Studies were undertaken to determine toxicological effects in a model species, Cyprinus carpio L. following hypoxic exposure either alone or in combination with representative heavy metal (i.e. copper; Cu) via a dietary route, at different levels of biological organisation (viz. biochemical, histological and individual level effects). Initially, the validation study of biological responses using a range of concentrations of dietary Cu as a relevant environmental contaminant was carried out (Chapter 3). The results showed a range of biological responses in exposed fish including significant genotoxic response as determined by induction of DNA strand breaks (i.e. the Comet assay) with bacterial enzymes Fpg and Endo-III (for detection of oxidative DNA damage) and reduction in growth rate suggesting the robustness of selected biomarkers. Subsequently, this approach was used initially to determine the biological responses following chronic hypoxic and hyperoxic exposure (Chapter 4). The results suggested that both hypoxic and hyperoxic conditions lead to a range of comparable biological responses. Following relative evaluation of chronic hypoxic and hyperoxic exposures, experiments were carried out to elucidate potential interactive effect of hypoxia in combination with dietary Cu (Chapter 5). The combined exposure of hypoxia and Cu induced a significantly higher level of DNA damage suggesting that DNA damage in fish can serve as a sensitive biomarker for changes in water quality as well as presence of genotoxic chemicals. The final sets of experiment were carried out to determine the biological responses in C. carpio following exposure to chronic hypoxic stress and subsequent recovery in normoxic condition for 7 days. Real-time PCR (qPCR) technology was used to examine the hypoxia inducible Factor-1 α (HIF-1α) gene expression pattern (Chapter 6). The results suggested that the expression levels of HIF-1α in response to hypoxia were significantly higher compared to normoxic controls, a high level of oxidative DNA damage under hypoxia and re-exposure to normoxic condition (i.e. recovery period). This will shed lights for development of adaptive response in higher vertebrates, which could also have significant clinical implications in human health.

Background and Methods: The involvement of iron in the risk for, and complications of, type 2 diabetes has generated substantial interest over the past 15 years, initially sparked by an association with raised serum ferritin, and the observation that people with iron overload diseases frequently develop diabetes. Considerable advances have since been made in understanding the effect glucose has on molecules, cells, and tissues; and the role that oxidative stress plays in the development of the pathologies of long-term diabetes. Poorly liganded iron is potentially both a contributor to, and consequence of, these complications. In vitro experiments with glucose-incubated transferrin by earlier workers have demonstrated loss of function with increasing glycation, leading to the suggestion that the failure of this key iron-binding protein may contribute to diabetic pathology, via the presence of redox active non-transferrin-bound iron (NTBI). In vitro glycated transferrin is examined here by ultrafiltration, to assess loss of function and possible oxidative fragmentation. Mass spectrometry is used to identify a range of amino acid glycation sites on in vitro glycated transferrin for the first time. Finally, several groups have previously measured NTBI in people with diabetes, finding little agreement in results. NTBI is measured here in a cohort of people with type 2 diabetes, using a new adaptation of earlier NTBI assays. NTBI is also assessed in pre-dialysis chronic kidney disease (CKD) stages I to III for the first time. Results and Conclusions: Experiments with glycated transferrin in vitro demonstrate oxidative fragmentation, explaining the loss of function reported by earlier groups. In vitro glycated transferrin examined by mass spectrometry reveals a substantial number and range of amino acids subject to glycation. Comparison with in vivo glycated transferrin suggests that many of the in vitro glycation sites are not glycated in vivo, and that there are many oxidized methionine residues which are potential artefacts, or likely to be repaired by methionine sulphoxide reductases in vivo. A study of people with type 2 diabetes finds no direct association between NTBI and protein glycation. Unexpected correlations between NTBI and LDL, and LDL and haemoglobin with increasing protein glycation, are reported for the first time. NTBI is suggested to be iron sourced from haemoglobin or haem, from erythrocyte haemolysis prior to sample collection. In people with pre-dialysis CKD stages I to III no significant difference in NTBI level compared to controls is seen, or correlations with markers of renal function. No link between NTBI and kidney function at this stage of disease is indicated.

No description available.

Oxidative stress is a feature of many chronic neurodegenerative diseases as well as a contributing factor in acute disorders including stroke. Fork head class of transcription factors (Foxos) play a key role in promoting oxidative stress-induced apoptosis in neurons through the upregulation of a number of pro-apoptotic genes. Here I demonstrate that synaptic NMDA receptor activity not only promotes Foxos nuclear exclusion but also suppresses the expression of Foxo1 in a PI3K-dependent fashion. I also found that Foxo1 is in fact, a Foxo target gene and that it is subject to a feed-forward inhibition by synaptic activity, which is thought to result in longerterm suppression of Foxo downstream gene expression than previously thought. The nuclear factor (erythroid 2-related) factor 2 (Nrf2) is another transcription factor involved in oxidative stress and the key regulator of many genes, whose products form important intrinsic antioxidant systems. In the CNS, artificial activation of Nrf2 in astrocytes has been shown to protect nearby neurons from oxidative insults. However, the extent to which Nrf2 in astrocytes could respond to endogenous signals such as mild oxidative stress is less clear. The data presented herein, demonstrate for the first time that endogenous Nrf2 could be activated by mild oxidative stress and that this activation is restricted to astrocytes. Contrary to the established dogma, I found that mild oxidative stress induces the astrocytic Nrf2 pathway in a manner distinct from the classical Keap1 antagonism employed by prototypical Nrf2 inducers. The mechanism was found to involve direct regulation of Nrf2's transactivation properties. Overall these results advance our knowledge of the molecular mechanism(s) associated with the control of endogenous antioxidant defences by physiological signals.

Ageing is the progressive decline in physiological performance with age, which is almost universal amongst multicellular organisms. While understanding ageing is an important aim in biological research, our current understanding of how and why we age is incomplete. In this thesis, I examine how sexual selection affects the evolution and mechanistic basis of ageing. I then explore how diet affects lifespan and reproduction in either sex. Finally, I test the hypotheses that oxidative stress, which occurs when cellular levels of Reactive Oxygen Species exceed circulating antioxidant defences causes ageing (i.e. the free radical theory of ageing) and/or constrains life-history strategies. To ask these questions, I employ quantitative genetics in decorated crickets Gryllodes sigillatus to examine the genetic co(variance) of ageing, lifespan, reproductive effort, oxidative damage and antioxidant protection. In the Australian field cricket, Teleogryllus commodus, I apply the geometric framework of nutrition to examine how lifespan, reproductive effort, oxidative damage and antioxidant capacity respond to dietary manipulation. In G. sigillatus, I found that sexual selection caused divergent strategies of age-dependent reproductive effort across the sexes and that this, in turn, promoted different rates of ageing in males and females. I found a trade-off between early reproductive effort and ageing rate in both sexes, although this trade-off was more pronounced in females (Chapter 3). I then explored the mechanistic basis of these sex-specific life-history strategies and, in support of the free radical theory of ageing, I found that oxidative damage was greatest in the shortest lived sex (females) and was negatively genetically correlated with lifespan. Additionally, oxidative damage was a cost of female reproductive effort that accelerated ageing, showing that oxidative stress may mediate sex-specific life-history strategies in decorated crickets (Chapter 4). If sexual selection affects reproduction and lifespan it should promote sex-specific life-history responses to dietary manipulation. In Australian black field crickets Teleogryllus commodus, I found that males and females have distinct dietary optima for lifespan and reproductive effort and that diet mediated a trade-off between these traits. I found that mating affected responses to dietary manipulation and caused sexual dimorphism in dietary intake under choice (Chapter 5). However, oxidative stress did not explain these life-history responses to dietary manipulation across the sexes (Chapter 6): although oxidative damage was greatest in the shortest lived sex (i.e. females), diets that extended lifespan did not reduce oxidative damage. My thesis illustrates the importance of considering sexual selection when considering the evolution and mechanistic basis of ageing. It offers equivocal support for the free radical theory of ageing but shows that oxidative stress may help underpin sex-specific life-history strategies. However, my results highlight that unravelling the relationship between oxidative stress and life-history strategies across the sexes will be a very difficult task.

M.Sc. (Nanoscience) / Please refer to full text to view abstract

No description available.

Oxidative stress is caused when there are more reactive oxygen species (ROS), than antioxidants to scavenge them, resulting in damage to cellular components. It has been implicated as a major player at multiple points in the disease process of Parkinson’s disease (PD) and many other conditions. For example, evidence suggests oxidative damage to the α-synuclein protein may affect its aggregation propensity. In addition, α-synuclein may increase ROS production. However, how this oxidative stress relates to neurodegeneration is not known. Therefore, there is a need for models of α-synucleinopathies and tools to assess the involvement of oxidative stress in the disease process. In order to model α-synucleinopathies, overexpression of the α-synuclein protein was used. A BacMam viral expression system containing human α-synuclein was generated and used to assess toxicity. α-Synuclein overexpression in undifferentiated or differentiated SH-SY5Y cells failed to show toxicity. However, the stability of α-synuclein protein expression and the cell line used may have influenced in the lack of toxicity. The current work provides important guidance for future experimental design. Flavonoids are found in plants and have antioxidant capability. AO-1-530 is a synthetic compound with a flavonoid head group and a long hydrocarbon tail. It is highly cell permeable and localises to the mitochondria. In order to investigate its protective properties, toxin-induced oxidative stress cell assays were established. AO-1-530, in the low micromolar range, was protective against high doses of tert-butyl hydroperoxide (tBHP), whereas natural antioxidants, such as myricetin and quercetin, showed limited protection or required at least 10-fold higher concentrations to achieve similar protection. The ability of AO-1-530 to directly scavenge radicals was assessed cell-free in solution and in a cell-based assay. In solution the mechanism of action was investigated by electron paramagnetic resonance (EPR) spectroscopy. AO-1-530 had similar scavenging ability to myricetin, but was a slightly stronger scavenger than quercetin. The intracellular scavenging ability was quantified by CellROX® Deep Red live imaging. Although the compounds had similar cell-free scavenging abilities, AO-1-530 significantly out-performed both myricetin and quercetin in the intracellular assay, suggesting the mitochondrial localisation is critical to its highly protective properties. AO-1-530 is a powerful, novel tool to study the involvement of oxidative stress in diverse disease models.

Oxidative stress can result in changes to many biomolecules and also affect their activities. We are interested in protein carbonylation, a type of unnatural oxidation which has been associated with numerous degenerative disease states and is also a consequence of the natural aging process. Protein carbonyls are stable species, but countless analytical barriers exist in terms of their identification. Thus, the main goal of this work was to develop and optimize analytical methods that could be used to help us better understand which, where, and how proteins are being carbonylated. Initial studies involved method validation for carbonylating, tagging, and enriching the model protein human serum albumin (HSA). We have developed a reproducible method of producing carbonylated protein in vitro in which HSA is treated with acrolein to carbonylate cysteines, histidines, and lysines. Protein carbonyls are compatible with various affinity labels and enrichment techniques. We strived to learn more about the efficiencies of various biotin affinity labels and avidin enrichment techniques using quantitative assays and mass spectrometry. Results showed a preference for different affinity labels based on their chemical properties and suggested that monomeric columns are selective for particular peptides. Most recently, method development and validation work was done involving a cleavable biotin tag that enables both enrichment and identification of protein carbonylation modification sites. This affinity tag offered the highest labeling efficiency of all tags tested in the past and greater coverage of modification sites than biotin hydrazide reagents. We applied our analytical methods to two sets of human blood samples. The first sample set was plasma taken from chronic kidney disease (CKD) patients. No carbonylation patterns were elucidated, but this project marked the beginning of blood analyses in which existing protocols were adapted to blood samples. The second sample set was serum/plasma taken from patients with traumatic injuries. We effectively applied our analytical methods to these sample sets and were able to visualize and quantitate temporal protein carbonylation patterns via Western blotting and iTRAQ-based mass spectrometry experiments. ProteoMiner experiments proved successful in that we were able to identify a larger and more diverse amount of carbonylated proteins via mass spectrometry.

Periodontal diseases are bacterially induced, inflammatory diseases which are responsible for loss of alveolar bone and connective tissue supporting the teeth which results in loss of teeth. Gram negative anaerobic bacteria are highly associated with these diseases. One of them is Porphyromonas gingivalis belonging to the phylum Bacteroidetes. Infection by P. gingivalis is recurrent after physical removal of the bacteria from the oral cavity and even after antibiotic treatment as development of resistance is not rare. Hence complete understanding the biology of this bacterium is of significance. This gram negative obligate anaerobe, being aerotolerant, manages to survive inside the oral cavity, where oxidative stress is ubiquitous. Genome sequence of P. gingivalis shows the presence of a transcriptional regulator OxyR which is a homologue of OxyR present in E. coli. P. gingivalis OxyR induces the expression of antioxidant defense genes like sod, ahpC-F, dps to protect the bacteria from oxidative stress. Expression of P. gingivalis OxyR regulon is not very well understood. Microarray studies carried out in our lab using P. gingivalis W83 to study gene regulation by OxyR, indicated that several genes in P. gingivalis are co-regulated by iron-and OxyR. Literature also supports that in iron deplete conditions genes involved in oxidative stress are down-regulated. These studies formed the basis of our hypothesis that OxyR might regulate the genes in P. gingivalis in an iron dependent manner. To study the mechanism of regulation by P. gingivalis OxyR and to determine whether OxyR regulation is iron dependent, two approaches were applied - in vitro characterization of binding and in vivo characterization. First step of in vitro characterization was to perform CHIP-chip assay to determine OxyR-binding sites present on the genomic DNA of P. gingivalis. As this assay was performed under completely anaerobic conditions, the target fragments to which OxyR was found to bind during this assay were not same as reported in literature. These and the fragments reported in literature were used for EMSA. EMSAs carried out using crude cell lysates and in vitro OxyR protein preparations showed expected results but the results were not reproducible. In vivo expressed and purified P. gingivalis OxyR never bound to the target fragments used. Preparation of a stable protein preparation and improvement in the parameters of EMSA is very important to further investigate the binding in vitro. The second approach is based on in vivo characterization of binding. This requires tagging the P. gingivalis OxyR at its C-terminus with fluorescent protein to observe its binding to the target DNA sequences. Fluorescently tagged OxyR, is expected to emit fluorescence from a highly localized area to produce sharp fluorescent spots when it is bound to its target sequences. Unbound OxyR is expected to emit a fluorescent signal which is spread over the entire area of the cell. This technique will help to determine the conditions under which OxyR binds to its target DNA sequences. This provides a means to confirm the results obtained from in vitro characterization instead of just extrapolating them.