Retinopathy of prematurity (ROP) commonly occurs in premature babies, with the first phase of vessel cessation followed by a second phase of vessel proliferation. In addition to vascular changes, neuronal abnormalities have also been observed. However, evidence for morphological changes of retinal neurons at the cellular level is lacking. Oxidative stress has been highly indicated in the pathogenesis of ROP. Increased oxidative stress level was demonstrated in preterm babies expecially in those with ROP. The activity of aldose reductase (AR), the first enzyme in the polyol pathway, has been found to contribute to oxidative stress. Therefore, the role of AR in ROP was examined using a mouse model of oxygen-induced retinopathy (OIR), which was a well-established model to mimic human ROP. Studies in examining the effects of AR on retinal vasculature showed that genetic deletion or pharmacological inhibition of AR reduced vaso-obliteration and neovascularization, possibly through regulating VEGF-induced pathway. In addition, morphological changes of various retinal neurons at different time points in the mouse model of OIR were also demonstrated. The degree of effects from hyperoxic and hypoxic exposure appeared to depend on the different stages of maturation of various retinal neurons. AR deficiency showed protective effects on retinal neurons including horizontal cells, rod bipolar cells and amacrine cells, possibly through attenuating the damage on blood vessels as well as facilitating blood vessel re-growth in the avascular area which provide more nutrients and supply to the retinal neurons. To elucidate the protective role of AR deficiency in ROP, the changes in oxidative stress and oxygen-dependent gene expression including HIF-1α and iNOS were investigated. AR deficiency attenuated oxidative stress induction to protect the neonatal retina. In addition, AR deficiency also showed attenuated HIF-1α expression and enhanced iNOS expression. This served to strictly control the HIF-1α level which in turn can tightly regulate VEGF induction in the mouse retinae after OIR. In order to further elucidate the role of AR in the pathogenesis of ROP, effects of AR deficiency on glial cells and microglia were investigated. AR deficiency reduced retinal astrocytic activation in hyperoxia and induced early M?ller cell gliosis in hypoxia. In addition, AR deficiency enhanced the specific function of microglia in different areas with facilitation of revascularization in avascular area and promotion of tufts regression in neovascular area. Moreover, AR deficiency also reduced the activation of a key inflammatory mediator NF-κB, which was considered to contribute to neovascularization. Therefore, AR deficiency demonstrated regulatory roles in reponses of glial cells, microglia and inflammation, contributing to the protective effects on neonatal retina in the mouse model of OIR. Taken together, AR deficiency reduced the vascular and neuronal changes possibly through attenuating oxidative stress and glial responses as well as modulating inflammatory responses, indicating a beneficial role of AR inhibition in OIR. These findings highly suggest the therapeutic potential of AR inhibition in the treatment of ROP. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Diabetic retinopathy (DR) was characterized by numerous hyperglycemia-dependent cellular and pathological changes in the retina, including retinal ischemia/reperfusion (I/R) injury. To determine the role of the 2nd enzyme of polyol pathway in relation with the pathogenesis in ischemic retinopathy, SDH deficient mice, C57BL/LiA, that lacked SDH activity, was used to study the pathogenesis of diabetic retinopathy, which also included I/R injury. Wild type and SDH-deficient mice were subjected to I/R injury by transiently occluding middle cerebral artery for two hours and twenty-two hour of reperfusion. The rationale of this study was to investigate the effect by blocking the conversion of sorbitol to fructose by SDH null mutation (SDH -/-), leading to accumulation of sorbitol level and reduction of oxidative stress, as demonstrated by the polyol pathway. Results: After induction with transient MCAO, there was increase in the thickness of OLM to ILM ipsilateral SDH+/+ compared with contralateral SDH+/+ (from 84 +/- 1 to 96 +/- 2 μm) while that of ipsilateral SDH-/- compared with contralateral SDH -/- (from 77 +/- 2 to 90 +/- 2 μm) suggested that there was edema after ischemic reperfusion injury. The result showed that there was increased cellular edema in ipsilateral retina of both SDH +/+ and SDH -/- retina after transient MCAO. The level of immunoreactivity against Aquaporin-4 and nitrotyrosine in studying the presence of oxidative stress; glutamine synthetase and glutamate in studying the toxicity of astrocyte glutamate; sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) in studying the regulation Ca2+ homeostasis was determined using immunohistochemistry. For all the antibodies, there was similar immunoreactivity level between the contralateral side of both SDH+/+ and SDH -/- mice. For the SDH+/+ group, there was increase in signal in the ipsilateral retina in comparison with the contralateral one. On the other hand, for the SDH-/- group, similar result was observed. There was increase in signal and it was found more in the ipsilateral retina in comparison with the contralateral retina. Finally, in the ipsilateral retina of both SDH +/+ and SDH -/- mice, increased immunoreactivity was found in both but their difference was not statistically significant. This concluded that SDH deletion and subsequent accumulation of sorbitol metabolites did not contribute significantly in the role of pathogenesis of ischemic retinopathy especially in mice after I/R injury. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

Risks of metabolic syndrome including cardiovascular disease and diabetes are significantly affected by maternal nutrition. This concept of developmental programming had been investigated in our laboratory and in an earlier study, it was established that maternal high-fat diet predisposed rat offspring to insulin resistance and higher triglyceride in serum, liver, skeletal muscle and adipose tissue. These abnormalities, however, were ameliorated by supplementing green tea extract to dam’s diet throughout gestation and lactation. The overall objective of this thesis was to examine lipid metabolism, oxidative stress and inflammatory responses in heart of offspring born to dams receiving high-fat diet with or without green tea supplementation during pregnancy and lactation. Female Sprague-Dawley rats were fed an obesogenic diet which was a high-fat diet (HF,30%), low-fat diet (LF,7%) or HF diet containing 0.75% green tea extract prior to conception and throughout gestation. During lactation, half of the dams had their diet switched from HF to GT and vice versa. Pups were weaned to the HF or LF diet, forming 10 offspring groups (gestation/lactation/postweaning): LF/LF/LF, LF/LF/HF, HF/HF/LF, HF/HF/HF, HF/GT/LF, HF/GT/HF, GT/GT/LF, GT/GT/HF, GT/HF/LF and GT/HF/HF. Except a larger fibrotic area, maternal HF diet did not affect lipid accumulation, oxidative status and inflammatory response in the heart of offspring. Analysis of variance revealed different, and even opposite, effects of GT supplementation during gestation and lactation. In offspring born to dams receiving GT supplementation during gestation, they had suppressed fatty acid oxidation (FAO) and higher triglyceride (TG) level in the heart. In contrast, when GT was supplemented to dams during lactation, offspring had elevated heart TG, cholesterol and free fatty acid levels but up-regulated FAO. Since FAO is associated with reactive oxygen species (ROS) production, modulation of FAO is believed to affect cellular stress responses in heart. Consistent with FAO, cardiac stress, apoptotic and inflammatory biomarkers including B-type natriuretic peptide (BNP), bcl 2 associated-x (bax) and interleukin-1β (IL1b) were down-regulated in offspring born to dams given GT during gestation, whereas GT supplementation during lactation increased the expression of pro-apoptotic markers: bax and caspase-3 (Cas3) concurrent with activation of antioxidant defense system: catalase, glutathione peroxidase (GPx) and glutathione S-transferase (GST) as adaptive mechanism against increased ROS. Uncoupling protein 2 (UCP 2) and subsequent higher bcl 2 /bax ratio has been reported to stimulate apoptosis. In agreement with this, mRNA expression of BNP, bax and Cas3 were found to correlate with that of UCP 2. This suggests UCP 2may play an important role in apoptosis under the impact of maternal GT supplementation. The present data suggest that the effect of maternal high-fat diet is organ specific causing apparently lesser damage to the heart. When GT is given in conjunction with a high-fat diet to dams during gestation, there is no clear cut advantage to the offspring. However, potential adverse effects could not be ruled out when GT is supplemented to dams during lactation possibly due to higher catechin exposure via milk. Future study should focus on establishing the benefits and safety use of GT during gestation. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

abstract / Biochemistry / Master / Master of Philosophy

The use of biomarkers is considered a promising alternative, or complement, to traditional ecotoxicological assays. Toxic effects are often initially manifested at the molecular or biochemical level, biomarkers are therefore used as sensitive indicators of toxic exposure. Ideally, biomarkers would also indicate reduced fitness and possible later effects at the individual or population levels. However, implementing biomarkers in ecotoxicology is challenging and few biomarkers have an established connection to reduced individual fitness. The aim of this thesis was to increase the value and improve the interpretation of biomarker responses in ecotoxicological studies by examining the impact of confounding factors and the relationship between oxidative biomarkers and reproductive effects in crustaceans. The sensitivity of biomarkers was confirmed in paper I as toxic effects of pharmaceuticals with conserved drug target orthologs were observed at the molecular and biochemical levels both earlier and at lower concentrations than effects on mortality and reproduction. No toxic effects were observed for the pharmaceutical without identified drug target orthologs, thus stressing the importance of considering toxic mechanisms and being aware of the most likely target when evaluating toxic effects also in non-target species. Many xenobiotics and environmental stressors interfere with oxidative processes, making oxidative biomarkers interesting to study in ecotoxicology and stress ecology. Still, feeding rate was identified as a confounding factor for antioxidant capacity (assayed as oxygen radical absorbance capacity, ORAC) and lipid peroxidation in ecotoxicological studies (paper II). However, ORAC normalized to protein was independent of altered feeding rates, hence it can be applied as a suitable exposure biomarker without considering alterations and effects of feeding rate. The connection between reproduction and oxidative stress is dual, as reproduction both can be inhibited by oxidative stress and induce pro-oxidative processes. Further, a positive association was found between ORAC and the occurrence of embryo aberrations in the benthic amphipod Monoporeia affinis (paper III). An association between antioxidant defense and reproduction was also observed for Daphnia magna (paper IV). Threshold values for identification of exposed individuals and prediction of possible later reproductive effects were established for ORAC. This thesis has contributed to diminishing some of the knowledge gaps limiting the use of oxidative biomarkers in ecotoxicology, by contributing to increased understanding of how oxidative biomarkers relate to important life-traits. Moreover, ORAC has been identified as a suitable biomarker of not only exposure, but also reproductive effects. Future research should continue to establish connections between biomarker responses and effects at higher levels, and focus on providing defined threshold values to enable predictions about later effects. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

In oviparous animals, maternal traits such as the investment of resources in eggs and oviposition site selection are often important determinants of offspring phenotypic quality, and may have an adaptive role in tailoring offspring phenotypes to local environmental conditions. This thesis examines the adaptive significance of two specific maternal traits in the green turtle (Chelonia mydas); namely the deposition of fat-soluble antioxidants in egg yolk, and the selection of nest sites via natal homing. (Please view 'front matter' file for full abstract)

Hyperoxia is an essential aid to life support in patients with severe respiratory failure. However, it is recognised as a contributor to the pathological consequences of oxidative stress including oxidative tissue damage, inflammation and cell death resulting in acute or chronic lung injury. The specific mechanisms behind this type of injury are still not completely understood. This study was undertaken with two main aims. Firstly, to evaluate the adverse effects of hyperoxia on the ciliary coverage using a novel large animal model. For the first time, an in vitro bronchus bovine tissue culture model was developed and used to quantify ciliary coverage loss over time. The protection role of antioxidant supplementation with α-tocopherol and ascorbate was also investigated. Secondly, the importance of the tight junction protein ZO-1 in hyperoxia-induced monolayer permeability was investigated using a human bronchial cell line (16HBE14o-) and the potential inflammation effects on bronchial tightness. Additionally studies were carried out in order to find out if antioxidant vitamin treatment can protect against or reduce these effects. Scanning electronic microscopy indicated that hyperoxia caused a time dependent decline (t½ = 3.4 d compared to 37.1 d under normoxia) in ciliary coverage (P < 0.0001). This was associated with an increase in the number of sloughed cells, many apparently intact, into the medium (p < 0.05). Several biochemical parameters were assessed to obtain evidence of oxidative stress caused by hyperoxia in this model including tissue damage (lactate dehydrogenase, LDH, in the medium), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), DNA damage (comet assay used for the first time with primary bronchus culture), protein oxidation (OxyBlot kit) and antioxidant status (total glutathione). Antioxidant vitamins had a significant protective effect on the hyperoxia-induced reduction in percentage ciliary coverage (P < 0.05). Moreover, an increase in the bronchial permeability was shown characterised by a significant decrease (P < 0.05) in transepithelial electrical resistance (TER) under hyperoxic conditions. The reduction of ZO-1 associated fluorescence (P < 0.01) is in compatible with the downregulation of ZO-1 expression assessed by RT-PCR. Levels of the pro-inflammatory cytokines IL-8, IL-6 and TNF-a concentration in the medium, as measured by ELISA, increased significantly (P < 0.001) under hyperoxia, and this was accompanied with a marked increase in the cytokine expression. However, the antioxidant vitamins E and C, partially reduced the impact effects of hyperoxia, both individually and in combination, whilst increases in ZO-1 expression and fluorescence intensity (P < 0.05), as well as the suppression of cytokine secretion and gene expression was modest. Use of these vitamins was not enough to reduce the epithelial permeability significantly compared to normoxia. The data implies that hyperoxia-induced damage to cultured bovine bronchial epithelium and the denudation of cilia over time with increased permeability was due, at least in part, to the decline in TJ protein expression and associated fluorescence intensity. The antioxidant vitamins vitamin E and C had partial protective effects against hyperoxia damage. However, additional studies are called for in order to further understand the possible associations between oxidative stress and inflammation caused by hyperoxia and tight junction proteins, also response to treatment with antioxidant individually or in combination.

The Ataxia-telangiectasia mutated (ATM) protein is regarded as the major regulator of the cellular response to DNA double Strand Breaks (DSBs). In response to DSBs, ATM dimers dissociates into active monomers in a process promoted by Mre11-Rad50-Nbs1 (MRN) complex. ATM-deficient cells exhibit signs of chronic oxidative stress, suggesting that ATM plays an important role in the regulation of reactive oxygen species (ROS). I show for the first time that ATM can be activated by oxidative stress directly in the form of exposure to H₂O₂. In vitro kinase assays with purified ATM suggest that the activation by H₂O₂ is independent of DSBs and the MRN complex. In 293T cells, H₂O₂ induces ATM autophosphorylation on serine 1981. p53 and Chk2 are also phosphorylated by ATM after H₂O₂ treatment but not histone H2AX and heterochromatin protein Kap1, indicating that ATM activation by H₂O₂ in human cells is independent of DNA damage. I also show that the cysteine residue 2991 is critical for ATM activation by H₂O₂ in vitro. / text

published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant inherited neurodegenerative disease for which there is no cure. SCA7 belongs to the group of polyglutamine disorders, which are all caused by the expansion of a polyglutamine tract in different disease proteins. Common toxic mechanisms have been proposed for polyglutamine diseases; however the exact pathological mechanism(s) are still unclear. The aim of this thesis was to identify and characterize the molecular mechanisms by which polyglutamine expansion in the ATXN7 protein cause SCA7 and how this can be counteracted. We found that mutant ATXN7 can be degraded by the ubiquitin proteasome system (UPS) and autophagy, the two main cellular degradation pathways. However aggregation stabilized the protein against degradation. Moreover, we found that mutant ATXN7 blocked the induction of autophagy by interfering with p53 and the ULK1-ATG13-FIP200 complex. Pharmacological stimulation of autophagy ameliorated aggregation, as well as toxicity. We also found that oxidative stress plays an important role in mutant ATXN7 toxicity and that the oxidative stress is generated by activation of NADPH oxidase 1 (NOX1) complexes. Furthermore, we showed that the increased NOX1 activity, together with polyQ expanded ATXN7 mediated disruption of the transcription factor p53, results in metabolic alterations in SCA7 cells. The expression of key p53 regulated metabolic proteins like AIF, TIGAR and GLUT1 was altered in SCA7 cells and resulted in reduced mitochondrial respiration, a higher dependence on glycolysis and reduced ATP levels. In summary, our data indicate that mutant ATXN7 mediated dysregulation of p53, resulting in autophagic and metabolic alterations, could play a key role in SCA7 and possibly other polyglutamine diseases.