Dissertations / Theses on the topic 'Cell Metabolism'
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Pat, Sze Wa. "Cell metabolism in cell death and cell growth." HKBU Institutional Repository, 2007. http://repository.hkbu.edu.hk/etd_ra/775.
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Iafelice, Bruno <1979>. "Miniaturized sensors for cell metabolism." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/397/1/Tesi_encripted.pdf.
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Iafelice, Bruno <1979>. "Miniaturized sensors for cell metabolism." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/397/.
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Chowdhury, Azazul Islam. "Role of Cell-cell Interactions and Palmitate on β-cells Function." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-230841.
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The islets of Langerhans secrets insulin in response to fluctuations of blood glucose level and efficient secretion requires extensive intra-islet communication. Secretory failure from islets is one of the hallmark in progression of type 2 diabetes. Changes in islet structure and high levels of saturated free fatty acids may contribute to this failure. The aim of this thesis is to study the role of cell-cell interactions and palmitate on β-cells functions. To address the role of cell-cell interactions on β-cells functions MIN6 cells were cultured as monolayers and as pseudoislets. Glucose stimulated insulin secretion was higher in pseudoislets compared to monolayers. Transcript levels of mitochondrial metabolism as well glucose oxidation rate was higher in pseudoislets. Insulin receptor substrate-1 (IRS-1) phosphorylation was altered when cells were grown as pseudoislets. Proteins expression levels related to glycolysis, cellular connections and translational regulations were up-regulated in pseudoislets. We propose the superior capacity of pseudoislets compared to monolayers depend on metabolism, cell coupling, gene translation, protein turnover and differential IRS-1 phosphorylation. To address the role of palmitate on β-cells human islets were cultured in palmitate. Long term palmitate treatment decreased insulin secretion which is associated with up-regulation of suppressor of cytokine signaling-2 (SOCS2) and protein inhibitor of activated STAT-1 (PIAS1). Up-regulation of SOCS2 decreased phosphorylation of Akt at site T308, whereas PIAS1 decreased protein level of ATP- citrate lyase (ACLY) and ATP synthase subunit B (ATP5B). We propose long term palmitate treatment reduces phosphatidylinositol 3-kinase (PI3K) activity, attenuates formation of acetyl-CoA and decreases ATP synthesis which may aggravate β-cells dysfunction.
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Board, Mary. "A study of energy metabolism in neoplastic cells." Thesis, University of Oxford, 1990. http://ora.ox.ac.uk/objects/uuid:d3e13e31-3fe8-4cd8-ad71-50d4e7df4d27.
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Sidiq, Karzan Rafiq. "Cell wall metabolism in Bacillus subtilis." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3243.
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Cell wall is a unique and essential component of bacterial cell. It defines cell shape and protects cell from bursting through its own internal osmotic pressure. It also represents a significant drain on the cells resources, particularly in Gram positives, where the wall accounts for more than 20 % of the dry weight of the cell, and approximately 50 % of ‘‘old’’ cell wall is degraded and new material made to permit cell growth. After the discovery of penicillin, there has been active study of bacterial cell wall structure and metabolism, as it represents the major target for antibacterial compounds. The biosynthetic pathways for cell wall precursors has been well investigated in bacteria generally, but the coordination of cell wall metabolic processes and the fate of turnover cell wall materials have only been well characterised in Gram-negative bacteria (e.g Escherichia coli). In Gram-positive bacteria, it has generally been accepted that the old wall is released from the surface and lost to the environment during growth, with apparent recycling of this material during stationary phase for Bacillus subtilis. It is also known that the Gram-positive wall is subject to significant post-synthetic processing, involving the linkage of wall teichoic acids and the cleavage of molecules from the structure, e.g. D-alanine, although the function of these is unclear. Understanding the importance of these processes has relevance for both the pathogenicity and biotechnological use of bacteria, as well as for understanding bacterial cell biology. As it is known that the peptidoglycan fragments (e.g muropeptides) induce the innate immune response in higher organisms and so act as a signal for infection, particularly for Gram-positive bacteria. Thus, understanding how they are generated and recycled by the bacteria may offer potential insights into novel therapeutics, also the accumulation of cell wall muropeptides should be avoided in biotechnological products. In this thesis, the D-alanine metabolism was manipulated to understand the mechanistic details of cell wall metabolism and D-alanine recycling in B. subtilis, using genetic, biochemical, bioinformatics and fluorescent microscopy approaches. Through these analyses, a D-alanine transporter (DatA, formerly YtnA) was identified by genetic screening. The roles of DatA and the carboxypeptidases, LdcB and DacA, in recycling of cell wall derived D-alanine have experimentally been confirmed. We also found that D-alanine aminotransferase (Dat) can act to synthesis D-alanine under certain conditions. From the data obtained a model for peptidoglycan assembly (coordinated synthesis and turnover) during growth of B. subtilis has been developed to take into account the various aspects of cell wall metabolism.
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Tilney-Bassett, Amanda L. "Phospholipid metabolism in T-cell activation." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239331.
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Tueller, Josephine Anna. "Investigation of Therapeutic Immune Cell Metabolism." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8704.
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This thesis addresses multiple approaches to investigating mechanisms of immune linked disease. There are four projects outlined below which describe the work of these investigations. First, educating students about techniques to study disease and therapies is an important area of research. Flow cytometry is a common technique in immunology and its versatility and high throughput abilities can be applied to many fields. While it is very useful, flow cytometry is a complex technique that requires training to operate and understand, and there are very few reports about administering effective training. This thesis outlines the first report of a full semester university course about flow cytometry. Students who completed the course reported increased confidence in their skill levels in conceptual, technical and analytical areas. Second, in the fight against cancer, immunotherapies may provide the necessary adaptability to successfully combat many cancer types. By strengthening and educating the immune system, clinicians can help patients fight cancer without resorting to harmful chemotherapeutics, or immunotherapies can be used in tandem with current treatments. Chimeric antigen receptor (CAR) T cells and checkpoint blockade are two of the most successful immunotherapies. CAR T cells combine the extraordinary binding ability of an antibody with T cell signaling molecules via genetic engineering, for a faster and more efficient cancer killing version of the patient's own T cells. These have been remarkably successful, but results depend on the specific signaling co-receptors that are included in the design. Increased understanding of co-receptor function could help in making CAR T cell design more specific, and enable CAR T cells to be effective against more types of cancers. Metabolic function is crucial in understanding T cell therapeutics because T cells need to use energy efficiently enough to compete with ravenous cancer cells. This thesis outlines an ongoing investigation into a co-receptor's effect on CAR T cell metabolism, suggesting that co-receptors can alter CAR T cell metabolism by increasing maximal respiration. Third, CD5 is a negative regulatory co-receptor on T cells that can modulate T cell activation. Related inhibitory co-receptors (PD-1 and CTLA-4) are currently being effectively blocked as checkpoint therapies to reactivate T cells towards cancerous cells. This thesis outlines ongoing work investigating CD5's impact on cellular metabolism. We have found that T cells without CD5 are hypermetabolic as compared to normal naïve T cells. CD5 deficient T cells also have higher maximal respiration, higher basal respiration and higher glycolytic capacity. These differences are also present transiently after non-specific activation. Thus, CD5 significantly regulates the ability of a T cell to use energy, suggesting that CD5 may be a good target for creating more efficient T cell immunotherapies. Fourth, in a separate project, this thesis examines environmental causes of disease. Asthma and allergies are common and growing problems in children and adults. Evaporative cooling can be a less expensive alternative to central cooling, but its effects on allergens and other bioaerosols in the home remains unclear. This project examines the relationship between evaporative cooling and bioaerosols (dust mites, bacterial endotoxin, and fungal β-(1→3)-D-glucans) in low income homes in Utah. We report significantly higher levels of these bioaerosols, particularly fungi in homes with evaporative cooling after adjusting for home-specific factors.
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Thomas, Geraint Mark Howard. "Lithium and phosphoinositide metabolism." Thesis, University of Wolverhampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238120.
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Hooper, Nigel Mark. "Metabolism of neuropeptides by cell-surface peptidases." Thesis, University of Leeds, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235486.
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Alakpa, Enateri V. "Cell metabolism in response to biomaterial mechanics." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/4970/.
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This project assessed the use of short chain peptide (F2/S) hydrogel biomaterial substrates as an instructional tool for driving stem cell differentiation through fine-tuning of the substrate mechanical properties (altered elasticity or stiffness) to mimic that of naturally occurring tissue types. By doing this, differentiation of mesenchymal stem cells (MSCs) into neuronal cells on a 2 kPa (soft) substrate, chondrocytes on 6 kPa (medium) substrate and osteoblasts on 38 kPa (rigid) substrates was achieved. This non-invasive procedure of influencing stem cell behaviour allows a means of exploring innate cell behaviour as they adopt different cell lineages on differentiation. As such, an LC-MS based metabolomics study was used to profile differences in cell behaviour. Stem cells were observed as having increased metabolic activity when undergoing differentiation compared to their ‘resting’ state when they are observed as metabolically quiescent or relatively inactive. As such, the metabolome, as a reflection of the current state of cell metabolism, was used to illustrate the observed divergence of phenotypes as differentiation occurs on each substrate F2/S type. The project further investigated the potential of endogenous small molecules (metabolites) identified using metabolomics, as effective compounds in driving or supporting cell differentiation in vitro. From this, the compounds cholesterol sulphate and sphinganine were found to induce MSC differentiation along the osteogenic and neurogenic routes respectively. A third compound, GP18:0, was observed to have influence on promoting both osteo- and chondrogenic development. These results highlight the potential role a broad based metabolomics study plays in the identification of endogenous metabolites and ascertaining the role(s) they play in cellular differentiation and subsequent tissue development. Lastly, the use of F2/S substrates as a potential clinical scaffold for the regeneration of cartilage tissue was explored. Long term differentiation of pericytes into chondrocytes cultured in 20 kPa F2/S substrates was assessed and the cellular phenotype of the resultant chondrocytes compared to the more conventionally used induction media method. Pericytes cultured within the biomaterial alone showed a balanced expressed of type II collagen and aggrecan with lessened type X collagen expression compared to the coupled use of induction media which showed a bias towards collagen (both type II and type X) gene expression. This observation suggests that in order to mimic native hyaline cartilage tissue in vitro, the use of biomaterial mechanics is potentially a better approach in guiding stem cell differentiation than the use of chemical cues.
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Gupta, Sneha Veeraraghavan. "Targeting Protein Metabolism in B-cell Malignancies." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343169973.
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Li, Zhaoqi Ph D. Massachusetts Institute of Technology. "Bioenergetics and metabolism of eukaryotic cell proliferation." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/130658.
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Thesis: Ph. D. in Biochemistry, Massachusetts Institute of Technology, Department of Biology, February, 2021Cataloged from the official PDF of thesis. "February 2021." Vita. Page 179 blank.
Includes bibliographical references.
Cellular growth and proliferation necessitates the transformation of cell-external nutrients into biomass. Strategies of biomass accumulation across the kingdoms of life are diverse and range from carbon fixation by autotrophic organisms to direct biomass incorporation of consumed nutrients by heterotrophic organisms. The goal of this dissertation is to better understand the divergent and convergent modes of metabolism that support biomass accumulation and proliferation in eukaryotic cells. We first determined that the underlying mechanism behind why rapidly proliferating cells preferentially ferment the terminal glycolytic product pyruvate is due to an intrinsic deficiency of respiration to regenerate electron acceptors. We tested this model across an assorted array of proliferating cells and organisms ranging from human cancer cells to the baker's yeast Saccharomyces cerevesiae. We next determined that a major metabolic pathway of avid electron acceptor consumption in the context of biomass accumulation is the synthesis of lipids. Insights from this work has led to the realization that net-reductive pathways such as lipid synthesis may be rate-limited by oxidative reactions. Lastly, we established the green algae Chlorella vulgaris as a model system to study the comparative metabolism of photoautotrophic and heterotrophic growth. We determined that heterotrophic growth of plant cells is associated with aerobic glycolysis in a mechanism that may be suppressed by light. Collectively, these studies contribute to a more holistic understanding of the bioenergetics and metabolic pathways employed by eukaryotic cells to accumulate biomass and lay the foundation for future studies to understand proliferative metabolism.
by Zhaoqi Li.
Ph. D. in Biochemistry
Ph.D.inBiochemistry Massachusetts Institute of Technology, Department of Biology
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Nunan, Kylie. "Cell wall metabolism in developing grape berries /." Title page, contents and abstract only, 1999. http://web4.library.adelaide.edu.au/theses/09APSP/09pspn972.pdf.
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Tejedor, Vaquero Sonia 1988. "Influence of metabolism in the regulation of T cell differentiation." Doctoral thesis, Universitat Pompeu Fabra, 2018. http://hdl.handle.net/10803/664638.
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Glucose is a key nutrient for T cells. Despite that T cell activation is impaired when they are deprived of glucose, it has also been shown that T effector responses can be elicited in vivo in glucose-poor environments, such as the intratumoral niche, which raises the question of how these cells can maintain their function in nutrient-restricted sites. In this work, we analyzed the ability of T effector cells to be activated by pro-inflammatory polarizing conditions under limiting glucose availability, using an in vitro model in which effector Th0 cells were restimulated to secondary effector T cells in glucose deficiency (0.3 mM). We found that secondary effector T cells could express characteristic Th1 and Th17 cytokines such as IL-17A and IFNγ when exposed to low glucose, but they lost expression of IL-22. Secondary effector T cells adapted to low glucose by reducing their rate of glucose consumption and expression of glycolysis genes, although they still kept using glucose as the main fuel for ATP production. In addition, we found that glucose limitation caused a mild, progressive impairment on mTORC1 activity in these cells that explained in part the downregulation of IL-22, an mTORC1-dependent cytokine. Our results also showed that secondary effector T cells that had experienced glucose stress acquired a nutrient-trained phenotype, and when they were later restimulated under glucose sufficiency they induced an altered cytokine expression pattern with exacerbated production of IL-22 and reduced IFNγ production. Finally, we observed that effector CD4+ T cells generated in different activation contexts in vivo exhibited different patterns of glucose-sensitive genes upon restimulation ex vivo, which suggested that the context in which T effector cells are induced might be a relevant determinant in shaping different response patterns to glucose limitation upon further stimulation. Altogether, our results uncover a previously unappreciated robustness of T cells to maintain effector function under nutrient-restricted conditions, also revealing that a prior history of nutrient stress can influence future effector T cell responses.La glucosa és un nutrient essencial per les cèl·lules T. Malgrat que l’activació T es veu disminuïda per la manca de glucosa, s’ha vist que respostes T efectores tenen lloc in vivo en entorns amb nivells baixos de glucosa, com són els tumors. Això planteja la incògnita de saber com aquestes cèl·lules poden mantenir les seves funcions en ambients pobres de nutrients. En aquest treball hem analitzat la capacitat de les cèl·lules T efectores (Th0) de ser activades en condicions pro-inflamatòries i nivells baixos de glucosa (0.3 mM). Hem vist que les cèl·lules T efectores secundàries poden induir citocines característiques de respostes Th1 i Th17 com la IL-17A i l’IFNγ en condicions de nivells baixos de glucosa, però perden la capacitat d’expressar la IL-22. Aquestes cèl·lules s’adapten a un entorn baix de glucosa reduint-ne el consum i reduint l’expressió de gens de la glicòlisi, malgrat tot, la glucosa segueix sent la seva principal font d’energia (ATP). A més a més, hem observat que nivells limitats de glucosa provoquen una lleu però progressiva deficiència en l’activitat d’mTORC1, necessària per la producció de la IL-22 i que explicaria en part la disminució dels nivells d’aquesta citocina. Els nostres resultats també mostren que les cèl·lules T efectores secundàries que han experimentat un estrès de glucosa adquireixen un fenotip de memòria que fa que responguin de manera alterada (producció exagerada de IL-22) a un segon estímul en presència de nivells normals de glucosa. Finalment, hem observat que les cèl·lules T CD4 efectores activades in vivo expressen diferencialment gens sensibles a glucosa quan són re-estimulades ex vivo. Això suggereix que el context d’activació d’una cèl·lula T és important per determinar la resposta d’aquestes cèl·lules a posteriors estimulacions en situació de baixa glucosa. En resum, els nostres resultats mostren que els limfòcits T son capaços de mantenir un ventall de funcions efectores en situacions de restricció de nutrients, però que el haver passat per una etapa d’estrès de nutrients pot condicionar els seus perfils d’expressió gènica en respostes efectores futures.
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Xue, Yue 1978. "Iron metabolism in mammalian cells." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79216.
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Iron, known for its versatility, is an essential element in the metabolism of mammalian cells. One of the most common iron disorders is autosomal recessive disease---hereditary hemochromatosis, which leads to the iron overload in population of northern European descent. During years of my graduate research, I focused on the study of Hemochromatosis gene Hfe and a point mutation C282Y that leads to more than 80% of all hemochromatosis cases.Iron Regulatory Proteins (IRPs), which serve as main posttranscriptional regulators of cellular iron homeostasis, are the other interest of research. Iron regulatory proteins reversibly interact with iron regulatory elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. The binding ability of IRPs is under tight control so that they respond to the changes in the intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Besides intracellular iron levels, some other stimuli, such as oxidative stress, are capable of regulating this RNA-protein interactions.
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Liu, Laura Xiaofei-Rose. "Maternal Cardiac Metabolism during Pregnancy." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17465316.
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Pregnancy profoundly alters maternal physiology in response to the demands of the fetus. Cardiac output increases in response to an increase in heart rate and stroke volume. Insulin resistance and fetal preference for glucose switches maternal usage to that of predominantly fat consumption. Previous studies have shown that the heart adopts these adaptions, but much still remains unanswered. We sought to fill this gap by exploring cardiac substrate utilization and metabolic regulation during late pregnancy in mice.
We found that by late gestation, serum triglycerides are elevated and serum glucose is unchanged. Furthermore, real-time bioluminescence imaging of late pregnant mouse hearts showed an increase in fatty acid uptake. But greater supply and uptake may not always equal greater oxidation. Using 13C-tracer analysis of Langendorff perfused ex vivo hearts, we observed ~30-50% less glucose usage in late pregnant mouse hearts. Fatty acid utilization, on the other hand, is increased.
The mechanisms regulating substrate switch in late pregnant mouse hearts have not been extensively studied. We determined that mitochondrial DNA copy number, morphology, expression of oxidative phosphorylation (OXPHOS) proteins, and respiratory capacity remain unaltered in late pregnancy. Furthermore, substrate transporter (GLUT4 and CD36) expression and localization are unchanged. Interestingly, pyruvate dehydrogenase kinase 4 (PDK4) is induced in late pregnant mouse hearts. PDK4 is a crucial regulatory enzyme that can decrease glycolysis by phosphorylation of pyruvate dehydrogenase (PDH). Consistent with this, we observed an increase in phospho-PDH in late pregnancy. Treatment of neonatal rat ventricular myocytes (NRVMs) with progesterone, a pregnancy hormone, induced PDK4 expression. Mifepristone, an antagonist of the progesterone receptor (PR), mitigated PDK4 induction, suggesting that PR-mediated pathway is responsible for this upregulation. Taken together, these studies indicate that the substrate switch in late pregnant mouse hearts resulting in decreased glucose oxidation and increased fatty acid utilization is likely by inhibition of PDH via PDK4 upregulation. Dysregulation of this switch could have implications for cardiac diseases during pregnancy.Medical Sciences
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Dolatshahi, Marjan. "Conformational changes of polyomavirus during cell entry." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111604.
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Similar to other non-enveloped viruses, the mechanism of cell entry for polyomaviruses is poorly understood. The polyomavirus capsid is an icosahedron composed of 72 pentamers of the major capsid protein VP1. There is one copy of minor capsid proteins, VP2 or VP3, at the center of each pentamer. According to previous studies, polyomavirus cell entry is a multi-step process which includes: 1) VP1 binding to sialic acid (SA) on the surface of host cells, 2) interaction of VP1 with alpha4beta1 integrin and 3) subsequent cell penetration. Biochemical studies have shown that SA alters polyomavirus protease sensitivity, suggesting a conformational change. The aim of this study was to determine these conformational changes at the molecular level. Therefore, we used single particle cryo-electron microscopy to construct 3D maps of wild type (WT) murine polyomavirus, WT bound to SA, a mutant with a disrupted integrin binding site, and the mutant bound to SA. Our results reveal that in both WT and mutant viruses, a significant conformational change happens after binding with SA which is seen as an additional ring of density inside the virus. Moreover some negative densities are seen in the difference map of WT and WT bound with SA, which suggests movement of some viral proteins after binding with SA.
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Breznan, Dalibor. "High-density lipoprotein metabolism in the kidney." Thesis, University of Ottawa (Canada), 2002. http://hdl.handle.net/10393/6120.
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The kidney is believed to play a major role in the clearance and re-absorption of high density lipoprotein (HDL) particles from the blood. Experiments were undertaken to explore the specific sites of renal HDL metabolism in vivo and to investigate in vitro the factors that regulate the renal re-absorption of HDL by HKC-8 human proximal tubule (PT) cells. Perfusion of a rabbit renal artery with [3H]cholesteryl ester (CE) and 125I-protein labeled HDL particles showed that the kidneys are capable of filtering both apolipoprotein A-I (apoA-I) and whole HDL. A fluorescent microscopic study with the HKC-8 cells showed that the PT cells can bind and take up HDL particles. (Abstract shortened by UMI.)
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Mashhedi, Haider. "Implicating insulin in neoplastic growth and metabolism." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=104681.
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In view of accumulating evidence that links obesity to increased cancer burden, there is interest in delineating the mechanisms by which obesity influences neoplastic growth. Increased insulin levels are commonly associated with obesity or ‘the metabolic syndrome' and thus the insulin receptor has been viewed as a potentially important molecular target for the treatment of certain cancers. To study the effects of insulin signaling attenuation on the growth of the experimental insulin-responsive mouse 4T1 breast cancer cells in vivo, we compared the effects of alloxan-induced insulin deficiency to that of BMS-536924, an inhibitor of the insulin and IGF-I receptor tyrosine kinases. Both interventions displayed anti-neoplastic activity, while metabolic toxicity resulted only from alloxan. Insulin receptor inhibition did not result in severe hyperglycemia and treatment with BMS-536924 was well tolerated. We attributed this to pharmacokinetic factors by measuring drug tissue accumulation and measuring glucose utilization in muscle to determine if BMS-536924 abolished insulin-dependent glucose uptake. Our data indicate that insulin dependent glucose uptake by muscle remained intact. Thus, tissue-specific distribution of BMS- 536924 may account for anti-neoplastic activity without severe metabolic toxicity, indicating that pharmacologic targeting of the insulin receptor in neoplastic disease may be practical.Population studies have shown that type II diabetic patients on the biguanide drug metformin have a reduced risk of cancer development or mortality from cancer compared to type II diabetic patients on other therapies. We previously showed that metformin acts as a growth inhibitor in tumor cells in vitro by increasing AMPK phosphorylation in a dose-dependent manner. AMPK activation by metformin, which is seen in tumor cells both in vitro and in vivo, also decreases circulating insulin levels as a secondary effect to lowering blood glucose levels in a setting of type II diabetes. Positron-emission tomography (PET) is an imaging technique that measures the glucose utilization rate that takes place in cancer cells by using the radiolabeled glucose analog 18F-2-Fluoro-2-Deoxy-D-Glucose (FDG). We were interested in the effects of metformin on tumor glucose uptake in mice harboring MC38 colon-adenocarcinoma allografts that were fed either a high-energy diet (that induces a type II diabetic phenotype), or a control diet. Our results show that metformin abolished diet-induced increases in serum insulin levels, tumor insulin receptor activation, and tumor FDG uptake associated with mice on the high-energy diet and that metformin had no effect on these measurements in mice on a control diet. This suggests that for a subset of neoplasms, diet and insulin influence tumor glucose uptake, and this may yield clinical relevance in upcoming trials evaluating metformin's anti-neoplastic activity.Compte tenu de l'accumulation de preuves liant l'obésité à un nombre accru de cancers, il y a un grand intérêt à définir les mécanismes par lesquels l'obésité influe sur la croissance néoplasique. Des niveaux d'insuline élevés sont couramment associés à l'obésité ou au «syndrome métabolique», ce qui fait que le récepteur de l'insuline est considéré comme une cible moléculaire potentiellement importante pour le traitement de certains cancers. Pour étudier les effets de l'atténuation de la signalisation de l'insuline sur la croissance du modèle expérimental du cancer du sein chez la souris, la lignée cellulaire insulino-sensible 4T1 in vivo, nous avons comparé les effets d'une déficience à l'insuline induite par l'alloxan à celle de BMS-536924, un inhibiteur des kinases tyrosine des récepteurs de l'insuline et d'IGF-I. Les deux interventions ont montré une activité anti-néoplasique, mais seulement l'alloxan a présenté une toxicité métabolique. L'inhibition des récepteurs d'insuline n'a occasionné qu'une faible hyperglycémie et le traitement avec BMS-536924 a été bien toléré. Nous avons attribué ce phénomène à des facteurs pharmacocinétiques en mesurant l'accumulation de drogue dans les tissus et pour déterminer si le BMS-536924 abolit l'absorption insulino-dépendante du glucose, nous avons mesuré la quantité de glucose utilisé dans le muscle. Nos données indiquent que la captation insulino-dépendante du glucose par le muscle est restée intacte. Ainsi, la distribution tissu-spécifique du BMS-536924 peut être responsable de l'activité anti-néoplasique sans toxicité métabolique grave, ce qui indique que le ciblage pharmacologique du récepteur de l'insuline dans la maladie néoplasique peut être efficace.Les études épidémiologiques ont montré que les patients diabétiques de type II prenant le médicament metformine (un biguanide) ont un risque réduit de développer un cancer ou d'un taux de mortalité due au cancer plus faible par rapport aux patients diabétiques de type II suivant d'autres thérapies. Nous avons déjà montré que la metformine agit comme un inhibiteur de la croissance des cellules tumorales in vitro en phosphorylant l'AMPK d'une manière dépendante de la dose. Outre l'activation de l'AMPK, qui est observée dans les cellules tumorales in vitro et in vivo, la metformine provoque aussi une diminution des taux d'insuline. Ceci est un effet secondaire de la réduction du taux de glycémie dans un contexte de diabète de type II. La tomographie par émission de positons (TEP ou PET) est une technique d'imagerie qui mesure le taux d'utilisation du glucose par les cellules cancéreuses à l'aide de l'analogue du glucose radiomarqué 18F-2-Fluoro-2-Désoxy-D-Glucose (FDG). Nous étions intéressés par les effets de la metformine sur la captation du glucose par les tumeurs d'adénocarcinome de côlon, MC38, allogreffées chez des souris qui ont été nourris avec une diète à haute teneur énergétique (induisant un phénotype diabétique de type II), ou un régime contrôle. Nos résultats montrent que la metformine abolie l'augmentation des niveaux sériques d'insuline, l'activation du récepteur d'insuline dans les tumeurs ainsi que l'absorption du FDG par les tumeur chez les souris ayant un régime riche en énergie et que la metformine n'a aucun effet sur ces mesures chez les souris ayant une diète contrôle. Ceci suggère que pour un sous-ensemble de néoplasmes, le régime alimentaire et le taux d'insuline influencent l'absorption du glucose par les cellules tumorales ce qui pourrait avoir une pertinence clinique dans les prochaines études clinique visant l'évaluation de l'activité anti-néoplasique de la metformine.
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Kensley, Joy A. "Glycogen metabolism in Corynebacterium glutamicum ATCC 13032." Doctoral thesis, University of Cape Town, 2004. http://hdl.handle.net/11427/4279.
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Includes bibliographical references (leaves 108-123).Corynebacterium glutamicum is a Gram-positive facultative aerobe particularly known for its industrial application in the synthesis of amino acids, such as L-glutamate and Llysine. The central metabolic pathways of this organism has been an area of much research by many groups. Linked to glycolysis is the synthesis of glycogen, previously considered a storage molecule of excess glucose. No information concerning the role of glycogen or its metabolism in C. glutamicum was known, and the aim of this work was to elucidate glycogen metabolism in this industrially important organism.
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Schulz, Anton A. "Nitrogen metabolism in Corynebacterium glutamicum ATCC 13032." Doctoral thesis, University of Cape Town, 2002. http://hdl.handle.net/11427/4329.
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Bibliography: leaves 125-146.Corynebacterium glutamicum is extensively used for the commercial production of a host of amino acids including lysine, glutamate, and threonine. Consequently, much research has been directed at analyzing nitrogen metabolism in this bacterium. In particular, our research focused on investigating the regulation of nitrogen assimilation. Initially, we searched for homologs of the Streptomyces glnR, glnII, and glnE genes in C. glutamicum. These studies, however, were met with limited success, and we therefore decided to use promoter probe vectors in order to identify nitrogen-responsive promoters.
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Johnson, Jenifer L. "Development of redox microphysiometry to assay cell signaling and metabolism /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8498.
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Jani, Klodiana. "The role of integrin-dependent cell matrix adhesion in muscle development /." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115688.
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Cell adhesion is essential to cell motility and tissue integrity and is regulated by the Integrin family of transmembrane receptors. Integrin binds to ligand extracellularly and provide anchor to the intracellular cytoskeleton via adhesion scaffolding proteins. In order to link cell to the surrounding matrix Integrin needs to be activated. Intracellular activation signals induce perturbations in Integrin cytoplasmic domain that are translated into a conformational change in extracellular region for high affinity ligand binding. Integrin engagement by matrix, in turn, triggers the assembly of adhesion complexes. Such early adhesions promote cytoskeletal organization with subsequent contractile activity that exerts forces against initial Integrin-matrix adhesions. In response to force, Integrin strengthens the interaction with matrix through its clustering and successive recruitment of additional adhesion components. These bidirectional regulatory loops mediated by such interactions are largely dependent on the unique function of Integrin adhesion components.We demonstrate a novel role for the PDZ/LIM domain protein Zasp as a core component of Integrin adhesions. Specifically, Zasp colocalizes with Integrins at focal adhesion in cultured cells and myotendinous junctions in Drosophila embryos. In both cases elimination of Zasp modifies Integrin function causing consequently defects in cell spreading and muscle attachment. Zasp supports Integrin adhesion to the extracellular matrix that is required to withstand tensile forces exerted during cell spreading and muscle contraction. Furthermore, we found that the distribution of Zasp in muscle Z-lines is essential to orchestrate the cross-linking of alpha-Actinin and Actin filaments. Disruption of Zasp leads to loss of muscle cytoarchitecture, pointing to a larger role for Zasp in sarcomere assembly. Finally, we demonstrate that Zasp, in addition to alpha-Actinin, physically interacts with the Integrin- and Actin-bound cytoskeletal protein Talin.
Collectively, our results point to a dual role for Zasp as a structural scaffold. First it regulates Integrin adhesion to the extracellular matrix by interacting with the head domain of Talin at the myotendinous junctions. Second, Zasp controls sarcomere assembly by tethering the presarcomeric alpha-Actinin component to the tail domain of Talin. Zasp finding as a crucial adhesion component provides further insights on the mechanism underlying Integrin-mediated adhesion.
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To, Wing Shu. "Effect of cellular redox and energy states on benzo[a]pyrene induced modes of death in the hepa and the HepG2 cell lines." HKBU Institutional Repository, 2010. http://repository.hkbu.edu.hk/etd_ra/1173.
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Surmann, Eva-Maria. "Connections between tumour suppression and cellular metabolism." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709136.
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Pinent, Armengol Montserrat. "Adipose cell metabolism modulation by red wine procyanidins." Doctoral thesis, Universitat Rovira i Virgili, 2005. http://hdl.handle.net/10803/8650.
Full textAbstract:
Flavonoids, and more specifically, red wine procyanidins, have many beneficial effectsagainst pathologies such as cardiovascular heart disease and related illnesses. Although
adipose tissue has a central role in some of these pathologies, including obesity and diabetes,
there is a lack of information about the effects of procyanidins on this tissue. This thesis
addresses this question. The effects of a grape seed procyanidin extract (GSPE) on the lipid
and glucose metabolism of adipocytes were evaluated by taking the 3T3-L1 cell line as a
model of study. Results show that the GSPE has insulinomimetic effects, stimulating glucose
uptake, glycogen synthesis and trigliceride synthesis. To achieve this, the GSPE shares some
of the mechanisms and intracellular mediators of the insulin-signalling pathways (such as
GLUT-4 translocation, PI3K and p38 MAPK) but it must also use other, complementary,
mechanisms. These results suggest that procyanidins have beneficial effects on diabetes
and/or insulin resistance. This is partially proven by in vivo studies that show that GSPE has
antihyperglycemic properties on streptozotozin-induced diabetic rats. Also analyzed in this
thesis are the molecular mechanisms used by GSPE to explain the already described lipolytic
effects. Protein kinase A and PPARã are shown to be involved in these effects. Some of
these results opened up another line of study into the effects of GSPE on the differentiation
process of the 3T3-L1. These studies showed that procyanidins alter the differentiation of
preadipocytes when added at the induction of differentiation. Since an increase in the
number of adipocytes has a negative effect on obesity, this is a promising characteristic of
GSPE that should be taken into account when its possible antiobesity properties are studied.
Als flavonoides, i més concretament a les procianidines del vi negre, se'ls han atribuït moltes
propietats beneficioses contra diverses patologies, com les malalties cardiovasculars i altres
patologies relacionades. Tot i que el teixit adipós juga un paper important en algunes
d'aquestes patologies, com la obesitat i la diabetis, la informació referent l'acció de les
procianidines en aquest teixit és escassa. Aquesta tesis estudia els efectes de les procianidines
derivades de pinyol de raïm (GSPE) en l'adipòcit, i per a dur-ho a terme es pren com a
model d'estudi la línia cel.lular 3T3-L1. Per una banda es descriuen els efectes del GSPE en
el metabolisme de lípids i glúcids de la cèl.lula adiposa. El GSPE fa un paper
insulinomimètic: estimula la captació de glucosa, la síntesi de glicògen i la síntesi de triacil
glicerols. L'anàlisi dels mecanismes moleculars per exercir aquests efectes mostra que GSPE
en part comparteix mecanismes i vies de senyalització propis de la insulina (translocació de
GLUT-4, PI3K, p38 MAPK); tanmateix, s'observa que GSPE ha d'usar també altres
mecanismes complementaris. Aquests resultats suggereixen que GSPE pot tenir efectes
positius en situacions de diabetis i/o resistència a insulina, donat que a més a més, els estudis
in vivo mostren que GSPE és antihiperglicèmic en condicions de diabetis induïda per
estreptozotocina. En aquesta tesis també s'analitzen els mecanismes moleculars que
explicarien els efectes lipolítics de les procianidines descrits en estudis previs, i s'ha trobat
que la proteina kinasa A i PPARã hi estan involucrats. Part d'aquests resultats han obert una
altra via d'estudi sobre els efectes de la GSPE en el procés de diferenciació de la cèl.lula
adiposa on s'ha observat que el tractament amb procianidines a l'inici de la diferenciació
dificulta aquesta transformació. Donat que l'augment del nombre d'adipòcits afecta
negativament la obesitat, aquest efecte de les procianidines és una característica
prometedora que caldrà tenir en compte en l'estudi del seu possible paper antiobesitat.
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Saridogan, Ertan. "Cell biology and metabolism of human Fallopian tube." Thesis, Queen Mary, University of London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286201.
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Bibby, Susan R. S. "Cell metabolism and viability in the intervertebral disc." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249331.
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Simon, Charles. "Novel resveratrol analogues : synthesis, metabolism and cell proliferation." Thesis, University of Leicester, 2011. http://hdl.handle.net/2381/9289.
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Resveratrol or trans-3,4’,5-trihydroxystilbene is a naturally occurring phytochemical contained in red grapes skin, nuts and berries. It has been shown over the years to have different biological properties particularly in the chemoprevention of cancer. However, it is metabolised in vivo to sulfates and glucuronides within 1h and is active against different targets in a dose dependant manner. A library of new analogues of resveratrol has been synthesised with the aim of stopping or at least slowing down the metabolism whilst keeping its activity on the inhibition of cancer cell proliferation. Seven new analogues of resveratrol were synthesised in which the phenol substituents were systematically replaced by benzylic alcohols and/or methoxy groups. The library was then assessed in in vitro enzymatic metabolism with mouse and human liver fractions in the presence of a cofactor. The compounds were also evaluated as inhibitors of HCA-7 colorectal cancer cell proliferation.
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Hung, Yin Pun. "Single Cell Imaging of Metabolism with Fluorescent Biosensors." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10147.
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Cells utilize various signal transduction networks to regulate metabolism. Nevertheless, a quantitative understanding of the relationship between growth factor signaling and metabolic state at the single cell level has been lacking. The signal transduction and metabolic states could vary widely among individual cells. However, such cell-to-cell variation might be masked by the bulk measurements obtained from conventional biochemical methods. To assess the spatiotemporal dynamics of metabolism in individual intact cells, we developed genetically encoded biosensors based on fluorescent proteins. As a key redox cofactor in metabolism, NADH has been implicated in the Warburg effect, the abnormal metabolism of glucose that is a hallmark of cancer cells. To date, however, sensitive and specific detection of NADH in the cytosol of individual live cells has been difficult. We engineered a fluorescent biosensor of NADH by combining a circularly permuted green fluorescent protein variant with a bacterial NADH-binding protein Rex. The optimized biosensor Peredox reports cytosolic \(NADH:NAD^+\) ratios in individual live cells and can be calibrated with exogenous lactate and pyruvate. Notably pH resistant, this biosensor can be used in several cultured and primary cell types and in a high-content imaging format. We then examined the single cell dynamics of glycolysis and energy-sensing signaling pathways using Peredox and other fluorescent biosensors: AMPKAR, a sensor of the AMPK activity; and FOXO3-FP, a fluorescently-tagged protein domain from Forkhead transcription factor FOXO3 to report on the PI3K/Akt pathway activity. With perturbation to growth factor signaling, we observed a transient response in the cytosolic \(NADH:NAD^+\) redox state. In contrast, with partial inhibition of glycolysis by iodoacetate, individual cells varied substantially in their responses, and cytosolic \(NADH:NAD^+\) ratios oscillated between high and low states with a regular, approximately half-hour period, persisting for hours. These glycolytic NADH oscillations appeared to be cell-autonomous and coincided with the activation of the PI3K/Akt pathway but not the AMPK pathway. These results suggest a dynamic coupling between growth factor signaling and metabolic parameters. Overall, this thesis presents novel optical tools to assess metabolic dynamics – and to unravel the elaborate and complex integration of glucose metabolism and signaling pathways at the single cell level.
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Singhal, Atul. "Growth and metabolism in homozygous sickle cell disease." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288009.
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Hudson, Michael John. "Monoterpene metabolism of Mentha and its cell cultures." Thesis, Imperial College London, 1985. http://hdl.handle.net/10044/1/37729.
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Roohi, Aysha. "Toxoplasma gondii infection and the host cell metabolism." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648369.
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Green, Martha Alexandra. "Apoplastic ascorbate metabolism in rose cell suspension cultures." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/14944.
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Endogenous intraprotoplasmic ascorbate in rose cell suspension cultures as a model system ranged from 0.05 mmol kg-1 in 0-d-old cultures to 1.1 mmol kg-1 in 5-d-old cultures. Apoplastic ascorbate was estimated as 0.5 and 8 μM in 0- and 5-d-old cultures respectively, indicating that ascorbate is endogenous to, and may be metabolised within, the apoplast. Exogenous (apoplastic) 1 mM L-[1-14C]ascorbate was almost completely consumed (metabolised and/or taken up) by rose cultures within 8 hours of administration. Total 14C was removed from medium but slower than ascorbate. The calculated concentration of metabolites of ascorbate showed that metabolites were formed in the medium and then removed from the medium in 5-d-old cultures. Removal of metabolites could be due to either uptake by or binding to cells. The nature of the metabolites of 0.5 mM [1-14C]ascorbate was examined in 5-d-old rose culture and spent medium by electrophoresis at pH 6.5. Ascorbate was metabolised both enzymically in spent medium and non-enzymically in boiled spent medium. Three 14C-metabolites were identified as dehydroascorbate, diketogulonate and oxalate. Other acidic 14C-metabolites (C, D, E and F) have not as yet been identified. F is highly mobile during electrophoresis at pH 2.0, showing that it has a low pK. C, D and E are also mobile at pH 2.0 but less so than F. E and C are interconvertible non-enzymically during storage and can E be regenerated by treatment with NaOH, suggesting that C is a lactone of E. 14C-F was converted to [14C]oxalate by whole culture and by spent medium but not by boiled spent medium, indicating an enzyme-catalysed reaction. The enzyme was partially inhibited by 100 mM azide but not by antioxidants. [14C]Oxalate was produced from 14C-F by alkali hydrolysis indicating the presence of an oxalyl ester group. The metabolism of apoplastic ascorbate, described in this thesis, is very different from its intraprotoplasmic metabolism. I have identified novel metabolites and propose a novel pathway for the metabolism of apoplastic ascorbate.
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Chaneton, Barbara Julieta. "Targeting cancer cell metabolism as a therapeutic strategy." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5762/.
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In the past 15 years the field of cancer metabolism has burst providing vast quantities of information regarding the metabolic adaptations found in cancer cells and offering promising hints for the development of therapies that target metabolic features of cancer cells. By making use of the powerful combination of metabolomics and 13C-labelled metabolite tracing we have contributed to the field by identifying a mitochondrial enzymatic cascade crucial for oncogene-induced senescence (OIS), which is a tumour suppressive mechanism important in melanoma, linking in this way OIS to the regulation of metabolism. Furthermore, we have identified the dependency on glutamine metabolism as an important adaptation occurring concomitantly with the acquisition of resistance to vemurafenib (BRAF inhibitor) in melanoma, which opens the possibility to combine therapies targeting glutamine metabolism with BRAF inhibitors, in order to overcome or avoid the onset of resistance in melanoma. Using the same strategy we have discovered an important mechanism of interregulation between glycolysis and amino acid metabolism, identifying the glucose-derived amino acid serine as an activator of the main isoform of pyruvate kinase present in cancer cells, PKM2. In addition, we provide new insights into the mechanism of allosteric regulation of this complex protein and a better understanding of the way it regulates central carbon metabolism. In summary, our results open new possibilities for the development of cancer therapies that manipulate metabolic adaptations found in cancer cells in order to kill them specifically or halt their growth.
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Wei, Hsi-Ju. "TARGETING DENDRITIC CELL METABOLISM TO INDUCE IMMUNE TOLERANCE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1538497998943838.
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Zancanaro, Krauss Maria Eduarda. "CD4+ T cell metabolism during Trichuris muris infection." Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/cd4-t-cell-metabolism-during-trichuris-muris-infection(24eb0cc7-db70-46ea-ba49-e4fe3d5a5d03).html.
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Trichuris trichiura is a gastrointestinal dwelling nematode that infects almost 500 million people worldwide. T. muris occurs naturally in mice and is very closely related the human whipworm, making it a suitable model to dissect the immune response against the parasite. Studies using the Trichuris muris system have identified CD4+ T cells as dictators of the outcome of infection. In wild type mice, infection with a high dose of T. muris eggs leads to resistance and worm expulsion, which are dependent on a Th2 response and the secretion of type 2 cytokines especially interleukin (IL) 13. Chronicity is dependent on a Th1 response and occurs when mice are infected with a low dose of T. muris eggs. It is well established that metabolic changes are essential to promoting T cell activation and effector function. Moreover, during chronic infection the host immune system is continuously exposed to parasite antigen, which represents a metabolic challenge. This thesis has investigated the importance of T cell metabolism during response against T. muris. Data presented here show that low and high dose T. muris infections promote upregulation of the glycolytic pathway in CD4+ T cells. During later stages of chronic infection, CD4+ T cells displayed supressed glycolysis and mitochondrial respiration, and may be due to metabolic modulation imposed by the parasite. Leucine uptake via the amino acid transporter Slc7a5 was previously shown to be required for mTORC1 activation and for T cell effector function. Data presented here show that in early stages following a high dose T. muris infection, mice that lack Slc7a5 in T cells have delayed worm expulsion, impaired production of antibodies, and lower levels of IL-13. Their CD4+ T cells present reduced glycolytic rates when compared to cells from cohoused infected wild type mice. However, at later stages of infection, antibody, IL-13 and glycolytic levels were restored together with worm expulsion. CD4+ T cells from the early stage of infection showed reduced phosphorylation of mTOR, which suggested that impairment of function was mTOR dependent. Indeed, mice lacking mTOR in T cells fail to expel a high dose of parasites. They showed abrogation of IL-13 production, impairment in antibody class switching and their CD4+ T cells failed to upregulate glycolysis. Thus, this thesis shows that mTOR is essential for the proper functioning of T cells during T. muris infection and efficient amino acid transport plays a significant role. Taken together, these data show that metabolic orchestration of T cell function influences the capacity to effectively control helminth infection and that even subtle changes in T cell metabolic control can have a major effect on response phenotype.
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CASATTA, NADIA. "Exploring the metabolism beyond cell aging in yeast." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/41494.
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All living organisms undergo a progressive physiological decline with age that results in an increased risk of the development of many diseases. Up to now, many factors have been shown to be involved in aging, like oxidative damage, telomere erosion, mitochondrial dysfunction, genomic instability or epigenetic changes, but, although many efforts made, none of them is commonly recognized as the primary molecular cause of this phenomenon. The budding yeast Saccharomyces cerevisiae has proven to be an experimental useful model for studying the aging process leading to the identification of pathways whose counterparts can be found in higher eukaryotes and in particular in humans. In yeast, the two paradigms of aging are described: the replicative aging and the chronological aging. The latter refers to the aging of cells in a quiescent-like state, with chronological lifespan (CLS) defined as the length of time that non-dividing yeast cells can maintain replicative potential. CLS is influenced by several factors, both intrinsic and extrinsic. The former group includes the signal transduction pathways involved in stress and nutrient responses, such as the TOR/Sch9 and the Ras/PKA pathways. Among the latter, in yeast two byproducts of the cell metabolism seem to play a determinant role: acetate and ethanol. In particular, their presence in the exhausted growth medium has a pro-aging effect, even though the exact role they play is still a matter of debate.
Moreover, a key role in lifespan regulation is widely recognized for Sir2, the founder of Sirtuins, a family of highly conserved NAD-dependent histone deacetylases. Unlike the other families of deacetylases, Sirtuins couple protein deacetylation with the cleavage of NAD+, a feature that makes them key elements for the interconnection between cell homeostasis and aging. In addition, Sirtuins also influence the activity of many metabolic enzymes in humans by modulating their acetylation state, strengthening the linkage between the metabolic status of the cell and Sirtuin function.
In this context, this work aims to deepen the knowledge on some extrinsic and intrinsic regulators of chronological aging. Among intrinsic factors, we analyzed Sir2, which plays a pro-aging role in CLS. We found that the lack of this deacetylase influences some aspects of the cell metabolism, with a particular regard to ethanol and acetate. Among the extrinsic factors, and particularly dealing with acetate, we focused on Ach1, a mitochondrial enzyme, whose function has not been well characterized yet but that may play a key role in the metabolism of acetic acid at mitochondrial level.
To analyze the interconnections among Sir2 activity, lifespan and the cell metabolism, we performed experiments in batch with fermentative and non-fermentative carbon sources, during chronological aging and in chemostat (glucose-limited cultures pulsed with ethanol and acetate) where growth parameters together with metabolite contents were analyzed. During chronological aging, CLS was also determined. Since in cells lacking Sir2 we determined an increase in acetate and ethanol catabolism, we focused on gluconeogenesis (in particular on Pck1, the rate limiting enzyme) and on the glyoxylate cycle (in particular on one of the two unique enzymes of this cycle, Icl1). In fact, both processes are fundamental when cells are growing on acetate and ethanol. By measuring the activity of Pck1 and Icl1, we found that in sir2D cells both these enzymatic activities were enhanced. In particular, the increased activity of Pck1 correlated with a higher acetylation level of this protein, giving also experimental evidence of a model where it has been proposed that Pck1, acetylated by Esa1, could be the target of Sir2- mediated deacetylation. Moreover, to check whether the activity of these enzymes was related with the CLS phenotype of sir2D cells, we inactivated ICL1 and PCK1 in sir2D cells to analyze the longevity and the metabolite level of the double mutant strains during chronological aging: both icl1D and pck1D mutants accumulated high level of extracellular acetate and ethanol and were short-lived mutants. In addition, ICL1 and PCK1 inactivation had epistatic effects on sir2D cells. Moreover, consistent with an increased gluconeogenetic flux, trehalose levels in sir2D stationary phase cells were higher compared with wild type cells. On the whole, we provided evidence that SIR2 inactivation positively affects acetate metabolism by enhancing the glyoxylate-requiring gluconeogenesis. In the aging context, this implies lower levels of negative extracellular factors and a major accumulation of protective trehalose which create a beneficial environment for long-term survival of non-dividing cells.
Then, we focused on acetate, and in particular on Ach1, a mitochondrial enzyme whose exact function has not been well characterized. In the ’90, Ach1 was identified as mitochondrial hydrolase, even though the physiological role of acetyl-CoA hydrolysis was not clear. Recently, in Aspergillus nidulans an enzyme with high sequence identity with Ach1 was identified which is involved in the process of propionate detoxification. On the basis of this, the hypothesis arose that in S. cerevisiae as well this enzyme could catalyze a transferase reaction by activating, rather than hydrolyzing, acetyl-CoA from acetate. In this work of thesis, we characterized the phenotype of ach1D cells, with the aim of better understanding the role of this protein in acetate metabolism and any potential implications on mitochondrial functionality and on CLS modulation. We found that chronologically aging ach1D cells accumulated a high amount of extracellular acetate which correlated with a short-lived phenotype. This phenotype was strictly dependent on extracellular acetate since, when the acid stress was abrogated either by a calorie restricted regimen (no acetic acid production) or by transferring chronologically aging ach1D cells to water, cell survival was restored. Moreover, the short-lived phenotype of chronological aging ach1D cells is accompanied by ROS accumulation, a compromised mitochondrial activity and a precocious activation of the Yca1-dependent apoptotic pathway. In agreement with this compromised mitochondrial activity, we also saw that ach1D cells had severe problems to grow in media containing acetate as carbon source, underlining a primary role of Ach1 enzymatic activity in acetic acid detoxification which is important for mitochondrial functionality. Mitochondrial functionality which also plays an essential role for chronological cell survival.
Since we found an inverse correlation between extracellular ethanol and acetic acid level and CLS, further experiments were performed to clarify the role played by these two pro-aging factors. Data obtained support the hypothesis that at physiological levels is not the mere presence of ethanol and acetic acid to influence the CLS but it is their metabolism. Thus, both these C2 compounds act as carbon sources that prevent entry of cells into a calorie restriction-like state, the only one in which cells are able to maintain a long term survival.
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BHARAT, ROHIT. "Targeting cancer cell metabolism: Gateway towards personalized medicine." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241161.
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Nell'ultimo decennio, uno dei messaggi chiave derivante dalle ricerche scientifiche sul cancro è la necessità di comprendere meglio il metabolismo delle cellule tumorali per lo sviluppo di una terapia personalizzata migliore e più efficace. Le cellule tumorali attuano un riarrangiamento metabolico che coinvolge diversi processi per supportare la loro natura proliferativa ed invasiva.
Per meglio comprendere l’entità del cambiamento metabolico, in questo studio abbiamo utilizzato un approccio systems level impiegando la metabolomica untargeted e la flussomica mediante isotopi stabili del carbonio (13C) in cellule tumorali esprimenti un K-Ras oncogenico.
Abbiamo testato gli effetti di farmaci inibitori del metabolismo di glucosio e glutammina per indagare eventuali vie metaboliche alternative attivate per la sopravvivenza delle cellule tumorali. Inoltre abbiamo investigato il ruolo del metabolismo cellulare nello sviluppo della resistenza alla terapia endocrina nel carcinoma mammario ERα positivo. I dati ottenuti hanno permesso di identificare specifici meccanismi metabolici di utilizzo della glutammina in cellule resistenti alla terapia, suggerendo l’utilizzo del farmaco metformina come adiuvante nel trattamento dei tumori resistenti alla terapia ormonale.
Infine, abbiamo contribuito alla comprensione del metabolismo delle cellule tumorali nel guidare la crescita e la proliferazione, esplorando il ruolo della glutammina oltre la nota funzione di fonte di carbonio e azoto; infatti sostituendo la glutammina con fonti alternative di carbonio e azoto, si osserva un fenotipo reverse Warburg.
I risultati di questa tesi aprono strade di ricerca per l'identificazione di nuovi potenziali obiettivi terapeutici e ci portano verso la progettazione di una strategia terapeutica migliore e più efficace per il trattamento dei pazienti oncologici.In the recent decade, one of the important keynote message derived through the summation of our global efforts against cancer is the need to better understand cancer cell metabolism for the development of better and efficacious personalized therapy. Cancer cells undertake a multifaceted rewiring of metabolic pathways in order to support their proliferative and invasive nature, which requires a systems level investigation to fully comprehend the scale of metabolic deregulation. In this study, we systematically investigated the metabolic differences using untargeted metabolomics and 13C flux omics approach in oncogenic K-Ras driven tumours. We tested the effects of drug inhibitors targeting glucose and glutamine metabolism to unravel the alternative metabolic pathways required for cancer cell survival. We further expanded our research towards understanding the role of cellular metabolism in driving resistance to endocrine therapeutic drugs in ERα positive breast cancer. We identified specific metabolic mechanisms of utilization of glutamine in resistant cells while also providing further basis for the use of metformin as an adjuvant in the treatment of endocrine therapyresistant cancers. Finally, we contributed to current understanding about cancer cell metabolism by exploring the role of glutamine beyond its role as a carbon and nitrogen source in driving growth and proliferation of cancer cells. Upon substitution of glutamine with appropriateiv nitrogen and carbon sources, cancer cells exhibited reverse Warburg phenotype. The findings from this thesis open up new avenues of research through the identification of new putative targets and bring us one step closer towards designing much better and efficacious therapeutic strategy for the treatment of cancer patients.
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Sujareerat, Charin. "Development of L1210 mutants in NAD metabolism." Thesis, University of Bath, 1989. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235333.
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Krzywinska, Ewelina. "Study of tumor cell metabolism and its relationship with NK cell-mediated immunotherapy." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON1T012/document.
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La formation et le développement d'une tumeur sont provoqués par une série de défauts qui se produisent à l'intérieur de la cellule cancéreuse et dans son microenvironnement. Ces anomalies permettent à la cellule de développer ses propres stratégies de croissance, de prolifération, de différenciation et de métabolisme. Toutes ces adaptations, ainsi que la création d'un micro-environnement unique favorisent la croissance de la tumeur et inhibent la réponse immunitaire anti-tumorale. Le métabolisme des cellules cancéreuses et l'évasion immunitaire sont des points très sensibles dans le développement des cancers et peuvent être utilisés en clinique. Les études récentes suggèrent que ces deux phénomènes sont liés, et que le métabolisme des cellules cancéreuses peut amener à l'échappement immunitaire par la tumeur. Le métabolisme des cellules tumorales a tendance à éviter l'activité mitochondriale et la phosphorylation oxydative, et est principalement basée sur la glycolyse pour la production d'énergie (effet Warburg). Mon travail de thèse est divisé en deux parties. Dans la première partie nous avons proposé un concept thérapeutique novateur avec une nouvelle thérapie combinatoire pour le traitement de cancers hématologiques. Cette thérapie est basée sur l'induction de changements métaboliques par le dichloroacétate (DCA), et elle est associée avec la chimiothérapie conventionnelle (doxorubicine, vincristine) pour réactiver les fonctions de p53. Les tumeurs avec p53 mutantes sont résistantes à cette combinaison. Dans ce cas, nous avons constaté que le DCA peut coopère avec 17-AAG (l'inhibiteur de Hsp90) pour éliminer spécifiquement les cellules cancéreuses. En conséquence, une meilleure compréhension des signaux et des mécanismes par lesquels le DCA sensibilise les cellules tumorales à la chimiothérapie est nécessaire pour en comprendre le mode d'action. En outre, l'identification de ce mécanisme permettra d'élucider les voies métaboliques impliquées dans la survie des cellules cancéreuses. La deuxième partie de ma thèse se concentre sur la biologie des cellules NK. Les cellules NK sont des lymphocytes du système immunitaire inné et possèdent une cytotoxicité naturelle contre les cibles, c'est à dire les cellules tumorales. L'utilisation optimale des cellules NK en clinique nécessite leur expansion et leur activation in vitro. Les cellules NK s'activent en présence de cytokines ou par le contact avec les cellules cibles. L'activation des cellules NK induit la prolifération, mais celle-ci dépend aussi de la présence d'autres cellules immunitaires. L'activation, par les cytokines et par les cellules cibles, induit un différent ARNm/microARN profil d'expression. L'analyse détaillée des isoformes de la protéine tyrosine phosphatase CD45 a permis de caractériser de nouvelles populations de cellules NK anti-tumorales humaines. L'identification de différentes populations de cellules NK est très importante pour la compréhension de leur physiologie et pour l'amélioration de leur utilisation en immunothérapie clinique. Cela peut également donner des informations précieuses sur l'état physiologique de l'hôte. En effet, l'augmentation des cellules CD45RAdim et CD45RO + dans le compartiment des cellules NK matures identifie clairement les patients avec des hémopathies malignes. Nous pensons que leur détection peut être utilisée comme un outil de diagnostic et également pour évaluer l'efficacité des traitements anti-tumoraux, car ces populations de cellules NK spécifiques devraient diminuer lors de l'élimination de cellules tumorales cibles. Dans l'avenir, nous voulons combiner le traitement du métabolisme de la tumeur avec la thérapie anti-tumorale basée sur les cellules NK. Sur la base de nos données préliminaires, nous pouvons proposer le traitement des cellules cancéreuses par des médicaments métaboliques pour augmenter la sensibilité et la reconnaissance par les cellules NK activéesTumor formation and development are caused by a range of defects that occur inside the cancer cell and in the external cellular microenvironment. These abnormalities allow developing tumors to establish their own strategies of growth, proliferation, differentiation and metabolism. All these adaptations, as well as the creation of a unique microenvironment, promote tumor growth and suppress the anti-cancer immune response. Tumor cell metabolism and immune evasion are sensitive points of cancer development that can be targeted in clinic. Recent studies suggest that these two phenomena are related and that cancer cell metabolism may propel tumor immune escape. Tumor cell metabolism tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS), and largely relies on glycolysis to produce energy (Warburg effect). My thesis work is divided into two parts. The first one proposes an innovative therapeutic strategy, which is the use of different combinatorial therapy depending on the p53 status for the treatment of hematological cancers. This is based on the induction of metabolic changes by dichloroacetate (DCA), combined with conventional chemotherapy (doxorubicin, vincristine) to reactivate wild type p53 functions. Mutant p53 tumors are resistant to this combination approach. However, we found that DCA synergized with the Hsp90 inhibitor 17-AAG to specifically eliminate these cells. Therefore, a clearer understanding of the signals and mechanisms by which DCA sensitize cancer cells to chemotherapy was needed to understand its mode of action. We uncovered it in our work. In addition, identification of this mechanism will help to elucidate metabolic pathways involved in cancer cell survival.The second part of my thesis is focused on the study of NK cell biology. NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity against targets, i.e. tumor cells. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and the encounter with target cells activate NK cells, induce their proliferation, and cause clearly different mRNA/miRNA expression profile. Detailed analysis of the leucocyte-specific phosphatase CD45 isoforms allowed us to characterize new human anti-tumor NK cell populations. The identification of the different NK cell populations is important for understanding their physiology and for improving their therapeutic use in the clinic. It can also give valuable information about the host physiological status. Indeed, the increase of CD45RAdim and CD45RO+ cells in the mature NK cell compartment clearly identifies patients with hematological malignancies. We thus hypothesize that their detection could be used as a diagnostic tool, and also to assess the efficacy of antitumor treatments, because these specific NK cell populations should decrease upon removal of the targeted tumor cells. Our future goal is to use a novel combinatorial therapy in hematological cancers that will combine metabolic drugs and NK cell-based therapy. Based on our preliminary data, we propose that the treatment of cancer cells with metabolic drugs could increase their sensitivity and recognition by activated NK cells
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Escoté, Miró Xavier. "Control of cell cycle progression by the last MAPK Hog1." Doctoral thesis, Universitat Pompeu Fabra, 2005. http://hdl.handle.net/10803/7186.
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Exposure of yeast to increases in extracellular osmolarity activates the stress-activated Hog1 MAP kinase, which is essential for cell survival upon osmotic stress. Activation of the Hog1 MAPK results in cell growth arrest, suggesting a possible role of the MAP kinase in the control of the cell cycle. Our results have shown that Hog1 activation resulted in accumulation of cells in the G1/S and G2/M transitions. At G1, Hog1 regulates the cell cycle progression by a dual mechanism that involves downregulation of G1 cyclin expression and direct targeting of the CDK-inhibitor protein Sic1. The MAPK interacts with Sic1, and phosphorylates a single residue of Sic1, which, in combination with the downregulation of cyclin expression, results in Sic1 stabilization and inhibition of cell cycle progression. Consistently, sic1_ cells, or cells containing a SIC1 allele mutated in the Hog1 phosphorylation site, are unable to arrest at G1 phase after Hog1 activation, and become sensitive to osmostress. Together, our data indicate that Sic1 is the molecular target for Hog1 that is required to modulate cell cycle progression in response to stress at G1. On the other hand, activation of the Hog1 MAPK also results in an increase of cells in the G2 phase. Arrested cells displayed down regulation of the Clb2-Cdc28 kinase activity and consequently enlarged buds, defects in spindle formation and orientation. These effects were prevented by deletion of the SWE1 gene. Thus, swe1Ä cells failed to arrest at G2, which resulted in a premature entry into mitosis and mislocalization of nuclei. Consistently, swe1Ä cells were osmosensitive. Swe1 degradation was reduced in response to activation of Hog1. Swe1 accumulation is mediated by the activity of the complex Hsl1-Hsl7. Hog1 phosphorylates a single residue at the regulatory domain of Hsl1, which leads to the mislocalization of Hsl7 from the bud neck, and consequent Swe1 accumulation. In addition, Hog1 downregulates G2 cyclin expression, reinforcing the inhibition of cell cycle progression at G2/M. These results indicate that Hog1 imposes a delay in critical phases of cell cycle progression necessary for proper cellular adaptation to new extracellular conditions.
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Dimasi, Don. "A mechanistic analysis of mammalian cell metabolism in continuous culture /." Thesis, Connect to Dissertations & Theses @ Tufts University, 1992.
Find full textAbstract:
Thesis (Ph.D.)--Tufts University, 1992.Submitted to the Dept. of Chemical Engineering. Adviser: Randall W. Swartz. Includes bibliographical references (leaves 243-247). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Lähdesmäki, Ilkka Johannes. "Flow injection methods for drug-receptor interaction studies, based on probing cell metabolism /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8590.
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Nilsson, Harriet. "The role of nitric oxide in cytoskeleton-mediated organelle transport and cell adhesion /." Linköping : Univ, 2001. http://www.bibl.liu.se/liupubl/disp/disp2001/med660s.pdf.
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Theret, Marine. "Cell and non-cell autonomous regulations of metabolism on muscle stem cell fate and skeletal muscle homeostasis." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB120/document.
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A l’état basal, les cellules souches musculaires sont quiescentes. Après blessure, ces cellules s’activent, s’amplifient et se différencient afin de réparer les myofibres lésées. Cependant, une petite population de ces cellules myogéniques activées ne va pas entrer dans la voie de la myogenèse, mais va retourner en quiescence par un phénomène appelé auto-renouvellement. Cette étape est cruciale afin de maintenir une réserve de cellules souches musculaires tout au long de la vie. Mais, les mécanismes cellulaires et moléculaires régulant ce phénomène sont peu décrits dans la littérature. La régénération musculaire est composée d’une série d’évènements complexes et bien orchestrés selon une cinétique précise. Le challenge de son étude est donc de pouvoir distinguer les évènements les uns des autres, tout en sachant qu’ils sont interconnectés. Bien que les cellules souches musculaires aient un fort potentiel de régénération, elles ont besoin d’interagir avec d’autres cellules au cours de la régénération, notamment avec les macrophages qui ont un rôle prépondérant dans ce processus. Après une blessure, les monocytes circulants sont recrutés sur le site de lésion et se différencient en macrophages inflammatoires. Ensuite, ces macrophages changent leur statut inflammatoire et acquièrent un profil anti-inflammatoire. Plusieurs études in vitro ont suggéré un rôle pour le métabolisme et son régulateur principal, la kinase activée par l’AMP (AMPK), dans la résolution de l’inflammation et dans le devenir des cellules souches adultes. Ainsi, j’ai étudié l’influence extrinsèque (via les macrophages) et intrinsèque du métabolisme sur le devenir des cellules souches musculaires au cours de la régénération. Pour cela, j’ai utilisé divers modèles déficients pour l’AMPK1 dans le macrophage, dans la cellule souche musculaire et dans la myofibre qui m’ont permis d’établir des cultures primaires de macrophages et de cellules musculaires. Dans un premier temps, grâce à ces outils, nous avons pu démontrer le rôle primordial de l’AMPK dans la résolution de l’inflammation au cours de la régénération musculaire et dans l’acquisition des fonctions anti-inflammatoires des macrophages. Dans ce contexte, l’activation de l’AMPK est dépendante de la kinase CAMKK et régule la phagocytose, principal phénomène cellulaire permettant le changement de statut inflammatoire des macrophages. Ce travail a été publié en 2013 dans le journal Cell Metabolism. Ensuite, j’ai mis en évidence un lien entre le métabolisme et le devenir des cellules souches musculaires. La suppression de l’AMPK dans les cellules souches musculaires augmente leur auto-renouvellement. Cette modification du devenir des cellules souches est due à un changement de métabolisme similaire à l’effet Warburg observé dans les cellules souches cancéreuses, qui s’effectue via la modulation de l’activité de l’enzyme Lactate Déshydrogénase, enzyme clé de la glycolyse. En conclusion, j’ai pu mettre en évidence deux nouveaux rôles de l’AMPK dans le devenir des cellules souches musculaires, établissant un lien de causalité entre métabolisme, inflammation et devenir des cellules souchesDuring skeletal muscle regeneration, muscle stem cells activate and recapitulate the myogenic program to repair the damaged myofibers. A subset of these cells does not enter into the myogenesis program but self-renews to return into quiescence for further needs. Control of muscle stem cell fate choice is crucial to maintain homeostasis but molecular and cellular mechanisms controlling this step are poorly understood. A difficulty of understanding muscle stem cell self-renewal is that skeletal muscle regeneration is a coordinated and non-synchronized process. Various and dissociated molecular and cellular mechanisms regulate muscle stem cell fate. Indeed, skeletal muscle regeneration requires the interaction between myogenic cells and other cell types, among which the macrophages. Macrophages infiltrate the muscle and adopt distinct and sequential phenotypes. They act on the sequential phases of muscle regeneration and resolving the inflammation by skewing their inflammatory profile to an anti-inflammatory state. Some in vitro studies suggested a role for the metabolism and the AMP-activated protein Kinase (AMPK), the master metabolic regulator of cells, in both inflammation and stem cell fate. Thus, I investigated the role of metabolism on muscle stem cell fate within the muscle stem cells (cell autonomous regulations) and through the action of macrophages (non-cell autonomous regulations) during skeletal muscle regeneration. To analyze muscle stem cell fate, I used in vitro (macrophages and muscle stem cell primary cultures), ex vivo (isolated myofibers) and in vivo (using specific mice model deleted specifically for AMPK1 in the myeloid lineage, in muscle stem cells or in myofibers) experiments. First, I highlighted that macrophagic AMPK1is required for the resolution of inflammation during skeletal muscle regeneration and for the trophic functions of macrophages on muscle stem cell fate. Moreover, CAMKK-AMPK1 activation regulates phagocytosis, which is the main cellular mechanism inducing macrophage skewing. This work was published in 2013 in Cell Metabolism. Second, I demonstrated that depletion of myogenic AMPK1 tailors muscle stem cell metabolism in a LKB1 independent manner, orients their fate to the self-renewal by promoting metabolic switch from an oxidative to a glycolytic metabolism pathway, through the over activation of a new molecular target, which is a key enzyme for glycolysis: the Lactate Dehydrogenase. To conclude, during my thesis, I established two new crucial roles of AMPK1 in muscle stem cell fate choice, linking for the first time metabolism, inflammation and fate choice
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Westermark, Pål. "Models of the metabolism of the pancreatic beta-cell." Doctoral thesis, KTH, Numerical Analysis and Computer Science, NADA, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-408.
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The pancreatic β-cell secretes insulin in response to a raised blood glucose level. Deficiencies in this control system are an important part of the etiology of diabetes. The biochemical basis of glucose-stimulated insulin secretion is incompletely understood, and a more complete understanding is an important component in the quest for better therapies against diabetes.
In this thesis, mathematical modeling has been employed in order to increase our understanding of the biochemical principles that underlie glucosestimulated insulin secretion of the pancreatic β-cell. The modeling efforts include the glycolysis in theβ-cell with particular emphasis on glycolytic oscillations. The latter have earlier been hypothesized to be the cause of normal pulsatile insulin secretion. This model puts this hypothesis into quantitative form and predicts that the enzymes glucokinase and aldolase play important roles in setting the glucose concentration threshold governing oscillations. Also presented is a model of the mitochondrial metabolism in the β-cell, and of the mitochondrial shuttles that connect the mitochondrial metabolism to the glycolysis. This model gives sound explanations to what was earlier thought to be paradoxical behavior of the mitochondrial shuttles during certain conditions. Moreover, it predicts a strong signal from glucose towards cytosolic NADPH formation, a putative stimulant of insulin secretion. The model also identifies problems with earlier interpretations of experimental results regarding the β- cell mitochondrial metabolism. As an aside, an earlier proposed conceptual model of the generation of oscillations in the TCA cycle is critically analyzed.
Further, metabolic control analysis has been employed in order to obtain mathematical expressions that describe the control by pyruvate dehydrogenase and fatty acid oxidation over different aspects of the mitochondrial metabolism and the mitochondrial shuttles. The theories developed explain recently observed behavior of these systems and provide readily testable predictions.
The methodological aspects of the work presented in the thesis include the development of a new generic enzyme rate equation, the generalized reversible Hill equation, as well as a reversible version of the classical general modifier mechanism of enzyme action.
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Pryjma, Mark Christopher. "Campylobacter jejuni metabolism in survival and host cell interactions." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/48597.
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Campylobacter jejuni is a leading cause of foodborne bacterial gastroenteritis in both developed and developing nations. Although C. jejuni is a common environmentally acquired pathogen, it is quite fastidious, rapidly losing viability in aerobic conditions. Genome sequence analyses have failed to identify classical virulence factors, making the pathogenic success of C. jejuni a mystery. Mutational analysis described herein identified novel metabolic factors that are important for infection of human epithelial cells as well as generation of oxidative stress in C. jejuni during aerobic incubation. Investigation of a novel operon, fdhTU, induced during C. jejuni epithelial infection, showed that FdhTU positively regulates formate dehydrogenase. Subsequent analyses found that fdhTU and formate dehydrogenase are important for recovery of C. jejuni from epithelial cells. Further work showed that intracellular C. jejuni are undergoing oxidative stress, and that neutralization of oxidative stress with sulfite or catalase could significantly enhance recovery of C. jejuni following epithelial cell infection. Analyses of other respiratory dehydrogenases failed to identify other systems important for recovery of C. jejuni from epithelial cells, but did identify a role for gluconate dehydrogenase in reducing necrosis in T84 epithelial cells in a reactive oxygen species- and calpain-dependent manner. In addition to the importance for epithelial cell infection, metabolic features were also found to be involved in causing oxidative stress in C. jejuni under aerobic conditions. C. jejuni was found to produce H₂O₂ when incubated in aerobic but not microaerobic conditions at 37ºC but not 4ºC, with formate dehydrogenase and sulfite oxidoreductase dependent respiration important for H₂O₂ production. Sulfite and cysteine could reduce C. jejuni loss of viability in aerobic conditions in a manner dependent on the sulfur assimilation pathway protein Atps. Atps was identified as important for aerobic survival, H₂O₂ resistance, and in reducing H₂O₂ produced by formate dehydrogenase dependent respiration. Characterization of the role of multiple respiratory systems in C. jejuni, a bacterial model that shares little with other common pathogenic bacteria, has identified a central role of respiration in epithelial cell infection and environmental survival.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Papas, Klearchos Kyriacos. "Bioenergetics, metabolism, and secretion of immunoisolated endocrine cell preparations." Thesis, Georgia Institute of Technology, 1996. http://hdl.handle.net/1853/11001.
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