Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+ homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production. One of the secondary consequences of such deficiencies is the adaptive transcriptional responses of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis. Additionally, several other genes that are involved in several other functions, such as metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses: firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The second hypothesis stated that genes involved in mitochondrial replication and transcription are differentially expressed in OXPHOS deficient cell lines. Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model was investigated. The increased expression of different MT isoforms in the presence of the complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening, apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated HeLa cells. Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes involved in OXPHOS function and regulation. Two experimental in vitro models were developed and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of complex I in 143B cells was developed, characterized and introduced. Then the effect of the knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding structural as well as functional proteins was determined. Additionally, to investigate the effect of stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex I, as well as the Rieske subunit of complex III were introduced and characterized. The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and transcription could not, without a doubt, be supported or contradicted by the data. It was determined from the data that an OXPHOS deficiency, which does not result in increased ROS levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless, increased ROS production in the presence of OXPHOS deficiency is probably not exclusively responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein involved early in this mechanism before any other regulatory proteins are affected. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references (leaves 166-191). / Microfiche. / xiii, 191 leaves, bound ill. 29 cm

Studying species at the molecular level can provide insights into how ecological and biological processes interrelate resulting in the diversity we see today. This information can be applied to conserve species at risk of extinction, or to better manage genetic diversity in species of economic importance. Species that inhabit freshwater riverine systems commonly exhibit population structures that are related to their relative dispersal capability, contemporary stream structure and/or historical stream structure. This thesis examined the populations genetic structure of wild and cultured stocks of the commercially farmed freshwater crayfish, C. quadricarinatus (von Martens), using genetic markers characterized by different modes of inheritance. C. quadricarinatus is distributed naturally in riverine systems in northern Australia, and southern Paupa New Guinea (PNG) and inhabits a variety of freshwater ecosystems ranging from ephemeral to permanent. Life history characteristics of C. quadricarinatus suggest a high level of genetic structuring among wild stocks might exist. However, seasonal flooding coupled with low topography across its distribution in northern Australia may promote sufficient gene flow among rivers to produce genetic homogeneity. Historical gene flow may also influence modern genetic structure as many distinct riverine catchments that C. quadricarinatus inhabits, were once connected at times of lower sea level. Insight into genetic relationships among C. quadricarinatus populations will allow for better management practices of wild populations in the future. The study investigated phylogenetic relationships among C. quadricarinatus representing 17 discrete natural drainages across the natural range in Australia and PNG, using 16s and COI gene sequences. Sequence analysis of both genes resolved two distinct genealogical lineages in Australia and three in PNG. The two divergent Australian lineages concur with original taxonomic descriptions of Reik (1969) based on external morphological differences. The three C. quadricarinatus populations sampled in PNG were all genetically distinct from each other, with one exhibiting a close association with an Australia lineage. The immense physical barriers (rugged mountain ranges) to gene flow in PNG will almost certainly have reduced dispersal capabilities for C. quadricarinatus. During times of lowered sea levels in the past, Australia and southern PNG were a single landmass with terrestrial and freshwater organisms theoretically able to disperse over associated land and via freshwater connections. The close genetic relationship between PNG and Australian C. quadricarinatus support a recent freshwater connection and hence gene flow between northern Australia and PNG C. quadricarinatus populations. Genetic differentiation among some C. quadricarinatus lineages exhibit as much genetic divergence at 16s RNA sequences as taxonomically recognised sub-species in the Cherax genus. Since C. quadricarinatus was originally described as different species based on external morphological differences (Reik, 1969), it is recommended that the taxonomy of C. quadricarinatus in Australia and PNG be re-evaluated. C. quadricarinatus specific microsatellite markers were developed for this study. Five variable loci were employed to investigate the extent of contemporary gene flow among fourteen C. quadricarinatus wild river populations in northern Australia. High FST and genetic distance estimates observed among pair wise comparisons of C. quadricarinatus populations are consistent with limited or no gene flow occurring among drainages. Speculation that C. quadricarinatus may disperse between adjacent or nearby drainages at times of flood, either across floodplains, or via flood plumes therefore seems highly unlikely among the populations examined in the current study. No significant correlation was observed between geographic distance and genetic distance among C. quadricarinatus populations here. C. quadricarinatus populations most closely resemble an island-like model, where gene flow is independent of geographic distance among populations and where genetic divergence occurs to a greater or lesser extent as a result of genetic drift within otherwise isolated populations. A significant number of C. quadricarinatus populations showed deviations from expected Hardy-Weinberg equilibrium (HWE). Samples sizes may not have been sufficiently large to reflect a true representation of genotypic proportions present in the sampled populations due to the highly variable nature of microsatellite loci. Deviations from HWE equilibrium, however, can also result from null alleles. Null allele estimates suggested a large proportion of null alleles were present in the C. quadricarinatus populations analysed. This may be a result of C. quadricarinatus populations confined to discrete drainages experiencing independent evolution, resulting in mutations in primer binding sites. The growing economic potential of C. quadricarinatus culture, both domestically and internationally, prompted expanding the current study to examine genetic diversity levels in commercial C. quadricarinatus stocks. The study employed five microsatellite markers to quantify genetic diversity in four Australian and three C. quadricarinatus culture stocks from overseas. Many C. quadricarinatus culture stocks also showed deviations from HWE expectations. This was not a surprising result given that the wild populations also deviated and domestication can also influence HWE. Relatively high levels of genetic diversity were observed. This probably results from intentional mixing of discrete river strains for production of the first commercial stock. Genetic differentiation estimates among culture stocks and assignment tests indicated that overseas culture stocks are most likely derived from the first commercial culture stock developed in Australia and then disseminated widely (the Hutchings stock). Robin Hutchings was a known supplier of live C. quadricarinatus to many international culture initiatives. Assignment of culture stocks back to their wild origins indicated that all C. quadricarinatus culture stocks sampled possess alleles that originate from the Flinders River (proportions ranged from 33-94%). Domestication of C. quadricarinatus to date has not resulted in significant reductions in levels of genetic diversity (heterozygosity or alleles richness) when compared to wild populations sampled in this study. Comparing culture stocks to wild populations to gauge their 'genetic health' may not be a suitable scale for evaluating genetic diversity in culture stocks. Wild populations are essentially evolving independently, are subjected to harsh seasonal environmental fluctuations resulting in periodic population crashes (genetic bottlenecks), with little or no recruitment from neighbouring drainages (gene flow). This study does however indicate that there is a large amount of genetic diversity distributed among wild populations that has yet to be exploited in culture. Genetic diversity in wild populations provides a resource for future stock improvement programs for C. quadricarinatus culture and thus requires careful conservation and appropriate management.

The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

Mitochondrial DNA encodes thirteen protein subunits in the oxidative phosphorylation system (OXPHOS) that is responsible for cellular energy production. Mitochondrial disorders have been identified to be associated with mtDNA mutations. However, the molecular mechanisms of specific mtDNA mutations are still being explored in order to establish causative links. This study tries to elucidate the mutational effects of mtDNA on OXPHOS complex activities and cell growths. Using mouse 3T3 fibroblasts as a cell model, single-cell clones with different growth rates were isolated. The entire mtDNA genome was sequenced for mutations. The enzymatic activities of OXPHOS complex I to V were analysed. Three growth patterns represented by five clones were identified. Three clones (clone #2, #3, and #6) had the shortest doubling times (11.5 - 14.9 hours). Clone #1 had a medium growth rate (19.2 hous); and clone #5 had a significantly slow growth rate (22 hours). MtDNA sequencing results revealed that clone #5 had several heteroplasmic mutations (one in 16S rRNA, two in tRNAser (UCN), three in tRNAasp, one in tRNAlys, one in COI, five in COII, and one in ATPase8) while the other four clones showed sequence homology. Enzymatic analyses showed that on average clone #5 had significantly low complex III, IV, and V activities (p < 0.05). Changes in biochemical properties and protein structure were analyzed to deduct possible mechanisms for reduced respiration. In conclusion, the slow growth rate is associated with reduced OXPHOS enzyme functions. It is most likely that the combination of COI and COII mutations resulted in the reduction of complex IV function. It is still unclear whether the ATPase8 mutation (T7869A) in the non-conserved region alone can have such a pronounced phenotypic effect. A reduction in complex III also cannot be explained since there were no mutations in the only mtDNA-encoded complex III gene, but it is possible that there are mutations in the nDNA-encoded complex III genes. Mutations in tRNA and rRNA genes may also be responsible for reduced protein syntheses and consequently reduced OXPHOS activities. It is unclear why complex I activity was not affected. Although the mutational effect of individual mtDNA mutation observed cannot be clearly identified, this study establishes a correlation between mtDNA mutation and cell energy production and growth.

Oxidative and nitrosative stress are deleterious physiological processes caused by an imbalance between reactants such as reactive oxygen and nitrogen species and antioxidants. Due to the links between oxidative and nitrosative stress and disease, there is much interest in accurately quantifying these in biological and physiological samples. There are numerous methods to quantify the in vivo oxidative and nitrosative damage to lipids, DNA and proteins however they are generally time-consuming, expensive and difficult. Furthermore, due to the complex nature of oxidative and nitrosative stress it would be appropriate to measure a number of different biomarkers, however this is rarely done. The first section of this thesis contains research aimed at developing a bioassay to simultaneously detect markers of oxidative and nitrosative stress. This includes; 1) a review of the studies investigating the ability of these biomarkers to predict the onset of disease, 2) a description of the attempts to develop the bioassay, 3) a study designed to test the sensitivity of the bioassay to detect changes in oxidative stress. Unfortunately, the attempts to develop the bioassay were not as successful as hoped and, in the interests of completing the PhD in the time allowed, the PhD changed focus to look at the effects of oxidant:antioxidant balance on mitochondrial biogenesis. The second section of the thesis contains a review of the literature on this topic and two original investigations. It is well documented that oxidative and nitrosative stress contributes to the progression of many diseases including; cardiovascular disease, type 2 diabetes, Alzheimer’s disease, kidney disease and cancer. To determine which biomarkers would have the greatest efficacy in the bioassay, a comprehensive review was undertaken. The aim of the review was to investigate studies which have measured oxidative and nitrosative biomarkers to determine whether they are independent predictors of cardiovascular events (Chapter two). From the review, fifty-one studies were identified with twenty-six of these measuring oxidised (Ox)-LDL, fifteen assessing myeloperoxidase (MPO), seven using lipid peroxidation measures and three quantifying protein oxidation in plasma/serum. The recommendation of the review was that all areas require further investigation, however, it was determined that Ox-LDL and MPO would be beneficial for inclusion in the bio-assay. Other biomarkers considered for the bio-assay were nitrotyrosine, superoxide dismutase and glutathione peroxidase. Chapter three outlines method development used to measure the oxidative and nitrosative markers simultaneously. Recent technology allows multiple analytes to be detected simultaneously from the one sample. The Mulit-plex system is used to detect analytes that have been sandwiched between primary capture and secondary biotinylated detection antibodies. The secondary antibody attaches to streptavidin-phycoerythrin and is used by the Mulit-plex analyser to quantify the analyte. During development of the bio-assay, clumping of microspheres, high background, no detection of standard curve or samples, matrix effects, mislabeling of antibodies by manufacturers and lack of commercial available antibodies were obstacles that limited the success of this method. MPO was the only biomarker that was successful. Chapter four contains a study that investigated the sensitivity of the MPO mulitplex bio-assay. Nine highly trained cyclists underwent an extensive exercise protocol designed to induce dehydration by 4 % body mass, rehydration of 150 % fluid loss and a performance time-trial. Plasma samples were taken at five time points; baseline, post dehydration, post rehydration, pre time-trial and post time-trial and analysed using the mulitplex bio-assay. The results showed that there was a significant increase in MPO post dehydration and post time-trial compared with all other time points (P<0.05), thereby demonstrating that the mulitplex bio-assay is sensitive to detect changes in exercise and appropriate rehydration reduces oxidative stress. The MPO mulitplex bio-assay requires further testing on patients with diseases to further validate its future applications. As mentioned above, due to time constraints it was decided to stop the attempts to create a multi-analyte bioassay and focus on another important area of cellular oxidative stress. Currently, there is much interest in the involvement of oxidant:antioxidant balance in mitochondrial biogenesis. The increase of mitochondrial content within the skeletal muscle, termed mitochondrial biogenesis, provides an increased capacity to generate ATP during exercise and is recognized as one of major cellular adaptations to exercise. Reactive oxygen species are produced during exercise and have been shown to induce mitochondrial biogenesis. One of the key instigators of mitochondrial biogenesis is peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α). PGC-1α is central to the transcription of mitochondrial and nuclear encoded genes, which regulate downstream pathways such as oxidative phosphorylation and fatty acid oxidation. Antioxidant supplementation is common among athletes and healthy individuals; however, antioxidant supplements suppress reactive oxygen species and could therefore could hinder mitochondrial biogenesis and the positive adaptations associated with exercise. To establish whether antioxidant supplementation reduced mitochondrial biogenesis in skeletal muscle, male Wistar rats were supplemented with α-tocopherol and α-lipoic acid for fourteen weeks (Chapter six). Animals were separated into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet and 4) exercise antioxidant diet. The exercise animals were trained 5 days/week for 14 weeks. Consistent with increased mitochondrial biogenesis and antioxidant defences following training, there were significant increases in PGC-1α mRNA and protein, COX IV and Cyt C protein abundance, citrate synthase activity, Nfe2l2 and SOD2 protein (P<0.05). Antioxidant supplementation reduced PGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity (P<0.05) in both sedentary and exercise-trained rats. In summary, antioxidants α-tocopherol and -lipoic acid supplementation suppresses beneficial adaptations in skeletal muscle such as markers of mitochondrial biogenesis and mitochondrial proteins, regardless of training status. The reduction in mitochondrial biogenesis may affect exercise training adaptations and reduce the ability of healthy individuals to attain optimal exercise adaptations. The last investigation (Chapter seven) studied the effect of reduced glutathione, through diethyl maleate (DEM) administration, on upstream regulators of mitochondrial biogenesis, markers of mitochondrial biogenesis and downstream signalling. Glutathione is a key antioxidant that reduces the amount of hydrogen peroxide. Male Wistar rats were divided into six groups 1) sedentary control, 2) sedentary DEM, 3) post-exercise control, 4) post-exercise DEM, 5) exercise-recovery and 6) exercise-recovery DEM. After an exercise bout to fatigue, animals were euthanized directly after exercise (post-exercise) or four hours post exercise (exercise-recovery). Exercising animals given DEM had significantly (P<0.05) decreased glutathione in skeletal muscle and had a significantly (P<0.05) greater increase in PGC-1α gene expression. There were also main interaction effects between exercise and DEM administration on SOD2 activity. Exercise altered the gene expression of GPx and the phosphorylation of p38 MAPK. Glutathione depletion decreased GPX activity and oxidised glutathione levels. These novel findings represent important in vivo evidence of the involvement of glutathione and oxidant:antioxidant balance in mitochondrial biogenisis. Overall this thesis has provided 1) the first comprehensive review on the prognostic ability of oxidative stress biomarkers to predict the onset of cardiovascular disease, 2) detailed information to assist in the further development of a multi-analyte bioassay to quantify oxidative and nitrosative stress, 3) data indicating that the MPO Mulit-plex bioassay is sensitive to detect physiological perturbations to oxidative stress, 4) evidence that antioxidant supplementation suppresses mitochondrial biogenesis and 5) proof that glutathione is important in the regulation of exercise-induced mitochondrial biogenesis.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 91-100). Also available via the World Wide Web.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 79-88). Also avialable via the World Wide Web.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol Inst., 2003. / Härtill 4 uppsatser.