O proteassomo é uma protease intracelular multimérica e multicatalítica responsável pela degradação de proteínas envolvidas no controle do ciclo celular, processos de sinalização, apresentação antigênica e no controle de síntese proteica. Ele é constituído por uma unidade catalítica central denominada 20S (20SPT) e por unidades regulatórias (19S) acopladas em uma ou ambas as extremidades para formar o 26S (26SPT). O 26SPT reconhece e direciona os substratos poliubiquitinados para a proteólise por um mecanismo dependente de ATP. Entretanto, o 20SPT também é ativo quando dissociado de unidades regulatórias, degradando proteínas independentemente de poliubiquitinação e consumo de ATP. Proteínas modificadas oxidativamente e outros substratos são degradados desta maneira. O 20SPT é composto por dois anéis heptaméricos centrais (β) onde estão localizados os sítios catalíticos e por dois anéis heptaméricos externos (α) que são responsáveis pela abertura da câmara catalítica. Anteriormente, foi observado pelo grupo que o 20SPT da levedura S.cerevisiae sofre S-glutatiolação na subunidade α5 nos resíduos de Cys76 e Cys221. Assim, foram obtidas neste projeto linhagens de levedura S. cerevisiae portando mutações sítio-específicas na subunidade α5 do 20SPT (α5-C76S ou α5-C221S), e posteriormente, ensaios comparativos quanto às consequências estruturais e funcionais dessas mutações foram realizados. Observamos um aumento na capacidade/velocidade de degradação nas atividades peptidásicas e proteolíticas da mutante C221, e também que, a população de proteassomo isolada dessa linhagem apresenta maior proporção da forma aberta da câmara catalítica, sendo esta imediatamente fechada pela remoção da glutationa do 20SPT na presença de DTT. Resultados opostos foram observados na linhagem mutante C76. Identificamos por espectrometria de massas o resíduo C76 do 20SPT glutationilado na mutante C221. Os ensaios fenotípicos mostraram um aumento da longevidade e resistência ao estresse oxidativo da C221, enquanto que a linhagem C76 mostrou uma dificuldade no crescimento. Contudo, a S-glutationilação do 20SPT é uma modificação química pós-traducional reversível de ocorrência fisiológica dependente do estado redox celular, e, o presente trabalho discute a modulação da câmara catalítica do 20SPT através da S-glutationilação de dois resíduos de cisteína localizados na subunidade α5, e as consequências desta modificação na função proteassomal / The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, signaling processes, antigen presentation, and control of protein synthesis. It comprises a central catalytic unit called 20S (20SPT) and regulatory units (19S) coupled at one or both ends to form 26S (26SPT). The 26SPT recognizes and directs polyubiquitinylated substrates targeted for proteolysis by an ATP-dependent mechanism. However, 20SPT is also active when dissociated from regulatory units, degrading proteins by a process independent of polyubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20SPT comprises two central heptamerics rings (β) where are located the catalytic sites and two external heptamerics rings (α) that are responsible for proteasomal gating. Previously, it was observed by our group that the 20SPT of yeast S. cerevisiae is modified by S-glutathionylation in the residues Cys76 and Cys221 of the α5 subunit. Thus, were obtained in this project strains of the S. cerevisiae carrying site-specific mutations in the α5 subunit of the 20SPT (α5-C76S or α5-C221S), and comparative assays as the structural and functional consequences of these mutations were performed. We observed an increase in capacity/speed of degradation in peptidase and proteolytic activity in the C221 strain and also that, the isolated population of the proteasome this strain presents the highest frequency of open catalytic chamber conformation, which is immediately closed by the removal of glutathione of the 20SPT in the presence of DTT. Opposite results were observed in the C76 strain. We identified by mass spectrometry the C76 residue of the 20SPT glutathionyilated the C221 mutant. Phenotypic assays show an increased longevity and resistance to oxidative stress of C221, while the C76 strain showed a difficulty in growth. However, the S-glutathionylation of the 20SPT is a reversible post-translational chemical modification of physiological occurrence dependent on the cellular redox state, and this project discusses the modulation of the catalytic chamber of 20SPT via S-glutationylation of the two cysteine residues located the α5 subunit, and the consequences of this change in proteosomal function

2,5-diaminoimidazolone (Iz) is an important product of a 4-electron oxidation of guanine. The present research focuses on the mechanisms of formation of Iz via pathways initiated by guanine oxidation by one-electron oxidants (OEOs) generated by X-ray radiolysis in aqueous solutions. The kinetics of formation and yields of Iz in reactions of native highly polymerized DNA with different OEOs have been compared using an HPLC-based quantitative analysis of low-molecular products generated from the reaction of DNA-bound Iz with primary amines. Mechanisms of Iz formation in DNA have been investigated including oxygen and superoxide dependence as well as the hypothesis that 8-oxo-G, another product of guanine oxidation, is not a major precursor to Iz. Results indicate Iz is produced in significant quantities in DNA from guanine oxidation and the efficiency of its formation correlates with the reduction potential and selectivity of a given OEO.

Regulators of G-protein signaling (RGS) proteins are a family of proteins that act as GTPase accelerating proteins (GAPs) through their interaction with GΑ subunits, including GΑo, GΑi, and GΑq but not GΑs. This increased rate of hydrolysis of GTP to GDP temporally regulates G-protein coupled receptor (GPCR) signaling. A member of this family, RGS4, has been implicated in several neurological disorders including Parkinson's Disease (PD). A hallmark of PD is the induction of oxidative stress within dopaminergic neurons. In this thesis, we evaluate the role of oxidative stress, including lipid peroxidation products with 4-hydroxy-2-nonenal (4HNE) as a model, in regulating RGS4 activity within neurons. Utilizing transfected RGS4, we evaluated whether RGS4 is readily modified by physiologically relevant concentrations of 4HNE by immuonoprecipitation of RGS4 from 4HNE treated cells. Further examination of recombinant RGS4 by mass spectrometry, revealed that RGS4 is readily modified at several cysteine residues by 4HNE, including C148. Modification at this residue has been shown to be a critical site for allosteric regulation of RGS4. This is confirmed through a malachite green based phosphate generation assay we developed to observe the GAP activity of RGS4 on its native binding partner GΑi. This malachite green based assay was then adapted for high throughput screening. The assay was successfully miniaturized to a 1536-well format. In a screen of 2300 compounds, 4 were identified as hits. The development of this simple and cheap assay can be adapted for usage with a variety of RGS proteins with little work to interrogate other pathways and identify novel RGS modulators. Finally, expansive study of PD has linked oxidative stress to the pathology of both diseases. What has not been discerned is the potential relationship between oxidative stress and the induction of RGS4. In support of the hypothesis, we evaluated the potential relationship between oxidative stress and RGS4 expression. This was accomplished by evaluating two striatal neuron like cell lines, SH-SY5Y and HCN-1A. After treatment with hydrogen peroxide, both cell lines showed increased RGS4 in response to oxidative stress. This response is not however related to mRNA expression, indicating this change is most likely an adjustment of proteasomal regulation of RGS4. This phenomenon may explain the rapid onset of Parkinsonian motor symptoms in reserpine treated animal models of PD, as excess dopamine in the cytoplasm may be rapidly metabolized in reactive products.

Maternal stress during pregnancy is associated with increased risk of psychiatric disorders in offspring, but embryonic brain mechanisms disrupted by prenatal stress are not fully understood. Our lab has shown that prenatal stress delays inhibitory neural progenitor migration. Here, we investigated redox dysregulation as a mechanism for embryonic cortical interneuron migration delay, utilizing direct manipulation of pro- and anti-oxidants and a mouse model of maternal repetitive restraint stress starting on embryonic day 12. Time-lapse, live-imaging of migrating GABAergic interneurons showed that normal tangential migration of inhibitory progenitor cells was disrupted by the pro-oxidant, hydrogen peroxide. Interneuron migration was also delayed by in utero intracerebroventricular rotenone. Prenatal stress altered glutathione levels and induced changes in both activity of antioxidant enzymes and expression of redox-related genes in the embryonic forebrain. Assessment of dihydroethidium (DHE) fluorescence after prenatal stress in ganglionic eminence, the source of migrating interneurons, showed increased levels of DHE oxidation. Maternal antioxidants (N-acetylcysteine and astaxanthin) normalized levels of DHE oxidation in ganglionic eminence, and ameliorated the migration delay caused by prenatal stress. In adult male offspring, prenatally-stressed mice exhibited anxiety-like behavior on the elevated plus maze, impaired motor learning on the rotarod, cognitive flexibility on the water T-maze task, and deficits in sensorimotor gating in the pre-pulse inhibition task. Prenatally-stressed adult female offspring showed anxiety-like behavior, deficits in sociability and impaired motor learning. Maternal antioxidants prevented anxiety-like behaviors and improved sensorimotor gating in both sexes, and improved habitual learning and cognitive flexibility in adult female mice. Lastly, prenatal stress led to increases in PV+/GAD67+ cell ratios in mFC in male mice, but decreases in female mice, and antioxidant treatments eliminated those differences. Hippocampal GAD67+ cell densities were reduced by prenatal stress and restored by astaxanthin in male mice, and PV+/GAD67+ cell ratio was reduced by prenatal stress and partially restored by N-acetylcysteine in female mice. GAD67+ cell densities across regions correlated significantly with anxiety-like behavior in both male and female mice and social behavior in female mice. Through convergent redox manipulations, delayed interneuron migration after prenatal stress was found to critically involve redox dysregulation. Redox biology during prenatal periods may be a target for protecting brain development.

Non-coding small RNAs (sRNAs) are integral to post-transcriptional gene regulation in bacteria. The function of an sRNA is dependent on both secondary structure and the sequence of its unstructured seed region. The sRNA seed region typically base-pairs with target mRNAs to down-regulate the expression of target genes by blocking the ribosome-binding site or by promoting RNase-mediated degradation of the sRNA-mRNA complex. sRNAs have also been shown to increase expression of target genes by releasing RNA secondary structures that block ribosome-binding sites. Selective pressure to maintain sRNA function conserves the sequence of the sRNA seed region, but mutations in mRNA sequences to match sRNA seed regions lead to the accumulation of new targets by an sRNA. In this study, the author identified a unique scenario where a 53-nucleotide insertion event occurred in the seed region of the sRNA MgrR in Escherichia fergusonii. This PhoP/PhoQ-regulated sRNA is conserved in most enteric bacteria and is known to increase bacterial resistance to the antimicrobial peptide polymyxin B by controlling the expression of the phosphethanolamine transferase gene eptB. The analyses shows that MgrR does not regulate the expression of eptB in E. fergusonii, as observed in E. coli. Instead, MgrR likely regulates the glycerol utilization pathway glpABCFKTQ-frdABCD, and the pentose phosphate sugar pathway ulaABÂ-tktC that produces NADPH. Cell sensitivity to oxidative stress is affected by the production of NADPH, and an mgrR-deletion strain of E. fergusonii was highly sensitive to hydrogen peroxide (H2O2) and deoxycholic acid in vitro and displayed a severe loss of fitness within murine gut. The ulaAB-tktC pathway is not present in E. coli MG1655 and deletion of MgrR did not cause any sensitivity to H2O2. This work demonstrates that sRNAs could evolve divergent functions in closely related bacteria.

PCB126 is the most potent agonist of the aryl hydrocarbon receptor (AhR) in the whole family of chlorinated biphenyls (PCBs), a class of persistent organic pollutants that are classified as probable human carcinogens. It exerts systematic toxicities in animals mainly through the AhR pathway and generates cellular oxidative stress which might damage cellular macromolecules. The goal of this thesis is to elucidate the mechanisms of PCB126 toxicity in the aspect of AhR-related oxidative stress. Reactive superoxide radicals could be generated as the result of electron leakage in the catalytic cycles of cytochrome P450 (CYP) enzymes which are super-induced by sustained AhR activation in response to PCB126. In addition, the disruption of mitochondrial electron transport chain caused by AhR ligands also contributes to the production of superoxide radicals. Correspondingly in this thesis, the impact of PCB126 on the expression of the recently-discovered, TCDD-inducible microsomal CYP2S1 enzyme was studied. Then the regulation of mitochondrial antioxidant enzyme MnSOD in response to PCB126 was investigated. Lastly, a microarray study on classic AhR ligands of β-naphthoflavone and 3-methylcholanthrene was conducted to explore possible mechanisms of AhR-associated toxicities and to find explanations for the regulation of CYP2S1 as well as MnSOD after PCB126 exposure. It was found that CYP2S1 was only weakly or not at all regulated by PCB126 in rats. As for MnSOD, although its messenger and protein levels were induced by PCB126, its enzymatic activity was significantly reduced in the liver, probably through post-translational mechanisms. Although dietary manganese supplementation did not reverse the loss of MnSOD activity due to PCB126 exposure, it significantly protected against liver hypertrophy caused by PCB126. Microarray study results were consistent with previous findings and indicated that in addition to changed expression of a number of CYPs, metal homeostasis as well as mitochondria functions were also affected by AhR ligands. Overall, both CYP enzymes and the mitochondria contribute to the AhR-mediated toxicities of PCB126 that are associated with a disturbance of cellular redox homeostasis.

Chilling-related postharvest rind breakdown of navel oranges is a significant economic problem worldwide. Chilling injury (CI) symptoms on navel orange fruit vary, and descriptive classification is generally ad hoc, making inter-study comparisons difficult. In this study, external symptoms of CI were related to patterns of cellular collapse in affected flavedo tissue, and a classification system developed to aid consistent symptom identification and improve communication within the supply chain. Potential markers of senescence were evaluated because older fruit were found to be more susceptible to CI. Electrolyte leakage, moisture content and protein content of flavedo tissue were ineffective indicators of both senescence and chilling stress. Rind colour and internal maturity were generally good indicators of fruit age, but lacked sensitivity over short time periods to be of use. Although there was a strong seasonal effect on CI incidence, pre-storage treatments, including hot water and methyl jasmonate, generally reduced the incidence of CI. Because these treatments elicit defence responses that protect tissue from chilling stress, the response and efficiency of plant defence systems is probably an important factor in chilling tolerance. The concentration of lipid hydroperoxides (LOOH) in flavedo tissue was lower in fruit that were stored at a chilling temperature (1�C) compared to fruit that were stored at a non-chilling temperature (12�C) and lipid peroxidation did not increase during storage at 1�C. There was also a lower concentration of LOOH in the chilling sensitive variety than in the chilling tolerant variety. Therefore, increased lipid peroxidation is not related to chilling stress and subsequent injury but the results do suggest a role for LOOH in stress signalling. Antioxidant activity in the lipophilic fraction of flavedo tissue extracts increased as fruit senesced and was strongly correlated with carotenoid content. LOOH concentrations in flavedo tissue also increased as fruit senesced. The antioxidant activity of both the lipophilic and hydrophilic fractions of flavedo tissue extracts was higher in fruit stored at 12�C than in fruit stored at 1�C.

Oxygen toxicity is considered significant in the poor survival of probiotic bacteria such as Lactobacillus acidophilus and Bifidobacterium spp. in yoghurts. This study investigated methods to protect these bacteria from oxygen exposure. To confirm the accuracy of the reported survival estimates of L. acidophilus or Bifidobacterium spp. in yoghurts, the reliability of several enumeration media was evaluated with different commercial yoghurts. None of the media however, was found reliable thereby casting doubts on the reported cell numbers of probiotic bacteria in yoghurts. After much research,it was found that although oxygen can be detrimental to L. acidophilus and Bifidobacterium spp.in culture broths, it may not be significant for their poor survival in yoghurts. Nevertheless, techniques such as oxidative stress stress adaption, alternative packaging materials and microencapsulation as investigated in this study, can serve as general protective techniques to help yoghurt manufacturers in maintaining the recommended numbers of probiotic bacteria in their products. This would eventually assist in the efficient delivery of probiotic health benefits to yoghurt consumers. / Doctor of Philosphy (PhD)

Probiotic bacteria are added to commercial yoghurts as adjunct cultures, to impart health benefits to consumers. To gain maximum therapeutic benefit, the bacteria must remain viable over the shelf life of the yoghurt. Studies have shown, however, that the viability of these bacteria decreases significantly over this period and in some products, is negligible prior to the expiry date. Some strains of probiotic bacteria are oxygen sensitive. Yoghurt has been found to contain a significant concentration of dissolved oxygen and it has been proposed that this has a negative effect on probiotic viability. In this research, several tests were conducted and observations made. Experiments were conducted with non-commercial probiotic cultures to observe the effect of low oxygen environment on probiotic viability. No significant difference existed in viability between probiotic bacteria stored in oxygen reduced yoghurt and regular yoghurt. All yoghurt stored in oxygen barrier packaging material displayed enhanced shelf-life properties, this was observed in replicated experiments. Oxygen barrier packaging combined with an oxygen scavenging material was found to be the most effective oxygen removal system, particularly when used with set type yoghurt. / Doctor of Philosophy (PhD)

Ageing is a complex phenomenon, characterised by progressive loss of function, decreasing resistance to age-associated pathologies and stress, and increasing rates of mortality. The Free Radical Theory of Ageing implicates reactive oxygen and nitrogen species (ROS/RNS) generation as being integral to the ageing process, subjecting the organism to oxidative stress. Oxidative damage to biomolecules is suggested to be causative in the formation of ageing-associated phenotypes, dysregulation and dysfunction. Mitochondria are responsible for the production of the majority of ROS/RNS through normal functioning of the respiratory chain. Previous studies have reported increasing mitochondrial dysfunction with age, including oxidative damage to protein, lipid and DNA. Thus, mitochondrial dysfunction is considered by many to be central to the Free Radical Theory of Ageing. However, there are conflicting data on changes in mitochondrial and cellular function and damage on ageing. To investigate the role of mitochondrial protein oxidative damage in ageing, the heart, brain and liver of young (~ 2 month-old) and old (24 month-old) Wistar rats were fractionated into homogenate, cytosolic and mitochondrial fractions. Mitochondrial function was evaluated by measuring activity of the oxidative phosphorylation Complexes I-V, and correlating activity with quantitation of Complex subunits. The activities of the electron transport chain Complexes (I-IV) were largely unchanged on ageing, and no significant differences were seen in the protein levels of nuclear-encoded Complex I-IV subunits. There was a ~ 40% decrease in ATP synthase activity in heart and liver mitochondria from old rats as compared to young, but no change in the level of the Complex V nuclear-encoded subunit. These results suggest the decreased activity is due to modification of Complex V in heart and liver mitochondria on ageing, rather than changes in expression. Oxidative stress is a common cause of mitochondrial dysfunction, and is often accompanied by an increase in cellular antioxidant defences. Expression of the mitochondrial antioxidant enzyme, MnSOD, was found to be increased in the liver (+ 74%) and heart (+ 82%), but not brain, in old rats, suggesting oxidative stress in these organs on ageing in rats. To investigate generalised protein oxidative damage accumulation on ageing, whole tissue homogenate, cytosol and mitochondria were isolated from young and old heart, brain and liver. These fractions were assayed for three markers of protein oxidative damage: protein carbonyl content (a marker for generalised oxidative damage occurring via attack by many ROS/RNS), and ortho-tyrosine and meta-tyrosine accumulation (two markers specific for hydroxyl radical attack on phenylalanine). There were no consistent age-related changes in these biomarkers in any tissues, and no consistent significant differences between cytosolic and mitochondrial protein oxidative damage for any of the three tissues in the two age cohorts. Mitochondria were further subfractionated into membrane-enriched and matrix-enriched subfractions, but again, protein oxidative damage markers were largely unchanged on ageing. These results suggest that there is no common pattern of mitochondrial dysfunction during ageing in rats. Increased mitochondrial oxidative stress is a feature of ageing, but generalised protein oxidative damage is neither necessary nor sufficient for development of the ageing phenotype.