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Mitochondria serve as the major source of reactive oxygen species (ROS) production in cells resulting in antioxidant systems and cell signaling pathways that are unique to mitochondria. Thioredoxin-2 (Trx-2) is the mitochondrial member of the thioredoxin superfamily, and acts specifically to reduce the mitochondrial peroxidase, peroxiredoxin-3. It has been proposed that Trx-2 associates with cytochrome c, which functions in mitochondrial respiration and apoptosis. Homozygous Trx-2 deletion in mice is embryonic lethal and it is hypothesized here that Trx-2 lethality is caused by loss of mitochondrial function and oxidative stress. Results of experiments investigating mitochondrial integrity, cell viability, and ROS levels in Trx-2(-/-) mouse embryonic fibroblasts (MEFs), and results from Trx-2 siRNA MEFs, are similar to findings of knockouts in previously reported proteins that function in mitochondrial respiration and support the involvement of Trx-2 in this process. Mitochondrial ROS have also been implicated as major secondary messengers in cell signaling. Results reported here using cancer cells and cancer cells depleted of mitochondrial DNA, which consequently produce few ROS, have indicated that mitochondrial ROS produced in hypoxia are necessary for HRE and ARE activation, and are fundamental in the activation of SP-1 during reoxygenation. However, mitochondrial ROS are not required for HIF-1α protein expression in hypoxia, indicating a unique relationship between HIF-1α, hypoxia, and mitochondrial ROS.

Poly(ADP)ribose (PAR) metabolism is essential to many cellular functions, including the maintenance of genomic integrity, the regulation of cell death mechanisms, as well as the regulation of gene expression. Recent work has uncovered many new players in the expanding effort to understand PAR metabolism and its cellular impact. PARP-1, the prototypical poly(ADP)ribose polymerase, was the first to be discovered, and has since been shown to be vital in the cellular response to DNA damage. Indeed, one report demonstrating that PARP-1 activation is required for apoptosis-inducing factor (AIF) release from mitochondria uncovered a novel link between DNA damage and signaling for cell death. The events following PARP activation, leading to signaling for AIF release, however, are still poorly understood. Based on our observations, we have developed a model to explain the nuclear/mitochondrial crosstalk that occurs following PARP activation. The work presented here answers several important questions regarding the relationship between ADP-ribose metabolism and mitochondria, including the role of PAR in signaling for the release of AIF, the presence of ADP-ribose metabolism protein members in mitochondria, and mitochondrial transcriptional effects following PARP activation. This work presents several novel findings, including the first report of a mitochondrial matrix isoform of poly(ADP-ribose) glycohydrolase (PARG) as well as direct evidence of mitochondria-associated PARP activity. Furthermore, it provides evidence for a novel effect of PARP-1 activation, in the specific transcriptional upregulation of the mitochondrial gene, NADH dehydrogenase, subunit 1 (ND1). Our data is consistent with the hypothesis that uncontrolled PARP activity results in energy metabolism dysfunction and cell death. Furthermore, it supports a model in which PARP activity is required for normal transcriptional responses in mitochondria following DNA damage. In total, this report adds to the body of work outlining the roles of PARP following DNA damage recognition and activation, demonstrating that ADP-ribose metabolism plays an important role in cell death regulation by both direct and indirect means.

Non-Hodgkin lymphoma is commonly associated with chronic infection and inflammation. Such conditions are characterized by chronic oxidative stress. Because apoptosis signaling is often mediated by reactive oxygen species, lymphoma arising in the context of oxidative stress may become resistant to these apoptosis signals. Resistance to oxidative stress could contribute to tumorigenesis and limit response to chemotherapy, as apoptosis induced by many drugs involves reactive oxygen species. We used a cell culture model to understand how changes in the ability to handle oxidative stress contribute to apoptosis resistance. WEHI7.2 murine thymic lymphoma cells transfected with catalase or selected for resistance to hydrogen peroxide acquire a concomitant resistance to apoptosis induced by glucocorticoids. Cytochrome c release is delayed in these variants, demonstrating that apoptosis resistance lies upstream, in the signaling phase, or in the mitochondria themselves. By comparing the apoptosis-sensitive WEHI7.2 parental cells with the oxidative stress- and apoptosis-resistant variant cells, we investigated the contribution of cytosolic and mitochondrial changes to glucocorticoid-induced apoptosis. We showed that neither JNK kinase signaling, nor GSTπ, a redox sensor protein which regulates JNK, is activated during glucocorticoid-induced apoptosis. Our work using isolated mitochondria and recombinant tBid protein in cell-free apoptosis assays showed that the apoptosisresistant variants are intrinsically resistant to the release of cytochrome c and other intermembrane space proteins. The resistance was mediated upstream and within the mitochondria, and occurred at both steps controlling cytochrome c release. Given that the resistant variants demonstrated alterations in mitochondrial apoptotic function, we investigated mitochondrial protein changes that could explain these differences. An increased expression of cytochrome c was observed in the resistant variants, but selective reduction of cytochrome c expression showed that this change alone was not sufficient to affect sensitivity. The balance of pro- and anti-apoptotic Bcl-2 family members in untreated cells also did not explain intrinsic resistance. Alterations in Bcl-2 protein levels following treatment could contribute to glucocorticoid resistance, but additional work to test Bcl-2 family interactions will be required. We have identified points of resistance that are important in glucocorticoid-induced apoptosis and may also contribute to resistance to novel mitochondrial-targeting drugs.

Mitochondria perform functions which are central to the life of most eukaryotic cells. These organelles can be considered the ultimate energy power house of a living cell. The role of mitochondria in cancer phenotype remains a fertile area of research. Several carcinogens are known to enter the mitochondria, resulting in impaired functioning and altered structure. Aflatoxin BI (AFB1) a primary type I mycotoxin elaborated by Aspergillus flavus and Aspergillus parasiticus, is carcinogenic for a wide species range. The epoxide is capable of binding to nucleic acids and proteins, resulting in induced mutations, cellular toxicity, and eventually carcinogenesis. Approximately 250 000 deaths occur annually in both China and Africa due to patients presenting with Hepatocellular Carcinoma (HCC). The causative agents being AFB1-ingestion via contaminated foods and feeds, and the Hepatitis B Virus infection. The toxin has a multifaceted mode of attack, capable of being activated to a highly reactive and carcinogenic derivative, the AFB1-8,9-epoxide, via the cytochrome P450 enzyme system of the microsomes, endoplasmic reticulum and also the mitochondria. The epoxide is capable of binding to nucleic acids and proteins, resulting in the formation of covalent adducts. The repeated occurrence of gold labelled toxin within mitochondria from hepatomas of patients presenting with HCC suggested that these organelles were direct sites of toxin binding. Despite observations that mitochondria appear as direct and perhaps preferential targets for attack by AFB1, the actual in vivo immunolocalisation and characterisation of bound AFB1 within liver mitochondria has not been reported previously. In addition the role of AFB1-protein binding within mitochondria was investigated to determine the mode of action of the toxin, within the mitochondrial system. Liver sections from rats treated with a single lethal dose of AFB1, showed distinct ultrastructural abnormalities viz. large nuclei, increased heterochromatin, and swollen mitochondria. Immunocytochemistry revealed for the first time, the selective localisation of conjugated gold labelled toxin within the mitochondria. Toxin was found in the intracristal and peripheral spaces and frequently within the mitochondrial matrix. The mitochondria isolated from treated rats revealed significant alterations and damage to the mitochondrial membranes. The cristae were also markedly swollen with the associated clearing of the mitochondrial matrix. Western blot immunoassays revealed the presence of five AFB1-bound proteins (150kDa, 50kDa, 25kDa, 18kDa, 14kDa) in the inner mitochondrial fraction of isolated mitochondria. High pressure liquid chromatography also revealed that a significant proportion (84%) of an initial dose of toxin, was absorbed by mitochondrial protein. This study is the first to show the presence of specific mitochondrial proteins involved in toxin binding. In addition, the presence of toxin within the mitochondria and the specific binding to inner mitochondrial proteins suggest that the toxin specifically targets the electron transport chain and hence effects ATP production. This study conclusively indicates that mitochondria are direct targets for attack by AFB1 during experimental carcinogenesis. Mitochondria therefore play an important role in AFB1-mediated carcinogenesis. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Mitochondria are eukaryotic organelles derived in evolution from within the ? subdivision of Proteobacteria. Although mitochondria are structurally and metabolically complex, modern-day mitochondrial genomes (mtDNA) encode only a small number of RNAs and proteins predominantly involved in adenosine triphosphate (ATP) formation through electron transport coupled to oxidative phosphorylation, as well as translation of mtDNA-encoded proteins. In humans, only 13 of the >1000 polypeptides that constitute the complete mitochondrial protein complement (proteome) are encoded in mtDNA; the remainder is encoded by nuclear DNA (nuDNA). It is therefore imperative to comprehensively catalog nuDNA-encoded mitochondrial proteins in order to understand holistically the evolution of mitochondria. Mitochondrial proteome investigations of animals, fungi and land plants have dramatically altered our conception of mitochondrial evolution: in contrast to mtDNA-encoded proteins, few nuDNA-encoded mitochondrial proteins are demonstrably derived from the eubacterial progenitor of mitochondria, and many are found only in eukaryotes. Notably, however, little is known about the mitochondria of eukaryotic microbes (protists), which constitute the bulk of biochemical and genetic diversity within the domain Eucarya. The proteomic characterization of protist mitochondria is therefore crucial to fully elucidating mitochondrial function and evolution. Employing tandem mass spectrometry (MS/MS), I have analyzed highly purified mitochondria from Acanthamoeba castellanii (Amoebozoa). In combination, nearly 750 nuDNA- and mtDNA-encoded proteins were identified. These data were used to catalog metabolic pathways and protein complexes, and to infer functional and evolutionary profiles of A. castellanii mitochondria. My analyses suggest that while A. castellanii mitochondria have many features in common with other eukaryotes, they possess several novel attributes and pronounced metabolic versatility. An analysis of the A. castellanii electron transport chain (ETC) was also performed, utilizing a combination of blue native polyacrylamide gel electrophoresis (BN-PAGE), MS/MS and bioinformatic queries. A significant proportion of A. castellanii ETC proteins was identified, yielding several insights into ETC evolution in eukaryotes. Lastly, I present two unusual cases of ‘split’ mitochondrial proteins: the iron-sulfur subunit SdhB of succinate:ubiquinone oxidoreductase (Complex II), in the phylum Euglenozoa and Cox1 of cytochrome c:O2 oxidoreductase (Complex IV) in various eukaryotes, including A. castellanii. Functional and evolutionary implications of these findings are discussed.

Programmed cell death (PCD) is pervasive in eukaryotes, playing a fundamental role in development. PCD in animals has been studied in detail, partly due to Caenorhabditis elegans, a worm whose anatomy allowed for the investigation of exactly 131 cells that die via PCD. Elucidating this complex pathway in this simple worm laid the foundation for further insights into mammalian PCD. Overall, less is known regarding PCD in plants, where cell death is broadly separated into developmentally regulated and environmentally induced. The lace plant (Aponogeton madagascariensis) undergoes developmentally regulated PCD to form perforations between longitudinal and transverse veins over its leaf surface. The optimization of protoplast isolation and induced cell death via heat shock (HS) in the lace plant is detailed here. Following HS, protoplasts displayed characteristics of PCD including: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei, increases in vesicles as well as Brownian motion, and plasma membrane blebbing. Additionally, mitochondrial dynamics were investigated, and a role for the mitochondrial permeability transition pore (MPTP) was indirectly established via cyclosporine A (CsA) experimentation. The main focus of this dissertation was to elucidate cellular dynamics during developmentally regulated PCD in the lace plant, which is visibly discernable during the window stage of leaf development. A single areole within a window stage leaf was further divided into three areas based on the progression of PCD; non-PCD (NPCD) cells, early PCD (EPCD) cells, and late PCD (LPCD) cells. Using this gradient, mitochondria were delineated into four stages based on distribution, motility, and membrane potential. Additionally, it was determined that the MPTP also played a role in developmental lace plant PCD, as inhibition of the pore with CsA not only reduced caspase-like proteases (CLPs) but also stopped perforation formation. Furthermore, the actin cytoskeleton was also investigated, with evidence suggesting it as a possible target for CLPs. The novel use of lace plant leaves for long-term live cell imaging allowed for the establishment of a timeline of cellular events that occur during developmental PCD. Major conclusions of this dissertation reveal various similarities between environmental induced and developmentally regulated PCD in this one plant species.

Mitochondrial content, an important determinant of muscle metabolic capacity, changes in individuals during development, and in response to physiological and environmental challenges. This phenotypic plasticity is attributed to the coactivator PPARγ coactivator-1α (PGC-1α) but it remains unclear if this transcriptional regulator accounts for evolutionary variation in mitochondrial content. In an attempt to explain why some species have higher muscle mitochondrial enzyme levels than other species, I examined if the transcriptional mechanisms that control mitochondrial content of a tissue in an individual are also responsible for differences between species. If PGC-1α creates differences between the mitochondrial content of species based on variation in promoter binding motifs, then cis-factor evolution may be the guiding force in scaling trends. In this thesis I explored the basis of size-dependent patterns by looking at layers of regulation, from catalytic activities to promoter evolution and regulation. A representative family, Rodentia, was used to collect muscle samples from a size range of approximately 20g up to 17 kg. As expected, in rodent lower limb muscles, mitochondrial and glycolytic enzyme activity exhibited reciprocal scaling patterns, though the scope differed between muscles. Very little of the variation was accounted for when the activity was related to DNA content. However, when COX activities were expressed relative to DNA, the scaling patterns were similar among the 3 muscles. To determine if interspecies differences were linked to transcriptional regulation, ~800bp of the PGC-1α promoter from 56 terrestrial mammals (5g-5000kg) was examined. The basal placental mammalian promoter possesses putative elements for Sp1, HNF3, myogenic factors and metabolic effectors, which have been retained in mammals with little change in order or spacing. To investigate the ability of these promoters to control PGC-1α expression, rodent promoters were cloned into luciferase reporter gene constructs and transfected into a common mouse myoblast background (Sol8 cells). Unlike mitochondrial content, promoter activity did not vary with body size across the rodent family. Likewise, PGC-1α transcript levels did not vary in rodent muscles in a way that would explain differences in COX activity. This suggests that though PGC-1α may be crucial for within species variation, transcriptional regulation of PGC-1α is not responsible for interspecies variation in mitochondrial content. / Thesis (Master, Biology) -- Queen's University, 2010-06-09 10:50:02.133

Many fish species respond to cold temperatures by inducing mitochondrial biogenesis, reflected in an increase in the activity of the mitochondrial enzyme cytochrome c oxidase (COX). COX is composed of 13 subunits, 3 encoded by mtDNA and 10 encoded by nuclear genes. I used thermal acclimation/winter acclimatization to explore how fish muscle controls the synthesis of COX. In this study, I used real-time PCR to measure mRNA levels for the 10 nuclear-encoded COX genes and several transcriptional regulators. I compared the thermal response of two cyprinid species, the tropical zebrafish (Danio rerio, acclimated to 11 and 30°C) and the temperate redbelly dace (Phoxinus eos, winter and summer acclimatized). I hypothesized that (i) there would be an increase in COX activity in the cold- versus warm-acclimated fish and (ii) changes in COX activity would be paralleled in the transcript levels of the nuclear-encoded COX subunits as well as the master-regulators and transcription factors of mitochondrial biogenesis. Zebrafish COX activity did not change in the cold but the transcript levels of some subunits decreased up to 70%. Redbelly dace COX activity was 2.9-fold higher in winter fish and though nuclear-encoded subunits had higher transcript levels the increases did not parallel enzyme activity, ranging from 1.7- to 21-fold higher in winter. There also did not appear to be parallel patterns in mRNA for the transcriptional regulators. In zebrafish, when COX activity did not change, there was no significant change in PGC-1α mRNA. In redbelly dace, when COX activity was 2.9-fold higher, PGC-1α mRNA was 6.3-fold higher. These observations suggest that coordination of COX subunit expression is imperfect, implying that subsets of these genes are more important in determining the COX activity. I assert that those genes that are most likely the candidates for regulating COX activity are COX4 and COX5A as they are the first regulatory subunits incorporated into the holoenzyme. Though arguments can also be made for COX5B, 6A and 7B based on the parallels between changes in enzyme activity and transcript abundance as well as the position in which they are assembled into the enzyme complex. / Thesis (Master, Biology) -- Queen's University, 2010-08-10 11:35:35.352