O trabalho tem como objetivo avaliar o perfil morfométrico e o metabolismo post mortem de bovinos Nelore com diferentes taxas de crescimento da desmama ao sobreano. Foram confinados 154 bovinos Nelore, machos não-castrados, com idade média de 18 meses e 350 kg de peso vivo. Os animais foram designados a um delineamento inteiramente casualizado com dois tratamentos (alta taxa de crescimento [ATC] e baixa taxa de crescimento [BTC] da desmama ao sobreano). Foram selecionados cinco animais extremos de ATC e cinco extremos de BTC (10 animais de cada ano) para realização das analises de cor (L*, a*, e b*), força de cisalhamento, perdas por cocção, e também analise do perfil morfométrico muscular. Amostras também foram coletadas para simulação da glicólise in vitro para a determinação de pH, glicose, glicose-6-fosfato e glicogênio nos pontos 0, 0,5, 2, 4, 8, 12 e 24 horas. Os dados foram analisados utilizando o software SAS 9.4. Não houve efeito da taxa de crescimento sobre as características de desempenho, carcaça nem qualidade de carne. Animais BTC apresentaram maior frequência de fibras do tipo I, enquanto animais ATC apresentaram frequência de fibras tipo IIa 4% maior. Em relação à área transversal das fibras musculares, fibras do tipo I provenientes de animais ATC foram 17% maiores. Os animais ATC apresentaram maior concentração de glicogênio nos pontos 0 e 8 horas. Os animais BTC apresentaram maiores concentrações de G6P nos postos 2 e 8 horas. Animais BTC apresentaram maiores concentrações de glicose nos pontos 8 e 24 horas. Em conclusão, a seleção de bovinos com alta taxa de crescimento no período da desmama ao sobreano promove um perfil de fibras mais glicolítico. Entretanto, essa mudança no perfil morfométrico não altera a queda do pH nem a qualidade da carne. / The objective of this study was to evaluate the morphometric profile and the postmortem metabolism of Nellore cattle with different growth rates from weaning to yearling. 154 non castrated Nellore males with 18 mo/old and 350 kg live weight were confined. The animals were assigned to a completely randomized design with two treatments (high growth rate [ATC] and low growth rate [BTC] from weaning to yearling). Five animals from ATC group and five animals from BTC group (10 animal each year) were selected. Analysis of color (L*, a*, and b*), shear force, morphometric profile, simulation of in vitro glycolysis for the determination of pH, glucose, glucose-6-phosphate and glycogen in 0, 0,5, 2, 4, 8, 12 e 24h were performed. The data were analyzed using SAS 9.4 software. There was no effect of growth rate on performance, carcass or meat quality characteristics. BTC Animals had a higher frequency of type I fibers, while ATC animals presented a 4% greater frequency of type IIa fibers. Regarding the cross-sectional area of muscle fibers, Type I fibers from ATC animals were 17% larger. ATC animals showed higher glycogen concentration at 0 h and 8 h. BTC animals had higher concentrations of G6P at 2h and 8h. BTC animals presented higher glucose concentrations at 8 h and 24 h. In conclusion, the selection of cattle with a high growth rate from weaning to yearling leads to a more glycolytic fiber profile. However, this change in the morphometric profile neither alters the pH drop nor the meat quality.

O trabalho tem como objetivo avaliar o perfil morfométrico e o metabolismo post mortem de bovinos Nelore com diferentes taxas de crescimento da desmama ao sobreano. Foram confinados 154 bovinos Nelore, machos não-castrados, com idade média de 18 meses e 350 kg de peso vivo. Os animais foram designados a um delineamento inteiramente casualizado com dois tratamentos (alta taxa de crescimento [ATC] e baixa taxa de crescimento [BTC] da desmama ao sobreano). Foram selecionados cinco animais extremos de ATC e cinco extremos de BTC (10 animais de cada ano) para realização das analises de cor (L*, a*, e b*), força de cisalhamento, perdas por cocção, e também analise do perfil morfométrico muscular. Amostras também foram coletadas para simulação da glicólise in vitro para a determinação de pH, glicose, glicose-6-fosfato e glicogênio nos pontos 0, 0,5, 2, 4, 8, 12 e 24 horas. Os dados foram analisados utilizando o software SAS 9.4. Não houve efeito da taxa de crescimento sobre as características de desempenho, carcaça nem qualidade de carne. Animais BTC apresentaram maior frequência de fibras do tipo I, enquanto animais ATC apresentaram frequência de fibras tipo IIa 4% maior. Em relação à área transversal das fibras musculares, fibras do tipo I provenientes de animais ATC foram 17% maiores. Os animais ATC apresentaram maior concentração de glicogênio nos pontos 0 e 8 horas. Os animais BTC apresentaram maiores concentrações de G6P nos postos 2 e 8 horas. Animais BTC apresentaram maiores concentrações de glicose nos pontos 8 e 24 horas. Em conclusão, a seleção de bovinos com alta taxa de crescimento no período da desmama ao sobreano promove um perfil de fibras mais glicolítico. Entretanto, essa mudança no perfil morfométrico não altera a queda do pH nem a qualidade da carne. / The objective of this study was to evaluate the morphometric profile and the postmortem metabolism of Nellore cattle with different growth rates from weaning to yearling. 154 non castrated Nellore males with 18 mo/old and 350 kg live weight were confined. The animals were assigned to a completely randomized design with two treatments (high growth rate [ATC] and low growth rate [BTC] from weaning to yearling). Five animals from ATC group and five animals from BTC group (10 animal each year) were selected. Analysis of color (L*, a*, and b*), shear force, morphometric profile, simulation of in vitro glycolysis for the determination of pH, glucose, glucose-6-phosphate and glycogen in 0, 0,5, 2, 4, 8, 12 e 24h were performed. The data were analyzed using SAS 9.4 software. There was no effect of growth rate on performance, carcass or meat quality characteristics. BTC Animals had a higher frequency of type I fibers, while ATC animals presented a 4% greater frequency of type IIa fibers. Regarding the cross-sectional area of muscle fibers, Type I fibers from ATC animals were 17% larger. ATC animals showed higher glycogen concentration at 0 h and 8 h. BTC animals had higher concentrations of G6P at 2h and 8h. BTC animals presented higher glucose concentrations at 8 h and 24 h. In conclusion, the selection of cattle with a high growth rate from weaning to yearling leads to a more glycolytic fiber profile. However, this change in the morphometric profile neither alters the pH drop nor the meat quality.

The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the ‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis respectively. In their human infective form in the bloodstream all three trypanosomatid parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6- bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set of structural and regulatory differences compared to the mammalian host enzyme. These differences, coupled with the availability of trypanosomatid PFK crystal structures present an opportunity for the structure-based design of specific inhibitors against the enzyme. Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’) were designed against T. brucei PFK with the aim of developing novel treatments against Human African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to trypanosomatid PFKs. We show that the compounds are specific; neither competing with the natural substrates ATP or F6P nor inhibiting the human PFK enzyme. We describe the development and testing of highly potent and specific low molecular weight PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage I HAT in mice models. We describe the tight, 1:1 binding of these compounds with trypanosomatid PFKs, and the thermodynamic characteristics of binding through various biophysical assays. We also show the unprecedented characterisation of the reverse PFK reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and inhibition by the drug-like compounds against trypanosomatid PFKs.

The metabolic pathways required for adenosine triphosphate (ATP) production within the cell are well understood, however recent publications suggest that metabolic pathways are closely linked to immune cell activation and inflammatory diseases. There has been little examination of the metabolic pathways that modulate mast cell activation and the feedback regulator lactic acid. Here we examine metabolic pathways and regulation within mast cells in the context of lipopolysaccharide (LPS) and interleukin (IL-33) activation, for which there has been little to no reported studies. First, we examine the effects of lactic acid, previously considered only a by-product of glycolysis and now understood to act as a negative feedback regulator of inflammation in the context of LPS activation and sepsis. Lactic acid is elevated in septic patients and associated with mortality, potentially due to suppressive effects on LPS signaling and contribution to late phase immunosuppression. By attenuating glycolysis and reducing ATP availability for signaling and cytokine transcription, lactic acid impairs the function of immune cells to fight the initial or subsequent infections. We support this with in vitro and in vivo data. Additionally, our lab has published that lactic acid can suppress IL-33 activation, potentially by metabolic modulation as with LPS activation; however there has been no study of the metabolic requirements for IL-33 activation. We report here that glycolysis is required for ATP and reactive oxygen species (ROS) production to augment signaling and cytokine production downstream of the IL-33 receptor. Together, these studies examine the contribution of metabolism to mast cell activation and may provide potential targets for treatments of diseases that involve LPS- or IL-33-dependent mast cell activation.

No description available.

Schweinfurthins are a family of natural products with significant anti-cancer activities. They were originally identified in the National Cancer Institute (NCI) human 60 cancer cell line screening. The growth inhibition profile of schweinfurthins is distinct from other clinically used anti-cancer agents, indicating that they have a novel mechanism of action or have a previously unrecognized protein target. Previous studies showed that schweinfurthins affect multiple cellular processes in cancer cells. For example, schweinfurthins can alter cytoskeleton organization, induce ER stress and apoptosis, and inhibit the mevalonate pathway. The mevalonate pathway is responsible for the production of isoprenoids and cholesterol, which have been shown to play regulatory roles in the Hedgehog (Hh) signaling pathway. In this study, we found that the Hh signaling pathway in NIH-3T3 and SF-295 cells was inhibited by schweinfurthins. The supplementation of mevalonate and cholesterol partially restored Hh signaling, indicating that schweinfurthins inhibit Hh signaling partially by down-regulating the products from the mevalonate pathway. Interestingly, schweinfurthins in combination with cyclopamine, an inhibitor of the Hh singaling pathway, synergistically decreased cell viability. In order to better understand the underlying mechanism of the anti-cancer action of schweinfurthins, we attempted to identify the protein target of schweifnurthins. Affinity chromatography was performed to pull down the protein target. We found that schweinfurhtins bound to the M2 isoform of pyruvate kinase (PKM2) and inhibit its pyruvate kinase activity. Knockdown of PKM2 by siRNA increased the sensitivity of SF-295 cells to schweinfurthins. The inhibition of PKM2 by schweinfurthins led to a reduction in the rate of glycolysis in cancer cells. Fructose 1,6-bisphosphate (FBP), an activator of PKM2, could alleviate schweinfurthin-mediated inhibition on PKM2 and glycolysis. Notably, FBP could also partially reverse the reduction of cell viability in the presence of schweinfurthins. Taken together, these studies revealed the mechanism by which schweinfurthins inhibit Hh signaling. In addition, we uncovered PKM2 as a schwienfurthin target and highlighted the importance of glycolysis suppression as a mechanism of the anti-cancer action of schweinfurthins.

Myc oncogenes encode transcription factors that bind to E-box sequences in DNA, driving the expression of a large number of target genes and are deregulated in approximately 70% of human cancers. Deregulated Myc expression cause enhanced proliferation (which is counteracted by apoptosis), angiogenesis and cancer. Though Myc’s importance in induction of S phase has been established, less is known about its functions in the G2 and M phases of the cell cycle. Paper I addresses the targeting of the Myc targets Aurora kinase A and B that have roles in G2/M transition and provide evidence that pharmaceutical Aurora kinase inhibition causes cell cycle arrest and apoptosis in a Myc-selective manner and is useful in treating Myc-induced lymphomas in vivo. The assumption that the important target genes responsible for the biological effects of Myc overexpression were those encoding components of the cell cycle machinery lead to little interest in other potentially important groups of target genes. However, recent work challenged this view by indicating that Myc target genes encoding metabolic enzymes may be critical for Myc-induced tumorigenesis. Importantly, the targeting of Myc target genes encoding metabolic enzymes has the potential of providing a new treatment strategy of Myc-induced cancers. Paper II covers the pharmaceutical targeting of the Myc-induced spermidine synthase (Srm) that shows promise as a tool for chemoprevention by affecting proliferation, but not for the treatment of established tumors. Paper III focuses on the negligible effect an Ldha mutation has on Myc- induced lymphomagenesis. Ldha has long been known to be a Myc target gene and in vitro experiments have recently indicated it to be important for transformation. It seems the negligible effect of the Ldh mutation can be explained by the high frequency of loss of either Arf or p53 in this mouse model, since enforced Ras-Myc oncogenic cooperation in soft agar assays of Ldh mutant MEFs effectively inhibits colony formation, and λ-Myc;Ldh mutant bone marrow infected with oncogenic Ras does not give rise to tumors when transplanted into wild-type mice. A role for Ldh in the ability of tumors to evade the immune system was also indicated in this study. The combined experiences and very different outcome of the three studies included in this thesis draw attention to the value of in vivo assessment of Myc downstream targets in Myc-induced lymphomagenesis.

Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarry analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameraic to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect.

<p>Functional imaging gives clinicians the ability to monitor breast cancer progression and response to therapy. Modern techniques such as Positron Emission Tomography (PET) has allowed for clinicians to visualize the metabolic need of breast cancer and track it longitudinally. However, these techniques are expensive, technologically complex and not easily implemented in rural areas. To add to the difficulty, breast cancer is a highly heterogeneous disease. The heterogeneity means that a single therapy is not always applicable to all patients and every patient requires an individual treatment plan. Being able to first diagnose breast cancer, and then monitor its response to therapy in a cost-effective manner is imperative to improve the survival of patients with this disease. </p><p>Optical techniques such as fluorescence are ideal for these applications since they can be fast and implemented with portable technology. These techniques use differences in light interaction with tissue to allow for abnormality detection. This dissertation tests the hypothesis that the fluorescent molecularly specific agents, protoporphyrin IX (PpIX) and 2-NBDG, which utilize metabolic alterations caused by cancer, can be used for ubiquitous breast cancer differentiation and therapy monitoring. Confocal microscopy is used to demonstrate the applicability of both agents in vitro to breast cancer cells regardless of phenotype. </p><p>First, 5-aminolevulinic acid (ALA) was incubated with cells causing an increased cellular production of the heme prequel, protoporphyrin IX (PpIX). In cancer cells, the production of PpIX is higher and allows for detection from normal after a 2 hour incubation period. The PpIX was then detected via confocal microscopy and the change in fluorescence intensity between ALA-induced PpIX and controls was measured. A spectroscopy measurement is also completed on a second experimental set of cells to demonstrate that collection of single spectra, post-ALA administration, can discriminate breast cancer cells from normal mammary epithelium. </p><p>2-NBDG is a fluorescent glucose analogue that is follows the metabolic pathway of glycolysis, similarly to D-glucose and fluorodeoxyglucose (FDG). Greater accumulation of 2-NBDG can occur in as little as 20 minutes in cells with higher glycolytic demand, which is commonly associated with cancer and hypoxic cells. The shorter incubation period required for 2-NBDG makes it ideal for clinical use, and 2-NBDG was therefore tested further. </p><p>2-NBDG uptake was used to detect changes in cellular glycolysis after anti-cancer and endocrine therapy. The anti-cancer therapies, lonidamine and a-cyano-hydroxycinnamate (a-Cinn), which increased and decreased glycolysis, respectively were tested on a subset of breast cancer cells. Lonidamine directly inhibits the metabolism of 2-NBDG and inhibited its uptake. a-Cinn stimulates glycolysis by inhibiting the monocarboxylate transporter 1 preventing lactate from entering as a source for oxidative phosphorylation. 2-NBDG was concurrently increased after a-Cinn treatment. Observation of changes in downstream glycolysis has been determined after the estrogen receptor therapy, tamoxifen, in breast cancer cells. Sixty percent of all breast cancers are estrogen receptor positive (ER+) and have the potential to respond. Known ER+ cells, MCF7, and ER- cells, MDA-MB-435, were treated with tam. 2-NBDG was used to determine therapeutic responders from non-responders by measureable differences in fluorescence uptake. </p><p>Finally, the effect of hypoxia, low oxygenation, on 2-NBDG uptake is discussed. The cellular response to hypoxia, known as the Pasteur Effect, causes an increase in glycolysis. Hypoxia is shown in vitro to increase 2-NBDG uptake. Simulated, chronic and cycling hypoxia were completed in vitro with subsequent increases in 2-NBDG as well. Cycling hypoxia has been previously shown to have a greater impact on tumor environment and was implemented in an in vivo murine dorsal window chamber mammary carcinoma model. The uptake of 2-NBDG in tumor and normal tumor-free tissue was tested and 2-NBDG discriminated normal from tumor in a normal oxygen environment. An increase in 2-NBDG was demonstrated after cycling hypoxia in tumor and normal tissue. However, by including hemoglobin saturation data, cycling hypoxic tumor tissue can be discriminated from cycling hypoxic normal tissue and normoxic tumor tissue. From these experiments, the applicability of 2-NBDG as a method to monitor changes in glycolysis and its increased potential by including hemoglobin</p><p> saturation measurements is demonstrated.</p> / Dissertation

ABSTRACT PURIFICATION AND CHARACTERIZATION OF HEXOKINASE ISOENZYMES FROM Rhizopus oryzae Dedeoglu, Didem MS., Department of Biotechnology Supervisor: Prof.Dr. Haluk Hamamci Co-supervisor: Dr. Seyda A&ccedil / ar February 2007, 116 pages Glycolysis is the central metabolic pathway for living organisms. Its regulation is important for the yield of the end products which are industrially important. These end products, like lactic acid produced by Rhizopus oryzae, are industrially important. Rhizopus oryzae is a filamentous fungus producing lactic acid and ethanol. The lactic acid yield of R. oryzae is low (&amp / #61566 / 70 %) compared to that of lactic acid bacteria (&amp / #61502 / 95 %) still it is noteworthy because R. oryzae produces only the L (+) form of lactic acid which can be metabolized in the human body. The yield of an industrial process should be high for the feasibility of the production of a particular product. If a way can be found increase the flux through the glycolysis the yield of lactic acid may increase as well. Keeping this in mind we wanted to focus on the first step of glycolysis, hexokinase of R. oryzae. Hexokinase catalyzes the reaction that converts glucose to glucose-6-phosphate. In this study for the first time the two isoenzymes of hexokinase of R. oryzae were purified and characterized by biochemically and kinetically Hexokinase has two isoenzymes. The purified enzymes (isoenzymes1 &amp / isoenzymes2) obeyed Michealis-Menten Kinetics. The Km value of purified isoenzyme 1 is 0.16 mM and isoenzyme 2, 0.21 mM at pH 7.70 for glucose. The Km value of isoenzyme1 for fructose was 28.8 mM. Essentially isoenzyme 2 can not utilize fructose. None of the isoenzymes were inhibited by trehalose-6-phophate.The monomer moleculer weight of isoenzymes were estimated SDS PAGE analysis. There were two different values for molecular weight of isoenzmye 1 / 62.9 and 42.5 kDa and two values for isoenzyme 2 / 56.2 and 41.6 kDa