This thesis extends the work of Crimmins (1995) on the control of glycolytic flux in yeast by the enzymes 6-phosphofructo-1-kinase and pyruvate kinase (Pyk1p). This study also examines the influence of Pf1kp and Pyk1p upon yeast resistance to the weak acid preservative, benzoic acid. In <I>Saccharomyces cerevisiae</I>, Pyk1p is encoded by <I>PYK1</I>, and the α and β subunits of Pf1kp are encoded by <I>PFK1</I> and <I>PFK2</I>, respectively. To test the influence of these genes upon glycolytic control, an isogenic set of <I>S. cerevisiae</I> mutants were utilised in which <I>PYK1, PFK1</I> and <I>PFK2</I> expression is dependent on the <I>PGK1</I> promoter. Increased Pf1k levels had little effect upon rates of glucose utilisation or ethanol production during fermentative growth. However, overexpressing Pyk1p resulted in an increased growth rate and an increase in glycolytic flux. This suggests that Pyk1p, but not Pf1kp, exerts some degree of control over the glycolytic flux under these conditions. The effects of reducing Pf1kp and Pyk1p levels were also studied by placing <I>PYK1, PFK1</I> and <I>PFK2</I> under the control of the weak <I>PGK1Δuas</I> promoter. The double Pf1kp mutant showed no significant changes in doubling time, ethanol production or glucose consumption. However, a mutant with a 3-fold reduction ion Pyk1p levels displayed slower growth rates and reduced glycolytic flux. In addition, there was an imbalance in the carbon flow in this mutant, with reductions in ethanol and glycerol production evident, along with increased TCA cycle activity. Hence, while Pf1kp levels did not affect cell physiology significantly under the conditions studied, reduced Pyk1p levels seemed to disturb glycolytic flux and carbon flow. Decreased Pf1kp levels caused an increase in the sensitivity of yeast cells to benzoate, whereas the Pyk1p mutant was not affected. This confirmed that benzoic acid specifically inhibits Pf1kp rather than glycolysis in general.

Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.

Most cancer cells rely on aerobic glycolysis to support the mitochondrial oxidative phosphorylation system (OXPHOS). The persistent oxic-anoxic cycle exerts selection pressures which lead to constitutive activation of glycolysis even in the presence of abundant oxygen. Expression of hypoxia-inducible factor 1 (HIF1) increases following hypoxia in neoplastic cells. This leads to the induction of pyruvate dehydrogenase kinase 1 (PDK1). The latter inactivates pyruvate dehydrogenase (PDH) that converts pyruvate to acetyl-coenzyme A for delivery to the tricarboxylic acid cycle (TAC). Dichloroacetic acid (DCA) is an inhibitor of PDK that forces cells into oxidative phosphorylation thereby suppressing cancer growth. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-3-ol-17-one (C9), along with a few other 17β-estradiol analogs, are a novel class of in silico-designed inhibitors of microtubule dynamics. These newly designed and synthesized antimitotic compounds induce G2/M arrest and apoptosis by docking to colchicine binding site between α- and β-tubulin. These compounds are 5 to 20 times more potent than their source molecule, 2-methoxyestradiol (2ME). To improve bioavailability C9 has been in silico-modified at carbon positions C2, C3 and C17 compared to 2ME. The approach to investigate the anticancer potential of the in silico-designed antimitotic C9 in combination with the glycolytic inhibitor DCA in vitro is novel. Human breast carcinoma cell line MCF-7 and non-tumorigenic breast cells MCF-12A were used as an experimental model system. The present study demonstrated that DCA (7.5 mM) in combination with C9 (130 nM) selectively inhibited half of MCF-7 cells‘ population (50.8%). Under the same treatment conditions, MCF-12A cells displayed high number of cell survival (70% cell growth). Qualitative morphological studies revealed decreased cell density in both cell lines, as well as hallmarks of apoptosis and autophagic processes including formation of apoptotic bodies, DNA fragmentation and autophagic vacuoles. Cell cycle- and apoptosis quantification analyses revealed C9+DCA treatment induced apoptosis in both cell lines and exhibited selectivity towards tumorigenic cells. Presence of autophagosome was observed and microtubule-associated protein 1 light chain 3 (II) (LC3-II) expression was elevated. Reduction of mitochondrial membrane potential depolarization in tumorigenic MCF-7 cells was demonstrated, but not in MCF-12A cells. Oxidative stress tests suggested the combination treatment C9+DCA is able to induce lysosomal rupture and/or mitochondrial damage in tumorigenic MCF-7 cells. Kinase inhibition studies revealed that transient activation of c-Jun N-terminal kinase (JNK) plays an important role in cell proliferation. However, C9+DCA stimulated prolonged JNK activation and, in turn, promoted Bcl-2 phosphorylation, thereby facilitating autophagic and apoptotic cell death. C9+DCA induced expression of a number of genes related to stress in MCF-7 treated cells including TP53BP1, MDM2 and BBC3/PUMA. Genes related to cell motility and maintenance of the cytoskeleton such as ACTG1, MAP7, TUBA1, TUBA6, TUBA8 and TUBB2A genes were down-regulated. In MCF-12A cells, treatment of C9+DCA induced expression of multidrug resistance gene ABCB1. Moreover, genes involved in reactive oxygen species metabolism FTH1, GSTA2, NOS2A, SMOX, SOD1 and SOD2 were also up-regulated. In conclusion, the novel 17β-estradiol derivative, C9, in combination with DCA is a potent antiproliferative treatment. This study addressed the mechanisms of combination treatment at the basis of molecular and cellular level, warranting further research projects to develop viable and functional combination treatment as clinically useable anticancer agents. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Physiology / PhD / Unrestricted

Regulation of Glucose metabolism is known to play an important role in pathogenesis of many diseases. Primarily because deregulation of this metabolic pathway can lead to either apoptosis or extended life span of the cells involved. Viruses are parasitic in nature, they utilize the host cellular pathways to support their own progeny; hence it is expected that viruses would regulate the central glucose metabolism of infected host cells. Human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome, and it uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+ T cells induces G2 arrest and cell death within 2-3 days, HIV-1 infection of macrophages results in longer survival of infected cells and low constitutive viral production, generating viral reservoirs. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of infected cells by altering the enzymatic activity and protein expression of various glycolytic components. The data suggests that the two HIV-1 target cell types exhibit very different metabolic outcomes. During viral replication in monocyte/macrophage lineage cells we observe increase in glycolytic protein expression and the same proteins show no modulation in T-cell lines post viral replication. Similar differential regulation is observed in case of enzymatic activity of glycolytic enzymes as well. We also conducted proteomic studies in collaboration with the proteomics core. HIV-1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Vpr is known to cause cell cycle block in infected cell and bring about cell death. However, macrophages are resistant to cell death and are viral reservoir, even Vpr over expression does not cause apoptosis in these cell types. The goal of the study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. We observed that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. We then focused on infected monocyte macrophages to identify if glycolytic components such as HK and G6PD were regulated by HIV-1 infection/replication. We report that Hexokinase-1 (HK-1) enzyme expression increases post infection of PBMCs where as the enzymatic activity of HK decreases. Similar effect is seen with HIV-1 replication in latently infected monocyte cell lines U1. The G6PD enzyme activity and expression both increases in infected PBMCs and in U1 cells post induction of viral replication with PMA. We also found that HK-1 translocate to the mitochondria of U1 cells post induction of HIV-1. It is known that the product of HK activity, Glucose 6-phosphate (G6P) releases HKI from the outer leaflet of mitochondria. Hence we conclude that the viral infection decreases HK activity to have less G6P produced in cell and increases G6PD enzyme activity ensuring the remaining G6P is quickly used up, supporting the adherence of outer mitochondrial membrane bound HK1. This sequence of cellular events ensures longer survival of infected cells supporting the viral progeny to propagate in the cell. We further show that suppressing the Pentose phosphate pathway (PPP) by blocking G6PD activity is not only detrimental to the survival of the infected cells it also suppresses viral replication and promoter level transactivation of the viral LTR. Next we sought to identify if glycolytic enzyme PKM2, that is also known to play a nonmetabolic dual role as a protein kinase regulating gene transcription has any effect on the transcription of HIV-LTR. Our study demonstrates upregulation of pyruvate kinase isoform M2 (PKM2) expression in whole cell extracts and nuclear extracts of HIV-1JRFL infected PBMCs and during reactivation of HIV-1 in chronically infected U1 cells. We then focused on understanding the potential role of PKM2 on HIV-1 LTR transactivation. Our studies demonstrate that over expression of PKM2 leads to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -120 bp to -80 bp to be essential for PKM2 mediated transactivation. This region contains the NFKB DNA binding site and mutation of NFKB binding site attenuated PKM2 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis confirmed interaction of PKM2 with HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional co-activator of HIV-1 LTR. Hence it opens up another possible target to curb HIV-1 replication at transcriptional level. This study sheds light on the regulation of glycolytic pathway of host cells by HIV-1 infection and its consequences for the virus, opening up new avenues to target viral replication and identify glycolytic markers of HIV-1 pathogenesis. / Biology

A percepção de cor e suas relações com água retida são aspectos importantes para definição do rendimento e qualidade final do processo produtivo da carne. A inter-relação entre as medidas objetivas de cor e a capacidade de retenção de água tem possibilitado a utilização de valores de atributos de cor para a predição de problemas com carne exsudativa. O objetivo do presente estudo foi identificar a relação entre pontos anatômicos de amostragem na secção transversal do bife de lombo suíno para obtenção das medidas de cor e medidas indiretas da capacidade de retenção de água, além do impacto nestes atributos devido à glicemia no momento do abate e no exsudado. Foram realizados dois ensaios, na seguinte ordem: 1) 19 amostras de lombo suíno foram adquiridas de forma aleatória no varejo, no dia seguinte ao abate do animal, e submetidas à determinação das variáveis de cor, pelo padrão CIElab, em seis pontos da superfície do bife, que posteriormente foram submetidos a medidas de exsudação e 2) 25 carcaças foram amostradas em abatedouro experimental, selecionadas com base nos níveis de glicemia plasmática dos animais no momento da sangria, formando duas categorias: normal-NO (74,8 ± 2,00 mg.dL-1) e alta-AL (134 ± 5,30 mg.dL-1). Três diferentes regiões (lateral, intermediária e medial) definidas com base nas observações do ensaio 1 foram analisadas para os parâmetros de cor, perdas por gotejamento em dois tempos e concentração de glicose no exsudado. No primeiro ensaio, a leitura de L* na região ventro-lateral do bife apresentou a maior correlação com a perda por gotejamento média geral do bife. A perda por gotejamento cresceu de maneira proporcional entre 48 e 72 horas após a determinação das variáveis de cor. O grupo AL, no segundo ensaio, apresentou perdas por gotejamento elevadas para a posição intermediária (11,0 ± 1,0 g.100-1) e lateral (9,0 ± 1,0 g.100-1). A coloração entre os grupos não diferiu, porém a L* da região intermediária foi mais alta (55,88 ± 1,55) e sua intensidade de vermelho foi menor (6,67 ± 0,35) em relação às regiões lateral (52,83 ± 1,65 e 7,68 ± 0,40) e medial (50,93 ± 1,65 e 7,67 ± 0,39), respectivamente. Maiores valores de glicose no exsudado também foram observados para as regiões intermediária e lateral. Os indicadores indiretos do metabolismo glicolítico muscular estão relacionados com cor ou perda por gotejamento e podem ser influenciados pela posição anatômica no músculo, com impactos sobre a capacidade de retenção de água, ocorrência de dupla tonalidade e qualidade do lombo suíno. / The perception of color and its relationship with retained water are important for defining the yield and quality of pork production process. The inter-relationship between objective measures of color and water holding capacity has been used for prediction of problems with exudative meat. The aim of this study was to identify the relationship between anatomical positions on cross section steak pork loin to obtain color measurements and indirect measurements of water holding capacity, and the impact in these attributes due to glucose at slaughter and the exudate. Two assays were conducted in the following order: 1) 19 samples of pork loin were randomly acquired in retail store, 24 hours after slaughter and subjected to the determination of color variables, following CIELab standard, in 6 positions of the steak surface, which subsequently underwent measurements of exudation; and 2) 25 carcasses were sampled at experimental abattoir selected based on plasma glucose levels of the animals at the time of bleeding, forming two categories: normal-NO (74.8 ± 2.00 mg.dL-1) and high-AL (134 ± 5.30 mg.dL-1). Three different regions (lateral, intermediate and medial) defined based on the observations of the assay 1 were analyzed for the color variables, drip loss in two times and glucose concentration in the exudate. In the first assay, the L* reading in the ventrolateral region of the steak had the highest correlation with drip loss average of all anatomical regions. The drip loss increased proportionally between 48 and 72 hours after determining the color parameters. The AL group from the second assay had higher drip loss, especially for the intermediate (11.0 ± 1.0 g.100g-1) and lateral position (9.0 ± 1.0 g.100g-1). The color did not differ between groups, but the L* of the intermediate region was higher (55.88 ± 1.55) and the intensity of red was lower (6.67 ± 0.35) compared to the lateral (52.83 ± 1.65 e 7.68 ± 0.40) and medial regions (50,93 ± 1,65 e 7,67 ± 0,39), respectively. Higher values of glucose exudate were also observed for the intermediate and lateral regions. The results confirm that indirect indicators of the muscle glycolytic metabolism are related to color or drip loss and can be influenced by the anatomical position of the muscle, impacting the ability to retain water, twotoning occurrence and overall pork loin quality.

A percepção de cor e suas relações com água retida são aspectos importantes para definição do rendimento e qualidade final do processo produtivo da carne. A inter-relação entre as medidas objetivas de cor e a capacidade de retenção de água tem possibilitado a utilização de valores de atributos de cor para a predição de problemas com carne exsudativa. O objetivo do presente estudo foi identificar a relação entre pontos anatômicos de amostragem na secção transversal do bife de lombo suíno para obtenção das medidas de cor e medidas indiretas da capacidade de retenção de água, além do impacto nestes atributos devido à glicemia no momento do abate e no exsudado. Foram realizados dois ensaios, na seguinte ordem: 1) 19 amostras de lombo suíno foram adquiridas de forma aleatória no varejo, no dia seguinte ao abate do animal, e submetidas à determinação das variáveis de cor, pelo padrão CIElab, em seis pontos da superfície do bife, que posteriormente foram submetidos a medidas de exsudação e 2) 25 carcaças foram amostradas em abatedouro experimental, selecionadas com base nos níveis de glicemia plasmática dos animais no momento da sangria, formando duas categorias: normal-NO (74,8 ± 2,00 mg.dL-1) e alta-AL (134 ± 5,30 mg.dL-1). Três diferentes regiões (lateral, intermediária e medial) definidas com base nas observações do ensaio 1 foram analisadas para os parâmetros de cor, perdas por gotejamento em dois tempos e concentração de glicose no exsudado. No primeiro ensaio, a leitura de L* na região ventro-lateral do bife apresentou a maior correlação com a perda por gotejamento média geral do bife. A perda por gotejamento cresceu de maneira proporcional entre 48 e 72 horas após a determinação das variáveis de cor. O grupo AL, no segundo ensaio, apresentou perdas por gotejamento elevadas para a posição intermediária (11,0 ± 1,0 g.100-1) e lateral (9,0 ± 1,0 g.100-1). A coloração entre os grupos não diferiu, porém a L* da região intermediária foi mais alta (55,88 ± 1,55) e sua intensidade de vermelho foi menor (6,67 ± 0,35) em relação às regiões lateral (52,83 ± 1,65 e 7,68 ± 0,40) e medial (50,93 ± 1,65 e 7,67 ± 0,39), respectivamente. Maiores valores de glicose no exsudado também foram observados para as regiões intermediária e lateral. Os indicadores indiretos do metabolismo glicolítico muscular estão relacionados com cor ou perda por gotejamento e podem ser influenciados pela posição anatômica no músculo, com impactos sobre a capacidade de retenção de água, ocorrência de dupla tonalidade e qualidade do lombo suíno. / The perception of color and its relationship with retained water are important for defining the yield and quality of pork production process. The inter-relationship between objective measures of color and water holding capacity has been used for prediction of problems with exudative meat. The aim of this study was to identify the relationship between anatomical positions on cross section steak pork loin to obtain color measurements and indirect measurements of water holding capacity, and the impact in these attributes due to glucose at slaughter and the exudate. Two assays were conducted in the following order: 1) 19 samples of pork loin were randomly acquired in retail store, 24 hours after slaughter and subjected to the determination of color variables, following CIELab standard, in 6 positions of the steak surface, which subsequently underwent measurements of exudation; and 2) 25 carcasses were sampled at experimental abattoir selected based on plasma glucose levels of the animals at the time of bleeding, forming two categories: normal-NO (74.8 ± 2.00 mg.dL-1) and high-AL (134 ± 5.30 mg.dL-1). Three different regions (lateral, intermediate and medial) defined based on the observations of the assay 1 were analyzed for the color variables, drip loss in two times and glucose concentration in the exudate. In the first assay, the L* reading in the ventrolateral region of the steak had the highest correlation with drip loss average of all anatomical regions. The drip loss increased proportionally between 48 and 72 hours after determining the color parameters. The AL group from the second assay had higher drip loss, especially for the intermediate (11.0 ± 1.0 g.100g-1) and lateral position (9.0 ± 1.0 g.100g-1). The color did not differ between groups, but the L* of the intermediate region was higher (55.88 ± 1.55) and the intensity of red was lower (6.67 ± 0.35) compared to the lateral (52.83 ± 1.65 e 7.68 ± 0.40) and medial regions (50,93 ± 1,65 e 7,67 ± 0,39), respectively. Higher values of glucose exudate were also observed for the intermediate and lateral regions. The results confirm that indirect indicators of the muscle glycolytic metabolism are related to color or drip loss and can be influenced by the anatomical position of the muscle, impacting the ability to retain water, twotoning occurrence and overall pork loin quality.

Metal toxicity is implicated in neurotoxicity, nephrotoxicity, aging and cancer. Protein oxidation resulting from oxidative stress is now known to be involved in metal toxicity. However, proteomic responses to metal induced oxidative stress have not been characterized. By using the yeast as a model, we characterized these changes occurring in response to sub-lethal doses of metals. Several proteins involved in protein synthesis, ribosome assembly decreased while antioxidant defenses, proteins involved in sulfur metabolism, and glutathione synthesis and ubiquitin increased following metal exposure. We also show that metals induced temporal and targeted protein oxidation independent of protein abundance. Among the targets were glycolytic enzymes and heat-shock proteins. As a consequence, glycolytic enzyme activities decreased whereas the levels and activities of the enzymes of the alternative pathway for glucose metabolism, pentose phosphate pathway (PPP) increased. True to prediction, we also found increased flow through the PPP as measured by elevated levels of NADPH and glutathione. NADPH and glutathione are crucial for maintaining the redox balance in the cell. Thus, rerouting of glucose metabolism into PPP is considered to be beneficial to the organism. Among the oxidation targets is a glycolytic protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that is required for apoptosis in neuronal cells. We show that not only is GAPDH required for metal induced apoptosis in yeast but also the levels of GAPDH transcript and protein increase in the cytosol and the nucleus in an isoform specific fashion. Such changes strongly implicate the role of GAPDH in yeast apoptosis. This work provides evidence for the involvement of targeted protein oxidation in metal toxicity, shows the overlaps and differences in the mechanism of copper and cadmium toxicity, allows comprehension of how metabolic processes respond to metal stress and explores the potential of GAPDH as a sensor of oxidative stress and mediator for apoptosis.

No description available.

Yes / Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional setting. / RMP and MS funded by Yorkshire Cancer Research (pump priming grant BPP 046). IJS and AL funded by NIHR Research & Innovation Division, Strategic Project Funding 2013 and Manchester Pharmacy School Fellowship.

O objetivo do presente estudo foi avaliar a relação entre indicadores de estresse pré-abate com características de qualidade e metabolismo pós-morte de bovinos terminados em pastagem. Foram colhidas amostras do músculo Longissimus de 55 bovinos da raça Nelore (Bos indicus), não castrados, terminados exclusivamente em pasto. Do total de animais abatidos, foram selecionadas 29 carcaças, de acordo com o pH e classificadas em dois tratamentos: DFD (pH &le; 5.8; n=13) e normal (pH &gt; 5.8; n=16). Durante a sangria, foram colhidas amostras de sangue para as análises de cortisol e de lactato plasmáticos. Foram colhidas amostras do músculo Longissimus thoracis no dia do abate para as análises de metabólitos e pH e 48h após foram retirados dois bifes de 2,5 cm de espessura cada um, para análises instrumentais de qualidade de carne após 0 ou 14 dias de maturação. Não foi observada diferença entre os tratamentos para lactato e cortisol sanguíneos. O pH reduziu com o tempo pós-morte para ambos o tratamentos, porém o tratamento DFD apresentou maiores valores em relação a carne com pH normal. Foi observada uma interação entre os tratamentos e os tempos avaliados para glicogênio (P = 0,006), lactato (P &lt; 0,0001), G-6-P (P &lt; 0,0001) e glicose (P &lt; 0,0001). A concentração de glicogênio diminuiu ao longo do tempo. Os valores de perdas por cocção e força de cisalhamento foram maiores para o tratamento DFD (P = 0,01). Com relação à cor, foi observado maiores valor de L* (P = 0,01) e b* (P = 0,01) para carne DFD. O valor de a* foi maior para a carne normal na carne não maturada, porém após 14 dias o tratamento DFD apresentou maior valor. Foram observadas maiores concentrações de glicogênio muscular nos tempos 8h e 24h, assim como maiores valores de glicose, glicose-6-fosfato e menores concentrações de lactato para o tratamento DFD (P &lt; 0,01). Os indicadores de estresse pré-abate não foram adequados para diferenciar carnes pelo pH final. Diferenças nos valores de pH final não recapitulam o mecanismo esperado de cortes mais escuros para as amostras de alto pH, enquanto que os metabólitos foram associados com diferenças encontradas no pH final, porém o mecanismo responsável por essa diferença não foi determinado. / The objective of the present study was to evaluate the relationship between pre-slaughter stress indicators with quality characteristics and post-mortem metabolism of cattle grazing. Samples of the Longissimus muscle were collected from 55 Nellore (Bos indicus) bovines, non-castrated, exclusively grassfed. From the total of slaughtered animals, 29 carcasses were selected according to pH and classified into two treatments: DFD (pH &le; 5.8, n = 13) and normal (pH&gt; 5.8, n = 16). During slaghter, blood samples were collected for plasma cortisol and lactate analyzes. Samples of the Longissimus thoracis muscle were collected on the day of slaughter for metabolite and pH analyzes and 48 hours after two steaks of 2.5 cm thickness were removed for instrumental analyzes of meat quality after 0 or 14 days of maturation. No difference was observed between treatments for blood lactate and cortisol. The pH reduced with the time after death for both treatments, however the DFD treatment presented higher values in relation to the meat with normal pH. It was observed an interaction between the treatments and the times evaluated for glycogen (P = 0.006), lactate (P &lt;0.0001), G-6-P (P &lt;0.0001) and glucose (P &lt;0.0001). The glycogen concentration decreased overtime. The values of cooking losses and shear force were higher for DFD treatment (P = 0.01). Regarding color, a higher value of L * (P = 0.01) and b * (P = 0.01) was observed for DFD meat. The value of a * was higher for normal meat in the unmated meat, but after 14 days the DFD treatment presented higher value. Higher concentrations of muscle glycogen were observed in 8h and 24h times, as well as higher glucose, glucose-6-phosphate and lower lactate concentrations for DFD treatment (P &lt; 0.01). The pre-slaughter stress indicators were not adequate to differentiate meats by the final pH. Differences in the final pH values did not recapitulate the expected mechanism of darker cuts for the high pH samples, while the metabolites were associated with differences found in the final pH, but the mechanism responsible for this difference was not determined.