Dichloroacetate (DCA) is an investigational drug for the treatment of genetic mitochondrial diseases. Its primary site of action is the pyruvate dehydrogenase (PDH) complex, which it stimulates by altering its phosphorylation state and stability. DCA is metabolized by and inhibits the bifunctional zeta-1 family isoform of glutathione transferase/maleylacetoacetate isomerase. Polymorphic variants of this enzyme differ in their kinetic properties toward DCA, thereby influencing its biotransformation and toxicity, both of which are also influenced by subject age. Results from open label studies and controlled clinical trials suggest chronic oral DCA is generally well-tolerated by young children and may be particularly effective in patients with PDH deficiency. Recent in vitro data indicate that a combined DCA and gene therapy approach may also hold promise for the treatment of this devastating condition.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22116号 / 医博第4529号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 妻木 範行, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Actinobacillus succinogenes cells were grown on Poraver® support particles in a packed-bed reactor. Dilution rates (D) of 0.054–0.72 h-1 were investigated. Glucose was used as substrate. CO2 (g) was bubbled into a complex medium to satisfy the fixation requirements and maintain anaerobic conditions. At D ≥ 0.31 h-1, an initial glucose concentration of 35 g.L-1 was used; at lower dilution rates, this was increased to 60 g.L-1 in order to avoid substrate limitations. By-product formation included acetic and formic acids. A maximum productivity of 10.7 g.L-1 was obtained at D = 0.7 h-1. It was found that the system provided repeatable results at a given D. The longest steady state period was maintained for about 97 h at D = 0.31 h-1. Steady state stability was maintained for > 72 h at D < 0.31 h-1. For periods longer than 75 h, however, inhibitory acid titres resulted in a gradual decline in productivity. At higher dilution rates, long-term stability could not be maintained. The low acid titres produced significant biofilm sloughing following aggressive biofilm growth, resulting in oscillatory system behaviour. For fermentation times < 115 h, the dilution rate was secondary to the attachment area in determining the total biomass at steady state. Total biomass values were then used to determine specific rates. A clear trend was observed, with the specific glucose consumption rate, and specific acid production rates, increasing with increasing D. This was explained by assuming a maintenance-driven system at all Ds studied. A product analysis indicated that at ΔS < 15 g.L-1, pyruvate formate lyase was the preferred oxidative route. A shift to the pyruvate dehydrogenase pathway occurred at higher ΔS values, so that the highest YSS values obtained exceeded 0.85 g.g-1. A decrease in C3 by-product formation resulted in high YSS values being maintained, indicating an additional, unknown source of nicotinamide adenine dinucleotide (NADH). It is recommended that any process utilising immobilised A. succinogenes cells should operate at an intermediate D, in order to maintain long-term reactor stability, high productivities and good yields. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

No description available.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23763号 / 医博第4809号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中島 貴子, 教授 戸井 雅和, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sink limitations in plants reduce the potential for photosynthesis and yield, particularly under conditions that favour enhanced source activity such as elevated CO2 (EC). Dark respiration, considered catabolic, has rarely been exploited to enhance sink activity in plants. Arabidopsis thaliana L. lines with partially-suppressed mitochondrial pyruvate dehydrogenase (mtPDH) kinase (mtPDHK), a negative post-translational regulator of the mtPDH complex, was shown previously to have both elevated mtPDH complex activity and increased seed weight and oil content at ambient CO2 (AC), suggesting an enhancement of sink activity. The mtPDH links glycolysis with the tricarboxylic acid (TCA) cycle. It was hypothesized that Arabidopsis having suppressed mtPDHK will display their greatest plant productivity at EC through a combined enhancement of source and sink activities. Control and transgenic Arabidopsis having either constitutive or seed-specific expression of antisense mtPDHK were grown at either AC or EC. Expression of mtPDHK and mtPDH complex activity in rosette leaves and reproductive tissues were measured, which required the development of an assay to quantify mtPDH activity. Vegetative and reproductive growth over time, seed oil parameters, and leaf net C exchange were also quantified. A parabolic relationship was found between mtPDHK expression and mtPDH activity, reflecting a role for mtPDH in balancing photosynthetic and respiratory processes. A number of growth and seed oil parameters were improved in transgenic lines, particularly at EC; many of these parameters showed a significant linear or quadratic correlation with mtPDHK expression and mtPDH activity. The proportion of very long chain fatty acids was increased in transgenic lines. Leaf net C exchange was enhanced at AC and EC, and particularly in lines showing repression of mtPDHK. The greatest enhancement in total seed and oil productivity was found for the constitutive lines 104 and 31 at EC (up to 2.8 times). These two lines exhibited a significant increase in inflorescence size, an increase in leaf water use efficiency, the lowest rate of mtPDH complex inactivation by ATP, and an intermediary enhancement of mtPDH complex activity in seeds. Thus, it is concluded that the mtPDH plays a key role in regulating sink and source activities in plants. / Natural Sciences and Engineering Research Council (NSERC) through the Green Crop Networks Research Network; Ontario Graduate Scholarship; Syngenta Graduate Scholarship; Ball Farm Services and Agrico Canada Ltd. Scholarship; Mrs. Fred Ball Scholarship; Arthur D. Latornell Scholarship; Hoskins Scholarship; Robb Travel Grant; Registrars and the Deans Scholarship and travel awards and bursaries from the University of Guelph, and the Ontario Agricultural College.