. Additionally, pincer ligands have become recognized as an important class of ligands for transition metal complexes. The unique steric and electronic tunability of pincer ligands has resulted in pincer-transition metal complexes being exploited as catalysts for a multitude of transformations. Both ligand classes, NHC and pincer, have been reported as stable organometallic catalysts that demonstrate high catalytic activity. The combination of these two ligand systems by incorporation of NHCs into pincer ligands has attracted considerable attention. NHC pincer systems have been reported as stable organometallic catalysts that demonstrate high catalytic activity. The expansion of -NHC ligand precursor methodologies and application of the newly reported methodologies in order to diversify -NHC ligand architectures is reported. Extension of the amine elimination methodology yielded unsymmetrical NHC Ta pincer complexes. Studies on the manipulation of the previously reported symmetrical -NHC pincer Ta complex’s coordination sphere to synthesize a rare Ta diimido complex with unique reactivity towards advantageous proton sources and oxidative amination of aminoalkenes are also expanded upon. These next generation catalysts developed from these architectures may prove useful in catalyzing a broad array of transformations not previously accessible through the four standard NHC containing pincer ligand architectures.