The syndrome of Malignant Hyperpyrexia in man follows administration of certain general anaesthetic agents, and, although rare, is fatal in 70% of cases (EDITORIAL, 1968). Following exposure to the anaesthetic, there is, in most instances of susceptible individuals, a rapid rise in body temperature, usually within a period of 10 minutes, often accompanied by muscular rigidity of the limbs~ Sometimes hyperthermia has.been delayed for hours and muscular rigidity not pronounced. The temperature reached maybe 43°C (115°F) or even somewhat above this. Halothane, CF3CHBrCl, a ha../o/~nated hydrocarbon, is thought to be responsible for most cases(WILSON, NICHOLS, DENT and ALLEN, 1966). Succinyl choline lfH2COOCH2CH2*(cH3 )~ 201_~H2COOCH2CH2~(cH3 )3 a skeletal muscle relaxant employed during anaesthesia, has also been implicated (BRITT and KALOW, 1970; HARRISON, 1971).

The gram negative bacterium Shigella flexneri is an etiological agent of bacillary dysentery, and causes destruction of the human intestinal epithelium. S. flexneri is primarily transmitted via the fecal-oral route to its primary infective site in the colon. The bacterium invades and replicates within colonic epithelial cells, ultimately ulcerating the mucosal epithelium. To successfully establish infection, S. flexneri must quickly adapt to different environments in the host, including adjusting metabolism in response to changes in available carbon sources. In this study, the importance of the glycolytic and gluconeogenic pathways in S. flexneri pathogenesis was examined. The metabolic regulators CsrA and Cra reciprocally regulate the glycolytic and gluconeogenic pathways. The post-transcriptional regulator Cra activates expression of genes involved in gluconeogenesis and represses glycolysis. Conversely, CsrA activates glycolysis and represses gluconeogenesis. The absence of Cra increased S. flexneri attachment and invasion of cultured epithelial cells. In contrast, the csrA mutant was significantly impaired in both adherence and invasion. Both the csrA and cra mutants formed small, turbid plaques, suggesting that both regulators are required for plaque formation. The opposing phenotypes of the csrA and cra mutants suggested a correlation between invasion and glycolysis. The role of glycolysis in S. flexneri pathogenesis was confirmed by directly examining the first committed step in the pathway. The glycolytic enzyme phosphofructokinase I (PfkI, encoded by pfkA) is repressed by Cra and activated by CsrA. Glycolysis was critical for S. flexneri pathogenesis, as a mutation in pfkA rendered the bacterium noninvasive. The invasion defect of the csrA and pfkA mutants was due to reduced expression and secretion of the Shigella invasion plasmid antigen (Ipa) effectors. Expression of the master virulence regulators virF and virB was significantly reduced in the pfkA mutant, and is the principle reason for decreased invasion. The data presented show that glycolysis is required for invasion, but that plaque formation requires both glycolysis and gluconeogenesis. Because expression of the master virulence regulators is repressed in the pfkA mutant, S. flexneri may use carbon as an environmental regulator of virulence gene expression. / text

No description available.

Cancer cells utilise glycolysis to produce lactate, even in the presence of sufficient levels of oxygen. Excess lactate is removed from cancer cells by MCT1 and MCT4, to prevent intracellular acidosis, apoptosis and to aid the continuous glycolytic flux. MCT1 and MCT4 are over-expressed in many types of human cancers, which correlates with reduced overall survival and increased treatment resistance. The potential role of MCT1 and MCT4 in two EOC cell lines (Skov3 and OV90) was investigated in this study. MCT1 was expressed at similar levels in Skov3 and OV90 cells. Therefore, stable cell lines over-expressing MCT1 were produced using both cell lines. MCT4 was expressed at high levels in Skov3 cells, but very low levels in OV90 cells. Therefore, a stable cell line with MCT4 silencing under the inducible control of doxycycline was produced using Skov3 cells, and a stable cell line to over-express MCT4 was produced using OV90 cells. The consequences of these genetic modifications on the metabolic phenotype, metastatic abilities and the sensitivity of cell lines to treatment with Carboplatin and Paclitaxel, were assessed in normoxia and 1 % hypoxia and 0.1 % hypoxia. Over-expressing MCT1 in Skov3 cells, had no effect on their metabolic phenotype or the sensitivity to treatment with Carboplatin and Paclitaxel. However, over-expressing MCT1 in Skov3 cells significantly enhanced their metastatic abilities, which correlated with reduced focal adhesion size. Silencing MCT4 in Skov3 cells, had no effect on the use of glycolysis, sensitivity to treatment with Carboplatin and Paclitaxel, or their metastatic abilities. However, following MCT4 silencing there was a significant increase in the levels of intracellular ROS. Over-expressing MCT1 in OV90 cells, had no effect on lactate levels or intracellular ROS. However, there was a significant reduction in both their glycolytic activity and mitochondrial mass. Furthermore, over-expressing MCT1 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with increased Pgp and LDHA expression. Over-expressing MCT4 in OV90 cells, caused an increase in the use of glycolysis and increased cell survival in hypoxia. There was also a significant enhancement in the metastatic abilities of these cells following the over- expression of MCT4, which correlated with reduced focal adhesion size. Furthermore, over-expressing MCT4 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with an increase in the expression of Pgp and LDHA.In summary, the findings of this study revealed that MCT1 and MCT4 play a significant role in the biological function of Skov3 and OV90 cells. High expression levels of MCT4 correlated with an increase in glycolysis and cell survival in hypoxia. Whereas high expression levels of MCT1 and MCT4 in correlated with an increase in the metastatic abilities, as well as with Paclitaxel resistance and increased Pgp expression.

No description available.

Abstract not available

The exponential growth of information demands the automated movement of data and software via new software models that are able to integrate data and components on their own without scientists’ direct involvement. However, current stand-alone software modeling environments do not support a secure software execution, nor do client server applications allow user customization of the software running on the servers. To address this problem, a biological pathway modeling environment was built as a stand-alone Rich Internet Application (RIA). The modeling environment was tested by constructing a simulation of the glycolysis pathways in the human erythrocytes, and the results were compared against one of the latest and richest erythrocyte metabolism models developed by Kuchel and Mulquiney. The working simulation was able to settle into a quasi-stable state, with substrate concentrations close to what Kuchel and Mulquiney presented. It was also found that while the browser environment does allow for dynamic applications to be developed, speed and performance do become major issues. In later versions, it is hoped that the performance of the simulator can be increased and that it will become possible to link models together and add collaboration tools.

The exponential growth of information demands the automated movement of data and software via new software models that are able to integrate data and components on their own without scientists’ direct involvement. However, current stand-alone software modeling environments do not support a secure software execution, nor do client server applications allow user customization of the software running on the servers. To address this problem, a biological pathway modeling environment was built as a stand-alone Rich Internet Application (RIA). The modeling environment was tested by constructing a simulation of the glycolysis pathways in the human erythrocytes, and the results were compared against one of the latest and richest erythrocyte metabolism models developed by Kuchel and Mulquiney. The working simulation was able to settle into a quasi-stable state, with substrate concentrations close to what Kuchel and Mulquiney presented. It was also found that while the browser environment does allow for dynamic applications to be developed, speed and performance do become major issues. In later versions, it is hoped that the performance of the simulator can be increased and that it will become possible to link models together and add collaboration tools.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.

Proliferating tissues, including embryonic and tumour tissues, preferentially employ aerobic glycolysis to support cell growth. This reliance on glycolysis even in the presence of oxygen, referred to as the “Warburg Effect”, may confer a proliferative, survival and invasive advantage and be exploited therapeutically. In this thesis, we demonstrate that the glycolytic enzyme Hexokinase 2 (HK2) is crucial for the “Warburg Effect” in human Glioblastoma Multiforme (GBM), the most common and therapeutically resistant malignant brain tumour. In contrast to normal brain and low-grade gliomas, GBMs exhibited a marked increase in HK2 expression, but not HK1, particularly in perinecrotic, hypoxic regions and its expression predicted poor overall survival of GBM patients. Stable loss of HK2 in GBM cells restored oxidative phosphorylation (OXPHOS)-mediated glucose metabolism, with increased oxygen consumption and decreased lactic acid production, an effect not seen with loss of glycolytic enzymes HK1 or PKM2. Furthermore, HK2 depletion resulted in decreased proliferation in vitro and in vivo and increased sensitivity to apoptotic inducers such as radiation and chemotherapy, both common adjuvant therapies of GBMs. Intracranial xenografts of GBM cells with reduced HK2 demonstrated significantly increased survival with decreased proliferation and angiogenesis yet enhanced invasiveness. In contrast, exogenous HK2 expression in GBM cells promoted proliferation, therapeutic resistance and intracranial growth. This was dependent partly on the PI3K/AKT dependent translocation of HK2 to the mitochondrial membrane. Stable loss of glycolytic enzymes HK2, HK1 and PKM2 reduced GBM proliferation but differentially altered the PI3K/AKT/mTOR and AMPK signaling pathways, the extent to which may influence whether a cell preferentially undergoes autophagy or apoptosis as the primary mode of cell death. Collectively, targeting enzymes employed by the tumour to modulate its energy metabolism, such as HK2 in GBMs, may favourably alter its therapeutic sensitivity to radiation and both classical and novel chemotherapeutic agents.