Oxidative stress is associated with development and progression of cardiovascular disease. Angiotensin II produces oxidative stress and endothelial dysfunction, and its actions may be attenuated by the activity of angiotensin converting enzyme type 2 (ACE2) which converts angiotensin II to the vasoprotective peptide angiotensin (1-7). Similarly, increased oxidative stress is associated with aortic valve stenosis in humans and mice. In my thesis studies, I explore mechanisms of modulation and generation of oxidative stress in cerebral arteries and heart valves. First, I tested the hypothesis that ACE2 deficiency increases oxidative stress and vasomotor dysfunction in cerebral arteries, and examined the role of ACE2 in vascular aging. Vasomotor function was assessed in the basilar artery ex vivo of adult and old ACE2 deficient (ACE2-/y) and wild type (WT or ACE2+/y) mice. ACE2 was present, but at relatively low levels in cerebral arteries. Systolic blood pressure was similar in adult and old ACE2-/y and ACE2+/y mice. Maximal dilatation to acetylcholine was impaired in the basilar artery from adult ACE2-/y mice compared to adult ACE2+/y. In old mice, maximal vasodilatation to acetylcholine was impaired in ACE2+/y mice and severely impaired in ACE2-/y mice. The antioxidant tempol improved responses to acetylcholine in adult and old ACE2-/y and ACE2+/y mice. Nitrotyrosine staining in the basilar artery was increased in adult ACE2-/y mice and in old ACE2-/y and ACE2+/y mice relative to adult ACE2+/y, which indicates that oxidative stress was higher in cerebral arteries from ACE2 deficient mice and old mice. Expression of NADPH oxidase subunits Nox2 and p47phox, and of pro-inflammatory molecules Rcan1 and TNF alpha; was increased in cerebral arteries from old ACE2-/y and ACE2+/y mice. Additionally, I tested the hypothesis that serotonin induces oxidative stress in human heart valves, and examined mechanisms by which serotonin may increase reactive oxygen species (ROS). Superoxide (O2.-) was measured in heart valves from explanted human hearts that were not used for transplantation. Superoxide levels (lucigenin-enhanced chemiluminescence) were increased in homogenates of cardiac valves and pulmonary artery after incubation with serotonin. A non-specific inhibitor of flavin-oxidases (DPI), or inhibitors of monoamine oxidase-MAO (tranylcypromine and clorgyline), prevented serotonin-induced increase in O2.-. Dopamine, another MAO substrate which is increased in patients with carcinoid syndrome, also increased superoxide levels in heart valves, and this effect was attenuated by clorgyline. Apocynin did not prevent increases in O2.- during serotonin treatment. Addition of serotonin to recombinant human MAO-A generated superoxide, and this effect was prevented by an MAO inhibitor. In conclusion, I have demonstrated that ACE2 deficiency impairs vasomotor function in cerebral arteries from adult mice and augments endothelial dysfunction during aging. Oxidative stress plays a critical role in cerebrovascular dysfunction induced by ACE2 deficiency and aging. I have also identified a novel mechanism whereby MAO-A can contribute to increased oxidative stress in human heart valves and pulmonary artery exposed to serotonin and dopamine.

Parkinson’s disease (PD) is a chronic and progressive movement disorder affecting an individual’s ability to move, and can become life threatening when it progresses to the point where an individual has difficulties swallowing, breathing, and chewing. PD is a neurodegenerative disorder caused by the damage of neurons, leading to the loss of nerve function and structure in the brain. Specifically, PD is characterized by the selective loss of the substania nigra, the dopamine (DA)-containing region of the brain. Due to loss of DAergic neurons, it has been suggested that DA serves as an endogenous toxin when there are alterations in the synthesis, metabolism, and regulation of DA. The pathogenesis of PD remains unclear, and many are working on determining what factors cause this neuronal death. Factors hypothesized to be important include: aging, genetics, endogenous toxins, and environmental toxicants. The aim of this work is to explore the role of endogenous neurotoxins, such as toxic dopamine metabolites, oxidative stress (OxS), and reactive oxygen species as contributors to the neurotoxicity relevant to PD, and to examine the potential for regulation of this toxicity by alterations in the antioxidant status of the cell. DA can undergo metabolism by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a highly toxic and reactive metabolite; that is hypothesized as a contributor to the neurotoxicity observed in PD. Subsequently, DOPAL can be further metabolized by aldehyde dehydrogenases or reductases to form 3,4- dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), respectively. When evaluating all of these metabolites, DOPAL displays the greatest toxicity both in vitro and in vivo. DOPAL contributes to cell toxicity through a variety of mechanisms; these include: 1) it is able to react with proteins, leading to covalent modification at Lys and Arg residues causing the formation of adducts 2) DOPAL can autooxidize to form quinone species, which are reactive with proteins 3) autooxidation and protein modification by DOPAL results in the generation of reactive oxygen species (ROS) (H₂O₂, O₂•−), which are also toxic. Of note, increasing ROS can impact the OxS levels, creating an imbalance that contributes to cell damage. This insult can include inhibiting the carbonyl metabolizing enzymes, further increasing DOPAL levels. During these interactions, damage occurs to proteins, enzymes, and DNA, causing an inability for the cell to perform properly, consequently leading to cell death. The initial work, described in Chapter 3, was determination of ROS and secondary insults that are produced during DOPAL-mediated neurotoxicity. Methodologies utilizing fluorescence detection were able to identify the production of both hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂•−). The formation of these ROS can result in an imbalance in oxidative status, contributing to augmented OxS in the cell. These ROS were produced both in purified protein assays, as well as, in cell based studies. These assays investigated formation of ROS during protein interaction, but were also tested in the presence of known toxins that have been correlated with PD. The work described in Chapter 4 explores conditions in these neurons that can impact the alteration of OxS through ROS. It was hypothesized that oxygen presence is necessary to catalyze the reaction of DOPAL with proteins. Therefore, work was completed to discover if oxygen deficiency could regulate DOPAL-protein interactions. Identification of protein modification, following oxygen eradication, confirmed that inhibition of DOPAL’s reactivity towards proteins succeeds the loss of oxygen. This led efforts to focus on other mechanisms by which to alter cellular oxidative status to influence DOPAL’s function in these cells. Additional work was completed to discover if radical scavengers similarly control resultant toxicity from DOPAL activity. As previously published, radical scavengers, such as tricine, exhibit a protective effect in regards to modification of proteins. Furthermore, we believe that oxidative status can serve as a target for mediation of DOPAL neurotoxicity. If affecting the capability of producing ROS species can impact OxS in DOPAL-mediated toxicity, it is believed that utilizing agents, such as antioxidants, can serve as a new potential treatment for PD. Chapters 5 (cellular models) and 6 (in vivo model) explore efforts to alter (+/-) antioxidant levels via addition of N-acetylcysteine (NAC), diamide (Dia), and buthionine sulfoxide (BSO). It was found that antioxidants, such as NAC, attenuate the adduction of proteins by DOPAL, alter DA metabolite levels, and inhibit behavioral characteristics of PD in the in vivo model. Conversely, oxidants Dia and BSO increased DOPAL and its subsequent modification of proteins. Finally, Chapter 7 includes a conclusion of the work documented here and addresses future potential directions. This project includes so new findings that need to be further characterized resulting in many future direction that can be explored. One major direction in which this project can be taken would be further validation of NAC to serve as a novel therapy for PD. The future directions will include all aspects of this project including a brief discussion of examining NAC analogs to increase bioavailability leading to a more potent drug model. To date there are limited answers into what is causing this neurodegeneration, and currently, there is no cure for PD. Therefore, my thesis research is making an impact in the field as it, has explored the ways in which a known toxic metabolite is leading to death of these neurons, has identified secondary products that are contributing to the toxicity observed, and has developed a potential new therapy for PD utilizing antioxidants. All of these will help advance research in the field to continue to identify new targets in this cellular pathway leading to a better understanding of the cause of PD.

Les modèles climatiques indiquent qu’il est très probable que les végétaux soient de plus en plus exposés à deux facteurs de stress environnementaux : l’ozone troposphérique (O3) et le déficit hydrique du sol, tous deux pouvant provoquer un stress oxydant pour le végétal. Dans des conditions naturelles, ces deux facteurs peuvent être concomitants ou se succéder. L’impact de l’O3 et de la sécheresse nécessite donc une attention particulière. Afin de déterminer les réponses de défense mises en place par les arbres, deux génotypes de Populus nigra x deltoides (Carpaccio et Robusta) ont été exposés aux contraintes séparées ou à leur combinaison en conditions contrôlées dans des chambres de culture. Pour explorer les effets des stress et l’interaction entre les deux contraintes, nous avons ciblé les deux premiers niveaux de défense des plantes que sont le contrôle de l’ouverture/fermeture des stomates et les processus de détoxication cellulaire. Nos résultats montrent que Carpaccio et Robusta sont tous deux relativement tolérants à une sécheresse modérée grâce à un contrôle efficient des stomates. Face à l’O3, cependant, les deux génotypes adoptent des stratégies de réponse différentes : un évitement important pour Carpaccio et une maximisation de l’assimilation au détriment des feuilles pour Robusta. Cela se traduit par une différence de fermeture des stomates. Les deux génotypes ne font alors pas face au même flux d’O3 entrant dans les feuilles, ce qui impacte la détoxication cellulaire, dans laquelle le glutathion semble jouer un rôle majeur. En lien avec les modifications de capacité antioxydante, l’activité des enzymes du cycle ascorbate-glutathion (MDHAR, DHAR et GR) et/ou l’expression des gènes codant pour ces protéines sont modifiées. En combinaison de stress, le déficit hydrique protège le végétal du stress oxydant induit par l’O3 en amplifiant la fermeture des stomates. En revanche, la croissance de l’arbre est impactée par l’effet additif des deux contraintes. De plus, l’induction de voies de régulation hormonales différentes par les deux contraintes pourrait modifier le « cross-talk » complexe régulant la réponse au stress combiné. Enfin, dans le cas d’une succession de stress, l’exposition à l’O3 avant un épisode de sécheresse impacte faiblement la réponse de l’arbre. Cependant, un ralentissement de la fermeture des stomates induit par l’O3 est observé malgré l’arrêt de la fumigation. Il est donc nécessaire de prendre en compte le ralentissement et la fermeture des stomates induit par l’O3 et le déficit hydrique dans les modèles de conductance stomatique utilisés pour calculer l’indicateur du flux d’O3 entrant, le PODy (Phytotoxic Ozone Dose above a threshold of y nmol O3 m-2.s-1). / Climate models indicate that it is very likely that plants will be more and more exposed to two environmental stressors: ground-level ozone (O3) and soil water deficit, both causing oxidative stress to the plant. Under natural conditions, these two factors can be concomitant or successive. Therefore, the impact of O3 and drought requires special attention. In order to determine the defensive responses adopted by trees, two genotypes of Populus nigra x deltoides (Carpaccio and Robusta) were exposed to separate or combined stresses under controlled conditions in growing chambers. To explore the effects of stresses and their interaction, we targeted the plant’s first two levels of defence: i) the control of stomatal opening and closing, ii) the cellular detoxification processes. Our results show that both Carpaccio and Robusta are relatively tolerant to moderate drought thanks to an efficient stomatal control. However, different response strategies were adopted by the two genotypes to cope with O3. For Carpaccio, the strategy is avoidance, and for Robusta, the strategy is maximization of net CO2 assimilation at the expense of leaves. This results in a difference in the stomatal closure. The two genotypes do not face the same flow of O3 entering the leaves. This impacts cellular detoxification in which glutathione seems to play a major role. Also, the activity of ascorbate-glutathione cycle enzymes (MDHAR, DHAR and GR) and/or the expression of genes encoding these proteins are modified. Under combined stresses, the water deficit protects the plant from the O3-induced oxidative stress by amplifying the stomatal closure. Nevertheless, the tree growth is impacted by the additive effect of the two stresses. Furthermore, the induction of different hormonal regulatory pathways by the two stressors could modify the complex "cross-talk" regulating the response to combined stress. Finally, in the case of a succession of stresses, exposure to O3 prior to a drought episode has a weak impact on the tree's response. However, O3 induced a stomatal sluggishness in closure despite the cessation of fumigation. It is therefore necessary to take into account stomatal closure and sluggishness induced by O3 and water deficit in the stomatal conductance models used to calculate the indicator of O3 flux inside the leaves, PODy (Phytotoxic Ozone Dose above a threshold of y nmol O3 m-2.s-1).

<p>Ispitivanja primene vodonika u kliničkim uslovima su prilično nova, međutim efikasnost<br />molekularnog vodonika u dosada&scaron;njim kliničkim ispitivanjima je evidentna. Cilj ovog<br />istraživanja bio je da se ispita uticaj suplementacije sa vodom obogaćenom molekularnim<br />vodonikom (HRW) na bazalne vrednosti parametara oksidativnog stresa kod vrhunskih<br />džudista. Eksperimentalni tretman odvijao se u dve faze. U prvoj fazi ispitanici<br />eksperimentalne grupe (N=6) su bili suplementirani sa HRW (4g) u trajanju od 28 dana,<br />dok je kontrolna grupa (N=6) koristila placebo. Nakon suplementacije ispitanici su imali<br />&bdquo;Washout&ldquo; period u trajanju od 28 dana, a zatim je ponovljen identičan protokol na<br />ispitanicima u trajanju od 28 dana. U drugoj fazi eksperimentalnog tretmana ispitivani su<br />akutni efekti suplementacije molekularnim vodonikom na acido baznu ravnotežu<br />vrhunskih džudiskinja. U ovoj fazi studije ispitanice eksperimentalne grupe (N=4) su<br />suplementirane sa HRW (4g) , dok je kontrolna grupa (N=4) koristila placebo. Nakon<br />suplementacije ispitanice su imale &bdquo;Washout&ldquo; period u trajanju od 4 dana, a zatim je<br />ponovljen identičan protokol na ispitanicama pri čemu su ispitanice eksperimentalne<br />grupe (N=4) koristile placebo, dok je kontrolna grupa (N=4) suplementirana sa HRW.<br />Rezultati ispitivanja aktivnosti antioksidativnih enzima kod Placebo i HRW grupe<br />ukazuju na pojavu statistički značajnih razlika u antioksidativnom statusu kod vrhunskih<br />džudista. Odgovor organizma usled unosa HRW ogleda se u povećanju aktivnosti enzima<br />SOD i sadržaja GSH usled sprovođenja programirane fizičke aktivnosti. Takođe, trend<br />opadanja sadržaja MDA u HRW grupi je bio detektabilan. Primena HRW nije izazvala<br />statistički značajne promene u biohemijsko hematolo&scaron;kim parametrima. Akutni efekti<br />suplementacije sa HRW uticali su statistički značajno na redukciju akumulacije laktata<br />kao i na povećanje pH krvi nakon primene specifičnog džudo fitnes testa (SJFT) kod<br />vrhunskih džudistkinja. Ipak, primena suplementa nije značajno uticala na pobolj&scaron;anje<br />performansi džudistkinja. Dobijeni rezultati ukazuju na ergogenu antioksidantnu moć<br />molekularnog vodonika, pri čemu su dalja istraživanja neophodna kako bi se podesili<br />uslovi u kojima bi molekularni vodonik ispoljio svoje maksimalno delovanje i u sportu.</p> / <p>Research of hydrogen application are quite new in clinical conditions, however the<br />efficiency of molecular hydrogen in previous clinical studies is evident. The aim of this<br />study was to investigate the effect of supplementation with hydrogen rich water (HRW)<br />on baseline oxidative stress parameters in elite male judokas. Experimental treatment was<br />carried out in two phases. In the first phase, experimental group (N=6) were<br />supplemented daily with HRW (4g) for 28 days, while the control group (N=6) was used<br />placebo. After supplementation intake all subjects had &quot;washout&quot; period for 28 days, after<br />which the same protocol was repeated for a period of 28 days. In the second stage of<br />experimental treatment, acute effects of supplementation with molecular hydrogen on<br />acid base balance of elite female judokas were examined. In this phase of the study,<br />subjects from experimental group (N=4) were supplemented daily with HRW (4g), while<br />the control group (N=4) used placebo. After supplementation all subjects had a<br />&quot;washout&quot; period for a 4 days, after which the same protocol was repeated wherein the<br />members of the experimental group (N=4) used placebo, while the control group (N=4)<br />was supplemented daily with HRW. The results of analyzed antioxidant enzymes activity<br />in placebo and HRW group indicated on the occurrence of statistically significant<br />differences in antioxidant status of elite judo athletes. Obtained results indicate that<br />response of the body due to intake HRW is reflected in increasing levels of SOD and<br />GSH due to the implementation of programmed physical activity. Also, trend of MDA<br />decreasing in HRW group was detectable. In addition, application of HRW did not cause<br />negative changes in the biochemical and hematological parameters. Acute effects of<br />supplementation were affected to a statistically significant reduction in lactate<br />accumulation and pH increase after applied special judo fitness test (SJFT) on the top<br />judoka. However, use of supplements had no significant effect on improving performance<br />in elite judokas. Obtained results confirm ergogenic antioxidant power of molecular<br />hydrogen, wherein further studies are necessary in order to adjust the conditions in which<br />molecular hydrogen will exerted its maximum in sport.</p>

Alzheimer’s disease (AD) is the most commonly diagnosed form of dementia in the aged, and is characterised by a progressive decline in memory, language and other cognitive functions, together with deterioration in behavioural, emotional and social skills. The earliest clinical symptoms include episodic memory loss and dysnomia. This is followed by other signs of cortical impairment including apraxia, agnosia, and visuospatial impairment. In advanced stages, victims become mute, cannot walk and are incontinent; they therefore become totally dependent on carers. AD is the third leading cause of death in the aging population after heart disease and cancer. The incidence of AD doubles every 5 years in subjects between the ages of 65 and 85 years, affecting one in three by the age of 80. AD is characterised by the existence of intracellular and extracellular amyloid deposits in the brain. Extracellular amyloid deposits consist of plaques, whereas the deposits within and around blood vessels are referred to as cerebral amyloid angiopathy (CAA). Neurofibrillary tangles (NFT) are characteristically found in AD; however, they are also found in some other neurodegenerative disorders such as tuberose sclerosis, amyotrophic lateral sclerosis, parkinson-dementia complex and dementia pugilistica.

The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietary selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.

In spite of impressive recent progress, the aetiopathogenesis of Alzheimer’s disease (AD) remains incompletely understood. The distinctive neuropathological features of AD, in particular the plaques and tangles, have been the particular focus of most aetiological theories. It is well accepted that AD is a multifactorial disease, with alterations to a variety of brain structures and cell types, including neurons, glia and the brain vasculature. Studies of risk factors have revealed a diversity of genetic variables that interact with health, diet and lifestyle-related factors in the causation of AD. These factors influence the structure, aggregation and function of a set of proteins that are increasingly the focus of research. The work in this thesis has focused on the pathophysiological aspects of some of these proteins in a number of cellular compartments and brain. Several assays have been established and techniques utilized in the completion of this work, including; differential detergent fractionation of brain tissue, 1D and 2D PAGE, western blotting with chemiluminescence detection, ELISA assays of Abeta 1-40 and 1-42, quantitative ECNI GCMS of o- and m-tyrosine as well as metabolites of the kynurenine pathway, quantitative MALDI-TOF assay of hemorphins and LCMSMS based proteomics, to identify proteins with altered expression levels in AD relative to control brain tissue. A variety of regional differences have been observed in the biochemistry of the AD cortex which are probably the outcome of local response variations to AD pathology. One of the most consistent threads throughout this work has been an apparent resilience of the occipital lobe relative to the other brain regions, as reflected in lower overall levels of oxidative stress and increased levels of proteins associated with metabolic processes, neuronal remodeling and stress reduction.

Oxidative damage has been suggested to play a key role in the pathogenesis of atherosclerosis and other cardiovascular disease. Increased free radical production induced by oxidative stress can oxidise low density lipoproteins, activates platelets, induces endothelial dysfunction and disturbs glucose transport by consuming endogenous antioxidants. Using a combination, of in vitro and in vivo experimental models, the primary aims of the studies undertaken for this thesis were to examine whether different antioxidants could negate risk factors leading to thrombosis, atherosclerosis and other cardiovascular diseases. The studies utilised the mechanisms involved in platelet activity and glucose uptake by skeletal muscle myotubes. The first study determined if olive leaf extract would attenuate platelet activity in healthy human subjects. Blood samples (n=11) were treated with five different concentrations of extract of Olea europaea L. leaves ranging from 5.4£gg/mL to 54£gg/mL. A significant reduction in platelet activity (pless than0.001) and ATP release from platelets (p=0.02) was observed with 54£gg/mL olive leaf extract. The next crossover study compared the effect of exercise and antioxidant supplementation on platelet function between trained and sedentary individuals. An acute bout of 1 hour exercise (sub maximal cycling at 70% of VO2max) was used to induce oxidative stress in 8 trained and 8 sedentary male subjects, before and after one week supplementation with 236 mg/day of cocoa polyphenols. Baseline platelet count and ATP release increased significantly (pless than0.05) after exercise in all subjects. Baseline platelet numbers in the trained were higher than in the sedentary (235¡Ó37 vs. 208¡Ó34 x109/L, p less than 0.05), whereas platelet activation in trained subjects was lower than sedentary individuals (51¡Ó6 vs. 59¡Ó5%, p less than0.05). Seven days of cocoa polyphenol supplementation did not change platelet activity compared to the placebo group. The third study determined the effect of 5 weeks of either 100mg/day £^-Tocopherol (n=14), 200mg/d £^-Tocopherol (n=13) or placebo (n=12) on platelet function, lipid profile and the inflammatory marker C-reactive protein. Blood £^-tocopherol concentrations increased significantly (pless than0.05) relative to dose. Both doses attenuated platelet activation (pless than0.05). LDL cholesterol, platelet aggregation and mean platelet volume were decreased by 100mg/d £^-tocopherol (all pless than0.05). The final study determined the effect of glucose oxidase induced oxidative stress and £^-tocopherol treatment on glucose transport and insulin signalling in cultured rat L6 muscle cells. One hour treatment with 100mU/mL glucose oxidase significantly decreased glucose uptake both with and without 100nM insulin stimulation (pless than0.05). Pre-treatment with 100ƒÝM and 200ƒÝM £^-tocopherol partially protected cells from the effect of glucose oxidase, whereas 200ƒÝM £^-tocopherol restored both basal and insulin stimulated glucose transport to control levels. Glucose oxidase-induced oxidative stress did not impair basal or insulin stimulated phosphorylation of Akt or AS160, but 200ƒÝM £^-tocopherol improved insulin-stimulated phosphorylation of these proteins. In summary, the results from the studies undertaken for this thesis provide evidence that antioxidant supplementation maintains normal platelet function, exerts a positive effect on blood lipid profile and improves glucose uptake in normal healthy asymptomatic population as well as under conditions of induced oxidative stress. Antioxidants including foods rich in cocoa, olive and gamma tocopherol have the potential to combat oxidative stress induced risk factors leading to cardiovascular diseases.

<p>Radiotherapy is one of the most common therapeutic methods for treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (< 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use.</p><p>8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids.</p><p>Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively.</p><p>Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption.</p><p>The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG.</p><p>In summary, our results confirm that extracellular 8-oxo-dG is a sensitive <i>in vivo</i> biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.</p>