Thesis (doctoral)--University of Tartu, 2006. / Thesis is based on 3 papers.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Aug 17.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Kenneth A. Johnson. Vita. Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

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Citrate synthase (CS) is a key mitochondrial enzyme in the tricarboxylic acid cycle (TCA). TCA provides NADH and FADH for the ETC to generate ATP through oxidative phosphorylation in muscle cells. The aim of this PhD project is to investigate the role of CS in skeletal muscle metabolism. The aim of the first study was to investigate the effects of a high fat diet (HFD, 45 % kcal fat) for 12 weeks on CS activity in the heart and gastrocnemius muscle of C57BL/6J (B6) mice and congenic (B6.A) characterised by 39% reduced CS activity. Spectrophotometric analysis of CS activity in the heart and gastrocnemius muscle revealed that HFD led to an increase in CS activity in gastrocnemius muscle but a decrease in the heart in both strains of mice. The aim of the second study was to investigate the effects of low CS activity on substrate metabolism in primary muscle cells established from B6 and B6.A mice. Primary muscle cells from both strains were incubated in radiolabelled glucose or palmitate to assess their oxidation in the mitochondria. The reduction of CS activity in B6.A muscle cells did not affect glucose and palmitate oxidation. The aim of the third study was to investigate the effects of D- and L-serine on CS activity in B6 muscle homogenates, primary muscle cells and purified CS from porcine heart. The muscle samples were incubated in D - or L-serine at 0.1 mM or 5 mM concentration and CS activity levels were assessed by spectrophotometer. D- or L-serine did not have any effect on CS activity in muscle samples. The aim of the fourth study was to investigate the effects of low CS activity on substrate metabolism in C2C12 muscle cells. Lentiviral transduction of C2C12 muscle cells with shRNA resulted in a reduction of CS activity and the metabolic pathways were assessed using XF24 Analyser, western blotting, Immunofluorescence and qRTPCR. Low CS activity was associated with a reduction in substrate oxidation by the mitochondria, an increase in glycolysis and ceramide accumulation in C2C12 muscle cells. The results highlight the significance of CS activity as a modulator of muscle metabolism.

Thesis (M.Sc.D.)--Boston University School of Graduate Dentistry, 1975. Pedodontics. / Bibliography included.

Cancer cells undergo a multifaceted rewiring of cellular metabolism to support their biosynthetic needs. Although the major determinants of this metabolic transformation have been elucidated, their broad biological implications and clinical relevance are unclear. In this study, I systematically analysed the expression of metabolic genes across 20 different cancer types and investigated their impact on clinical outcome. I found that cancers undergo a tissue-specific metabolic rewiring, which converges towards a common metabolic landscape. Of note, downregulation of mitochondrial genes is associated with the worst clinical outcome across all cancer types and correlates with the expression of epithelial-to-mesenchymal transition (EMT) gene signature, a feature of invasive and metastatic cancers. Consistently, suppression of mitochondrial genes is identified as key metabolic signature of metastatic melanoma and renal cancer, and metastatic cell lines. This comprehensive analysis reveals unexpected facets of cancer metabolism, with important implications for cancer patients stratification, prognosis, and therapy. I then investigated how mitochondrial dysfunction could affect cell behaviour. I capitalised on a recently developed in vitro cell model with increasing levels of m.8993T > G mutation heteroplasmy. I found that impaired utilisation of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). This metabolic coupling is facilitated by the formation of a multienzymatic complex between MDH1 and GAPDH. Importantly, such metabolic coupling between glutamine metabolism and cytosolic NADH recycling is able to support increased glycolytic flux, an important hallmark of cells with dysfunctional mitochondria, as well as cancer cells. Finally, increased glycolysis in cells with mitochondrial dysfunction is associated with enhanced cell migration, in an MDH1-dependent fashion. These results describe a novel link between glycolysis and mitochondrial dysfunction, and uncover potential targets for cells that rely on aerobic glycolysis for proliferation and migration, such as cancer cells.

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